the j biological c vol. 279, no. 37, issue of...

Nuclear Factor-inducing Kinase Plays a Crucial Role inOsteopontin-induced MAPK/I�B� Kinase-dependent NuclearFactor �B-mediated Promatrix Metalloproteinase-9 Activation*

Received for publication, April 27, 2004, and in revised form, July 2, 2004Published, JBC Papers in Press, July 7, 2004, DOI 10.1074/jbc.M404674200

Hema Rangaswami, Anuradha Bulbule, and Gopal C. Kundu‡

From the National Center for Cell Science, Pune 411 007, India

We have recently demonstrated that osteopontin(OPN) induces nuclear factor �B (NF�B)-mediatedpromatrix metalloproteinase-2 activation through I�B�/I�B� kinase (IKK) signaling pathways. However, the mo-lecular mechanism(s) by which OPN regulates proma-trix metalloproteinase-9 (pro-MMP-9) activation, MMP-9-dependent cell motility, and tumor growth and theinvolvement of upstream kinases in regulation of theseprocesses in murine melanoma cells are not well de-fined. Here we report that OPN induced �v�3 integrin-mediated phosphorylation and activation of nuclear fac-tor-inducing kinase (NIK) and enhanced the interactionbetween phosphorylated NIK and IKK�/� in B16F10cells. Moreover, NIK was involved in OPN-induced phos-phorylations of MEK-1 and ERK1/2 in these cells. OPNinduced NIK-dependent NF�B activation through ERK/IKK�/�-mediated pathways. Furthermore OPN en-hanced NIK-regulated urokinase-type plasminogen acti-vator (uPA) secretion, uPA-dependent pro-MMP-9activation, cell motility, and tumor growth. Wild typeNIK, IKK�/�, and ERK1/2 enhanced and kinase-negativeNIK (mut NIK), dominant negative IKK�/� (dn IKK�/�),and dn ERK1/2 suppressed the OPN-induced NF�B acti-vation, uPA secretion, pro-MMP-9 activation, cell motil-ity, and chemoinvasion. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibodydrastically inhibited the OPN-induced cell migrationand chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinctpathways. The level of active MMP-9 in the OPN-in-duced tumor was higher compared with control. To ourknowledge, this is the first report that NIK plays a cru-cial role in OPN-induced NF�B activation, uPA secre-tion, and pro-MMP-9 activation through MAPK/IKK�/�-mediated pathways, and all of these ultimately controlthe cell motility, invasiveness, and tumor growth.

Osteopontin (OPN)1 is a secreted, non-collagenous, sialicacid-rich phosphoglycoprotein (1, 2). OPN acts both as che-

mokine and cytokine. It has an N-terminal signal sequence, ahighly acidic region consisting of nine consecutive asparticacid residues, and a GRGDS cell adhesion sequence predictedto be flanked by the �-sheet structure (3). This protein has afunctional thrombin cleavage site and is the substrate fortissue transglutaminase (2). It is produced by osteoclast,macrophages, T cells, hematopoietic cells, and vascularsmooth muscle cells (4). OPN binds with type I collagen (5),fibronectin (6), and osteocalcin (7). Several highly metastatictransformed cells synthesize higher levels of OPN comparedwith non-tumorigenic cells (8). It has been shown that OPNalso interacts with several integrins and CD44 variants in anRGD sequence-dependent and -independent manner (9, 10).OPN is involved in normal tissue remodeling processes suchas bone resorption, angiogenesis, wound healing, and tissueinjuries as well as certain diseases such as restenosis, ath-erosclerosis, tumorigenesis, and autoimmune diseases (10–12). OPN causes cell adhesion, migration, extracellular ma-trix (ECM) invasion, and cell proliferation by interaction withits receptor �v�3 integrin in various cell types (10). Integrinsare noncovalently associated, heterodimeric, cell surface gly-coproteins with �- and �-subunits.

The NF�B family consists of several members including p65,p50, Rel B, and c-Rel molecules (13). The activity of NF�B istightly regulated by its inhibitors, the I�B family of proteins(14). These inhibitory proteins bind to NF�B dimers, maskingtheir nuclear localization sequence resulting in cytoplasmicretention of NF�B (15). Upon stimulation, I�B is phosphoryl-ated and degraded via the ubiquitination and proteosome-me-diated pathway leading to the nuclear import of NF�B where itbinds to cognate sequence in promoter regions of multiplegenes. I�B� kinase (IKK) is a multisubunit protein kinase, theactivation of which phosphorylates I�B. Nuclear factor-induc-ing kinase (NIK) is a member of the mitogen-activated proteinkinase kinase kinase family that may either directly or indi-rectly phosphorylate or activate IKK�/�, leading to the phos-phorylation and degradation of I�B� followed by NF�B activa-tion (16). Previous data also has indicated that NIK regulatesdifferentiation of PC-12 cells through phosphorylation and ac-tivation of MEK-1 (17). The data also show that constitutivelyexpressed or overexpressed wild type NIK in the presence ofTNF, interleukin-1, TRAF-2, and TRAF-6 induced, and kinase-negative NIK (mut NIK) in the presence of the same stimulisuppressed, the NF�B activation (18, 19). Our previous resultsindicate that OPN induces cell motility, tumor growth, NF�B-

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ To whom correspondence should be addressed: National Center forCell Science (NCCS), NCCS Complex, Pune 411 007, India. Tel.: 91-20-25690951 (ext. 203); Fax: 91-20-25692259; E-mail: [email protected].

1 The abbreviations used are: OPN, osteopontin; NIK, nuclear factor-inducing kinase; NF�B, nuclear factor �B; I�B�, inhibitor of nuclearfactor �B; IKK, I�B� kinase; MAPK, mitogen-activated protein kinase;ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase;

EMSA, electrophoretic mobility shift assay; Luc, luciferase; uPA, uroki-nase-type plasminogen activator; MMP, matrix metalloproteinase;ECM, extracellular matrix; mut, kinase-negative; dn, dominant nega-tive; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor;p-, phospho-; wt, wild type; DTT, dithiothreitol; MT1, membrane type-1.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 37, Issue of September 10, pp. 38921–38935, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 38921

mediated urokinase-type plasminogen activator (uPA) secre-tion, and promatrix metalloproteinase-2 (pro-MMP-2) activa-tion through the phosphatidylinositol 3-kinase/IKK/Aktsignaling pathways (20–22). However, the signaling pathwayby which OPN regulates NIK activation and NIK-dependentMAPK/IKK-mediated NF�B activation through phosphoryla-tion and degradation of I�B� in B16F10 cells is not welldefined.

uPA is a serine protease that interacts with uPA receptorand facilitates the conversion of inert zymogen plasminogeninto widely acting serine protease plasmin and activation ofMMPs (23, 24). These proteases then degrade the surround-ing matrix components (collagen, gelatin, fibronectin, andlaminin) and allow cancer cells to migrate to distant sites. Itis established that uPA plays a significant role in tumorgrowth and metastasis (25, 26). NF�B-response element ispresent in the promoter region of uPA, which plays a key rolein cancer metastasis. However, the molecular mechanism bywhich OPN regulates NIK activation and NIK-dependentMAPK- and IKK-mediated NF�B activation and uPA secre-tion, which lead to the activation of pro-MMP-9 and controlcell migration, ECM invasion, and tumor growth, is not welldocumented.

MMPs are ECM-degrading enzymes that play a critical rolein embryogenesis, tissue remodeling, inflammation, and angio-genesis (27). We have recently reported that OPN inducesNF�B-mediated pro-MMP-2 activation through I�B�/IKK sig-naling pathways (20, 21). MMP-9 (also called type IV collagen-ase or gelatinase B) is another important contributor to theprocess of invasion, tumor growth, and metastasis (28–31).MMP-9 can efficiently degrade native type IV and V collagens,fibronectin, ectactin, and elastin. The regulation of activationof MMP-9 is more complex than that of most of the other MMPsbecause most of the cells do not express a constitutively activeform of MMP-9, but its activity is induced by different stimulidepending on cell types (32–34) thereby contributing to thespecific pathological events. MMP-9 is not only associated withinvasion and metastasis but also has been implicated in angio-genesis, rheumatoid arthritis, retinopathy, and vascular steno-sis, and hence MMP-9 is considered to be a therapeutic targetof high priority (35). Earlier reports indicate that transgenicmice lacking MMP-9 show reduced keratinocyte hyperprolif-eration and decreased incidence of invasive tumors (36). NF�B-responsive element is present in the promoter region ofMMP-9, which plays a key role in cancer metastasis. However,the molecular mechanism by which OPN regulates NF�B-me-diated pro-MMP-9 activation and controls cell migration andtumor growth is not well understood.

In this study, we found that OPN induced phosphorylationand activation of NIK and the interaction between phosphoryl-ated NIK and IKK in B16F10 cells. OPN enhanced NIK- andIKK-dependent NF�B activation. OPN also induced NIK-me-diated NF�B activation by modulating the phosphorylation ofERK1/2. Moreover OPN induced uPA secretion and uPA-de-pendent pro-MMP-9 activation, cell motility, chemoinvasion,and tumor growth. The OPN-induced NIK-dependent ERK/IKK-mediated NF�B transactivation, pro-MMP-9 activation,and cell motility were enhanced when cells were transfectedwith wild type NIK, IKK�/�, and ERK1/2 and suppressed whencells were transfected with mut NIK, dn IKK�/�, or dn ERK1/2.Taken together, these data demonstrated that OPN enhancescell motility, chemoinvasion, and tumor growth and inducesNIK-dependent NF�B-mediated uPA secretion and uPA-regu-lated pro-MMP-9 activation through MAPK/IKK-mediated sig-naling pathways.

EXPERIMENTAL PROCEDURES

Materials—The rabbit polyclonal anti-phospho-NIK (Thr-559), anti-NIK, anti-IKK�/�, anti-NF�B p65 X TransCruz, anti-p-MEK-1, anti-MEK-1, anti-ERK1/2, anti-I�B�, anti-uPA, anti-MMP-9, and anti-actinand the mouse monoclonal anti-phospho-ERK1/2, anti-phospho-I�B�antibodies, and I�B� recombinant protein were purchased from SantaCruz Biotechnology. Mouse monoclonal anti-MMP-9 was from Onco-gene. Mouse monoclonal anti-human �v�3 integrin antibody was fromChemicon International. LipofectAMINE Plus, GRGDSP, and GRGESPwere obtained from Invitrogen. PD98059 was from Calbiochem. TNF�was from R&D Systems. The dual luciferase reporter assay system andNF�B consensus oligonucleotide were purchased from Promega. Boy-den type cell migration chambers were obtained from Corning, andBioCoat MatrigelTM invasion chambers were from Collaborative Bio-medical. The [�-32P]ATP was purchased from Board of Radiation andIsotope Technology (Hyderabad, India). The human OPN was purifiedfrom milk as described previously and used throughout these studies(20). The nude mice (NMRI, nu/nu) were from National Institute ofVirology (Pune, India). All other chemicals were of analytical grade.

Cell Culture—The B16F10 cells were obtained from American TypeCulture Collection (Manassas, VA). These cells were cultured in Dul-becco’s modified Eagle’s medium supplemented with 10% fetal calfserum, 100 units/ml penicillin, 100 �g/ml streptomycin, and 2 mM

glutamine in a humidified atmosphere of 5% CO2 and 95% air at 37 °C.Plasmids and DNA Transfection—The wild type NIK (wt pcDNA

NIK) and kinase-negative NIK (mut pcDNA NIK, NIK-K429A/K430A)in pcDNA3 were generous gifts from Prof. David Wallach (WeizmannInstitute of Science, Rehovot, Israel). The wild type and dominantnegative constructs of IKK� (wt IKK� and dn IKK�) and IKK� (wtIKK� and dn IKK�) in pRK were kind gifts from Prof. D. V. Goeddel(Tularik Inc., San Francisco, CA). The wt ERK1 and dn ERK1 in pCEP4and wt ERK2 and dn ERK2 in p3XFLAG-CMV-7.1 were from Dr.Melanie Cobb (Department of Pharmacology, University of TexasSouthwestern Medical Center, Dallas, TX). The luciferase reporter con-struct (pNF�B-Luc) containing five tandem repeats of the NF�B bind-ing site was a generous gift from Dr. Rainer de Martin (University ofVienna, Vienna, Austria). The super-repressor form of I�B� cDNAfused downstream to a FLAG epitope in an expression vector (pCMV4)was a gift from Dr. Dean Ballard (Vanderbilt University School ofMedicine, Nashville, TN). B16F10 cells were split 12 h prior to trans-fection in Dulbecco’s modified Eagle’s medium containing 10% fetal calfserum. These cells were transiently transfected with cDNA using Lipo-fectAMINE Plus according to manufacturer’s instructions. BrieflycDNA (8 �g) was mixed with Plus reagent, and then the cDNA ReagentPlus were incubated with LipofectAMINE. The LipofectAMINE PluscDNA complex was added to the cells and incubated further at 37 °C for12 h. The control cells received LipofectAMINE Plus alone. The cellviability was detected by a trypan blue dye exclusion test. After incu-bation, the medium was removed, and the cells were refed with freshmedium and maintained for an additional 12 h. These transfected cellswere used for interaction studies between phosphorylated NIK andIKK, I�B� phosphorylation, IKK assay, MEK-1 and ERK1/2 phospho-rylations, EMSA, NF�B luciferase reporter gene assay, and detection ofMMP-9 by zymography and Western blot and uPA secretion by Westernblot, cell migration, and chemoinvasion assays.

In Vitro Kinase Assay—To examine the effect of OPN on NIK activ-ity, the semiconfluent B16F10 cells were treated with 5 �M OPN for 5min at 37 °C. The cells were lysed in kinase assay lysis buffer (20 mM

Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM

�-glycerophosphate, 10 mM NaF, 10 mM p-nitrophenyl phosphate, 300�M Na3VO4, 1 mM benzamidine, 2 �M phenylmethylsulfonyl fluoride, 10�g/ml aprotinin, 1 �g/ml leupeptin, 1 �g/ml pepstatin, 1 mM DTT, and0.25% Nonidet P-40). The supernatant was obtained by centrifugationat 12,000 � g for 10 min at 4 °C. The cell lysates containing an equalamount of total proteins were immunoprecipitated with rabbit anti-NIK antibody. Half of the immunoprecipitated samples were incubatedwith IKK as substrate in kinase assay buffer (20 mM Hepes (pH 7.7), 2mM MgCl2, 10 mM �-glycerophosphate, 10 mM NaF, 10 mM p-nitrophe-nyl phosphate, 300 �M Na3VO4, 1 mM benzamidine, 2 �M phenylmeth-ylsulfonyl fluoride, 10 �g/ml aprotinin, 1 �g/ml leupeptin, 1 �g/mlpepstatin, and 1 mM DTT) containing 10 �M ATP and 3 �Ci of[�-32P]ATP at 30 °C. The kinase reactions were stopped by addition ofSDS sample buffer. The samples were resolved by SDS-PAGE, dried,and autoradiographed. The remaining half of the immunoprecipitatedsamples were subjected to SDS-PAGE and analyzed by Western blotusing anti-NIK antibody. A fraction of equal volume of samples from the

NIK Regulates OPN-induced MAPK/IKK-mediated MMP-9 Activation38922

kinase reaction mixture was analyzed by Western blot using anti-IKK�/� antibody.

To investigate the role of NIK in OPN-induced IKK activity, the cellswere treated with 5 �M OPN for 10 min at 37 °C. In separate experi-ments, cells were transfected with wild type NIK or kinase-negativeNIK in the presence of LipofectAMINE Plus and then treated with 5 �M

OPN. The cells were lysed in kinase assay lysis buffer. The cell lysatescontaining an equal amount of total proteins (300 �g) were immuno-precipitated with anti-IKK�/� antibody. Half of the immunoprecipi-tated samples were incubated with recombinant I�B� (4 �g) in kinasebuffer (20 mM Hepes (pH 7.7), 2 mM MgCl2, 10 �M ATP, 3 �Ci of[�-32P]ATP, 10 mM �-glycerophosphate, 10 mM NaF, 10 mM p-nitrophe-nyl phosphate, 300 �M Na3VO4, 1 mM benzamidine, 2 �M phenylmeth-ylsulfonyl fluoride, 10 �g/ml aprotinin, 1 �g/ml leupeptin, 1 �g/mlpepstatin, and 1 mM DTT) at 30 °C. The kinase reaction was stopped byaddition of SDS sample buffer. The sample was resolved by SDS-PAGE,dried, and autoradiographed. The remaining half of the immunoprecipi-tated samples were analyzed by Western blot using anti-IKK�/� anti-body. A fraction of equal volume of samples from the kinase reactionmixture was analyzed by Western blot using anti-I�B� antibody. Thelevel of NIK in the immunoprecipitated samples was analyzed by West-ern blot using anti-NIK antibody.

To check whether OPN regulates the interaction between phospho-rylated NIK and IKK�/�, cells were treated with 5 �M OPN at 37 °C for10 min. Cell lysates were immunoprecipitated with rabbit anti-IKK�/�antibody. The immunoprecipitated samples were analyzed by Westernblot using rabbit anti-phospho-NIK antibody. The same blots werereprobed with anti-IKK�/� antibody as loading control.

Western Blot Analysis—To delineate the role of OPN in regulation ofNIK phosphorylation, the cells were treated with 5 �M OPN for 0–30min. In separate experiments, cells were pretreated with anti-�v�3

integrin antibody (20 �g/ml) or GRGDSP or GRGESP peptide (10 �M)and then treated with 5 �M OPN for 7 min at 37 °C. In other experi-ments, cells were treated with TNF� (20 ng/ml) for 5 min as positivecontrol. The cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4),150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxy-cholate, 0.1% SDS, 5 mM iodoacetamide, and 2 mM phenylmethylsulfo-nyl fluoride), and the lysates containing an equal amount of totalproteins were analyzed by Western blot with rabbit anti-phospho-NIKantibody. The same blots were reprobed with rabbit anti-NIK or anti-actin antibody as loading controls and detected by the ECL detectionsystem (Amersham Biosciences).

To examine the effect of NIK on OPN-induced I�B� phosphorylationand degradation, cells were transfected with either wild type NIK orkinase-negative NIK and then treated with 5 �M OPN for 10 min. Thecell lysates were analyzed by Western blot using mouse anti-phospho-I�B� antibody. The same blots were reprobed with rabbit anti-I�B�antibody. As loading control, the expression of actin was also detectedby reprobing the blot with anti-actin antibody.

To detect whether NIK regulates OPN-induced MEK-1 and ERK1/2phosphorylations, cells were transfected with wild type or kinase-negativeNIK and then treated with 5 �M OPN for 10 min. Cell lysates wereanalyzed by Western blot using anti-p-MEK-1 or anti-p-ERK1/2 antibody.The same blots were reprobed with anti-MEK-1 or anti-ERK1/2 antibody.

To check whether �v�3 integrin is involved in OPN-induced MEK-1phosphorylation, the cells were pretreated with anti-�v�3 antibody orRGD peptide (GRGDSP or GRGESP) and then treated with OPN. Thecell lysates were subjected to Western blot analysis using anti-p-MEK-1antibody. The same blots were reprobed with anti-MEK-1 antibody.

To investigate the role of OPN on pro-MMP-9 activation, the cellswere treated with 5 �M OPN for 24 h. In separate experiments, cellswere pretreated with anti-�v�3 integrin antibody (20 �g/ml), RGD/RGEpeptide (10 �M GRGDSP or GRGESP), anti-uPA antibody (25 �g/ml), oranti-MMP-2 antibody (0–50 �g/ml) for 1 h and then treated with 5 �M

OPN. The conditioned medium was collected, concentrated, and dia-lyzed. The samples containing an equal amount of total proteins wereanalyzed by Western blot using rabbit anti-MMP-9 antibody.

