Ligands of Macrophage Scavenger Receptor Induce CytokineExpression via Differential Modulation of Protein KinaseSignaling Pathways*

Received for publication, December 11, 2000, and in revised form, May 18, 2001Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.M011117200

Hsien-Yeh Hsu‡§, Show-Lan Chiu¶, Meng-Hsuan Wen‡, Kuo-Yen Chen‡, and Kuo-Feng Hua‡

From the ‡Faculty of Medical Technology, Institute of Biotechnology in Medicine, National Yang-Ming University,Taipei 112, and the ¶National Laboratory of Foods and Drugs, National Health Administration, Taipei 115, Taiwan

Our previous works demonstrated that ligands ofmacrophage scavenger receptor (MSR) induce proteinkinases (PKs) including protein-tyrosine kinase (PTK)and up-regulate urokinase-type plasminogen activatorexpression (Hsu, H. Y., Hajjar, D. P., Khan, K. M., andFalcone, D. J. (1998) J. Biol. Chem. 273, 1240–1246). Tocontinue to investigate MSR ligand-mediated signaltransductions, we focus on ligands, oxidized low densitylipoprotein (OxLDL), and fucoidan induction of the cy-tokines tumor necrosis factor-a (TNF) and interleukin1b (IL-1). In brief, in murine macrophages J774A.1, Ox-LDL and fucoidan up-regulate TNF production; addi-tionally, fucoidan but not OxLDL induces IL-1 secretion,prointerleukin 1 (proIL-1, precursor of IL-1) protein,and proIL-1 message. Simultaneously, fucoidan stimu-lates activity of interleukin 1-converting enzyme. Wefurther investigate the molecular mechanism by whichligand binding-induced PK-mediated mitogen-activatedprotein kinase (MAPK) in regulation of expression ofproIL-1 and IL-1. Specifically, fucoidan stimulates activ-ity of p21-activated kinase (PAK) and of the MAPKs ex-tracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. Combined with PKinhibitors and genetic mutants of Rac1 and JNK in PKactivity assays, Western blotting analyses, and IL-1 en-zyme-linked immunosorbent assay, the role of individ-ual PKs in the regulation of proIL-1/IL-1 was extensivelydissected. Moreover, tyrosine phosphorylation ofpp60Src as well as association between pp60Src andHsp90 play important roles in fucoidan-induced proIL-1expression. We are the first to establish two fucoidan-mediated signaling pathways: PTK(Src)/Rac1/PAK/JNKand PTK(Src)/Rac1/PAK/p38, but not PTK/phospho-lipase C-g1/PKC/MEK1/ERK, playing critical roles inproIL-1/IL-1 regulation. Our current results indicateand suggest a model for MSR ligands differentially mod-

ulating specific PK signal transduction pathways, whichregulate atherogenesis-related inflammatory cytokinesTNF and IL-1.

Overexpression of macrophage scavenger receptors (MSR)1

on activated macrophages and macrophage-derived foam cellsin atherosclerotic lesions has been reported (2–5). Scavengerreceptors mediate the high affinity binding and internalizationof modified lipoproteins including OxLDL (6–8), implying animportant role in lipid-laden foam cell formation during ather-osclerosis development and progression. In addition to OxLDL,a diverse group of polyanionic compounds has been listed asligands for MSR (9, 10). The broad nature of ligand specificityin MSR may explain receptor-multifaceted functions of macro-phages such as adhesion (11, 12), clearance of pathologic sub-stances (13, 14), host defenses (9, 15), and cytokine production(this paper and Refs. 9 and 16). However, the molecularmechanisms for MSR on macrophages possessing the capacityof cytokine induction remain unclear and need furtherinvestigation.

The activated macrophages not only transform into foamcells via uptake of OxLDL, but also aberrantly produce a bat-tery of inflammatory cytokines that play pivotal roles in theprocess of atherogenesis. It has been demonstrated that inter-leukin-1b (IL-1) and tumor necrosis factor-a (TNF) (2, 17–19)are the major inflammatory cytokines (2, 18, 19) altering thefunction of macrophages in ways that appear to promote ath-erosclerosis (20). Recent studies show that cytokines secretedfrom activated macrophages can be initiated or potentiated byone of the MSR ligands, OxLDL (21), thus dysregulating ex-pression of MSR and likely promoting the development of lo-calized inflammatory reactions. Indeed, the macrophage-de-rived inflammatory cytokines IL-1 and TNF cause adjacentendothelial cellular expression of procoagulant activity,

* This work was supported in part by Research Grants NSC 89-2320-B-010-083 from the National Science Council, Taiwan (to H.-Y. H.),NHRI-GT-EX89S937L from the National Health Research Institutes,Taiwan (to H.-Y. H.), and by Grant 89-B-FA22–2-4 from the Programfor Promoting Academic Excellence of Universities, Ministry of Educa-tion, Taiwan (to H.-Y. H.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Dr. Chi-Shuen Tsou.§ Recipient of an award from the Medical Research and Advancement

Foundation. To whom correspondence should be addressed: Faculty ofMedical Technology, Institute of Biotechnology in Medicine, NationalYang-Ming University, 155 Li-Nong St., Shih-Pai, Taipei 112, Taiwan.Tel.: 011-886-2-2826-7252; Fax: 011-886-2-2826-4092; E-mail: hyhsu@ym.edu.tw.