To ascertain the role of NIK in OPN-induced uPA secretion, the cellswere either treated with 0–5 �M OPN or transfected with wild type NIKor mut NIK and then treated with 5 �M OPN for 24 h. To examinewhether ERK1/2 plays any roles in OPN-induced uPA secretion, cellswere transfected with wild type or dn ERK1/2 and then treated withOPN. The cell lysates containing an equal amount of total proteins weresubjected to Western blot analysis using rabbit polyclonal anti-uPAantibody. As loading controls the expression of actin was also detectedby reprobing the blots with rabbit anti-actin antibody.

Zymography Experiments—The gelatinolytic activity was measuredas described previously (20). To further check the effect of OPN on

pro-MMP-9 activation, the cells were treated with 0–10 �M OPN for24 h or pretreated with anti-uPA antibody (25 �g/ml) for 1 h and thentreated with 5 �M OPN. The conditioned medium was collected bycentrifugation. The samples containing an equal amount of total pro-teins were mixed with sample buffer in the absence of reducing agentand loaded onto zymography SDS-polyacrylamide gels containing gel-atin (0.5 mg/ml) as described previously (20). The gels were incubated inincubation buffer (50 mM Tris-HCl (pH 7.5) containing 100 mM CaCl2, 1�M ZnCl2, 1% (v/v) Triton X-100, and 0.02% (w/v) NaN3) for 16 h. Thegels were stained with Coomassie Blue and destained. Negative stain-ing showed the zones of gelatinolytic activity. In another experiment,cells were also transfected with wild type NIK, kinase-negative NIK, orthe super-repressor form of I�B� in the presence of LipofectAMINEPlus and then treated with OPN for 24 h. In separate experiments, cellswere transfected with wild type or dominant negative IKK� and IKK�and then treated with 5 �M OPN. The conditioned medium was col-lected, and the level of active MMP-9 was detected by zymography asdescribed above.

EMSA—To check whether NIK and IKK play any role in OPN-induced NF�B-DNA binding, cells were either treated with 5 �M OPNor transfected with wild type NIK, kinase-negative NIK, or wild typeand dn IKK� and then treated with OPN for 6 h at 37 °C, and EMSAwas performed as described previously (37). To investigate whetherMEK-1 or ERK1/2 is involved in OPN-induced NF�B-DNA binding andwhether it is regulated by NIK, cells were either pretreated withPD98059 or transfected with wt NIK or kinase-negative NIK and thentreated with PD98059 or transfected with wild type and dn ERK1 andERK2. Transfected or treated cells were further treated with OPN. Thenuclear extracts were prepared as described earlier (37). Briefly thecells were resuspended in hypotonic buffer (10 mM Hepes (pH 7.9), 1.5mM MgCl2, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5mM dithiothreitol). The nuclear pellet was extracted in nuclear extrac-tion buffer (20 mM Hepes (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl2, 0.2 mM

EDTA, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM

DTT). The supernatant was used as a nuclear extract. The nuclearextracts (10 �g) were incubated with 16 fmol of 32P-labeled double-stranded NF�B oligonucleotide (5�-AGT TGA GGG GAC TTT CCC AGGC-3�) in binding buffer (25 mM Hepes buffer (pH 7.9), 0.5 mM EDTA, 0.5mM DTT, 1% Nonidet P-40, 5% glycerol, 50 mM NaCl) containing 2 �g ofpolydeoxyinosinic deoxycytidylic acid (poly(dI-dC)). The DNA-proteincomplex was resolved on a native polyacrylamide gel and analyzed byautoradiography. For supershift assay, the OPN-treated nuclear ex-tracts were incubated with anti-NF�B p65 (TransCruz) antibody for 30min at room temperature and analyzed by EMSA.

NF�B Luciferase Reporter Gene Assay—The semiconfluent cellsgrown in 24-well plates were transiently transfected with a luciferasereporter construct (pNF�B-Luc) containing five tandem repeats of theNF�B binding site using LipofectAMINE Plus reagent (Invitrogen). Inseparate experiments, cells were individually transfected with wildtype NIK, kinase-negative NIK, the super-repressor form of I�B�, wildtype and dn IKK� and IKK�, or wild type and dn ERK1 and ERK2along with pNF�B-Luc. The transfection efficiency was normalized bycotransfecting the cells with pRL vector (Promega) containing a full-length Renilla luciferase gene under the control of a constitutive pro-moter. After 24 h of transfection, cells were treated with 5 �M OPN for6 h. Cells were harvested in passive lysis buffer (Promega). The lucif-erase activities were measured by luminometer (Lab Systems) usingthe dual luciferase assay system according to the manufacturer’s in-structions (Promega). Changes in luciferase activity with respect tocontrol were calculated.

Cell Migration Assay—The migration assay was conducted using aTranswell cell culture chamber according to the standard procedure asdescribed previously (20–22). Briefly the confluent monolayers ofB16F10 cells were harvested with trypsin-EDTA and centrifuged at800 � g for 10 min. The cell suspension (5 � 105cells/well) was added tothe upper chamber of the prehydrated polycarbonate membrane filter.The lower chamber was filled with fibroblast-conditioned medium,which acted as a chemoattractant. Purified OPN (5 �M) was added tothe upper chamber. In another experiment, cells were individuallypretreated with PD98059 (0–50 �M), anti-uPA antibody (25 �g/ml),anti-MMP-9 antibody (25 �g/ml), anti-MMP-2 antibody (25 �g/ml), or acombination of anti-MMP-2 and anti-MMP-9 antibodies (25 �g/ml) at37 °C for 6 h. In other experiments, cells were individually transfectedwith wild type and kinase-negative NIK, the super-repressor form ofI�B�, wild type and dn IKK� and IKK�, or wild type and dn ERK1 andERK2 and used for migration assays. These treated or transfected cellswere incubated in a humidified incubator in 5% CO2 and 95% air at37 °C for 16 h. The non-migrated cells on the upper side of the filter

NIK Regulates OPN-induced MAPK/IKK-mediated MMP-9 Activation 38923

were scraped, and the filter was washed. The migrated cells on thereverse side of the filter were fixed with methanol and stained withGiemsa. The migrated cells on the filter were counted under an invertedmicroscope (Olympus). The experiments were repeated in triplicate.Preimmune IgG served as a nonspecific control.

Chemoinvasion Assay—The chemoinvasion assay was performed us-ing a Matrigel-coated invasion chamber as described previously (20–22). The cell suspension (5 � 105 cells/well) was added to the upperportion of the prehydrated Matrigel-coated chamber. The lower cham-ber was filled with fibroblast-conditioned medium, which acted as achemoattractant. Purified OPN (5 �M) was added to the upper chamber.In another experiment, cells were individually pretreated with anti-MMP-9 antibody (25 �g/ml), anti-MMP-2 antibody (25 �g/ml), a combi-nation of anti-MMP-2 and anti-MMP-9 antibodies, and anti-uPA anti-body (25 �g/ml) at 37 °C for 6 h. In other experiments, cells wereindividually transfected with wild type and kinase-negative NIK, wildtype and dn IKK� and IKK�, or the super-repressor form of I�B� asdescribed above and used for invasion assays. OPN (5 �M) was used inthe upper chamber. The cells were incubated at 37 °C for 16 h. Thenon-migrated cells and Matrigel from the upper side of the filter werescraped and removed using a moist cotton swab. The invaded cells onthe lower side of the filter were stained with Giemsa and washed withphosphate-buffered saline (pH 7.6). The invaded cells were thencounted, and photomicrographs were taken under the inverted micro-scope (Olympus). The experiments were repeated in triplicate. Preim-mune IgG served as a nonspecific control.

In Vivo Tumorigenicity Experiments—The tumorigenicity experi-ments were performed as described previously (20, 21). The cells weretreated in the absence or presence of purified human OPN (10 �M) at37 °C for 20 h. After that, the cells (5 � 106/0.2 ml) were detached andinjected subcutaneously into the flanks of male athymic NMRI (nu/nu)mice (6–8 weeks old). Four mice were used in each set of experiments.The mice were kept under specific pathogen-free conditions. OPN (10�M) was again injected into the tumor sites twice a week for up to 4weeks. After 4 weeks, the mice were killed, and the tumor weights weremeasured. The tumor tissues were homogenized and lysed in lysis

buffer (50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1% NonidetP-40, 15 �g/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride)and centrifuged at 12,000 � g for 10 min. The clear supernatants werecollected, and the levels of pro- and active MMP-9 were detected byWestern blot analysis. Briefly the samples containing an equal amountof total proteins were resolved by SDS-PAGE and analyzed by Westernblot using anti-MMP-9 antibody. The levels of pro- and active MMP-9 intumor samples were also analyzed by zymography as described above.

RESULTS

OPN Induces �v�3 Integrin-dependent NIK Phosphoryla-tion—Because we have reported earlier that OPN inducesNF�B-mediated pro-MMP-2 activation through IKK/I�B�-me-diated pathway (20), we first examined whether any upstreamkinase(s) such as NIK plays any role in OPN-induced NF�Bactivation in B16F10 cells. Accordingly these cells were treatedwith 5 �M OPN for 0–30 min at 37 °C. The cell lysates contain-ing an equal amount of total proteins were resolved by SDS-PAGE, and the level of phosphorylated NIK was detected byWestern blot analysis using anti-phospho-NIK (Thr-559) anti-body (Fig. 1, upper panel A, lanes 1–5). The data revealed thatthe maximum level of OPN-induced NIK phosphorylation oc-curred at 7 min (upper panel A, lane 3) and also suggested thatThr-559 of NIK is crucial for OPN-induced NIK phosphoryla-tion. The same blots were reprobed with anti-NIK antibody asloading control, and the data showed that there was no changein expression of non-phospho-NIK in these cells upon treat-ment with OPN, confirming the equal loading of samples (mid-dle panel A, lanes 1–5). Actin was also used as a loading control(lower panel A, lanes 1–5). To further check whether �v�3

integrin or the RGD/RGE peptide is involved in OPN-inducedNIK phosphorylation, cells were pretreated with anti-�v�3 in-

FIG. 1. Panel A, OPN stimulates NIK phosphorylation. B16F10 cells were treated with 5 �M OPN for 0–30 min. The cell lysates containing anequal amount of total proteins were analyzed by Western blot using anti-phospho-NIK antibody (upper panel A, lanes 1–5). The blots were reprobedwith anti-NIK (middle panel A) or anti-actin antibody (lower panel A) as loading controls. Panel B, OPN induces NIK phosphorylation through �v�3integrin-mediated pathways. The cells were individually pretreated with anti-�v�3 integrin antibody, GRGDSP, or GRGESP and then treated with5 �M OPN. The cell lysates were analyzed by Western blot using anti-phospho-NIK antibody (upper panel B, lanes 1–5). The same blots werereprobed with anti-NIK antibody (lower panel B). Panel C, TNF�, as control, enhances NIK phosphorylation. Cells were treated with TNF�, andlevels of phospho- and non-phospho-NIK were detected by Western blot (panel C). Panel D, OPN induces the interaction between phosphorylatedNIK and IKK. The cells were treated with 5 �M OPN. In separate experiments, cells were transfected with wt or kinase-negative NIK and thentreated with OPN. Cell lysates were immunoprecipitated with anti-IKK�/� antibody. Half of the samples were immunoblotted with anti-phospho-NIK antibody (upper panel D, lanes 1–4), and the other half were analyzed by anti-IKK�/� antibody (lower panel D, lanes 1–4). All these bandswere analyzed densitometrically, and the -fold changes were calculated. The data shown here represent three experiments exhibiting similareffects. IB, immunoblot; pNIK, phospho-NIK; IP, immunoprecipitation; Ab, antibody.


tegrin antibody or RGD/RGE peptide (GRGDSP or GRGESP)and then treated with OPN. The level of phosphorylated NIKwas detected by Western blot analysis. The data showed that�v�3 integrin antibody and RGD (GRGDSP) but not RGE(GRGESP) peptide suppressed the OPN-induced NIK phospho-rylation in these cells (upper panel B, lanes 1–5). The level ofnon-phospho-NIK was unchanged (lower panel B, lanes 1–5).As a control, cells were treated with TNF�, and the levels ofphosphorylated and non-phosphorylated NIK were detected byWestern blot using anti-p-NIK and anti-NIK antibodies, re-spectively. The data revealed that OPN stimulates NIK phos-phorylation in these cells (panel C, lanes 1 and 2). Western blotdata were quantified by densitometric analysis, and the -foldchanges were calculated. These results demonstrated thatOPN binds with �v�3 integrin receptor and induces NIKphosphorylation.

OPN Stimulates the Interaction between Phosphorylated NIKand IKK—To delineate whether OPN plays any role in regu-lating the interaction between phosphorylated NIK and IKK,B16F10 cells were treated with 5 �M OPN. In separate exper-iments, cells were individually transfected with wild type NIKor kinase-negative NIK in the presence of LipofectAMINE Plusand then treated with OPN. Cell lysates were immunoprecipi-tated with rabbit polyclonal anti-IKK�/� antibody. Half of theimmunoprecipitated samples were analyzed by Western blotusing anti-phospho-NIK antibody, and the remaining half of

the samples were immunoblotted with anti-IKK�/� antibody.The results indicated that cells transfected with wild type NIKfollowed by treatment with OPN showed maximum interactionbetween phosphorylated NIK and IKK�/� compared with cellstreated with OPN alone or untreated cells (Fig. 1, panel D,lanes 1–3). Cells transfected with kinase-negative NIK sup-pressed the OPN-induced interaction between phosphorylatedNIK and IKK�/� in these cells (lane 4). All these bands werequantified densitometrically, and the -fold changes were calcu-lated. These results suggested that OPN enhances the interac-tion between phosphorylated NIK and IKK�/�.

OPN Induces NIK Activity and NIK-dependent IKK Activitythrough Phosphorylation and Degradation of I�B�—To ascer-tain the role of OPN on NIK activity, the cells were treated with5 �M OPN, and the lysates were immunoprecipitated withrabbit anti-NIK antibody. Half of the immunoprecipitated sam-ples were incubated with IKK as substrate in kinase assaybuffer containing [�-32P]ATP. The samples were resolved bySDS-PAGE and autoradiographed. The radiolabeled, phospho-rylated IKK-specific band was detected in OPN-treated cellsdemonstrating that OPN induces NIK activity (Fig. 2, upperpanel A, lane 2). The NIK activity was not detected in theuntreated cells (lane 1). The remaining half of the immunopre-cipitated samples were analyzed by Western blot using anti-NIK antibody as loading control (middle panel A). A fraction ofequal volume of samples from the kinase reaction mixture was

FIG. 2. Panel A, OPN induces NIK activity. Cells were treated with 5 �M OPN, cell lysates were immunoprecipitated with anti-NIK antibody,and half of the immunoprecipitated samples were used for the kinase assay using IKK as substrate (upper panel A, lanes 1–2). The remaining halfof the immunoprecipitated samples were immunoblotted with anti-NIK antibody (middle panel A). The fraction containing an equal volume ofkinase reaction mixture was analyzed by Western blot using anti-IKK antibody (lower panel A). Panel B, OPN stimulates NIK-dependent IKKactivity. The cells were treated with 5 �M OPN or transfected with wild type and kinase-negative NIK and then treated with OPN. Cell lysateswere immunoprecipitated with anti-IKK�/� antibody, and half of the immunoprecipitated samples were used for the kinase assay using I�B� assubstrate (panel B, a). The remaining half of the immunoprecipitated samples were analyzed by Western blot using anti-IKK�/� antibody (panelB, b). The fraction containing an equal volume of kinase reaction mixture was immunoblotted with anti-I�B� antibody (panel B, c). The level ofexpression of NIK was also analyzed by Western blot using anti-NIK antibody (panel B, d). Panel C, NIK is required for OPN-induced I�B�phosphorylation and degradation. The cells were either treated with 5 �M OPN or transfected with wild type or kinase-negative NIK and thentreated with OPN as described under “Experimental Procedures.” Cell lysates were analyzed by Western blot using anti-phospho-I�B� (pI�B�)antibody (upper panel C, lanes 1–4). The same blots were reprobed with anti-I�B� (middle panel C) or anti-actin (lower panel C) antibody. All thesebands were analyzed densitometrically, and the -fold changes were calculated. The data shown here represent three experiments exhibiting similareffects. IP, immunoprecipitation; IB, immunoblot.


analyzed by Western blot using anti-IKK�/� antibody as con-trol (lower panel A). These data demonstrated that OPN in-duces NIK activity in these cells.

To further check whether NIK plays any direct role in OPN-induced IKK activity, cells were transfected with wild type orkinase-negative NIK and then treated with 5 �M OPN. The celllysates were immunoprecipitated with rabbit anti-IKK�/� an-tibody. Half of the immunoprecipitated samples were incu-bated with I�B� as substrate in kinase assay buffer containing[�-32P]ATP. The samples were resolved by SDS-PAGE andautoradiographed. Cells transfected with wild type NIK fol-lowed by treatment with OPN showed maximum IKK activity(Fig. 2, panel B, a, lane 3) compared with untreated cells (lane1) or cells induced with OPN alone (lane 2). Cells transfectedwith kinase-negative NIK followed by treatment with OPNsuppressed the IKK activity significantly (lane 4) indicatingthat the kinase domain of NIK plays a crucial role in OPN-induced IKK activity. The remaining half of the immunopre-cipitated samples were analyzed by Western blot using anti-IKK�/� antibody (panel B, b, lanes 1–4). A fraction of equalvolume of samples from the kinase reaction mixture was ana-lyzed by Western blot using anti-I�B� antibody (panel B, c,lanes 1–4). The level of NIK was also analyzed by Western blotusing anti-NIK antibody (panel B, d). These results suggestedthat NIK plays a significant role in OPN-induced IKK activity.

Since we showed that OPN-induced IKK activity was regu-lated by NIK, we therefore sought to determine whether NIKplays any role in OPN-induced phosphorylation and degradationof I�B�. Accordingly cells were either treated with 5 �M OPN ortransfected with wild type or kinase-negative NIK and thentreated with OPN. The level of phosphorylated I�B� in cell ly-sates was detected by Western blot using anti-phospho-I�B�antibody. The data demonstrated that cells transfected with wildtype NIK enhanced OPN-induced I�B� phosphorylation com-pared with untreated cells (Fig. 2, upper panel C, lanes 1–3). Cellstransfected with kinase-negative NIK suppressed OPN-inducedI�B� phosphorylation (lane 4). The blot was reprobed with anti-I�B� antibody. The low level of I�B� was observed in cellstreated with OPN or transfected with wild type NIK followed by

OPN treatment indicating the degradation of I�B� (middle panelC, lanes 1–4). The same blots were reprobed with anti-actinantibody as loading control (lower panel C, lanes 1–4). These datasuggested that NIK plays a crucial role in OPN-induced I�B�phosphorylation and degradation.

OPN Stimulates �v�3 Integrin-mediated NIK-dependentMEK-1/ERK1/2 Phosphorylations—To examine whether NIKplays any role in OPN-induced MEK-1 and ERK1/2 phospho-rylations, cells were treated with 5 �M OPN alone, pretreatedwith �v�3 integrin antibody and RGD/RGE peptide, or trans-fected with wild type and kinase-negative NIK and thentreated with OPN. Cell lysates were analyzed by Western blotusing anti-phospho-MEK-1 or anti-phospho-ERK1/2 antibody.Wild type NIK enhanced whereas kinase-negative NIK sup-pressed the OPN-induced MEK-1 and ERK1/2 phosphoryla-tions in these cells (Fig. 3, upper panels A and C, lanes 1–4).The data also indicated that OPN-induced MEK-1 phosphoryl-ation was inhibited by �v�3 integrin and RGD but not RGEpeptide (upper panel B, lanes 1–5). The same blots were rep-robed with anti-MEK-1 and anti-ERK1/2 antibodies as loadingcontrols (lower panels A–C). These data suggested that OPNinduces �v�3 integrin-dependent MEK-1 and ERK1/2 phospho-rylations through an NIK-mediated pathway.

NIK Plays a Crucial Role in OPN-induced ERK/IKK-depend-ent NF�B-DNA Binding—We have reported earlier that OPNinduces pro-MMP-2 activation through activation of NF�B. Inthis study, we first examined whether NIK regulates OPN-in-duced NF�B-DNA binding in B16F10 cells. Accordingly cellswere treated with 5 �M OPN or transfected with wild type andkinase-negative NIK or wild type and dn IKK� and then treatedwith OPN. The nuclear extracts were prepared and used forEMSA using 32P-labeled NF�B oligonucleotides. Wild type NIKenhanced and kinase-negative NIK suppressed OPN-inducedNF�B-DNA binding (Fig. 4, panel A, lanes 1–4). Similarly wildtype IKK� induced and dn IKK� inhibited OPN-enhanced NF�B-DNA binding (panel B, lanes 1–4). The same results were ob-tained in cells transfected with wild type and dn IKK� (data notshown). These data suggested that OPN induces NF�B-DNAbinding through NIK/IKK-mediated pathways.

FIG. 3. OPN induces �v�3 integrin-mediated NIK-dependent MEK-1 and ERK1/2 phosphorylations. Panels A–C, cells were transfectedwith wild type and kinase-negative NIK or pretreated with anti-�v�3 integrin antibody or RGD/RGE peptide (GRGDSP or GRGESP) and thenstimulated with OPN. Cell lysates were analyzed by Western blot using anti-phospho-MEK-1 (upper panels A and B) or anti-phospho-ERK1/2(upper panel C) antibody. The same blots were reprobed with anti-MEK-1 (lower panels A and B) or anti-ERK1/2 (lower panel C) antibody. Thebands were analyzed densitometrically, and the -fold changes were calculated. The data shown here represent three experiments exhibiting similareffects. IB, immunoblot; Ab, antibody.


To examine the effect of ERK1/2 on OPN-induced NF�B-DNA binding, cells were pretreated with two doses of PD98059or transfected with wild type and dominant negative constructsof ERK1 and ERK2 and then treated with OPN. The nuclearextracts were prepared and used for EMSA. The data revealedthat overexpression of wild type ERK1 or ERK2 resulted in anincrease in OPN-induced NF�B-DNA binding, whereas dnERK1 or ERK2 reduced the OPN-induced NF�B-DNA binding(Fig. 4, panel E, lanes 1–6). PD98059, an MEK-1 inhibitor,suppressed OPN-induced NF�B-DNA binding (panel C, lanes1–4). To ascertain whether OPN-induced ERK-mediatedNF�B-DNA binding is NIK-dependent, cells were transfectedwith wild type and kinase-negative NIK followed by treatmentwith PD98059 and then stimulated with OPN. The OPN-en-hanced NF�B-DNA binding caused by overexpression of wildtype NIK was also suppressed by PD98059 (panel D, lanes1–4). These results demonstrated that OPN induces NF�B-DNA binding through an NIK/ERK1/2-mediated pathway. Todetermine whether the band obtained by EMSA was indeedNF�B, the nuclear extracts were incubated with anti-p65 an-tibody and then analyzed by EMSA. The results showed theshift of the NF�B-specific band to a higher molecular weightwhen the nuclear extracts were treated with anti-p65 antibody,suggesting that the OPN-activated complex consists of the p65subunit in these cells (panel F, lanes 1 and 2).

OPN Induces NIK-dependent ERK/IKK-mediated NF�BTransactivation—To further investigate whether NIK regu-lates ERK/IKK-dependent OPN-induced NF�B transcriptionalactivity, a luciferase reporter gene assay was performed. Cells

were transiently transfected with NF�B luciferase reporterconstruct (pNF�B-Luc) and then treated with OPN (5 �M). Inseparate experiments, cells were individually transfected withwild type and kinase-negative NIK, the super-repressor form ofI�B�, wild type IKK�/� and dn IKK�/�, or wild type ERK1/2and dn ERK1/2 along with pNF�B-Luc and then treated withOPN (5 �M). The transfection efficiency was normalized bycotransfecting the cells with pRL vector. Changes in luciferaseactivity with respect to control were calculated. The -foldchanges were calculated, and the results are expressed as themeans � S.E. of three determination. The values were alsoanalyzed by Student’s t test (p � 0.001). The data showed thatwild type NIK enhanced but kinase-negative NIK or the super-repressor form of I�B� suppressed OPN-induced NF�B activityin these cells (Fig. 5, panel A). Wild type IKK� and especiallyIKK� enhanced OPN-induced NF�B activity (panel B). Both dnIKK� and IKK� suppressed OPN-induced NF�B activity (panelB). Similarly wt ERK1 and wt ERK2 enhanced whereas dnERK1 and dn ERK2 suppressed OPN-induced NF�B activity(panel C). These data further suggested that NIK regulatesOPN-induced ERK/IKK-dependent NF�B activation.

OPN Stimulates �v�3 Integrin-mediated NIK- and ERK/IKK-dependent Pro-MMP-9 Activation—To examine the effect ofOPN on pro-MMP-9 activation, the B16F10 cells were treatedwith increasing concentrations of OPN (0–10 �M). The condi-tioned media were collected, and the gelatinolytic activity ofMMP-9 was detected by zymography. Increased levels ofMMP-9 expression and activation (92-kDa pro- and 86-kDaactive forms) were observed when cells were treated with two

FIG. 4. Panels A and B, OPN induces NIK/IKK-dependent NF�B-DNA binding. Cells were treated with 5 �M OPN. In other experiments, cellswere individually transfected with wild type and kinase-negative NIK or wild type and dn IKK� and then treated with OPN. The nuclear extractswere prepared and analyzed by EMSA. Panels C–E, ERK1/2 is involved in OPN-induced NIK-mediated NF�B-DNA binding. Cells were eitherpretreated with PD98059 or transfected with wild type and kinase-negative NIK, treated with PD98059, and then treated with OPN. In otherexperiments, cells were transfected with wt and dn ERK1 and ERK2 and then treated with OPN. The nuclear extracts were analyzed by EMSA.Panel F, supershift assay. The nuclear extracts from OPN-treated cells were incubated with anti-p65 antibody and analyzed by EMSA. The resultsshown in A–F represent three experiments exhibiting similar effects. SS, supershift; Ab, antibody.


different concentrations of OPN (Fig. 6, upper panel A, lanes 2and 3). Almost no pro- and active MMP-9-specific bands weredetected in the untreated cells (lane 1). The levels of pro- andactive MMP-9 expression (gelatinolytic activity) were quanti-fied densitometrically and analyzed statistically. As comparedwith controls, there were about 3- and 5-fold increases inMMP-9 activation when the cells were treated with 5 and 10 �M

OPN, respectively (lower panel A).To check whether �v�3 integrin or RGD peptide is involved in

OPN-induced pro-MMP-9 activation, cells were pretreated withanti-�v�3 integrin antibody, GRGDSP, or GRGESP and thentreated with 5 �M OPN. The conditioned media were collected,and the levels of pro- and active MMP-9 were detected byWestern blot using anti-MMP-9 antibody. The level of OPN-induced MMP-9 activation (Fig. 6, upper panel B, lane 2) wasreduced significantly when cells were individually treated withanti-�v�3 integrin antibody (lane 3) or with GRGDSP peptide(lane 4) but not with GRGESP peptide (lane 5). No detectablelevel of MMP-9 was observed in OPN-untreated cells (lane 1).The intensities of the MMP-9-specific bands were quantified

densitometrically and analyzed statistically (lower panel B).These data suggested that �v�3 integrin and RGD peptide playan important role in OPN-induced MMP-9 activation.

To investigate the role of NIK, IKK, or ERK1/2 in OPN-induced MMP-9 activation, cells were individually transfectedwith wild type NIK, kinase-negative NIK, the super-repressorform of I�B�, wild type and dn IKK� and IKK�, or wild typeand dn ERK1/2 and then treated with 5 �M OPN. The level ofMMP-9 was detected by zymography as described above. Theresults indicated that wild type NIK enhanced whereas kinase-negative NIK or the super-repressor form of I�B� suppressedthe OPN-induced MMP-9 activation (Fig. 6, upper panel C,lanes 2–5). Wild type IKK� enhanced but dn IKK� suppressedOPN-induced MMP-9 activation (upper panel D, lanes 2–4). NoMMP-9 was detected in OPN-untreated cells (panels C and D,lane 1). Similar results were obtained in cells transfected withIKK� (data not shown). These bands were quantified densito-metrically and analyzed statistically (lower panels of panels Cand D). Similarly wild type ERK1/2 enhanced and dn ERK1/2inhibited OPN-induced pro-MMP-9 activation (data not

FIG. 5. Panels A and B, OPN enhances NF�B transactivation through an NIK/IKK-mediated pathway. Cells were transiently transfected withluciferase reporter construct (pNF�B-Luc). In other experiments, cells were individually transfected with wild type and kinase-negative NIK, wildtype and dn IKK�/�, or the super-repressor form of I�B� along with pNF�B-Luc. The transfected cells were treated with 5 �M OPN. Cell lysateswere used to measure the luciferase activity. The values were normalized to Renilla luciferase activity. Panel C, ERK1/2 is required inOPN-induced NF�B transactivation. Cells were transfected with wild type and dn ERK1 and ERK2 along with pNF�B-Luc. The transfected cellswere treated with 5 �M OPN. Cell lysates were used to measure the luciferase activity. The values were normalized to Renilla luciferase activity.The -fold changes were calculated, and means � S.E. of triplicate determinations are plotted. The values were also analyzed by Student’s t test(*, p � 0.001). sup. rep., super-repressor.


shown). These results demonstrated that NIK regulates OPN-induced pro-MMP-9 activation through ERK/IKK-mediatedpathways in these cells.

OPN Induces NIK-dependent IKK- and ERK1/2-mediateduPA Secretion and uPA-dependent Pro-MMP-9 Activation—Wefirst examined whether NIK, IKK, and ERK1/2 are involved inOPN-induced uPA secretion in B16F10 cells. Accordingly cellswere either treated with various concentrations of OPN (0–5�M) or transfected with wild type NIK, kinase-negative NIK,wild type and dn IKK�/�, wild type ERK1/2, or dn ERK1/2 andthen treated with OPN. The cell lysates were analyzed byWestern blot using rabbit polyclonal anti-uPA antibody. Thedata showed that OPN-induced uPA secretion was enhancedwhen cells were transfected with wild type NIK and suppressedwhen transfected with kinase-negative NIK (Fig. 7, upperpanel A, lanes 1–5, and upper panel B, lanes 1–4). Wild typeERK1/2 stimulated and dn ERK1/2 blocked OPN-induced uPAsecretion (upper panel C, lanes 1–6). Similarly wild typeIKK�/� enhanced and dn IKK�/� suppressed OPN-induceduPA secretion (data not shown). All these blots were reprobed

with anti-actin antibody (lower panels A–C), bands were quan-tified by densitometric analysis, and the -fold changes werecalculated (panels A–C). These data suggested that OPN in-duces NIK-dependent uPA secretion through ERK/IKK-medi-ated pathways.

To examine whether uPA plays any role in OPN-inducedMMP-9 activation, the cells were pretreated with anti-uPA anti-body and then treated with OPN (5 �M). The level of MMP-9 wasdetected by zymography and Western blot analysis. The resultsindicated that uPA antibody suppressed the OPN-inducedMMP-9 activation as shown by zymography (Fig. 7, panel D,lanes 1–3) and Western blot (panel E, lanes 1–3), indicating thatuPA is involved in OPN-induced MMP-9 activation.

uPA and MMP-9 Play Crucial Roles in OPN-induced NIK-dependent IKK-mediated Cell Migration and Chemoinvasion—Because NIK in the presence of OPN regulates ERK- andIKK-dependent NF�B-mediated uPA secretion and uPA-regu-lated pro-MMP-9 activation, we therefore sought to determinewhether these signaling molecules play any role in OPN-in-duced cell migration and chemoinvasion. Accordingly cells were

FIG. 6. Panel A, OPN induces pro-MMP-9 activity as shown by zymography. Cells were treated in the absence or presence of OPN (0–10 �M).The conditioned media were collected, and MMP-9 activity was analyzed by gelatin zymography. The arrows indicate both 92-kDa (pro-) and86-kDa (active) MMP-9-specific bands (upper panel A, lanes 1–3). Panel B, OPN induces �v�3 integrin-mediated pro-MMP-9 activation. Cells werepretreated with anti-�v�3 integrin antibody (20 �g/ml) or GRGDSP or GRGESP peptide (10 �M) and then treated with 5 �M OPN. The levels of pro-and active MMP-9 in the conditioned media were detected by Western blot using anti-MMP-9 antibody (upper panel B, lanes 1–5). Panels C andD, OPN stimulates NIK/IKK-dependent pro-MMP-9 activation. Cells were individually transfected with wild type and kinase-negative NIK, wildtype and dn IKK�, or the super-repressor form of I�B� and then treated with 5 �M OPN. The conditioned media were collected, and MMP-9 activitywas analyzed by gelatin zymography (upper panels C and D, lanes 1–5, lanes 1–4). All these bands were quantified by densitometry and arerepresented in the form of a bar graph (lower panels A–D). The data shown here represent three experiments exhibiting similar effects. sup. rep.,super-repressor; Ab, antibody; IB, immunoblot.


treated with anti-MMP-9 or anti-uPA antibody or transfectedwith wild type NIK, kinase-negative NIK, the super-repressorform of I�B�, or wild type and dn IKK� and IKK� and thenused for migration or chemoinvasion assay. OPN (5 �M) wasused in the upper chamber. The data indicated that anti-MMP-9 or anti-uPA antibody suppressed the OPN-induced cellmigration (Fig. 8, panel A) and chemoinvasion (panel B). Wildtype NIK enhanced whereas kinase-negative NIK or the super-repressor form of I�B� suppressed the OPN-induced cell mi-gration (panel A) and chemoinvasion (panel B). Similarly wildtype IKK� and IKK� stimulated and dn IKK� and IKK� in-hibited the OPN-induced cell migration (panel C) and invasion(panel D). These data demonstrated that OPN induces uPA-and MMP-9-dependent cell migration and chemoinvasionthrough NIK/IKK/NF�B-mediated pathways.

ERK1/2 Plays Critical Roles in OPN-induced NIK-regulatedCell Migration—To examine the role of ERK1/2 in OPN-in-duced NIK-dependent cell migration, the cells were individu-ally pretreated with PD98059, an MEK-1 inhibitor, or trans-fected with wild type NIK and kinase-negative NIK and thentreated with PD98059. In separate experiments, cells weretransfected with wild type ERK1/2 or dn ERK1/2. These trans-fected or treated cells were used for the migration assay. Thedata indicated that PD98059 suppressed OPN-induced cell mi-gration in the absence or presence of NIK (Fig. 9, panel D).Similarly wild type ERK1 and wild type ERK2 enhanced anddn ERK1 and dn ERK2 inhibited OPN-induced cell migration(panel E). These data demonstrated that ERK1/2 plays animportant role in OPN-induced NIK-dependent cell migration.

Both MMP-2 and MMP-9 Play Important Roles in OPN-induced Cell Migration and Chemoinvasion—We have reported

earlier that OPN induces pro-MMP-2 activation, which ulti-mately regulates cell motility, invasiveness, and tumor growth(20). In this study, we showed that OPN stimulates NIK-de-pendent uPA secretion and uPA-regulated pro-MMP-9 activa-tion. Therefore, we sought to determine whether both OPN-induced pro-MMP-2 and pro-MMP-9 activations exert anyindependent roles in regulating OPN-induced cell migrationand chemoinvasion. Accordingly cells were pretreated with ei-ther anti-MMP-2 antibody or anti-MMP-9 antibody alone or amixture of both. These cells were used for a migration orinvasion assay. The data indicated that MMP-2 or MMP-9antibody suppressed OPN-induced cell migration and chemo-invasion (Fig. 9, panels A and B). Pretreatment of cells with amixture of anti-MMP-2 and anti-MMP-9 antibodies had anadditive effect and hence drastically reduced OPN-induced cellmigration and chemoinvasion (panels A and B).

To ascertain whether there is any cross-talk between OPN-induced pro-MMP-2 and pro-MMP-9 activations, cells werepretreated with two different concentrations of anti-MMP-2antibody followed by treatment with OPN. The conditionedmedia were collected, and the level of MMP-9 was detected byWestern blot analysis using anti-MMP-9 antibody. The datashowed that OPN-induced MMP-9 activation was unaffectedwhen cells were pretreated with anti-MMP-2 antibody (Fig. 9,panel C, lanes 1–4). These data clearly indicated that OPN-induced activations of MMP-2 and MMP-9 occurred throughtwo distinct signaling mechanisms, and both played independ-ent roles in regulating cell migration and chemoinvasion inB16F10 cells.

OPN Induces Pro-MMP-9 Activation in Tumor of NudeMice—The in vitro data prompted us to examine whether

FIG. 7. Panels A–C, OPN stimulates NIK- and ERK1/2-dependent uPA secretion. The cells were treated with OPN (0–5 �M). In separateexperiments, cells were transfected with wild type and kinase-negative NIK or wild type and dn ERK1 and ERK2 and then treated with OPN. Thelevel of uPA in the cell lysates was detected by Western blot using anti-uPA antibody. The arrow indicates the uPA-specific band (upper panelsA–C). The same blots were reprobed with anti-actin antibody as loading control (lower panels A–C). Panels D and E, uPA is required inOPN-induced pro-MMP-9 activation. Cells were pretreated with anti-uPA antibody (25 �g/ml) and then treated with 5 �M OPN. The conditionedmedia was collected, and the MMP-9 activities were analyzed by zymography (panel D, lanes 1–3) and by Western blot (panel E, lanes 1–3). Thearrows indicate both 92- and 86-kDa MMP-9-specific bands. The results shown here represent three experiments exhibiting similar effects. IB,immunoblot; Ab, antibody.


OPN plays any role in MMP-9 activation in tumor of nudemice. Accordingly B16F10 cells were treated with OPN (10�M) and were injected subcutaneously into the flanks of nudemice. Fig. 10, panel A, shows typical photographs of tumorsgrown in 4-week-old nude mice (a and b). After 4 weeks, themice were killed, and tumor weights were measured. Theweights of the OPN-induced tumors were increased at least3-fold compared with the tumors of the non-OPN-injectedmice (Table I). Four mice were used in each set of experi-ments. The changes in tumor weights were analyzed statis-tically by Student’s t test (p � 0.002). These data are consist-ent with our previous data.

To examine the levels of pro- and active MMP-9 in tumors,the samples were lysed, and MMP-9 expression was analyzedby zymography (Fig. 10, panel B). The levels of both pro- andactive MMP-9 in the tumors produced by OPN (10 �M) weresignificantly higher (lane 2) compared with the levels of MMP-9in the tumors of the non-OPN-injected mice (lane 1). The levelsof MMP-9 in tumors were further confirmed by Western blotanalysis (panel C). Both pro- and active MMP-9 expressionswere higher in the tumors produced by OPN (panel C), andthese are corroborated by the zymography data (panel B).

These data demonstrated that OPN induces pro-MMP-9 acti-vation in tumor of nude mice, and it correlates with tumorgrowth in nude mice.