1 The abbreviations used are: MSR, macrophage scavenger receptortype A; LDL, low density lipoprotein; OxLDL, oxidized LDL; IL-1,interleukin 1b; proIL-1, prointerleukin 1b; TNF, tumor necrosis fac-tor-a; PTK, protein-tyrosine kinase; PI 3-kinase, phosphatidylinositol3-kinase; PKC, protein kinase C; HB, herbimycin A; GA, geldanamycin;Hsp90, heat-shock protein 90; NRPTK, non-receptor protein-tyrosinekinase; PAK, p21-activated kinase; MAPK, mitogen-activated proteinkinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal protein kinase; p38, p38 mitogen-activated protein kinase;ICE (caspase 1), interleukin 1-converting enzyme; RT-PCR, reversetranscription-polymerase chain reaction; HRP, horseradish peroxidase;GAPDH, glyceraldehyde phosphate dehydrogenase; DN-JNK, domi-nant negative JNK construct; DN-Rac1, dominant negative Rac1 con-struct; CA-Rac1, constitutive activated Rac1 construct; ELISA, enzyme-linked immunosorbent assay; MEK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; SB, SB203580.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 31, Issue of August 3, pp. 28719–28730, 2001© 2001 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 28719

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

changing the endothelial cell surface from antithrombotic tothrombotic.

On the other hand, fucoidan, the principal polysaccharidesulfate ester occurring in the various species of Phaeophyceae(brown seaweed), although it is a non-lipoprotein, is recognizedas an MSR ligand and effectively competes with OxLDL inMSR ligand binding studies (9, 10). Up-regulation of MSRexpression in human THP-1 monocytic macrophages uponphorbol 12-myristate 13-acetate priming has been reported (16,22, 23). Binding of fucoidan to MSR stimulates urokinase-typeplasminogen activator secretion in human THP-1 macrophages(1), in activated peritoneal murine macrophages, and in murinemacrophage cell lines (J774A.1 and RAW264.7) (24, 25). Theexpression of uPA via MSR ligand-mediated signaling mole-cules include protein-tyrosine kinases (PTK), phospholipaseC-g1, phosphatidylinositol 3-kinase (PI 3-kinase), and proteinkinase C (PKC), indicating a molecular model for regulation ofurokinase-type plasminogen activator expression by the spe-cific ligand of MSR (1). Furthermore, herbimycin A (HB) inhib-its fucoidan-induced tyrosine phosphorylation of phospholipaseC-g1 (1), suggesting that an unidentified PTK is involved infucoidan-induced signaling. In this paper, we demonstrateagain that geldanamycin (GA) or HB inhibits fucoidan-inducedphosphorylation of various protein tyrosines and expression ofIL-1. It is known that GA or HB inhibits the activation ofcertain tyrosine kinases; however, their cellular target is heat-shock protein 90 (Hsp90) (26), and its expression is induced bycell stress. Obviously, the MSR-induced cytokines IL-1 andTNF could lead to cellular stress including inflammation. BothGA and HB, benzoquinone ansamycin antibiotics, inhibit var-ious signal transduction proteins including non-receptor pro-tein-tyrosine kinase (NRPTK) pp60Src. Pharmacologically, HBor GA binds in a specific manner to Hsp90 and inhibitspp60SrczHsp90 heterocomplex formation (26–28). Although li-gands of MSR such as fucoidan and polyinosinic acid induceIL-1 production in human THP-1 macrophages (16), they areunable to examine systematically the MSR ligand-regulatedsignaling cascade for IL-1 production in macrophages. Hence,we propose and investigate pp60Src and Hsp90, which mayplay certain roles in fucoidan-mediated effects such as theassociation between pp60Src and Hsp90 in forming a complexin the regulation of fucoidan-induced prointerleukin 1b

(proIL-1) protein expression.In studies reported here, we observe tha both OxLDL and

fucoidan up-regulate TNF production; additionally, fucoidanbut not OxLDL induces proIL-1 protein and IL-1 secretion inmurine macrophages J774A.1. We demonstrate for the firsttime that fucoidan differentially stimulates the signaling ma-chinery including NRPTK, pp60Src, p21-activated kinase(PAK), and mitogen-activated protein kinases (MAPKs; i.e.ERK, JNK, and p38). Recent studies indicate that IL-1 secre-tion is post-transcriptionally regulated via IL-1 converting en-zyme (ICE, caspase 1) (29–32). In here, we find that fucoidan-mediated specific signals regulate IL-1 secretion, andsimultaneously, fucoidan induces ICE activity during alterna-tion of proIL-1/IL-1 expression. Moreover, combining withpharmacological inhibitors and genetic mutants in protein ki-nase assays, we investigate the molecular mechanism for MSRligand-mediated signal transduction pathways in the regula-tion of proIL-1/IL-1 expression. Specifically, we further dissectand confirm the role of several key signaling molecules ininduction of proIL-1, and we establish two signaling cascades,i.e. the PTK(Src)/Rac1/PAK/JNK pathway and the PTK(Src)/Rac1/PAK/p38 pathway, in the regulation of proIL-1/IL-1expression.