DISCUSSION

In this study, we investigated whether OPN regulates pro-MMP-9 activation and MMP-9-dependent cell motility, inva-siveness, and tumor growth. Moreover we examined whetherany upstream kinase such as NIK is involved in OPN-inducedNF�B activation, NF�B-mediated uPA secretion, pro-MMP-9activation, and cell motility through activation of MAPK/IKKin B16F10 cells. We showed that OPN induced phosphorylationand activation of NIK and enhanced the interaction betweenphosphorylated NIK and IKK�/� in these cells. The data re-vealed that OPN induced NIK- and IKK-dependent NF�B ac-tivation through phosphorylation and degradation of I�B�.Moreover NIK in the presence of OPN enhanced MEK-1 andERK1/2 phosphorylations and MEK/ERK-dependent NF�B ac-tivation. OPN induced NIK-dependent ERK/IKK-mediateduPA secretion, uPA-dependent pro-MMP-9 activation, cell mi-gration, and chemoinvasion. OPN induced the pro-MMP-9 ac-tivation in tumor of nude mice. These data demonstrated that

FIG. 8. Panels A–D, NIK and IKK�/� are involved in OPN-stimulated uPA and MMP-9-dependent cell migration and chemoinva-sion. The migration assay was conducted either by using untreated cells (5 � 106cells/well) or cells pretreated with anti-MMP-9 antibody (25�g/ml) or anti-uPA antibody (25 �g/ml). In separate experiments, cells were transfected with wild type and kinase-negative NIK, wild type and dnIKK� and IKK�, or the super-repressor form of I�B�. The purified human OPN (5 �M) was added in the upper chamber. The treated or transfectedcells were used for migration assays. Note that OPN-induced cell migration was blocked by anti-MMP-9 antibody, anti-uPA antibody, kinase-negative NIK, and dn IKK�/� and enhanced by wild type NIK and wild type IKK�/� (panels A and C). The same results were obtained in thechemoinvasion assay (panels B and D). The results are expressed as the means � S.E. of three determinations. Ab, antibody; sup. rep.,super-repressor.


OPN induces NIK-dependent cell motility, tumor growth,NF�B-mediated uPA secretion, and uPA-regulated pro-MMP-9activation by activating ERK/IKK signaling pathways.

Recent studies have demonstrated that mitogen-activatedprotein kinase kinase kinases including NIK and MEK kinases1–3 are involved in the activation of IKK complex (38–40).Kouba et al. (41) have shown that NIK regulates NF�B activa-tion pathways in epidermal keratinocytes. Thus we examinedwhether NIK has any effect in OPN-induced NF�B activationin B16F10 cells. The data showed that OPN stimulated NIKphosphorylation and its kinase activity in these cells. Pretreat-ment of cells with anti-�v�3 integrin antibody or RGD but notRGE peptide inhibited OPN-induced NIK phosphorylation in-

dicating that �v�3 integrin is involved in this process. Previousdata has also suggested that NIK strongly interacts with bothIKK� and -� (42, 43). NIK also interacts with TRAF proteinsincluding TRAF-3 as shown by yeast two-hybrid systems (44).Our data revealed that OPN enhances the interaction betweenphosphorylated NIK and IKK�/� in B16F10 cells. Previousstudies have also indicated that IKK activation alone could notaccount for the total NF�B activity in HS294T cells (45). Foehret al. (17) have recently demonstrated that NIK regulates dif-ferentiation of PC-12 cells through MEK/ERK pathways. Theseresults prompted us to investigate whether OPN regulatesNIK-dependent NF�B activation through the MAPK pathwayin B16F10 cells. Our data indicated that transient overexpres-

FIG. 9. Panels A and B, both MMP-2 and MMP-9 play important roles in OPN-induced cell migration and chemoinvasion. The migration assaywas performed either by using untreated cells or cells pretreated with anti-MMP-2 antibody (25 �g/ml) or anti-MMP-9 antibody (25 �g/ml) or acombination of both. The purified human OPN was added to the upper chamber. These cells were used for migration assays (panel A). The sameresults were obtained in the invasion assay (panel B). Panel C, OPN-induced pro-MMP-9 activation is distinct from pro-MMP-2 activation. Cellswere pretreated with anti-MMP-2 antibody (0–50 �g/ml) and then treated with 5 �M OPN. The level of MMP-9 was detected by Western blot usinganti-MMP-9 antibody (panel C, lanes 1–4). Note that OPN-induced MMP-9 activation is unaffected by anti-MMP-2 antibody. Panels D and E,ERK1/2 is involved in OPN-stimulated NIK-dependent cell migration. Cells were either pretreated with PD98059 or transfected with wild type orkinase-negative NIK and then treated with PD98059. In separate experiments, cells were transfected with wild type and dn ERK1 and ERK2.These treated or transfected cells were used for the cell migration assay (panels D and E). The results are expressed as the means � S.E. of threedeterminations. Ab, antibody.


sion of wild type NIK up-regulates OPN-induced MEK andERK1/2 phosphorylations, whereas kinase-negative NIK abro-gated these processes. We also showed that OPN-induced NIK-dependent MEK-1 phosphorylation was suppressed by anti-�v�3 integrin blocking antibody and RGD peptide but not RGEpeptide indicating that OPN regulates NIK-mediated MAPKactivation through �v�3 integrin-mediated pathways.

Several reports have indicated that NF�B is involved incontrol of large number of cellular processes such as inflamma-tory and immune response, developmental processes, cellgrowth, and apoptosis. In addition, NF�B is activated in sev-eral pathological conditions like arthritis, inflammation,asthma, neurodegenerative disease, heart disease, and cancers(46). In our previous report, we have demonstrated that OPNstimulated NF�B activation by inducing the phosphatidylinosi-tol 3-kinase/IKK activity in murine melanoma and breast can-cer cells (21, 22). However, it was not clear whether NIK isinvolved in regulation of OPN-induced NF�B activation andwhether MAPK/IKK plays any role in this activation process inB16F10 cells. In this study, we found that OPN induces NIK-dependent IKK activity through interaction between phospho-rylated NIK and IKK. Transfection of these cells with wild typeNIK and IKK�/� but not with kinase-negative NIK or dnIKK�/� enhanced OPN-induced NF�B-DNA binding and NF�Btransactivation. Moreover PD98059, an MEK-1 inhibitor,down-regulated NF�B-DNA binding and NF�B activation(data not shown) in cells transfected with wild type NIK fol-lowed by treatment with OPN. Overexpression of wild typeERK1/2 but not dn ERK1/2 dramatically enhanced OPN-in-duced NF�B activation. These data suggested a link betweenOPN-induced NIK/IKK/NF�B and NIK/ERK/NF�B pathwaysin B16F10 cells.

Signals transduced by cell adhesion molecules play an im-portant role in tumor cell attachment, motility, and invasion,all of which regulate tumor metastasis. Cell-matrix interac-tions play a major role in tissue remodeling, cell survival, andtumorigenesis. OPN, an ECM protein, plays a significant rolein cell adhesion, migration, and metastasis. The overexpression

of OPN is linked with various cancers and their metastaticpotentials (8). MMPs are a family of Zn2�-dependent endopep-tidases that are responsible for remodeling of the extracellularmatrix and degradation of ECM proteins. MMP-9 is known todegrade basement membrane, which normally separates theepithelial from the stromal compartment. Elevated levels ofMMP-9 have been reported in various cancers. Several studieshave shown the correlation between MMP-9 expression andmetastatic potential of tumor (47). MMP-9 is not only associ-ated with invasion/metastasis but also reported to be involvedin a number of angiogenic relevant diseases such as rheuma-toid arthritis, retinopathy, and vascular stenosis and is consid-ered to be a therapeutic target of priority (35, 36). Kim et al.(48, 49) have also demonstrated that MMP-9 activity but notMMP-2 activity significantly affects tumor intravasation intoblood vessel and that uPA is required for pro-MMP-9 activa-tion. Several other reports have indicated the correlation be-tween uPA expression and metastatic potential and shown thatuPA plays a major role in regulating MMP activation (22, 50).Therefore, we sought to determine whether OPN regulates uPAsecretion and whether uPA plays any role in regulation ofpro-MMP-9 activation. In this study, we showed that OPNinduces uPA secretion and uPA-dependent pro-MMP-9 activa-tion. Pretreatment of cells with anti-�v�3 integrin antibody orRGD but not RGE peptide inhibited OPN-induced pro-MMP-9activation indicating that OPN induces pro-MMP-9 activationthrough �v�3 integrin-mediated pathways. Overexpression ofwild type NIK and IKK�/� enhanced and kinase-negative NIK,dn IKK�/�, or the super-repressor form of I�B� suppressed theOPN-induced pro-MMP-9 activation demonstrating that OPNregulates pro-MMP-9 activation through NIK/IKK/NF�B-me-diated pathways. Recent data also indicated that the expres-sion of MMP-9 is down-regulated in ERK-mutated stable trans-fectants; this inhibits glioma invasion in vitro (51). In thisstudy, we examined whether MAPK, especially ERK1/2, regu-lates OPN-induced pro-MMP-9 activation in B16F10 cells. Theresults showed that overexpression of wild type ERK1/2 but notdn ERK1/2 up-regulated OPN-induced uPA secretion leadingto the activation of pro-MMP-9 indicating that OPN inducesuPA-dependent pro-MMP-9 activation through NIK/MAPK-mediated pathways.

Previous studies have indicated that uPA and MMP-9 ex-pressions are inversely related to MT1-MMP expression inesophageal carcinoma (52). It has been implicated that thereare two pathways involved in esophageal carcinogenesis, one isinvolved in the MT1-MMP/MMP-2 activation pathway and theother one is the uPA/MMP-9 activation pathway, and bothpathways are critical in regulation of cancer cell motility, in-

FIG. 10. OPN induces tumor growthand MMP-9 activation in tumors ofnude mice. Panel A, typical photographsof tumors in nude mice. The cells weretreated in the absence or presence of OPN(10 �M) and then injected subcutaneouslyinto the flanks of nude mice. a, cells withphosphate-buffered saline; b, cells withOPN. The results show the representativeof four mice used in each set of experi-ments. Panels B and C, detection of activeMMP-9 in tumors of nude mice by zymog-raphy (panel B) and Western blot (panelC). The tumor samples were lysed, andthe levels of MMP-9 in these sampleswere analyzed by zymography and West-ern blot as described earlier. The arrowsindicate the 92- and 86-kDa MMP-9-spe-cific bands.

TABLE IOPN induces tumor growth in nude mice

B16F10 cells were treated with 10 �M OPN for 16 h and injected intonude mice (NMRI). The injection was performed twice a week for 4weeks. The mice were killed, and the tumor weights were measured andanalyzed statistically by Student’s t test (p � 0.002). Mice injected withcells in phosphate-buffered saline (PBS) were used as controls.

No. nude mice Treatment Tumor weight (-fold change)

4 Control (PBS) 1.0 � 0.154 OPN (10 �M) 3.2 � 0.17


vasiveness, tumor growth, and angiogenesis. We have reportedearlier that OPN induces pro-MMP-2 activation throughNF�B-mediated induction of MT1-MMP in murine melanomacells (20). However, the mechanism by which OPN regulatesuPA-dependent pro-MMP-9 activation and controls cell migra-tion, invasion, and tumor growth are not well defined. Pretreat-ment of cells with anti-uPA antibody suppressed OPN-inducedpro-MMP-9 activation indicating that uPA plays a crucial rolein this process. Moreover pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody dramati-cally suppressed the OPN-induced cell migration and chemo-invasion, whereas pretreatment of cells with anti-MMP-2antibody had no effect on OPN-induced pro-MMP-9 activation.These data suggested that both MMP-2 and MMP-9 are in-volved in OPN-induced cell migration and chemoinvasion, butOPN-induced MMP-2 activation is distinct from MMP-9 acti-vation. Wild type NIK, IKK�/�, and ERK1/2 enhanced andkinase-negative NIK, dn IKK�/�, and dn ERK1/2 suppressedOPN-induced cell migration and chemoinvasion. OPN also in-duced the pro-MMP-9 activation in tumor of nude mice. Thesedata demonstrated that OPN induces NIK-regulated NF�B-mediated uPA-dependent pro-MMP-9 activation, cell motility,and tumor growth via ERK/IKK-mediated signaling pathways.

In summary, we demonstrated for the first time that OPNinduces activation and phosphorylation of NIK through interac-tion between IKK and phosphorylated NIK. OPN also enhancesNIK-dependent IKK�/�- and ERK1/2-mediated NF�B activation,uPA secretion, and pro-MMP-9 activation. Taken together, OPNinduces cell migration, tumor growth, and NIK-dependentNF�B-mediated uPA secretion and pro-MMP-9 activation by ac-tivating IKK/ERK signaling pathways (Fig. 11). These findingsmay be useful in designing novel therapeutic interventions that

block the OPN-regulated NIK-dependent IKK- and ERK1/2-me-diated NF�B activation resulting in reduction of uPA secretionand pro-MMP-9 activation and consequent blocking of cell motil-ity, invasiveness- and melanoma growth.

Acknowledgments—We thank Prof. David Wallach for providing wildtype NIK (wt pcDNA NIK) and kinase-negative NIK (mut pcDNA NIK,NIK-K429A/K430A) in pcDNA3 and Prof. D. V. Goeddel for wild typeand dominant negative constructs of IKK� (wt IKK� and dn IKK�) andIKK� (wt IKK� and dn IKK�) in pRK. We also thank Dr. Melanie Cobbfor wt ERK1 and dn ERK1 in pCEP4 and wt ERK2 and dn ERK2 inp3XFLAG-CMV-7.1, Dr. Rainer de Martin for the luciferase reporterconstruct (pNF�B-Luc) containing five tandem repeats of NF�B bindingsite, and Dr. Dean Ballard for the super-repressor form of I�B� cDNAfused downstream to a FLAG epitope in an expression vector (pCMV4).

REFERENCES

1. Butler, W.T. (1989) Connect. Tissue Res. 23, 123–1362. Denhardt, D., and Guo, X. (1993) FASEB J. 7, 1475–14823. Prince, C. W. (1989) Connect. Tissue Res. 21, 15–204. Weber, G. F. (2001) Biochim. Biophys. Acta 1552, 61–855. Chen, Y., Bal, B. S., and Gorski, J. P. (1992) J. Biol. Chem. 267, 24871–248786. Singh, K., Devouge, M. W., and Mukherjee, B. B. (1990) J. Biol. Chem. 265,

18696–187017. Ritter, N. M., Farach-Carson, M. C., and Butler, W.T. (1992) J. Bone Miner.

Res. 7, 877–8858. Craig, A. M., Bowden, G. T. Chambers, A. F., Spearman, M. A., Greenberg,

A. H., Wright, J. A., and Denhardt, D. T. (1990) Int. J. Cancer 46, 133–1379. Weber, G. F., Akshar, S., Glimcher, M. J., and Cantor, H. (1996) Science 271,

509–51910. Panda, D., Kundu, G. C., Lee, B. I., Peri, A., Fohl, D., Chackalaparampil, I.,

Mukherjee, B. B., Li, X. D., Mukherjee, D. C., Seides, S., Rosenberg, J.,Stark, K., and Mukherjee, A. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,9308–9313

11. Liaw, L., Birk, D. E., Ballas, C. B., Whitsitt, J. S., Davidson, J. M., and Hogan,B. L (1998) J. Clin. Investig. 101, 1468–1478

12. Rittling, S. R., and Novick, K. E. (1999) Cell Growth Differ. 8, 1061–106913. Bauerle, P. A., and Baltimore, D. (1996) Cell 87, 13–2014. Karin, M. (1999) J. Biol. Chem. 274, 27339–2734215. Baldwin, A.S. (1996) Annu. Rev. Immunol. 14, 649–68116. Malinin, N. L., Boldin, M. P., Kovalenko, A.V., and Wallach, D. (1997) Nature

FIG. 11. Molecular mechanism ofOPN-induced NIK-regulated NF�B-mediated uPA secretion and pro-MMP-9 activation through MAPK/IKK pathways. Binding of OPN to �v�3integrin receptor induces phosphorylationand activation of NIK and its subsequentinteraction with IKK�/�, which activatesNF�B through phosphorylation and deg-radation of I�B�. In addition, NIK in thepresence of OPN induces MEK-1/ERK1/2phosphorylation, which also activatesNF�B. OPN stimulates NIK-dependentIKK/MAPK-mediated uPA secretion,which regulates pro-MMP-9 activation,cell motility, invasion, and tumor growth.


385, 540–54417. Foehr, E. D., Bohuslav, J., Chen, L.-F., DeNoronha, C., Geleziunas, R., Lin, X.,

O’Mahony, A., and Greene, W. C. (2000) J. Biol. Chem. 275, 34021–3402418. Song, H. Y., Regnier, C. H., Kirschning, C. J., Goeddel, D. V., and Rothe, M.

(1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9792–979619. Nasuhara, Y., Adcock, I. M., Caitley, M., and Barnes, P. J., and Newton, R.

(1999) J. Biol. Chem. 274, 19965–1997220. Philip, S., Bulbule, A., and Kundu, G. C. (2001) J. Biol. Chem. 276,

44926–4493521. Philip, S., and Kundu, G. C. (2003) J. Biol. Chem. 278, 14487–1449722. Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278,

28593–2860623. Collen, C, (1999) Thromb. Haemostasis 82, 259–27024. Blasi, F. (1999) Thromb. Haemostasis 82, 198–20425. MacDougall, J. R., and Matrisian, L. M. (1995) Cancer Metastasis Rev. 14,

351–36226. Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278,

6209–622127. Shapiro, S. D. (1998) Curr. Opin. Cell Biol. 10, 602–60828. Scorilas, A., Karameris, A., Amogiannaki, N., Ardavanis, A., Bassilopoulos, P.,

Trangas, T., and Talleri, M. (2001) Br. J. Cancer 84, 1488–149629. Hrabec, E., Strek, M., Nowak, D., and Hrabec, Z. (2001) Respir. Med. 95, 1–430. Sakamoto, Y., Mafune, K., Mori, M., Shiraishi, T., Imamura, H., Mori, M.,

Takayama, T., and Makuuchi, M. (2000) Int. J. Oncol. 17, 237–24331. Shen, K. H., Chi, C. W., Lo, S. S., Kao, H. L., Lui, W. Y., and Wu, C. W. (2000)

Anticancer Res. 20, 1307–131032. Dubois, B., Masure, S., Hurtenbach, U., Paemen, L., Heremans, H., Van den

Oord, J., Sciot, R., Meinhardt, T., Hammerling, G., Opdenakker, G., andArnold, B. (1999). J. Clin. Investig. 104, 1507–1515

33. Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer,J. L., Kronberger, A., He, C. S., Bauer, E. A., and Goldberg, G. I. (1988)J. Biol. Chem. 263, 6579–6587

34. Houde, M., de Bruyne, G., Bracke, M., Ingelman-Sundberg, M., Skoglund, G.,Masure, S., van Damme, J., and Opdenakker, G. (1993) Int. J. Cancer. 53,

395–40035. Folkman, J. (1999) Nat. Biotechnol. 17, 74936. Fini, M. E., Cook, J. R., Mohan, R., and Brinckerhoff, C. E. (1998) in Matrix

Metalloproteinases (Parks, W. C., and Mecham, R. P., eds) pp. 300–356,Academic Press, New York

37. Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2004) J. Biol. Chem. 279,11051–11064

38. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science278, 866–869

39. Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., andOkumura, K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3537–3542

40. Nemoto, S., DiDonato, J. A., and Lin, A. (1998) Mol. Cell. Biol. 18, 7336–734341. Kouba, D. J., Nakano, H., Nishiyama, T., Kang, J., Uitto, J., and Mauviel, A.