EXPERIMENTAL PROCEDURES

Cell Cultures

Murine macrophage J774A.1 cells were obtained from ATCC (Rock-ville, MD), propagated in RPMI 1640 medium supplemented with 10%heated-inactivated fetal bovine serum and 2 mM L-glutamine (Life Tech-nologies, Inc.), and cultured in a 37 °C, 5% CO2 incubator. Using theHistopaque®-1077 method, human monocyte-derived macrophageswere obtained from the Taiwan Blood Center (Taipei, Taiwan) from theblood of healthy persons.

Materials

Histopaque®-1077, fucoidan, sodium orthovanadate, phenylmethyl-sulfonyl fluoride, bovine serum albumin (fraction V), GA, and curcuminwere purchased from Sigma. LipofectAMINE PLUS® reagent was pur-chased from Life Technologies, Inc. An Immobilon® polyvinylidene di-fluoride membrane was purchased from Millipore Inc. (Bedford, MA).non-radioactive Western blot chemiluminescence Reagent, Renais-sance®, and 10 Ci/mmol [g-32P]ATP were purchased from PerkinElmerLife Sciences. REZOl®C&T was from PROtech Technology Co. (Taipei,Taiwan). A GeneAmp® RNA PCR kit for RT-PCR amplification waspurchased from PerkinElmer Life Sciences. OxLDL was supplied by Dr.Ming-Shi Shiao (Veteran General Hospital, Taipei, Taiwan).

Growth Factors and Antibodies—Anti-IL-1b, 3ZD monoclonal anti-body (a gift from the National Institutes of Health, Bethesda, MD),anti-PAK, rabbit polyclonal IgG, anti-rabbit IgG-HRP, anti-mouse IgG-HRP and protein A/G plus agarose were obtained from Santa CruzBiotechnology (Santa Cruz, CA). Anti-phosphotyrosine clone 4G10(mouse monoclonal IgG2bk) was purchased from Upstate Biotechnol-ogy, Inc. Monoclonal anti-Hsp90 and monoclonal anti-Src (OncogeneInc.) were obtained from Calbiochem-Novabiochem Corp. (La Jolla,CA). The CaspACE® Assay System, Fluorometric was purchased fromPromega Co.

Kinase Assay Kits—The p44/42 MAP kinase assay Kit, SAPK/JNKassay kit, and p38 MAP kinase assay kit were purchased from CellSignaling Technology (Beverly, MA).

Protein Kinase Inhibitors—PD98059 was from Cell Signaling Tech-nology. PP1 was from Biomol (Plymouth meeting, PA). Calphostin C,PP2, and HB were from Calbiochem-Novabiochem Corp. Wortmannin,LY294002, and SB203580 were from Sigma.

Oligonucleotides—Primers for TNF, proIL-1/IL-1, and glyceralde-hyde phosphate dehydrogenase (GAPDH) were synthesized from localMD Bio. Inc. (Taipei, Taiwan). The dominant negative JNK construct(DN-JNK) was a gift from Dr. Michael Karin (UCSD, San Diego, CA)(33). The dominant negative Rac1 construct (DN-Rac1) and constitutiveactivated Rac1 construct (CA-Rac1) were gifts from Dr. S. Bagrodia(Cornell University, Ithaca, NY) (34).

Isolation of LDL for Preparation of OxLDL, RNA Isolation, RTand PCR Amplification for Detecting the Expression of TNF orProIL-1/IL-1, Protein Assay (Determined by Bio-Rad Protein

Assay), Western Blotting Analysis, and Enzyme-linkedImmunosorbent Assay for Measurement of TNF and IL-1

All detail methods and procedures have been presented previously(22, 35).

Analysis of ICE (Caspase 1) Activity

To analyze the ICE activity in J774A.1 cells we basically followed theprotocols from the CaspACE® assay system, fluorometric. Briefly,the cells were incubated with 25 mg/ml fucoidan or 5 mg/ml OxLDL. Atthe indicated time, the cells were washed twice with ice-cold phosphate-buffered saline (without Ca12, Mg12), harvested in 300 ml of cell sus-pension buffer (25 mM pH 7.5 HEPES, 5 mM MgCl2, 5 mM EDTA, 5 mM

dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatinA, 10 mg/ml leupeptin, from Promega Co.). The suspended cells werestored in 1.5-ml centrifugation tubes, frozen and thawed three times,and then centrifuged at 12,000 3 g at 4 °C for 15 min; the pellets werediscarded. The protein concentration of supernatant in cell extract wasdetermined, and the rest of the supernatant was saved at 280 °C forfuture use. For assaying ICE activity, 32 ml of ICE-like enzyme assaybuffer, 2 ml of dimethyl sulfoxide, and 10 ml of 100 mM dithiothreitolwere added to 75 mg of protein of cell extract from tested samples atvarious times, and then adjusted with water to 98 ml. For each testedsample there was a negative control (the same as above tested samplewith the addition of 2 ml of 2.5 mM ICE inhibitor (Ac-YVAD-CHO)) anda blank control (32 ml of ICE-like enzyme assay buffer, 10 ml of 100 mM

dithiothreitol, 2 ml of dimethyl sulfoxide, and 54 ml of water). All testedsamples were incubated at 30 °C for 30 min. After the incubation, 2 ml

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-128720

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

of 2.5 mM ICE substrate (Ac-YVAD-AMC) was added to each sample,followed by an additional 60-min incubation at 30 °C; then the intensityof the fluorescence of the tested sample was measured at an excitationwavelength of 360 nm and an emission wavelength of 460 nm. Calcu-lation of the relative fluorescence units and the caspase activity for eachsample, as well as construction of standard curve and AMC calibrationcurves were as described in the Technical Bulletin from Promega Co.