(2001) J. Biol. Chem. 276, 6214–622442. Ling, L., Cao, Z., and Goeddel, D. V. (1998) Proc. Natl. Acad. Sci. U. S. A. 95,

3792–379743. Delhase, M., Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284,

309–31344. Takaori-Kondo, A., Hori, T., Fukunaga, K., Morita, R., Kawamata, S., and

Uchiyama, T. (2000) Biochem. Biophys. Res. Commun. 272, 856–86345. Devalaraja, M. N., Wang, D. Z., Ballard, D. W., and Richmond, A. (1999)

Cancer Res. 59, 1372–137746. Rayet, B., and Gelinas, C. (1999) Oncogene 18, 6938–694747. Davies, B., Waxman, J., Wasan, H., Abel, P., Williams, G., Krausz, T., Neal, D.,

Thomas, D., Hanby, A., and Balkwill, F. (1993) Cancer Res. 53, 5365–536948. Kim, J., Yu, K., Kovalski, K., and Ossowski, L. (1998) Cell 94, 353–36249. Sehgal, G., Hua, J., Bernhard, E. J., Sehgal, I., Thompson, T. C., and Muschel,

R. J. (1998) Am. J. Pathol. 152, 5591–559650. Aguirre Ghiso, J. A., Kovalski, K., and Ossowski, L. (1999) J. Cell Biol. 147,

89–10351. Lakka, S. S., Jasti, S. L., Gondi, C., Boyd, D., Chandrasekar, N., Dinh, D. H.,

Olivero, W. C., Gujrati, M., and Rao, J. S. (2002) Oncogene 21, 5601–560852. Yamashita, K., Tanaka, Y., Miniori, K., Inoue, H., and Mori, M. (2004) Int. J.

Cancer 110, 201–207


JNK1 Differentially Regulates Osteopontin-induced Nuclear Factor-inducing Kinase/MEKK1-dependent Activating Protein-1-mediatedPromatrix Metalloproteinase-9 Activation*

Received for publication, December 17, 2004, and in revised form, February 15, 2005Published, JBC Papers in Press, March 9, 2005, DOI 10.1074/jbc.M414204200

Hema Rangaswami, Anuradha Bulbule, and Gopal C. Kundu‡

From the National Center for Cell Science, Pune 411 007, India

We have recently demonstrated that nuclear factor-in-ducing kinase (NIK) plays a crucial role in osteopontin(OPN)-induced mitogen-activated protein kinase/I�B�kinase-dependent nuclear factor �B (NF�B)-mediatedpromatrix metalloproteinase-9 activation (Rangaswami,H., Bulbule, A., and Kundu, G. C. (2004) J. Biol. Chem.279, 38921–38935). However, the molecular mecha-nism(s) by which OPN regulates NIK/MEKK1-dependentactivating protein-1 (AP-1)-mediated promatrix meta-lloproteinase-9 activation and whether JNK1 plays anyrole in regulating both these pathways that controlthe cell motility are not well defined. Here we reportthat OPN induces �v�3 integrin-mediated MEKK1 phos-phorylation and MEKK1-dependent JNK1 phosphoryla-tion and activation. Overexpression of NIK enhancesOPN-induced c-Jun expression, whereas overexpressedNIK had no role in OPN-induced JNK1 phosphorylationand activation. Sustained activation of JNK1 by overex-pression of wild type but not kinase negative MEKK1resulted in suppression of ERK1/2 activation. But thisdid not affect the OPN-induced NIK-dependent ERK1/2activation. OPN stimulated both NIK and MEKK1-dependent c-Jun expression, leading to AP-1 activation,whereas NIK-dependent AP-1 activation is independentof JNK1. OPN also enhanced JNK1-dependent/independent AP-1-mediated urokinase type plasmino-gen activator (uPA) secretion, uPA-dependent pro-matrix metalloproteinase-9 (MMP-9) activation, cell mo-tility, and invasion. OPN stimulates tumor growth, andthe levels of c-Jun, AP-1, urokinase type plas-minogen activator, and MMP-9 were higher in OPN-induced tumor compared with control. To our knowl-edge this is first report that OPN induces NIK/MEKK1-mediated JNK1-dependent/independent AP-1-mediatedpro-MMP-9 activation and regulates the negative cross-talk between NIK/ERK1/2 and MEKK1/JNK1 pathwaysthat ultimately controls the cell motility, invasiveness,and tumor growth.

Osteopontin (OPN),1 a non-collagenous, sialic acid-rich andglycosylated phosphoprotein, is a member of the extracellular

matrix (ECM) protein family (1, 2). OPN acts both as chemo-kine and cytokine. It is produced by osteoclast, macrophages, Tcells, hematopoietic cells, and vascular smooth muscle cells (3).It has an N-terminal signal sequence, a highly acidic regionconsisting of nine consecutive aspartic acid residues, and aGRGDS cell adhesion sequence predicted to be flanked by the�-sheet structure (4). This protein has a functional thrombincleavage site and is a substrate for tissue transglutaminase (2).It binds with several integrins and CD44 variants in an RGDsequence-dependent and -independent manner (5, 6). This pro-tein is involved in normal tissue remodeling process such asbone resorption, angiogenesis, wound healing, and tissue in-jury as well as certain diseases such as tumorigenesis, resteno-sis, atherosclerosis, and autoimmune diseases (6–8). OPN ex-pression is up-regulated in several cancers and is reported toassociate with tumor progression and metastasis (9–11). OPNregulates cell adhesion, cell migration, ECM-invasion, and cellproliferation by interacting with its receptor �v�3 integrin invarious cell types (6). Previous data indicated that OPN in-duces MT1-MMP-mediated pro-matrix metalloproteinase-2(pro-MMP-2) activation and urokinase type plasminogen acti-vator (uPA)-mediated pro-matrix metalloproteinase-9 (pro-MMP-9) activation, cell motility, ECM invasion, and tumorgrowth (12–16).

Integrins are non-covalently associated, heterodimeric, cell-surface glycoproteins with �- and �-subunits. The various com-binations of the �- and �-subunits form integrin dimers withdiverse ligand specificity and biological activities. The interactionof cell surface integrin with ECM proteins can lead to the regu-lation of cell growth, differentiation, adhesion, and migration.

MEKK1, a member of the mitogen-activated protein 3-kinasefamily, is a mammalian serine/threonine protein kinase ini-tially identified on the basis of its homology with STE11 thatactivates the pheromone-responsive mitogen-activated proteinkinase cascade in yeast (17). Previous data indicated that over-expression of constitutively active forms of MEKK1 leads toJNK activation via phosphorylation of its upstream kinase,mitogen-activated protein kinase kinase 4 (18). The data alsoshowed that MEKK1 has the ability to activate ERK, but itseffect is less potent (19). These results suggest that MEKK1 isan upstream kinase in the mitogen-activated protein kinasecascade. Nuclear factor-inducing kinase (NIK) is another mem-ber of the mitogen-activated protein 3-kinase family that hasbeen implicated in NF�B activation. Few reports indicate thatNIK may also be involved in the regulation of transcriptionfactor, AP-1, as its activation leads to the induction of c-Fos

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ To whom correspondence should be addressed: National Center forCell Science, NCCS Complex, Pune 411 007, India. Tel.: 91-20-25690951 (ext. 203); Fax: 91-20-25692259; E-mail: [email protected] [email protected].

1 The abbreviations used are: OPN, osteopontin; NIK, nuclear factor-inducing kinase; MEKK1, mitogen-activated protein kinase kinase ki-nase; ERK, extracellular signal regulated kinase; JNK, c-Jun N-termi-nal kinase; AP-1, activating protein-1; EMSA, electrophoretic mobility

shift assay; DTT, dithiothreitol; Luc, luciferase; ECM, extracellularmatrix; uPA, urokinase type plasminogen activator; MMP-9, matrixmetalloproteinase-9; IKK, I�B kinase complex; wt, wild type; dn, dom-inant negative; mut, mutant.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 19, Issue of May 13, pp. 19381–19392, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 19381

that associates with c-Jun to form an AP-1 heterodimeric com-plex that can promote targeted gene expression (20). However,the molecular mechanism by which OPN regulates NIK andMEKK1-mediated AP-1 transactivation and whether JNK isinvolved in both these pathways is not clearly understood.Various mitogen-activated protein kinase cascades (e.g.ERK1/2, JNK, p38) are often portrayed as linear cascades, andindications of cross-talk between the various cascades are lim-ited (21, 22). In this respect, the present study also examineswhether any cross-talk exists between OPN-induced NIK/ERK-and MEKK1/JNK-signaling pathways.

uPA is a member of serine protease family that interactswith the uPA receptor and facilitates the conversion of inertzymogen plasminogen into widely acting serine protease plas-min (23). Plasmin regulates cell invasion by degrading matrixproteins such as type IV collagen, gelatin, fibronectin, andlaminin or indirectly by activating MMPs (24, 25). It is estab-lished that uPA plays a significant role in tumor growth andmetastasis (26–28). It is regulated at the transcriptional levelby a number of transcription factors. AP-1 transcription factorduplex also plays a major role in the regulation of uPA expres-sion through binding to its promoter (29). However, the molec-ular mechanism by which OPN regulates NIK and MEKK1-mediated JNK-dependent/independent AP-1 activation anduPA secretion in murine melanoma (B16F10) cells is notwell defined.

MMPs are ECM degrading enzymes that play a critical rolein embryogenesis, tissue remodeling, inflammation, and angio-genesis (30). We have recently reported that OPN inducesNF�B-mediated pro-MMP-2 activation through I�B�/IKK sig-naling pathways (12, 13). MMP-9, also referred to as type IVcollagenase or gelatinase B, efficiently degrades native type IVand V collagens, fibronectin, ectactin, and elastin and, hence,plays a major role in invasion, tumor growth, and metastasis(31–34). The regulation of activation of MMP-9 is more complexthan most of the other MMPs because most of the cells do notexpress a constitutively active form of MMP-9, but its activityis induced by different stimuli depending on cell types (35–37),thereby contributing to the specific pathological events. MMP-9is not only associated with invasion and metastasis but also hasbeen implicated in angiogenesis, rheumatoid arthritis, retinop-athy, and vascular stenosis and, hence, is considered to beprioritized therapeutic target (38). However the molecularmechanism by which OPN regulates AP-1-mediated pro-MMP-9 activation and controls cell migration and tumorgrowth is not well defined.

In this paper we have demonstrated that OPN induces �v�3integrin-mediated NIK- and MEKK1-dependent c-Jun expres-sion, leading to AP-1 activation and uPA secretion in B16F10cells. This OPN-induced MEKK1- and NIK-mediated AP-1transactivation occurs through both JNK-dependent and -inde-pendent pathways. OPN also induces a negative cross-talkbetween NIK/ERK and MEKK1/JNK1 pathways. Moreover,OPN also induces uPA secretion and uPA-dependent pro-MMP-9 activation, cell motility, invasion, and tumor growth.Taken together, these data demonstrated that OPN induces�v�3 integrin-mediated NIK and MEKK1 kinase activities thatultimately enhance c-Jun expression through JNK-dependentand -independent pathways. OPN regulates cross-talk betweenJNK and ERK that leads to the induction of uPA secretion anduPA-dependent pro-MMP-9 activation, cell motility, invasion,and tumor growth.

EXPERIMENTAL PROCEDURES

Materials—The rabbit polyclonal anti-NIK, anti-MEKK1, anti-IKK�/�, anti-JNK1, anti-c-Jun, anti-ERK1/2, anti-uPA, anti-MMP-9and anti-actin, mouse monoclonal anti-phospho-ERK1/2, anti-phospho-

tyrosine antibodies, recombinant MEK-1, p42 MAPK, and c-Jun pro-teins were purchased from Santa Cruz Biotechnology. Mouse mono-clonal anti-MMP-9 was from Oncogene. Mouse monoclonal anti-human�v�3 integrin antibody was from Chemicon International. Lipo-fectamine Plus, GRGDSP, and GRGESP were obtained from Invitro-gen. Mouse monoclonal anti-phosphoserine detection kit and SP600125were from Calbiochem. Myelin basic protein was from Sigma. The dualluciferase reporter assay system and AP-1 consensus oligonucleotidewere purchased from Promega. Boyden type cell migration chamberswere obtained from Corning, and BioCoat MatrigelTM invasion cham-bers were from Collaborative Biomedical. The [�-32P]ATP was pur-chased from the Board of Radiation and Isotope Technology (Hyder-abad, India). The human OPN was purified from milk as describedpreviously and used throughout these studies (12). The nude mice(NMRI, nu/nu) were from National Institute of Virology (Pune, India).All other chemicals were of analytical grade.

Cell Culture—The B16F10 cells were obtained from American TypeCulture Collection (Manassas, VA). These cells were cultured in Dul-becco’s modified Eagle’s medium supplemented with 10% fetal calfserum, 100 units/ml penicillin, 100 �g/ml streptomycin, and 2 mM

glutamine in a humidified atmosphere of 5% CO2 and 95% air at 37 °C.Plasmids and DNA Transfection—The wild type NIK (wt pcDNA

NIK) and kinase-negative NIK (mut pcDNA NIK, NIK-K429A/K430A)in pcDNA3 were generous gifts from Prof. David Wallach (WeizmannInstitute of Science, Rehovot, Israel). The wild type and kinase negativeconstructs of MEKK1 (pcDNA3 MEKK1 and pcDNA3 FlagMEKK1K432M) were kind gifts from Prof. Tom Maniatis (Harvard University,Cambridge). The wild type c-Jun in pRJB10B and dominant negativec-Jun in pELFIN were gifts from Dr. Jalam (Ochsner Clinic Founda-tion, New Orleans, LA). The wild type and dominant negative forms ofJNK1 in pcDNA3 were kind gifts from Dr. Roger Davis (University ofMassachusetts Medical School, Worcester, MA). The luciferase reporterconstruct (pAP-1-Luc) containing seven tandem repeats of the AP-1binding site was from Stratagene. The B16F10 cells were split 12 hbefore transfection in Dulbecco’s modified Eagle’s medium containing10% fetal calf serum. These cells were transiently transfected withcDNA using Lipofectamine Plus according to manufacturer’s instruc-tions as described (Invitrogen). These transfected cells were used forJNK1 and ERK1/2 phosphorylation and kinase assay, c-Jun expression,AP-1-DNA binding, AP-1 luciferase reporter gene assay, and detectionof MMP-9 by zymography and Western blot, uPA secretion by Westernblot, cell migration, chemoinvasion, and in vivo experiments.

Western Blot Analysis—To ascertain the role of MEKK1 and JNK-1in OPN-induced uPA secretion, the cells were transfected with wildtype and kinase negative MEKK1 or wild type and dominant negativeJNK-1 or pretreated with 50 �M SP600125 (JNK-1 inhibitor) and thentreated with 5 �M OPN for 24 h. To examine whether c-Jun plays anyrole in OPN-induced uPA secretion, cells were transfected with wildtype and dominant negative c-Jun and then treated with OPN. The celllysates containing equal amount of total proteins were subjected toWestern blot analysis using rabbit polyclonal anti-uPA antibody anddetected by ECL detection system (Amersham Biosciences). As loadingcontrols, the expression of actin was also detected by reprobing the blotswith rabbit anti-actin antibody.

Immunoprecipitation—To examine the effect of OPN in regulation ofMEKK1 phosphorylation, cells were treated with 5 �M OPN at 37 °C for0–60 min. In separate experiments, cells were pretreated with anti-�v�3 integrin antibody (20 �g/ml), GRGDSP or GRGESP peptide (10�M) and then treated with 5 �M OPN. The cells were lysed in lysis buffer(50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, and 2mM phenylmethylsulfonyl fluoride), and cell lysates containing equalamount of total proteins were immunoprecipitated with rabbit poly-clonal anti-MEKK1 antibody. The immunocomplexes were analyzed byWestern blot using mouse monoclonal anti-phospho-serine antibody. Asloading control, same blots were reprobed with rabbit polyclonal anti-MEKK1 antibody.

To delineate the role of OPN in regulation of JNK-1 phosphorylation,the cells were treated with 5 �M OPN for 0–90 min at 37 °C. In separateexperiments cells were pretreated with anti-�v�3 integrin antibody (20�g/ml), GRGDSP or GRGESP peptide (10 �M) and then treated with 5�M OPN. Cell lysates were immunoprecipitated with rabbit polyclonalanti-JNK-1 antibody. The immunocomplexes were analyzed by Westernblot using mouse monoclonal anti-phosphotyrosine antibody. The sameblots were reprobed with rabbit anti-JNK-1 antibody as loading control.

To detect whether NIK and MEKK1 are involved in the regulation ofOPN induced JNK-1 phosphorylation, cells were transfected with wildtype and kinase negative NIK or wild type and kinase negative MEKK1

Cross-talk between OPN-induced JNK and ERK Pathways19382

and then treated with 5 �M OPN for 15 min. Cell lysates were immu-noprecipitated with rabbit polyclonal anti-JNK-1 antibody and ana-lyzed by Western blot using anti-phosphotyrosine antibody. The sameblots were reprobed with anti-JNK1 antibody.

Nuclear Extracts and Western Blot—To check the level of c-Junexpression in the nucleus, cells were treated with 5 �M OPN for 0–4 hat 37 °C. In separate experiments, cells were pretreated with anti-�v�3integrin antibody (20 �g/ml), GRGDSP or GRGESP peptide (10 �M) ortransfected with wild type and kinase negative NIK or wild type andkinase negative MEKK1 and then treated with 5 �M OPN. The nuclearextracts were prepared as described (16). Briefly, cells were incubatedin hypotonic buffer (10 mM Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM KCl,0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) andallowed to swell on ice for 10 min. Cells were homogenized in a Douncehomogenizer. The nuclei were separated by spinning at 3300 � g for 5min at 4 °C. The nuclear pellet was extracted in nuclear extractionbuffer (20 mM Hepes (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride and 0.5 mM dithi-othreitol) and centrifuged at 12,000 � g for 30 min. The supernatantwas used as nuclear extract. The protein concentration was measuredby the Bio-Rad protein assay. The nuclear extracts were resolved bySDS-PAGE, and the level of c-Jun was detected by Western blot usingrabbit polyclonal anti-c-Jun antibody.

EMSA—To check whether NIK and MEKK1 play any role in OPN-induced AP-1-DNA binding, cells were either treated with 5 �M OPN ortransfected with wild type and kinase negative NIK or wild type andkinase negative MEKK1 and then treated with OPN for 1 h at 37 °C.The nuclear extracts were prepared as described earlier (16). To inves-tigate whether JNK1 is involved in OPN-induced AP-1-DNA bindingand whether it is regulated by NIK, cells were either pretreated withSP600125 (JNK-1 inhibitor) or transfected with wild type NIK and thentreated with SP600125. Transfected or treated cells were furthertreated with OPN. The nuclear extracts (10 �g) were incubated with 16fmol of 32P-labeled double-stranded AP-1 oligonucleotide (5�-CGC TTGATG ACT CAG CCG GAA-3�) in binding buffer (25 mM Hepes (pH 7.9),0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol, 50 mM NaCl)containing 2 �g of poly(dI-dC). The DNA-protein complex was resolvedon a native polyacrylamide gel and analyzed by autoradiography. Forsupershift assay, the OPN-treated nuclear extracts were incubatedwith anti-c-Jun antibody for 30 min at room temperature and analyzedby EMSA.

AP-1 Luciferase Reporter Gene Assay—The semi-confluent cellsgrown in 24-well plates were transiently transfected with a luciferasereporter construct (pAP-1-Luc) containing seven tandem repeats of theAP-1 binding site using Lipofectamine Plus reagent (Invitrogen). Inseparate experiments, cells were individually transfected with wildtype and kinase negative NIK or wild type and kinase negative MEKK1or wild type and dn JNK1 along with AP-1-Luc. The transfection effi-ciency was normalized by cotransfecting the cells with pRL vector(Promega) containing a full-length Renilla luciferase gene under thecontrol of a constitutive promoter. After 24 h of transfection, cells wereeither pretreated with SP600125 for 1 h and/or treated with 5 �M OPNfor 6 h. Cells were harvested in passive lysis buffer (Promega). Theluciferase activities were measured by luminometer (Lab Systems) us-ing the dual luciferase assay system according to the manufacturer’sinstructions (Promega). Changes in luciferase activity with respect tocontrol were calculated.