Assay Activity of ERK, JNK, p38, and PAK in Fucoidan-treatedJ774A.1 Cells with or without PK Inhibitors

Methods for assaying these PKs, including cell lysate preparation,immunoprecipitation of PK, in vitro PK reaction, analysis of PK activ-ity, and quantification of PK activity were as described previously (35).

Transfection of DN-JNK, DN-Rac1, and CA-Rac1 into J774A.1Cells and Assay of JNK and p38 Activity as well as Assay of

ProIL-1 Protein Expression in the Transfected Cells uponFucoidan Stimulation

Methods for transfection and MAPK activity assays were as de-scribed (35). Briefly, the conditions for growing J774A.1 cells to betransfected were identical to those of regular J774A.1 cells, except thecells were subpassaged the day before transfection and replaced withfresh medium. The transient transfection of DN-JNK, DN-Rac1, CA-Rac1, or control (i.e. the empty expression vector) at 10 mg of DNA/100-mm plate into cells was conducted by using LipofectAMINE PLUS®

reagent (Life Technologies, Inc.) as described previously (35). The effi-ciency of both transfection and expression was monitored by the he-magglutinin tag expression. DN-Rac1-transfected cells were stimulatedwith fucoidan for 120 min and 240 min, respectively, then used to assaythe activity of JNK and p38 as described above. DN-JNK-transfectedcells and DN-Rac1-transfected cells were harvested after 24, 48, and72 h (each sample was treated with fucoidan for 8 h), as well asCA-Rac1-stably transfected cells were harvested after fucoidan treat-ment for 4, 12, or 24 h, respectively. ProIL-1 protein expression in cellswas detected by Western blotting analysis.

Statistical Analysis

Statistical differences between the experimental groups were exam-ined by analysis of variance, and statistical significance was deter-mined at p , 0.05. The experiments were conducted three times or asindicated, and all data are expressed as the mean 6 S.E.

RESULTS

Ligands of MSR, Lipoprotein (i.e. OxLDL), and Non-lipopro-tein (i.e. Fucoidan) Up-regulate Inflammatory Cytokine TNFExpression in J774A.1 Cells—The effect of OxLDL and fu-coidan on expression of TNF protein expression by J774A.1cells was initially determined. Cells were grown at varioustimes in media with 5 mg/ml native LDL, 5 mg/ml OxLDL, 25mg/ml fucoidan, or with no supplement (i.e. as control). Thereleased TNF in conditioned media was measured by enzyme-linked immunosorbent assay (ELISA). Specifically, between 4and 12 h, OxLDL-treated cells produced about 600 pg/ml TNF,or about 100% more TNF than those of LDL-treated or controlcells (the background of TNF is about 300 pg/ml); after 24 h, thereleased TNF in OxLDL-treated cells returned to basal level(Fig. 1A). In contrast, cells treated with fucoidan for 4 h beganto increase the TNF released to about 15,000 and 30,000 pg/mlat 8 and 24 h, respectively (Fig. 1B). We next determinedwhether TNF secretion in conditioned medium was reflected byincreasing TNF message stimulated by OxLDL and fucoidan inJ774A.1 cells. Using RT-PCR, we detected the alternation ofTNF message between 2 and 24 h and found that no significantincrease of TNF mRNA was observed in either fucoidan-treatedcells (Fig. 1C) or OxLDL-treated cells (data not shown).

Fucoidan Up-regulates IL-1 and, ProIL-1 Message and Stim-ulates Activity of ICE in J774A.1 Cells—To detect the effect ofMSR ligands on another important inflammatory cytokine,IL-1 protein secretion, we used ELISA to quantitate matureIL-1 secretion in the conditioned medium of J774A.1 cells. Asshown in Fig. 2A, approximately 10 pg/ml IL-1 protein wasdetected in conditioned medium in 8-h fucoidan-treated cellscompared with that of control cells or of native LDL-incubated

cells (both below detectable level). The IL-1 concentration in-creased with prolonged fucoidan incubation time; at 48 h, theaccumulated concentration of IL-1 in fucoidan-treated cells wasup to 50 pg/ml. In contrast, OxLDL barely stimulated IL-1secretion in the cells for all testing periods (Fig. 2A). Resultsindicate that in J774A.1 cells, ligation of different MSR ligandsleads to differentially induced IL-1 protein secretion.