Zymography Experiments—The gelatinolytic activity was measuredas described (12, 13). To ascertain the role of MEKK1 and JNK1 onOPN-induced pro-MMP-9 activation, the cells were transfected withwild type and kinase negative MEKK1 or wild type and dn JNK1 orpretreated with SP600125 (0–50 �M) for 1 h and then treated with 5 �M

OPN. The conditioned medium was collected, and the samples contain-ing equal amount of total proteins were mixed with sample buffer in theabsence of reducing agent and loaded onto zymography-SDS-PAGE-containing gelatin (0.5 mg/ml) as described (12, 13). The gels wereincubated in incubation buffer (50 mM Tris-HCl (pH 7.5) containing 100mM CaCl2, 1 �M ZnCl2, 1% (v/v) Triton-X100, and 0.02% (w/v) NaN3) for16 h. The gels were stained with Coomassie Blue and destained. Neg-ative staining showed the zones of gelatinolytic activity.

In Vitro Kinase Assay—To examine the effect of OPN on JNK activ-ity, the semiconfluent cells were treated with 5 �M OPN for 15 min at37 °C. To investigate the role of NIK and MEKK1 in OPN-induced JNKactivity, in separate experiments the cells were transfected with wildtype and kinase negative NIK or wild type and kinase negative MEKK1in presence of Lipofectamine plus and then treated with 5 �M OPN. Thecells were lysed in kinase assay lysis buffer (20 mM Tris-HCl (pH 8.0),500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM �-glycerophosphate, 10

mM NaF, 10 mM p-nitrophenyl phosphate, 300 �M Na3VO4, 1 mM ben-zamidine, 2 �M phenylmethylsulfonyl fluoride, 10 �g/ml aprotinin, 1�g/ml leupeptin, 1 �g/ml pepstatin, 1 mM DTT, and 0.25% NonidetP-40). The supernatant was obtained by centrifugation at 12,000 � g for10 min at 4 °C. The cell lysates (300 �g) containing equal amount oftotal proteins were immunoprecipitated with rabbit polyclonal anti-JNK1 antibody. Half of the immunoprecipitated samples were incu-bated with recombinant c-Jun as substrate in kinase assay buffer (20mM Hepes (pH 7.7), 2 mM MgCl2, 10 mM �-glycerophosphate, 10 mM

NaF, 10 mM p-nitrophenyl phosphate, 300 �M Na3VO4, 1 mM benzami-dine, 2 �M phenylmethylsulfonyl fluoride, 10 �g/ml aprotinin, 1 �g/mlleupeptin, 1 �g/ml pepstatin, and 1 mM DTT) containing 10 �M ATP and3 �Ci of [�-32P]ATP at 30 °C. The kinase reactions were stopped by theaddition of SDS sample buffer. The samples were resolved by SDS-PAGE, dried, and autoradiographed. The remaining half of the immu-noprecipitated samples were subjected to SDS-PAGE and analyzed byWestern blot using anti-JNK1 antibody. The levels of MEKK1 and NIKexpressions in the transfected cell lysates containing equal amount oftotal proteins were detected by Western blot using anti-MEKK1 oranti-NIK antibody.

To analyze the effect of overexpressed MEKK1 on OPN-inducedERK1/2 activity, cells were transfected with wild type and kinase neg-ative MEKK1 and then treated with OPN. In separate experiments, thewild type MEKK1-transfected cells were also cotransfected with wildtype or dominant negative JNK-1 or treated with 20 �M SP600125(JNK1 inhibitor) and then treated with OPN. The cells lysates contain-ing equal amount of total proteins were immunoprecipitated with anti-ERK1/2 antibody. Half of the immunocomplexes were incubated with 2�g of myelin basic protein in kinase assay buffer supplemented with 10�M ATP and 3 �Ci of [�-32P]ATP at 30 °C for 10 min. The samples wereresolved on SDS-PAGE and autoradiographed. The remaining half ofthe immunoprecipitated samples were analyzed by Western blot usinganti-ERK1/2 antibody.

NIK-coupled Kinase Assay—NIK-coupled kinase activity was as-sayed as described previously (39). Briefly, cells were treated with 5 �M

�PN. In separate experiments, cells were transfected with wild typeNIK and then treated with OPN. In other experiments the wild typeNIK-transfected cells were cotransfected with wild type and kinasenegative MEKK1 and then treated with OPN. Cell lysates were immu-noprecipitated with anti-NIK antibody. Half of the immunocomplexeswere incubated with 0.5 �g of recombinant MEK-1 protein in kinaseassay buffer containing 100 �M ATP, 10 �Ci of [�-32P]ATP for 20 min at30 °C. After that, 2 �g of recombinant kinase inactive ERK (p42) pro-tein was added to the reaction mixture, and the samples were incubatedfor an additional 30 min at 30 °C. The samples were resolved on SDS-PAGE and autoradiographed. The remaining half of the immunopre-cipitated samples was analyzed by Western blot using anti-NIK anti-body. The NIK activity was also assayed under the same conditionsusing IKK as substrate as described previously (16).

Cell Migration and Chemoinvasion Assays—The migration and in-vasion assays were conducted using non-coated or MatrigelTM-coatedTranswell cell culture chambers according to the standard procedure asdescribed previously (12–16). Briefly, cells were transfected with wildtype and kinase negative MEKK1 or wild type and dn JNK1 or wildtype and dn c-Jun or pretreated with SP600125. In separate experi-ments cells were transfected with wild type and kinase negative NIKfollowed by treatment with SP600125. The transfected or treated cellswere harvested with trypsin-EDTA and centrifuged at 800 � g for 10min. The cell suspension (5 � 105 cells/well) was added to the upperchamber of the uncoated or MatrigelTM-coated prehydrated polycarbon-ate membrane filter. The lower chamber was filled with fibroblastcondition medium, which acted as a chemoattractant. Purified OPN (5�M) was added to the upper chamber. The cells were incubated in ahumidified incubator in 5% CO2 and 95% air at 37 °C for 16 h. Thenon-migrated cells and/or the MatrigelTM from the upper side of thefilter were scraped and removed using moist cotton swab. The migratedor invaded cells in the reverse side of the filter were fixed with methanoland stained with Giemsa. The migrated or invaded cells on the filterwere counted under an inverted microscope (Olympus). The experi-ments were repeated in triplicate. Preimmune IgG served as nonspe-cific control.

In Vivo Tumorigenicity Experiments—The tumorigenicity experi-ments were performed as described previously (12, 13, 16). The cellswere treated in the absence or presence of purified human OPN (10 �M))at 37 °C for 20 h. In separate experiments cells were transfected withwild type and kinase negative NIK or wild type and kinase negativeMEKK1 in the presence of Lipofectamine plus and then treated with 5�M OPN. After that, the cells (5 � 106/0.2 ml) were detached and

Cross-talk between OPN-induced JNK and ERK Pathways 19383

injected subcutaneously into the flanks of male athymic NMRI (nu/nu)mice (6–8 weeks old). Four mice were used in each set of experiments.The mice were kept under specific pathogen-free conditions. OPN (10�M) was again injected into the tumor sites twice a week for up to 4weeks. After 4 weeks the mice were killed, and the tumor weights weremeasured. The tumor tissues were homogenized and lysed in lysisbuffer (50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1% NonidetP-40, 15 �g/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride)and centrifuged at 12,000 � g for 10 min. The clear supernatants werecollected, and the levels of uPA and MMP-9 were detected by Westernblot analysis using specific antibodies. To check the level of c-Junexpression and AP-1-DNA binding, the tumor tissues were homoge-nized in buffer A (10 mM Hepes buffer (pH 7.9) containing 10 mM KCl,0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 10% Nonidet P-40, 2 �g/mlaprotinin, 2 �g/ml leupeptin, 0.5 mg/ml benzamidine), and the nucleiwas separated by spinning at 13,000 � g for 10 min at 4 °C. The nuclearpellet was extracted in Buffer C (20 mM Hepes buffer (pH 7.9) contain-ing 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenyl-methylsulfonyl fluoride, 2 �g/ml aprotinin, 2 �g/ml leupeptin) for 2 h onice and centrifuged at 13,000 � g for 5 min at 4 °C. The supernatantwas used as nuclear extract. The nuclear extracts were subjected toWestern blot analysis using anti-c-Jun antibody. The AP-1-DNA bind-ing in the nuclear extracts was performed by EMSA as described above.

RESULTS

OPN Induces �v�3 Integrin-dependent MEKK1 and JNK1Phosphorylations—To investigate the role of OPN on MEKK1and JNK1 phosphorylations and to demonstrate the involve-ment of �v�3 integrin in this activation process, B16F10 cellswere treated with 5 �M OPN at 37 °C or pretreated with anti-�v�3 integrin antibody or RGD/RGE peptide (GRGDSP orGRGESP) and then treated with OPN. The cell lysates wereimmunoprecipitated with anti-MEKK1 antibody and analyzedby Western blot using anti-phosphoserine antibody. For JNKphosphorylation studies, cell lysates were immunoprecipitatedwith anti-JNK1 antibody and analyzed by Western blot using

anti-phosphotyrosine antibody. The data revealed that maxi-mum level of OPN-induced MEKK1 and JNK1 phosphoryla-tions occurred at 5 and 15 min, respectively (Fig. 1, upperpanels of A and C, lanes 1–5). As loading controls, same blotswere reprobed with anti-MEKK1 or anti-JNK1 antibody (lowerpanels of A and C, lanes 1–5). Pretreatment of cells with anti-�v�3 integrin antibody or RGD (GRGDSP) but not RGE(GRGESP) peptide suppressed the OPN-induced MEKK1 andJNK1 phosphorylations in these cells (upper panel of B and D,lanes 1–5). Same blots were reprobed with anti-MEKK1 oranti-JNK1 antibody (lower panels of B and D, lanes 1–5). Allthe bands were quantified by densitometric analysis, and the-fold changes were calculated. These results demonstrated thatOPN induces MEKK1 and JNK1 phosphorylations through�v�3 integrin-mediated pathway.

MEKK1 but Not NIK Is Required for OPN-induced �v�3Integrin-mediated JNK1 Phosphorylation—Because we havereported earlier that OPN induces NIK-dependent NF�B-me-diated pro-MMP-9 activation through ERK/IKK-mediatedpathways, therefore we sought to examine whether NIK/MEKK1 plays any role in OPN-induced JNK1 phosphorylationin B16F10 cells. Accordingly, cells were transfected with wildtype and kinase negative NIK or wild type and kinase negativeMEKK1 and then treated with OPN. Cell lysates were immu-noprecipitated with anti-JNK1 antibody and analyzed by West-ern blot using anti-phosphotyrosine antibody. Wild typeMEKK1 enhanced, whereas kinase negative MEKK1 sup-pressed the OPN-induced JNK phosphorylation (Fig. 1, upperpanel of E, lanes 1–4). The data also indicated that OPN-induced JNK1 phosphorylation was unaffected upon transfec-tion of cells with both wild type and kinase negative NIK (lanes5 and 6). The same blots were reprobed with anti-JNK1 anti-

FIG. 1. Panel A, OPN stimulates MEKK1 phosphorylation (pMEKK1). B16F10 cells were treated with 5 �M OPN for 0–60 min. Cell lysates wereimmunoprecipitated (IP) with anti-MEKK1 antibody and analyzed by Western blot (IB) using anti-phosphoserine (p-Ser) antibody (Ab) (upperpanel A, lanes 1–5), and the same blots were reprobed with anti-MEKK1 antibody (lower panel A). Panel B, OPN induces �v�3 integrin-mediatedMEKK1 phosphorylation. The cells were individually pretreated with anti-�v�3 integrin antibody, GRGDSP, or GRGESP and then treated with5 �M OPN. The cell lysates were immunoprecipitated with anti-MEKK1 antibody and analyzed by Western blot using anti-phosphoserine antibody(upper panel B, lanes 1–5), and same blots were reprobed with anti-MEKK1 antibody (lower panel B). Panels C and D, OPN stimulates �v�3integrin-mediated JNK1 phosphorylation (pJNK1). Cells were treated with 5 �M OPN for 0–90 min or pretreated with anti-�v�3 integrin antibody,GRGDSP, or GRGESP and then treated with 5 �M OPN for 15 min. The cell lysates were immunoprecipitated with anti-JNK1 antibody andanalyzed by Western blot using anti-phosphotyrosine (p-Tyr)antibody (upper panels C and D, lanes 1–5). The same blots were reprobed withanti-JNK1 antibody (lower panels C and D). Panel E, OPN-induced JNK1 phosphorylation is enhanced by MEKK1 but not by NIK. Cells weretransfected with wild type and kinase negative MEKK1 or wild type and kinase negative NIK and then stimulated with OPN. Cell lysates wereimmunoprecipitated with anti-JNK1 antibody and analyzed by Western blot using anti-phosphotyrosine antibody (upper panel E, lanes 1–6). Sameblots were reprobed with anti-JNK1 antibody (lower panel E). All these bands were analyzed densitometrically, and the -fold changes werecalculated. The data shown here represent three experiments exhibiting similar effects.


body as loading control (lower panel of E, lanes 1–6). Thesedata suggested that MEKK1 but not NIK plays a crucial role inOPN-induced JNK1 phosphorylation.

MEKK1 but Not NIK Enhances the OPN-induced JNK1 Ac-tivity—To ascertain the role of OPN on JNK1 activity, the cellswere treated with 5 �M OPN, and the cell lysates were immu-noprecipitated with rabbit anti-JNK1 antibody. Half of theimmunoprecipitated samples were incubated with recombinantc-Jun as substrate in kinase assay buffer. The samples wereresolved by SDS-PAGE and autoradiographed. The radiola-beled, phosphorylated c-Jun-specific band is detected in OPN-treated cells, demonstrating that OPN induces JNK1 activity(Fig. 2, upper panel of A, lane 2). The JNK1 activity is notdetected in the untreated cells (lane 1). To further checkwhether MEKK1 and NIK play any direct role in OPN-inducedJNK1 activity, in separate experiments cells were transfectedwith wild type and kinase negative MEKK1 or wild type andkinase negative NIK and then treated with OPN, and a JNK1kinase assay was performed. Cells transfected with wild typeMEKK1 followed by treatment with OPN showed maximumJNK1 activity (lane 3) compared with untreated cells (lane 1).The cells transfected with kinase negative MEKK1 followed bytreatment with OPN showed reduced level of JNK1 activity(lane 4), indicating that the kinase domain of MEKK1 plays a

crucial role in OPN-induced JNK1 activity. The data also indi-cated that OPN-induced JNK1 activity was unaffected uponoverexpression of both wild type and kinase negative NIK(upper panel B, lanes 1–4). The remaining half of the immu-noprecipitated samples was analyzed by Western blot usinganti-JNK1 antibody (middle panels A and B, lanes 1–4). Thelevels of MEKK1 and NIK were also analyzed by Western blotusing anti-MEKK1 and anti-NIK antibodies, respectively(lower panels A and B, lanes 1–4). These results suggested thatMEKK1 but not NIK plays a significant role in modulatingOPN-induced JNK activity.

Overexpression of Active MEKK-1 Attenuates OPN-inducedERK1/2 Activation—MEKK-1 functions as a mitogen-activatedprotein kinase kinase kinase in the JNK pathway; however,several reports have suggested that MEKK-1 may also affectthe ERK pathway (21). To determine the effect of MEKK1 onOPN-induced ERK activation, cells were transfected with wildtype and kinase negative MEKK1 and then treated with OPN.The cell lysates were immunoprecipitated with anti-ERK1/2antibody, and kinase activity was measured using myelin basicprotein as the substrate. The data indicated that overexpres-sion of wild type MEKK1 almost completely attenuates OPN-induced ERK activation (Fig. 2, upper panel C, lanes 1–4). Thisabrogation depends on MEKK-1 kinase activity because ERK

FIG. 2. Panels A and B, OPN induces MEKK1 (panel A)- but not NIK (panel B)-dependent JNK1 activity. Cells were treated with 5 �M OPN ortransfected with wild type and kinase negative MEKK1 or wild type and kinase negative NIK and then stimulated with OPN. Cell lysates wereimmunoprecipitated (IP) with anti-JNK1 antibody, and half of the immunoprecipitated samples were used for JNK kinase assay using recombinantc-Jun as substrate (upper panels A and B, lanes 1–4). The remaining half of the immunoprecipitated samples were immunoblotted (IB) withanti-JNK1 antibody (middle panels A and B, lanes 1–4). The levels of expressions of MEKK1 and NIK in the cell lysates were detected by Westernblot using anti-MEKK1 (lower panel A, lanes 1–4) or anti-NIK antibody (lower panel B, lanes 1–4). Panel C, overexpression of MEKK1 attenuatesOPN-induced ERK activation. Cells were transfected with wild type and kinase negative MEKK1 and then treated with OPN. Cell lysates wereimmunoprecipitated with anti-ERK1/2 antibody, and half of the immunoprecipitated samples were used for ERK kinase assay using myelin basicprotein as substrate (upper panel C, lanes 1–4). Half of the immunoprecipitated samples were immunoblotted with anti-ERK1/2 antibody (lowerpanel C, lanes 1–4). Panel D, JNK1 inhibition enhances OPN-induced ERK activation. Cells were cotransfected with wild type MEKK1 along withwild type or dn JNK1 and then treated with OPN. In separate experiments cells were transfected with wild type MEKK1 and treated withSP600125 followed by OPN. Cell lysates were immunoprecipitated with anti-ERK1/2 antibody and used for ERK kinase assay (upper panel D, lanes1–6). Half of the immunoprecipitated samples were analyzed by Western blot using anti-ERK1/2 antibody (lower panel D). MBP, myelin basicprotein. Panel E, OPN-induced NIK-dependent ERK activation is unaffected by overexpression of MEKK1. Cells were cotransfected with wild typeNIK along with wild type or kinase negative MEKK1 and then treated with OPN. Cell lysates were immunoprecipitated with anti-NIK antibodyand used for NIK coupled kinase assay using MEK and ERK as substrates as described under “Experimental Procedures” (upper panel E, lanes1–5). Half of the immunoprecipitated samples were immunoblotted with anti-NIK antibody (middle panel E). The NIK activity was also assayedunder the same conditions using IKK as substrate (lower panel E, lanes 1–5). The data shown here represent three experiments exhibiting similareffects.


activation is not affected by kinase negative MEKK-1 (lane 3).Half of the immunoprecipitated samples were immunoblottedwith anti-ERK1/2 antibody (lower panel C, lanes 1–4). Theseresults suggested that MEKK1 negatively regulates OPN-in-duced ERK activation.

JNK1 Plays a Crucial Role in OPN-induced MEKK1-depend-ent ERK1/2 Inactivation—Previous results indicated thatJNK�/� mice showed enhanced phosphorylation of ERK lead-ing to tumor growth (40); therefore, we have speculated thatactivation of JNK1 by OPN may play a role in suppression ofERK1/2 activation. Accordingly, cells transfected with wildtype MEKK1 were cotransfected with either wild type ordominant negative JNK1 and then treated with OPN. Inseparate experiments cells transfected with wild typeMEKK1 were treated with JNK1 inhibitor, SP600125, andthen treated with OPN. Overexpression of wild type MEKK1alone or with wild type JNK1 suppressed the OPN-inducedERK activation (upper panel D, lanes 1–4), whereas domi-nant negative JNK1 or SP600125 along with wild typeMEKK1 reversed this effect (lanes 5 and 6). Half of theimmunoprecipitated samples were immunoblotted with anti-ERK1/2 antibody (lower panel D, lanes 1–6). These datasuggested that JNK1 acts as negative regulator in OPN-induced MEKK1-dependent ERK1/2 activation.