FIG. 1. Effect of MSR ligands, OxLDL and fucoidan, on TNFproduction and TNF mRNA in murine macrophage J774A.1cells. Panel A, effect of OxLDL on TNF production in cells. CumulativeTNF is presented as the concentration of TNF in cell-conditioned me-dium treated with 5 mg/ml OxLDL collected at the indicated time within24 h and measured using an ELISA as described under “ExperimentalProcedures.” The data are reported as representative of three experi-ments (n 5 3). Panel B, effect of fucoidan on TNF production in cells.Experiments and measurement for TNF production in 25 mg/ml fu-coidan-treated cells were similar to those described for panel A. One ofthree experiments is presented (n 5 3). Panel C, RT-PCR analysis ofexpression of TNF mRNA in cells. Total RNA was isolated from cellsgrown in serum-free medium in the presence of 5 mg/ml OxLDL or 25mg/ml fucoidan for various times as indicated. For fucoidan treatment,ethidium bromide-stained agarose gel with amplified TNF cDNA at 692base pairs (bp) and normalized by comparison with RT-PCR of mRNA ofGAPDH at 450 bp, a constitutively expressed gene, are indicated witharrows for TNF and GAPDH, respectively. The detailed method isdescribed under “Experimental Procedures.” One of three experimentsis presented (n 5 3). Results in OxLDL-treated cells were similar to theresults of RT-PCR for TNF mRNA (data not shown).

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-1 28721

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

To investigate further the molecular mechanism of the se-cretion of mature IL-1 (17–18 kDa), initially the fucoidan-induced IL-1 precursor, proIL-1 (34 kDa) was detected by West-ern blotting analysis. As shown in Fig. 2B, fucoidan-inducedproIL-1 protein was detected between 2 and 4 h and peaked at8 h. After 12 h, proIL-1 protein decreased, and it graduallyreturned to basal level around 24 h. There was no detectableproIL-1 protein in OxLDL-treated cells for all periods (data notshown), consistent with no IL-1 secretion in OxLDL-treatedcells. Moreover, using RT-PCR, we demonstrated 2-h fucoidanincubation, but not OxLDL-induced proIL-1 mRNA expression,compared with native LDL-treated cells or with control cells.The fucoidan-induced proIL-1 message peaked between 6 and8 h (Fig. 2C); at 12 h, it remained higher than that of controlcells. After 24 h, the induced proIL-1 message returned to basallevel. The similar up-regulation of proIL-1 mRNA detected byNorthern analysis is comparable to the message detected byRT-PCR (data not shown).

Post-transcriptional regulation and processing of proIL-1protein into mature IL-1 secretion via ICE has been reported(31, 32). Because incubation of cells with fucoidan inducedproIL-1 protein and simultaneously led to IL-1 secretion in atimely fashion, we examined whether fucoidan stimulates ICEactivity during IL-1 secretion. As shown in Fig. 2D, ICE activ-

ity increased 2-fold compared with control cells after a 6-hfucoidan stimulation; in contrast, there is no detectable ICEactivity in OxLDL-treated cells. Hence, we focus on fucoidan-mediated signal transductions including protein kinase in theregulation of proIL-1/IL-1 in J774A.1 cells.

Fucoidan Activates ERK in J774A.1 Cells—Our previousresults demonstrated that fucoidan transduces PK signalingpathways in the up-regulation of urokinase-type plasminogenactivator (1). To examine fucoidan-mediated signal transductionpathways in the regulation of IL-1 gene expression, first wetested whether fucoidan stimulates MAPK. Incubation ofJ774A.1 cells with fucoidan led to a modest phosphorylation ofElk-1, a transcriptional factor, evidencing the activation of ERKinduced by fucoidan (Fig. 3A). Experiments for the time course offucoidan-induced ERK activity were further conducted. As de-tected by Western blot analysis with anti-phospho-Elk-1 anti-body, which recognizes the activated, serine 383-phosphorylatedform of Elk-1 by activated ERK (36), ERK activity in untreatedcells barely exists. Upon fucoidan stimulation, the activity ofERK was detected around 30 min, and it reached the maximallevel of about 2-fold at 120 min; after 240 min, ERK activitygradually returned to basal level (Fig. 3, A and B).

Role of PKC and MEK1 in Fucoidan-induced ProIL-1 ProteinExpression—Fucoidan induces activity of PTK, PKC, and ERK