To examine whether NIK plays any role in regulation ofOPN-induced MEKK-dependent JNK-mediated ERK1/2 inac-tivation, cells were transfected with wild type NIK and thencotransfected with either wild type or kinase negative MEKK1and then treated with OPN. The NIK kinase activity wasmeasured by a coupled kinase assay using MEK and ERK assubstrates. The data indicated that expression of active ormutant MEKK-1 had no effect on OPN-induced NIK activity(upper panel E, lanes 1–5), suggesting that overexpressed NIKeven in the presence of MEKK up-regulates OPN-induced ERKactivation. Half of the immunoprecipitated samples were im-munoblotted with anti-NIK antibody (middle panel E, lanes1–5). The level of NIK activity was also detected by using IKKas substrate (lower panel E, lanes 1–5).

OPN Induces �v�3 Integrin-mediated NIK and MEKK1-de-pendent c-Jun Expression—Earlier reports have demonstratedthat MEKK in the presence of stimulus induces JNK-depend-ent c-Jun phosphorylation and enhances AP-1 activation (41).

Therefore, we sought to determine whether OPN induces c-Junexpression and whether MEKK1/NIK is involved in this proc-ess. Accordingly, cells were treated with 5 �M OPN for 0–4 h.The nuclear extracts were prepared, and the level of c-Junexpression was detected by Western blot analysis using anti-c-Jun antibody. The results indicated that OPN induces c-Junexpression, and maximum expression was observed at 1 h (Fig.3, panel A, lanes 1–5).

To further confirm that this OPN-induced c-Jun expressionoccurs through �v�3 integrin-mediated pathway, cells werepretreated with anti-�v�3 antibody, RGD/RGE peptide, andthen treated with OPN for 1 h. The data revealed that �v�3antibody and RGD but not RGE suppressed OPN-induced c-Jun expression (panel B, lanes 1–5).

To examine further whether NIK and MEKK1 play impor-tant roles in OPN-induced c-Jun expression, cells were trans-fected with wild type and kinase negative NIK or wild type andkinase negative MEKK1 and then treated with OPN for 1 h.The nuclear extracts were prepared, and the level of c-Junexpression was detected by Western blot using anti-c-Jun an-tibody. Wild type NIK enhanced and kinase negative NIKsuppressed OPN-induced c-Jun expression (panel C, lanes1–4). Similarly, kinase negative MEKK1 inhibited and wildtype MEKK1 induced the OPN-induced c-Jun expression (lanes5 and 6). Moreover, overexpression of wild type NIK, whichdoes not affect OPN-induced JNK phosphorylation and kinaseactivity (Fig. 1, panel E and Fig. 2, panel B) significantlyup-regulate c-Jun expression. These data indicated that OPNinduces c-Jun expression through both NIK- and MEKK1-de-pendent pathways; however, NIK-mediated c-Jun expressionoccurs in a JNK1-independent manner.

NIK and MEKK1 Play Important Roles in OPN-induced AP-1-DNA Binding—We have reported earlier that OPN inducesAP-1-mediated secretion of uPA through c-Src-dependent trans-activation of epidermal growth factor receptor in breast cancercells (15). Therefore, in this paper we have first examinedwhether NIK and MEKK1 regulate OPN-induced AP-1-DNAbinding in B16F10 cells. Accordingly, cells were either treatedwith 5 �M OPN or transfected with wild type and kinase negativeNIK or wild type and kinase negative MEKK1 and then treatedwith OPN. The nuclear extracts were prepared and used forEMSA using 32P-labeled AP-1 oligonucleotides. Wild type NIK

FIG. 3. Panels A and B, OPN enhances �v�3 integrin-mediated c-Jun expression. Cells were treated with 5 �M OPN for 0–4 h or pretreated withanti-�v�3 integrin antibody, RGD/RGE peptide (GRGDSP or GRGESP), and then stimulated with OPN. The nuclear extracts were prepared asdescribed under “Experimental Procedures.” The level of c-Jun in the nuclear extracts was analyzed by Western blot (IB) using anti-c-Jun antibody(panels A and B, lanes 1–5). Panel C, NIK and MEKK1 play independent roles in OPN-induced c-Jun expression. Cells were transfected with wildtype and kinase negative NIK or wild type and kinase negative MEKK1 and then treated with OPN. The nuclear extracts were prepared andanalyzed by Western blot using anti-c-Jun antibody (panel C, lanes 1–6). All these bands were analyzed densitometrically, and the -fold changeswere calculated. The data shown here represent three experiments exhibiting similar effects.


enhanced and kinase negative NIK suppressed OPN-inducedAP-1-DNA binding (Fig. 4, panel A, lanes 1–4). Similarly, wildtype MEKK1 induced and kinase negative MEKK1 inhibitedOPN-enhanced AP-1-DNA binding (panel B, lanes 1–4). Thesedata suggested that OPN induces AP-1-DNA binding throughboth NIK- and MEKK1-mediated pathways.

To examine the role of JNK1 on OPN-induced-AP-1-DNAbinding, cells were pretreated with 0–50 �M SP600125 (JNK1inhibitor) and then treated with OPN. The nuclear extractswere prepared and used for EMSA. SP600125 suppressedOPN-induced AP-1-DNA binding in a dose-dependent manner(panel C, lanes 1–4). To ascertain whether OPN-induced JNK1mediated AP-1-DNA binding is NIK-dependent, cells weretransfected with wild type NIK followed by treatment withSP600125 and then stimulated with OPN. The OPN-enhancedAP-1-DNA binding caused by overexpression of wild type NIKwas unaltered by SP600125, suggesting that OPN-inducedNIK-mediated AP-1-DNA binding is JNK-independent (panelD, lanes 1–4). Whether the band obtained by EMSA is indeedAP-1, the nuclear extracts were incubated with anti-c-Jun an-tibody and then analyzed by EMSA. The results showed theshift of the AP-1-specific band to a higher molecular weightwhen the nuclear extracts were treated with anti-c-Jun anti-body (panel E, lanes 1 and 2).

OPN Induces NIK- and MEKK1-regulated JNK1-mediatedAP-1 Transactivation—To further investigate whether NIKand MEKK1 regulate OPN-induced JNK1-mediated AP-1transactivation, luciferase reporter gene assay was performed.Cells were transiently transfected with AP-1 luciferase re-porter construct (pAP-1-Luc) and then treated with OPN. Inseparate experiments, cells were individually transfected withwild type and kinase negative NIK or wild type and kinasenegative MEKK1 or wild type and dominant negative JNK1along with pAP-1-Luc and then treated with OPN. In separateexperiments, wild type NIK-transfected cells were cotrans-fected with pAP-1-Luc, treated with SP600125, and thentreated with OPN. The transfection efficiency was normalizedby cotransfecting the cells with pRL vector. Changes in lucif-erase activity with respect to control were calculated. The -foldchanges were calculated, and the results are expressed as themeans � S.E. of three determinations. The values were alsoanalyzed by Student’s t test (p � 0.002). The data showed that

wild type NIK enhanced but kinase negative NIK suppressedOPN-induced AP-1 activity in these cells (Fig. 5, panel A).Similarly, wild type MEKK1 enhanced and kinase negativeMEKK1 inhibited OPN-induced AP-1 activity (panel B). Wildtype JNK enhanced, whereas dn JNK1 moderately suppressedOPN-induced AP-1 activity (panel C). The enhanced AP-1transactivation caused by overexpression of wild type NIKfollowed by OPN treatment was unaffected upon treatmentwith JNK1-specific inhibitor, SP600125 (panel C). These dataindicated that OPN induces AP-1 transactivation through NIK-and MEKK/JNK-mediated pathways and further suggestedthat OPN induces a shift in balance toward activation of ERKfollowed by AP-1 activation.

OPN Stimulates NIK- and MEKK1-mediated c-Jun-depend-ent uPA Secretion and uPA-dependent MMP-9 Activation—Wehave recently demonstrated that NIK plays a crucial role inOPN-induced uPA secretion and uPA-dependent MMP-9 acti-vation in B16F10 cells (16). Therefore, we have examinedwhether MEKK1, JNK1, and c-Jun are involved in OPN-in-duced uPA secretion. Accordingly, cells were transfected withwild type and kinase negative MEKK1 or wild type and dnJNK1 or wild type and dn c-Jun and then treated with OPN. Inseparate experiments cells were pretreated with SP600125 (50�M) and then stimulated with OPN. The cell lysates wereanalyzed by Western blot using rabbit polyclonal anti-uPAantibody. The data showed that OPN-induced uPA secretionwas enhanced when cells were transfected with wt MEKK1 andwt c-Jun and suppressed when transfected with kinase nega-tive MEKK1 and dn c-Jun (Fig. 6, upper panel A, lanes 1–6).Wild type JNK1 stimulated and dn JNK1 or JNK1 inhibitor(SP600125) moderately reduced OPN-induced uPA secretiondue to up-regulation of ERK-mediated c-Jun expression lead-ing to activation of AP-1 (upper panel B, lanes 1–5). All theseblots were reprobed with anti-actin antibody (lower panels Aand B). All bands were quantified by densitometric analysis,and the -fold changes are calculated (panels A and B). Thesedata further demonstrated that OPN induces uPA secretionthrough both NIK/ERK as well as MEKK1/JNK-mediatedpathways.

To examine whether OPN-induced NIK/MEKK1-mediateduPA secretion leads to MMP-9 activation, cells were trans-fected with wild type and kinase negative MEKK1 or wild type

FIG. 4. Panels A and B, OPN inducesNIK (panel A)- and MEKK1 (panel B)-de-pendent AP-1-DNA binding. Cells wereeither treated with OPN or transfectedwith wild type and kinase negative NIKor wild type and kinase negative MEKK1and then treated with OPN. The nuclearextracts were prepared and analyzed byEMSA (panels A and B, lanes 1–4). PanelC, JNK is involved in OPN-induced AP-1-DNA binding. Cells were pretreated with0–50 �M SP600125 (JNK inhibitor) andthen treated with OPN. The nuclear ex-tracts were analyzed by EMSA (lanes1–4). Panel D, OPN-induced NIK-medi-ated AP-1-DNA binding is independent ofJNK. Cells were either treated with OPNor transfected with wild type NIK, treatedwith 0–50 �M SP600125 (JNK inhibitor),and then treated with OPN. The nuclearextracts were analyzed by EMSA (lanes1–4). E, supershift (SS) assay. The nu-clear extracts from OPN treated cellswere incubated with anti-c-Jun antibody(Ab) and analyzed by EMSA (lanes 1 and2). The results shown here representthree experiments exhibiting similareffects.


FIG. 5. Panels A and B, OPN enhances NIK (panel A)- and MEKK1 (panel B)-dependent AP-1 transactivation. Cells were transiently transfectedwith luciferase reporter construct (pAP-1-Luc). In separate experiments cells were individually transfected with wild type and kinase negative NIKor wild type and kinase negative MEKK1 along with pAP-1-Luc. The transfected cells were treated with 5 �M OPN. Cell lysates were used tomeasure the luciferase activity (panels A and B). The values were normalized to Renilla luciferase activity. Panel C, JNK is differentially regulatedin OPN-induced NIK-dependent AP-1 transactivation. Cells were transfected with wild type and dominant negative JNK1 along with pAP-1-Lucand treated with 5 �M OPN. In other experiments cells were transfected with wild type NIK along with pAP-1-Luc and treated with 0–50 �M

SP600125 and then with OPN. Cell lysates were used to measure the luciferase activity (panel C). The -fold changes were calculated, and means �S.E. of triplicate determinations are plotted. The values were also analyzed by Student’s t test (*, p � 0.002).

FIG. 6. Panel A, OPN stimulates MEKK1- and c-Jun-mediated uPA secretion. Cells were either treated with OPN or transfected with wild typeand kinase negative MEKK1 or wild type and dn c-Jun and then treated with OPN. The levels of uPA in the cell lysates were analyzed by Westernblot (IB) using anti-uPA antibody (upper panel A, lanes 1–6). The same blots were reprobed with anti-actin antibody (lower panel A, lanes 1–6).Panel B, JNK plays a crucial role in OPN-induced uPA secretion. Cells were transfected with wild type and dn JNK1 or pretreated with 50 �M ofSP600125 (JNK inhibitor) and then treated with OPN. The level of uPA in the cell lysates was analyzed by Western blot using anti-uPA antibody(upper panel B, lanes 1–5). The same blots were reprobed with anti-actin antibody (lower panel B, lanes 1–5) as the loading control. All these bandswere quantified densitometrically. Panels C–E, JNK is differentially regulated in OPN-induced MEKK1-dependent pro-MMP-9 activation. Cellswere individually transfected with wild type and kinase negative MEKK1or wild type and dn JNK1 or pretreated with SP600125 and then treatedwith OPN. The conditioned media were collected, and the activity of MMP-9 was examined by gelatin zymography (panels C–E, lanes 1–4). Thedata shown here represent three experiments exhibiting similar effects.


and dn JNK1 or pretreated with SP600125 and then treatedwith OPN. The conditioned media were collected and used forgelatinolytic activity by zymography. Increased levels ofMMP-9 activation (86 kDa) were observed when cells weretreated with OPN (panels C–E, lane 2). Almost no MMP-9-specific band was detected in the untreated cells (lane 1). Wildtype MEKK1 enhanced, whereas kinase negative MEKK1 sup-pressed OPN-induced MMP-9 activation (panel C, lanes 1–4).Wild type JNK1 enhanced, and dn JNK1 moderately reducedOPN-induced MMP-9 activation (panel E, lanes 1–4). JNK1inhibitor SP600125 retained OPN-induced MMP-9 activationdue to up-regulation of ERK activation (panel D, lanes 1–4).These data suggested that OPN induces uPA expression anduPA-dependent MMP-9 activation through both NIK/ERK1/2-and MEKK1/JNK1-mediated pathways and further demon-strated that OPN induces a shift in balance toward activationof ERK, thereby retaining the uPA secretion and MMP-9activation.

MEKK1, JNK1, and c-Jun Play Crucial Roles in OPN-in-duced �v�3 Integrin-mediated Cell Migration and Chemoinva-sion—We have shown that OPN induces �v�3 integrin-medi-ated NIK/ERK and MEKK1/JNK1-dependent c-Jun expressionleading to uPA secretion and uPA-dependent MMP-9 activa-tion. Therefore, we have examined whether these OPN-inducedNIK/MEKK1-dependent MMP-9 activations play any role incell migration and chemoinvasion. Accordingly, cells were ei-ther transfected with wild type and kinase negative MEKK1 orwild type and dn JNK1 or wild type and dn c-Jun in thepresence of Lipofectamine Plus and then used for migration orchemoinvasion assay. In separate experiments cells were pre-treated with JNK1 inhibitor (SP600125) or transfected withwild type and kinase negative NIK and then treated with JNK1inhibitor. OPN was used in the upper chamber. The datashowed that wild type MEKK1, JNK1, and c-Jun enhanced andmutant MEKK1 and c-Jun suppressed OPN-induced cell mi-gration (Fig. 7, panels A, C, and E) and chemoinvasion (panelsB, D, and F). The data also indicated that dn JNK1 andSP600125 unaltered the OPN-induced cell migration (panel C)

and chemoinvasion (panel D). The enhanced migration andinvasion caused by overexpression of wild type NIK is unaf-fected by cells treated with JNK1 inhibitor (panels C and D).However, cells transfected with mutant NIK followed by treat-ment with JNK1 inhibitor suppressed OPN-induced migrationand invasion (panel C and D), suggesting that NIK-regulatedmigration and invasion are independent of JNK, and both thepathways synergistically contribute the OPN-induced cell mi-gration and chemoinvasion. These data demonstrated thatOPN-induced uPA secretion and uPA-dependent pro-MMP-9activation are regulated by NIK/ERK and MEKK1/JNK1 path-ways, and all of these ultimately control the motility and inva-siveness of B16F10 cells.

OPN Induces NIK/MEKK1-dependent c-Jun Expression, AP-1-DNA Binding, uPA Secretion, and MMP-9 Activation in Tu-mor of Nude Mice—The in vitro data prompted us to examinewhether NIK and MEKK1 play any role in OPN-induced c-Junexpression, AP-1-DNA binding, uPA secretion, and MMP-9 ac-tivation in the tumors of nude mice. Accordingly, cells wereeither treated with OPN or transfected with wild type and

TABLE INIK and MEKK1 enhances OPN-induced tumor

growth in nude miceB16F10 cells were treated with 10 �M OPN for 16 h and injected into

nude mice (NMRI). The injection was performed twice a week for 4weeks. In separate experiments cells were transfected with wild typeand kinase negative NIK or wild type and kinase negative MEKK1followed by treatment with OPN and then injected into nude mice. Themice were killed, and the tumor weights were measured and analyzedstatistically by Student’s t test (p � 0.002). Mice injected with cells inphosphate-buffered saline were used as controls.

No. nudemice Transfection/Treatment Tumor weight

(-fold changes)

4 Control (PBS) 1.0 � 0.174 OPN (10 �M) 3.1 � 0.154 Wt NIK � OPN (10 �M) 6.1 � 0.144 Mut NIK � OPN (10 �M) 1.4 � 0.164 Wt MEKK1 � OPN (10 �M) 5.4 � 0.124 Mut MEKK1 � OPN (10 �M) 1.2 � 0.15

FIG. 7. Roles of MEKK1, JNK1, and c-Jun in OPN-stimulated cell migration and chemoinvasion. The migration assay was conductedeither by using untreated cells or cells transfected with wild type and kinase negative MEKK1 or wild type and dn JNK1 or wild type and dn c-Jun.The purified human OPN (5 �M) was added in the upper chamber. The treated or transfected cells were used for migration assay. Note thatOPN-induced migration was suppressed by kinase negative MEKK1 and dn c-Jun and enhanced by wt MEKK1, wt JNK1, and wt c-Jun. Inseparate experiments cells were treated with 0–25 �M SP600125 or transfected with wild type or kinase negative NIK and then treated with 25�M SP600125. These transfected or treated cells were used for migration assay (panels A, C, and E). dn JNK-1 and SP600125 did not alterOPN-induced cell migration (panel C). Note that JNK1 plays a differential role in OPN-induced NIK/MEKK1-dependent cell migration. The sameresults were obtained in chemoinvasion assays (panels B, D, and F). The results are expressed as the means � S.E. of three determinations.


kinase negative NIK or wild type and kinase negative MEKK1followed by treatment with OPN and then injected subcutane-ously into the flanks of nude mice. Table I shows the -foldchange of tumor weight grown in 4-week-old nude mice. Therewere at least 6- and 5.4-fold increased in tumor weight whenwild type NIK or wild type MEKK1-transfected cells wereinjected, respectively. Four mice were used in each set of ex-periments. The changes in tumor weights were analyzed sta-tistically by Student’s t test (p � 0.002). The tumor sampleswere lysed, and the level of c-Jun expression in the nuclearextract was detected by Western blot using anti-c-Jun anti-body. The AP-1-DNA binding in the nuclear extract was per-formed by EMSA. Both wild type NIK and wild type MEKK1showed significantly higher levels of c-Jun expression (Fig. 8,panel A, lanes 1–6) and AP-1-DNA binding (panel B, lanes 1–6)compared with cells treated with OPN alone or transfectedwith kinase negative NIK (mut NIK) or mut MEKK1.

To further examine the levels of uPA and MMP-9 in thesetumors, the samples were lysed, and the levels of uPA andMMP-9 were analyzed by Western blot using anti-uPA andanti-MMP-9 antibody, respectively. The results indicated thattumor generated by injecting the mice with wild type NIK andMEKK1-transfected cells showed higher level of uPA expres-sion (panel C, lanes 1–6) and MMP-9 activation (panel D, lanes1–6) compared with cell treated with OPN alone or transfectedwith mutant NIK or MEKK1. These data demonstrated thatOPN induces both NIK- and MEKK-1-mediated AP-1 activa-tion leading to uPA secretion and pro-MMP-9 activationthrough JNK1-dependent/independent pathways in tumor ofnude mice and these data corroborates with in vitro data.