FIG. 2. Effect of fucoidan and OxLDL on expression of IL-1, proIL-1 protein, and proIL-1 mRNA as well as on stimulation of ICE(caspase 1) activity in J774A.1 cells. Panel A, effect of fucoidan and OxLDL on IL-1 secretion in cells. Cells were treated with 25 mg/ml fucoidanor 5 mg/ml OxLDL, and conditioned media were harvested at the time indicated within 48 h. Concentrated media were assayed for IL-1concentration using an IL-1-specific ELISA; one of four experiments is presented (n 5 4). Panel B, Western blotting analysis of proIL-1 proteinexpression in fucoidan-treated cells. Cells were treated with fucoidan or OxLDL for the various times as indicated, and whole cell lysates wereanalyzed by Western blot with anti-IL-1 antiserum, as described under “Experimental Procedures.” The proIL-1 (34 kDa) and a-tubulin (as aninternal control) are indicated as arrows on the right; one of four experiments is presented (n 5 4). No proIL-1 protein was found in OxLDL-treatedcells (data not shown). Panel C, RT-PCR analysis of proIL-1 mRNA expression in cells. Total RNA was isolated from cells treated with fucoidanor OxLDL within 24 h. Ethidium bromide-stained agarose gel with amplified proIL-1 mRNA at 563 base pairs (bp) and normalized by comparisonwith RT-PCR of GAPDH mRNA is indicated with arrows for proIL-1 and GAPDH. One of four experiments is presented. No proIL-1 mRNA wasfound in OxLDL-treated cells (data not shown). Panel D, time-dependent activation of ICE activity by OxLDL and fucoidan in J774A.1 cells. Cellswere treated with OxLDL and fucoidan for the indicated times. Cell extracts (75 mg of protein) were incubated in the presence of 50 mM fluorescentcaspase 1 substrate Ac-YVAD-CHO for 1 h at 30 °C. Caspase 1 activity was measured fluorometrically after substrate cleavage with excitation at360 nm and emission at 460 nm. The detailed method is described under “Experimental Procedures” or as in the instruction manual of theCaspACE® assay system, fluorometric, from Promega. Experiments were repeated three times, and a representative result is shown.

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-128722

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

and simultaneously stimulates proIL-1 protein. We examinedwhether the PKC/MEK1/ERK pathway is one of fucoidan-in-duced PTK downstream signaling cascades in the regulation ofproIL-1. Initially, experiments were conducted for dose re-sponses of calphostin C (PKC inhibitor, 0.25, 0.50, 1.0, and 5.0mM) and of PD98059 (MEK1 inhibitor, 10, 25, 50, and 100 mM)in inhibiting ERK activity. J774A.1 cells were exposed to theindicated concentrations of inhibitors, followed by incubationwith fucoidan, and the appropriate concentrations of calphostinC and PD98059 inhibiting ERK activity were determined as 1.0and 50 mM, respectively (data not shown). Results of Westernblotting analysis show that at the indicated concentration,neither calphostin C (Fig. 4A) nor PD98059 (Fig. 4B) blocksfucoidan-induced proIL-1 protein, although calphostin C andPD98059 effectively inhibit fucoidan-stimulated ERK activity,suggesting that the PKC/MEK1/ERK pathway is less involvedin fucoidan-induced proIL-1 protein expression.

Fucoidan Stimulates JNK Activity and the Role of JNK inFucoidan-induced ProIL-1 Protein—The inflammatory re-sponse of J774A.1 cells to fucoidan resulting in induction ofIL-1 expression suggested to us one possibility for fucoidanactivation of the stress-related JNK pathway. We examinedwhether fucoidan activates JNK. Cells incubated with fucoidanled to JNK activation (Fig. 5A) as determined by Western blotanalysis via anti-phospho-c-Jun, an antibody that recognizes

the activated, serine 63-phosphorylated form of c-Jun (37, 38).As in a time-course fashion, JNK activity gradually increasedaround 60 min, and the maximal activity of JNK was at 120min, about 13-fold of untreated cells (Fig. 5, A and B). After 240min, the induced JNK activity gradually returned to basallevel. To investigate the role of fucoidan-induced JNK activity,initially, we chose and examined curcumin (a JNK inhibitor) inthe regulation of proIL-1 protein expression. As shown in Fig.6A, increasing curcumin above 0.1 mM gradually reduces fu-coidan-induced proIL-1 protein, and completely inhibitsproIL-1 protein at concentrations above 1 mM. Alternatively,experiments of transient transfection of DN-JNK into J774A.1cells were conducted, and we studied directly the role of in-duced JNK in the regulation of proIL-1. Results of Westernblotting analyses indicate that upon fucoidan stimulation,there is about 10-fold less proIL-1 protein in DN-JNK-trans-fected cells than in control cells (Fig. 6B); in addition, noproIL-1 protein could be detected in the 48- and 72-h DN-JNK-transfected cells. Consistently, there is no detectable JNK ac-tivity in the fucoidan-treated DN-JNK cells (data not shown).

Fucoidan Stimulates p38 Activity and the Role of p38 inFucoidan-induced ProIL-1 Protein—To explore additional fu-coidan-mediated signal transduction pathways, we further ex-amined whether fucoidan induces p38 activity, another impor-tant stress-related MAPK member. Upon fucoidan stimulation,p38 activity gradually increased as detected by Western blot-ting analysis with anti-phospho-ATF-2, an antibody that spe-

FIG. 3. Time course of fucoidan-induced p44/42 ERK activity inJ774A.1 cells. Panel A, analysis of p44/42 ERK activity in fucoidan-treated J774A.1 cells. The ERK-induced phosphorylation of Elk-1 wasmeasured by quantitative immunoblotting with phospho-Elk-1 (Ser-383) antibody and the Phototope®-HRP Western detection system.Briefly, whole cell lysates (200 mg of protein) at various times wereincubated with 20 ml of immobilized Phospho-p44/42 ERK (Thr-202 andTyr-204) monoclonal antibody (50% slurry) at 4 °C for 24 h. The immu-noprecipitated active (phosphorylated) ERK was centrifuged at 4 °C for10 min, pellet washed twice with 13 lysis buffer and 13 kinase buffer,respectively, then resuspended in 13 kinase buffer. The kinase reac-tions were performed in the presence of 200 mM cold ATP and 2 mg ofElk-1 fusion protein at 30 °C for 30 min. Elk-1 phosphorylation wasmeasured by Western blotting of nonradioactive labeled samples usingphospho-Elk-1 (Ser-383) antibody plus Phototope-HRP Western detec-tion system via chemiluminescence. The detailed method is describedunder “Experimental Procedures” or in the NEB instruction manual.Panel B, histograms represent quantification by PhosphorImager ofphospho-Elk-1 (Ser-383) for fucoidan-activated ERK activity in eachsample with using ImageQuaNT® software from Molecular Dynamics.All data of relative activity are expressed as a comparison with un-treated J774A.1 cells (i.e. t 5 0; activity of control cells defined as 1).Similar experiments were repeated four times, and a representativeresult is shown.

FIG. 4. Effect of PKC inhibitor (calphostin C) and of MEK1inhibitor (PD98059) on fucoidan-regulated proIL-1 protein ex-pression. Panel A, effect of calphostin C on fucoidan-induced proIL-1protein expression. First, the dose response of the inhibitor of PKC,calphostin C (various concentrations: 0.1, 0.25, 0.50, and 1.0 mM), onfucoidan-induced proIL-1 protein was studied, and the effective concen-tration of calphostin C was determined as 1 mM (data not shown). Afterincubation with or without 1 mM calphostin C for 1 h, cells were dividedinto two groups, fucoidan-untreated (samples 1 and 3) and fucoidan-treated (samples 2 and 4). For fucoidan treatment, cells were incubatedwith 25 mg/ml fucoidan for an additional 8 h. After incubation, sampleswere subjected to Western blot analysis of proIL-1 as described in thelegend of Fig. 2B. The arrows on the right represent the positions ofproIL-1 and a-tubulin, respectively. This experiment is a representativeof three similar experiments. Panel B, effect of the inhibitor of MEK1,PD98059, on fucoidan-induced proIL-1 protein expression. As in panelA, the effective concentration of PD98059 for MEK1 was determined as50 mM. After incubation with or without 50 mM PD98059 for 1 h, cellswere divided into two groups, fucoidan-untreated (samples 1 and 3) andfucoidan-treated (samples 2 and 4). For fucoidan treatment cells wereincubated with 25 mg/ml fucoidan for an additional 8 h. After treat-ments, the samples were subjected to Western blot analysis as de-scribed in the legend of panel A. This experiment is a representative ofthree similar experiments.

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-1 28723

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

cifically recognizes the activated, Thr-71-phosphorylated formof ATF-2 (39) (Fig. 7A). The results of a time-course study offucoidan-induced p38 activity indicated that at 60 min, p38activity increased to 6-fold that of control cells. At 240 min, p38activity of treated cells reached to about 10-fold that of controlcells; after 480 min, it returned to basal level (Fig. 7, A and B).

To dissect the effect of fucoidan-induced p38 activity inATF-2 phosphorylation, we designed a series of experiments.Using a specific p38 inhibitor, SB203580 (SB), we initiallyconducted the inhibitory dose-response study of SB in fucoidaninduction of p38 activity. J774A.1 cells were exposed to variousconcentrations of SB (0.1, 1.0, and 10 mM) followed by incuba-tion with fucoidan for various times. In the time-course study,SB effectively inhibits fucoidan-induced p38 activity at concen-trations above 1 mM compared with control untreated cells,although 0.1 mM SB reduces p38 activity slightly (Fig. 8A).Moreover, we further examined the potential role of fucoidan-induced p38 activation and ATF-2 phosphorylation in the reg-ulation of proIL-1 protein. In J774A.1 cells pretreated with orwithout SB, followed by incubation with fucoidan, Westernblotting analyses were conducted to study the SB inhibitorydose response (concentrations were 0.1, 1.0, and 10 mM) inproIL-1 protein expression. SB completely inhibited fucoidan-induced proIL-1 protein at concentrations above 1 mM comparedwith control cells (Fig. 8B, samples 6 and 8 versus sample 2);but no inhibition below 0.1 mM (Fig. 8B, sample 4).

Fucoidan Stimulates PAK Activity—To investigate furtherthe possible upstream signaling networks involved in regula-

tion of JNK or p38 activity, one of the relevant signaling mol-ecules, PAK, was chosen. PAK activity in the time-course studywas examined using an immunoprecipitated radioactive pro-tein kinase activity assay, with phosphorylation of substrate,myelin basic protein (40). As shown in Fig. 9, A and B, themaximal fucoidan-stimulated PAK activity in phosphorylationof myelin basic protein occurred at 30 min, and after 60 min thestimulated PAK activity returned to basal level.