DISCUSSION

In a recent study (16) we have demonstrated that OPNstimulates NIK-dependent NF�B-mediated uPA secretion anduPA-dependent pro-MMP-9 activation that controls cell motil-ity and tumor growth through both IKK and ERK1/2-mediatedpathways in murine melanoma cells. In this paper we have

delineated the molecular mechanism by which OPN regulatesNIK/MEKK1-dependent c-Jun expression and AP-1 transacti-vation and the differential role of JNK1 in these activationprocesses in murine melanoma cells. We have shown that OPNinduces �v�3 integrin-mediated MEKK1 phosphorylation lead-ing to c-Jun activation in a JNK-dependent manner. The dataalso revealed that OPN induces NIK activation, which furtherenhances c-Jun expression, leading to AP-1 transactivation in aJNK-independent pathway. Overexpression of MEKK1 leads tosustained activation of JNK, resulting in a negative cross-talkbetween MEKK1/JNK and NIK/ERK pathways. OPN bindingto �v�3 integrin induced NIK/MEKK1-dependent c-Jun ex-pression, which ultimately stimulates uPA secretion and uPA-dependent pro-MMP-9 activation that enhances cell migration,chemoinvasion, and tumor growth.

OPN plays a significant role in tissue remodeling processessuch as bone resorption, angiogenesis, wound healing, andtissue injury as well as certain diseases such as restenosis,atherosclerosis, tumorigenesis, and autoimmune diseases (6–8). Integrins are cell surface glycoproteins that bind to theextracellular matrix proteins. Recent studies have demon-strated that down-regulation of NIK activation does not affecttumor necrosis factor �-induced JNK activation (42). It hasbeen also reported that ERK and JNK pathways play crucialroles in regulating MMP-9 activation and cell motility ingrowth factor-stimulated human epidermal keratinocytes (43).These results prompted us to investigate whether binding ofOPN to �v�3 integrin receptors regulates JNK1 activation andwhether NIK is involved in this activation process. In thisstudy we have demonstrated that OPN induces �v�3 integrin-mediated MEKK1 and JNK1 phosphorylations in B16F10 cells.Pretreatment of cells with anti-�v�3 integrin antibody andRGD but not RGE peptide inhibited OPN-induced MEKK1 andJNK1 phosphorylations, indicating that �v�3 is involved inthis process. Furthermore, OPN-induced JNK1 activation isMEKK1-dependent but NIK-independent. This was confirmed

FIG. 8. Panels A and B, OPN enhances NIK/MEKK-dependent c-Jun expression and AP-1-DNA binding in tumors of nude mice. The samplesobtained from tumors generated by wild type and kinase negative NIK or wild type and kinase negative MEKK1 were lysed, and nuclear extractswere prepared and subjected to Western blot using anti-c-Jun antibody (panel A, lanes 1–6). The same nuclear extracts were used for EMSA assays(panel B, lanes 1–6). Panels C and D, OPN induces NIK/MEKK-dependent uPA expression and MMP-9 activation in same tumors of nude mice.The samples obtained from tumors generated by wild type and kinase negative NIK or MEKK1 were lysed, and the levels of uPA were analyzedby Western blot using anti-uPA antibody (panel C, lanes 1–6). The level of MMP-9 in these samples was also analyzed by Western blot usinganti-MMP-9 antibody (panel D, lanes 1–6). The data shown here represent three experiments exhibiting similar effects.


by the fact that transient overexpression of wild type MEKK1enhanced and kinase negative MEKK1 suppressed OPN-in-duced JNK1 phosphorylation and kinase activity, whereasoverexpression of wild type and kinase negative NIK does notaffect OPN-induced JNK1 activation.

MEKK1, a Ser/Thr protein kinase has been reported as amitogen-activated protein kinase kinase kinase that activatesJNK via phosphorylation of its downstream kinase mitogen-activated protein kinase kinase 4 (18). Shen et al. (44) haverecently reported that sustained activation of JNK blocks ERKactivation in response to mitogenic factors like epidermalgrowth factor and phorbol 12-myristate 13-acetate. Growingevidence also indicated that cross-regulation between JNK andERK may play an important role in determining cell survival ordeath. These results prompted us to examine whether overex-pression of MEKK1 leads to enhanced JNK activation andwhether this activation may affect OPN-induced NIK-mediatedERK activation. Our data demonstrated a negative cross-talkbetween OPN-induced NIK/ERK and MEKK1/JNK activationand further suggested that sustained activation of JNK re-sulted in the attenuation of ERK activation. Previous studieshave indicated that MEKK1 also has the ability to activateERK, but the effect is less potent (19). This may be implicatedto the short and long phase of MEKK1 activation, which resultsin a different cellular response; that is, a short phase activationthat leads to ERK activation and a long phase activation thatresults in inhibition of ERK activation. Also, the inhibition ofOPN-induced NIK-mediated ERK activation caused by overex-pression of wild type MEKK1 involves the ability of MEKK1 toactivate the JNK pathway. These implications delineate amechanism in which treatment with the same agonist mayresult in a different cellular outcome depending on the durationof treatment. These data are consistent with the recent reportthat JNK1 deficiency stimulates 12-O-tetradecanoylphorbol-13-acetate-induced ERK phosphorylation, leading to enhancedskin tumorigenesis (40).

It is well established that JNK, a member of the mitogen-activated protein kinase family, could be phosphorylated afterexposure to ultraviolet irradiation, growth factors, or cyto-kines, which in turn phosphorylates specific serine residues(serine 63 and serine 73) of c-Jun and enhances the AP-1transcriptional activity. AP-1, a family of transcription factors,consists of homo or heterodimers of Jun, Fos, or activatingtranscription factor protein (44–46). Previous reports havedemonstrated that AP-1 is involved in several cellular pro-cesses such as cell growth, apoptosis, and cell motility (45). Inaddition, AP-1 activity is elevated in a number of pathologicalconditions. Natoli et al. (42) have reported that overexpressionof NIK, which does not activate JNK, strongly activates tran-scription directed by a canonical AP-1 site. Because we haveshown that OPN induces MEKK1-dependent but NIK-inde-pendent JNK phosphorylation and AP-1 response element ispresent in the promoter region of MMP-9 gene, we sought todetermine the level of c-Jun expression upon OPN stimulation.OPN enhances the expression of c-Jun, resulting in enhance-ment of AP-1-DNA binding activity. Our data also indicatedthat OPN induces both NIK- and MEKK1-mediated c-Jun ex-pression, leading to AP-1-DNA binding and AP-1 transactiva-tion. The enhanced AP-1-DNA binding caused by overexpres-sion of wild type NIK was unaffected upon inhibition of JNKactivation by SP600125, a specific JNK inhibitor. These datasuggested that overexpression of NIK, which does not affectJNK activation significantly, up-regulates AP-1-DNA bindingand transcriptional activity, indicating that OPN induces NIK-dependent AP-1 activation, which is independent of JNK.

Signals transduced by cell adhesion molecules play an im-

portant role in tumor cell attachment, motility, and invasion,all of which regulate metastasis. OPN, an ECM protein, playsa significant role in cell adhesion, migration, and metastasis.MMPs are a family of Zn2�-dependent endopeptidases that areresponsible for remodeling of the extracellular matrix and deg-radation of ECM proteins. MMP-9 is known to degrade base-ment membrane, which normally separates the epithelial fromstromal compartment. Elevated levels of MMP-9 have beenreported in various cancers. Several studies have shown acorrelation between MMP-9 expression and the metastatic po-tential of tumor (47). Kim et al. (48) also demonstrated thatMMP-9 activity but not MMP-2 activity significantly affectstumor intravasation into blood vessel, and uPA is required forpro-MMP-9 activation (49). Several other reports have indi-cated the correlation between uPA expression and metastaticpotential and shown that uPA plays major role in regulatingMMPs activation (16, 50). In this study we have reported thatOPN induces uPA secretion and uPA-dependent pro-MMP-9activation through NIK/ERK- and MEKK1/JNK-mediated AP-1-dependent pathways. Overexpression of wild type MEKK1and c-Jun enhanced, and kinase negative MEKK1 and dn c-Junsuppressed the OPN-induced uPA secretion, demonstratingthat OPN regulates uPA secretion through MEKK1/c-Jun-me-diated pathways. Similarly, overexpression of wild type JNK1enhanced OPN-induced uPA secretion and MMP-9 activation.Moreover, cells transfected with wild type NIK followed bytreatment with JNK1 inhibitor enhanced, whereas cells trans-fected with kinase negative NIK followed by treatment withJNK1 inhibitor suppressed the OPN-induced cell migrationand invasion, indicating that OPN regulates these effectsthrough both NIK- and JNK-mediated pathways. Wild typeMEKK1, JNK1, and c-Jun enhanced, and kinase negativeMEKK1 and dn c-Jun suppressed OPN-induced cell migrationand ECM invasion. However, transfection of cells with thedominant negative form of JNK1 or treatment with SP600125moderately inhibits OPN-induced uPA secretion or uPA-de-pendent pro-MMP-9 activation, cell migration, and ECM inva-

FIG. 9. Molecular mechanism of OPN-induced NIK/MEKK1-de-pendent AP-1 activation, AP-1-mediated uPA secretion, andMMP-9 activation through differential activation of JNK1. Bind-ing of OPN to �v�3 integrin induced the phosphorylation and activationof MEKK1 which induces c-Jun-mediated AP-1 activation in a JNK1-dependent manner. In addition, OPN also induced NIK-dependent c-Jun-mediated AP-1 activation through JNK1-independent pathway.These lead to a cross-talk between NIK/ERK and MEKK1/JNK1 path-ways. Both NIK and MEKK1 in the presence of OPN regulate AP-1-de-pendent uPA secretion and MMP-9 activation, and all of these controlthe cell motility, invasion, and tumor growth.


sion. This may be due to the cross-talk between MEKK1/JNKand NIK/ERK pathways. These data are consistent with therecent data reported by Chen et al. (40) that disruption of theJNK1 gene resulted in an increase in ERK phosphorylationleading to enhancement of skin tumorigenesis. The in vitrodata are also supported by in vivo data which showed that OPNinduced both NIK- and MEKK1-mediated c-Jun expression,leading to uPA-dependent pro-MMP-9 activation in tumors ofnude mice. These data demonstrated that OPN induces NIK/MEKK1-regulated AP-1-mediated uPA-dependent pro-MMP-9activation, cell motility, and tumor growth through differentialactivation of JNK1 in B16F10 cells.

In summary, we have demonstrated for the first time thatOPN induces both NIK- and MEKK1-mediated c-Jun expres-sion, leading to AP-1 transactivation in B16F10 cells. Ligationof OPN to �v�3 integrin receptor induces the phosphorylationand kinase activity of JNK1. This was blocked by kinase neg-ative MEKK1 but unaffected by kinase negative NIK, indicat-ing that OPN-induced NIK-mediated AP-1 transactivation isJNK-independent. OPN also induces a negative cross-talk be-tween MEKK1/JNK and NIK/ERK pathways. Overexpressionof wild type MEKK1 attenuates OPN-induced NIK-mediatedERK activation, which is restored upon JNK inhibition. Takentogether, OPN-induced uPA-dependent pro-MMP-9 activation,cell motility, and tumor growth through both NIK- andMEKK1-mediated c-Jun expression and JNK1 plays a differ-ential role in modulating these processes (Fig. 9). These find-ings may be useful in designing novel therapeutic interventionsthat block the OPN-regulated NIK- and MEKK1-dependentc-Jun expression and AP-1 transactivation through differentialactivation of JNK1, resulting in reduction of uPA secretion andMMP-9 activation and consequent blocking of cell motility,invasiveness. and metastatic spread of malignant melanoma.

Acknowledgments—We thank Prof. David Wallach for providing wildtype NIK (wt pcDNA NIK) and kinase negative NIK (mut pcDNA NIK;NIK-KK429/430AA) in pcDNA3, and the wild type and dominant neg-ative constructs of MEKK1 (pcDNA3-MEKK1 and pcDNA3-FlagMEKK1 K432M) were kind gifts from Prof. Tom Maniatis (HarvardUniversity, Cambridge). The wild type c-Jun in pRJB10B and dominantnegative c-Jun in pELFIN were gifts from Dr. Jalam (Ochsner ClinicFoundation, New Orleans, LA). The wild type and dominant negativeform of JNK1 in pcDNA3 were kind gifts from Dr. Roger Davis (Uni-versity of Massachusetts Medical School, Worcester, CA).

REFERENCES

1. Butler, W. T. (1989) Connect. Tissue Res. 23, 123–1362. Denhardt, D., and Guo, X. (1993) FASEB J. 7, 1475–14823. Weber, G. F. (2001) Biochim. Biophys. Acta 1552, 61–854. Prince, C. W. (1989) Connect. Tissue Res. 21, 15–205. Weber, G. F., Akshar, S., Glimcher, M. J., and Cantor, H. (1996) Science 271,

509–5196. Panda, D., Kundu, G. C., Lee, B. I., Peri, A., Fohl, D., Chackalaparampil, I.,

Mukherjee, B. B., Li, X. D., Mukherjee, D. C., Seides, S., Rosenberg, J.,Stark, K., and Mukherjee, A. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,9308–9313

7. Liaw. L., Birk, D. E., Ballas, C. B., Whitsitt, J. S., Davidson, J. M., and Hogan,B. L. (1998) J. Clin. Investig. 101, 1468–1478

8. Rittling, S. R., and Novick, K. E. (1999) Cell Growth Differ. 8, 1061–10699. Senger, D. R., Aseh, B. B., Smith, B. D., Perruzzi, C. A., and Dvorak, H. F.

(1983) Nature 302, 714–71510. Brown, L. F., Papadopoulos-Sergiou, A., Berse, B., Manseau, E. J., Tognazzi,

K., Perruzzi, C. A., Dvorak, H. F., and Senger, D. R. (1994) Am. J. Pathol.145, 610–623

11. Craig, A. M., Bowden, G. T. Chambers, A. F., Spearman, M. A., Greenberg,A. H., Wright, J. A., and Denhardt, D. T. (1990) Int. J. Cancer 46, 133–137

12. Philip, S., Bulbule, A., and Kundu, G. C. (2001) J. Biol. Chem. 276,44926–44935

13. Philip, S., and Kundu, G. C. (2003) J. Biol. Chem. 278, 14487–1449714. Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278,

28593–2860615. Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2004) J. Biol. Chem. 279,

11051–1106416. Rangaswami, H., Bulbule, A., and Kundu, G. C. (2004) J. Biol. Chem. 279,

38921–3893517. Lange-Carter, C. A., Pleiman, C. M., Gardner, A. M., Blumer, K. J., and

Johnson, G. L. (1993) Science 260, 315–31918. Yan, M., Dai, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and

Templeton, D. J. (1994) Nature 372, 798–80019. Minden, A., Lin, A., McMahon, M., Lange-Carter, C., Derijard, B., Davis, R. J.,

Johnson, G. L., and Karin, M. (1994) Science 266, 1719–172320. Karin, M. (1996) Philos. Trans. R. Soc. Lond-Biol. Sci. 351, 127–13421. Gallo, K.A., and Johnson, G. L. (2002) Nat. Rev. Mol. Cell Biol. 3, 663–67222. Garrington, T. P., and Johnson, G. L. (1999) Curr. Opin. Cell Biol. 11, 211–21823. Besser, D., Verde, P., Nagamine, Y., and Blasi, F. (1996) Fibrinolysis 10,

214–23724. Dano, K., Andreasen, P. A., Grondahl-Hansen, J., Kristensen, P., Neilsen,

L. S., and Skriver, L. (1985) Adv. Cancer Res. 44, 139–16625. Murphy, G., Willenbrock, F., Crabbe, T., O’Shea, M., Ward, R., Atkinson, S.,

O’Connell, J., and Docherthy, A. (1994) Ann. N. Y. Acad. Sci. 732, 31–4126. Blasi, F. (1997) Immunol. Today 18, 415–41727. Mukhina, S., Stepanova, V., Traktouev, D., Poliakov, A., Beabealashvilly, R.,

Gursky, Y., Minashkin, M., Shevelev, A., and Tkachak, V. (2000) J. Biol.Chem. 275, 16450–16458

28. Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278,6209–6221

29. DeCasare, D., Vallone, D., Caracciolo, A., Sassone-Corsi, P., Nerlov, C., andVerde, P. (1995) Oncogene 11, 365–376

30. Shapiro, S. D. (1998) Curr. Opin. Cell Biol. 10, 602–60831. Scorilas, A., Karameris, A., Amogiannaki, N., Ardavanis, A., Bassilopoulos, P.,

Trangas, T., and Talleri, M. (2001) Br. J. Cancer. 84, 1488–149632. Hrabec, E., Strek, M., Nowak, D., and Hrabec, Z. (2001) Respir. Med. 95, 1–433. Sakamoto, Y., Mafune, K., Mori, M., Shiraishi, T., Imamura, H., Mori, M.,

Takayama, T., and Makuuchi, M. (2000) Int. J. Oncol. 17, 237–24334. Shen, K. H., Chi, C. W., Lo, S. S., Kao, H. L., Lui, W. Y., and Wu, C. W. (2000)

Anticancer Res. 20, 1307–131035. Dubois, B., Masure, S., Hurtenbach, U., Paemen, L., Heremans, H., Van den O.

J, Sciot R., Meinhardt, T., Hammerling, G., Opdenakker, G., and Arnold, B.(1999). J. Clin. Investig. 104, 1507–1515

36. Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer,J. L., Kronberger, A., He, C. S., Bauer, E. A., and Goldberg, G. I. (1988)J. Biol. Chem. 263, 6579–6587

37. Houde, M., de Bruyne, G., Bracke, M., Ingelman-Sundberg M., Skoglund, G.,Masure, S., van Damme, J., and Opdenakker, G. (1993) Int. J. Cancer. 53,395–400

38. Folkman, J. (1999) Nat. Biotechnol. 17, 74939. Tzivion, G., Luo, Z., and Avruch, J. (1998) Nature 394, 88–9240. She, Q., Chen, N., Bode, A. M., Flavell, R. A., and Dong, Z. (2002) Cancer Res.

62, 1343–134841. Devary, Y., Gattlieb, R. A., Smeal, T., and Karin, M. (1992) Cell 71, 1081–109142. Natoli, G., Costanzo, A., Moretti, F., Fulco, M., Balsano, C., and Levrero, M.

(1997) J. Biol. Chem. 272, 26079–2608243. Zeigler, M. E., Chi, Y., Schmidt, T., and Varani, J. (1999) J. Cell. Physiol. 180,

271–28444. Shen, Y. H., Godlewski, J., Zhu, J., Sathyanarayana, P., Leaner, V., Birrer,

M. J., Rana, A., and Tzivion, G. (2003) J. Biol. Chem. 278, 26715–2672145. Karin, M., Liu, Z. G., and Zandi, E. (1997) Curr. Opin. Cell Biol. 9, 240–24646. Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072, 129–15747. Davies, B., Waxman, J., Wasan, H., Abel, P., Williams, G., Krausz, T., Neal, D.,

Thomas, D., Hanby, A., and Balkwill, F. (1993) Cancer Res. 53, 5365–536948. Kim, J., Yu, K., Kovalski, K., and Ossowski, L. (1998) Cell 94, 353–36249. Sehgal, G., Hua, J., Bernhard, E. J., Sehgal, I., Thompson, T. C., and Muschel,

R. J. (1998) Am. J. Pathol. 152, 5591–559650. Aguirre Ghiso, J. A., Kovalski, K., and Ossowski, L., (1999) J. Cell Biol. 147,

89–103


the j biological c vol. 279, no. 37, issue of...

Documents