Effect of Rac1 Transfection on Fucoidan-stimulated JNK andp38 Activity and the Effect of Rac1 Transfection on Fucoidan-induced ProIL-1 Protein Expression—Activation of Rac1GTPase stimulates PAK activity in some cells (34). To dissectthe relationship of fucoidan-induced signal transduction amongRac1 and JNK and p38, experiments for transfection of theDN-Rac1 construct were conducted and examined JNK and p38activity, respectively. Upon fucoidan stimulation, there was10-fold lower JNK activity (Fig. 10A, sample 2 versus sample 1)and 5-fold lower p38 activity (Fig. 10B, sample 2 versus sample1) in DN-Rac1-transfected cells than those of empty expressionvector-transfected control cells (sample 1 in Fig. 10, A and B).Obviously, without fucoidan stimulation, no JNK and p38 ac-tivity is detected in cells transfected with empty expressionvector (sample 3 in Fig. 10, A and B). To investigate further therole of Rac1 in proIL-1 protein expression, experiments for theeffect of DN-Rac1 and CA-Rac1 on fucoidan-induced proIL-1protein were conducted. Moreover, the effects of DN-Rac1 andCA-Rac1 on fucoidan-induced proIL-1 protein were investi-gated further. As shown in Fig. 10C, upon 8-h fucoidan stimu-lation, proIL-1 was expressed much less in DN-Rac1-trans-fected cells (i.e. the 24-, 48-, and 72-h post-DN-Rac1-transfectedcells) than that of empty expression vector-transfected controlcells. In contrast, upon 12- and 24-h fucoidan stimulation, therewas more proIL-1 expression (a superinduction) in CA-Rac1-transfected cells than in control cells (Fig. 10D). All of theseresults indicate that in fucoidan-induced signal transduction

FIG. 5. Time course of fucoidan-activated JNK activity. PanelA, analysis of JNK activity in fucoidan-treated J774A.1 cells by quan-titative Western blotting using phospho-c-Jun (Ser-63) antibody andthe Phototope-HRP Western detection system. Briefly, whole cell ly-sates (250 mg of protein) were incubated overnight with 2 mg of c-Jun(1–89) fusion protein beads at 4 °C for 24 h. The active complex ofJNKzc-Jun fusion protein beads was centrifuged at 4 °C for 10 min, andthe pellet was washed twice with 13 lysis buffer and 13 kinase buffer,respectively, and then resuspended in 13 kinase buffer. The kinasereactions were performed in the presence of 100 mM cold ATP at 30 °Cfor 30 min. The phosphorylation of c-Jun at Ser-63 was measured byWestern blotting of nonradioactive labeled samples using phospho-c-Jun (Ser-63) antibody plus the Phototope-HRP Western detection sys-tem via chemiluminescence. The detailed method is described under“Experimental Procedures” or in the NEB instruction manual. Panel B,histograms represent quantification by PhosphorImager of phospho-c-Jun (Ser-63) for fucoidan-activated JNK activity in each sample withusing ImageQuaNT software from Molecular Dynamics. All data ofrelative activity are expressed in comparison with untreated cells (t 50; activity of control cells defined as 1). Similar experiments wererepeated four times, and a representative result is shown.

FIG. 6. Effect of curcumin and DN-JNK transfection on fu-coidan-regulated proIL-1 protein expression. Panel A, effect ofcurcumin on fucoidan-regulated proIL-1 protein. Cells were treatedwith various concentrations (0.1, 1.0, 10, and 100 mM) of curcumin asdescribed under “Experimental Procedures.” After a 24-h curcumintreatment, cells were treated with fucoidan for 8 h, and Western blot-ting analyses of proIL-1 protein expression were conducted as describedin the legend of Fig. 2B. Similar results were obtained in three separateexperiments. Panel B, effect of DN-JNK transfection on fucoidan-regu-lated proIL-1 protein expression. Cells were transient transfected withDN-JNK or with the empty expression vector as control (Control) forvarious times, i.e. 24, 48, and 72 h as indicated and described under“Experimental Procedures.” After a 24-, 48-, or 72-h DN-JNK transfec-tion, cells were treated with fucoidan for additional 8 h, and thenWestern blotting analyses of proIL-1 expression were conducted asdescribed in the legend of Fig. 2B. The data are representative of threeseparate experiments.

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-128724

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

networks, Rac1 mediates JNK and p38 activity and furtherregulates proIL-1 protein expression.

Effect of the PI3-kinase Inhibitor, Wortmannin, on Fucoidan-induced JNK and p38 Activity and on Fucoidan Regulation ofProIL-1 Protein and IL-1 Secretion—As demonstrated above,the PKC/MEK1/ERK pathway plays less of a role in fucoidanregulation of proIL-1 expression. To explore other fucoidan-mediated signal transduction pathways involving in proIL-1expression, we used wortmannin, an inhibitor of PI 3-kinase, toexamine the potential role of PI 3-kinase in fucoidan activationof JNK or p38. In J774A.1 cells preincubated with or withoutwortmannin for 60 min, followed by treatment with fucoidanfor an additional 120 or 240 min, the activity of JNK and p38was examined. In the absence of wortmannin, fucoidan inducedactivity of JNK and p38 approximate 13-fold (Fig. 11A, sample2 versus sample 1) and 10-fold (Fig. 11B, sample 2 versussample 1), respectively, compared with control untreated cells(sample 1). Interestingly, in cells preincubated with wortman-nin followed by treatment of fucoidan, JNK and p38 activityincreased approximately 16-fold (Fig. 11A, sample 4 versussample 1) and 23-fold (Fig. 11B, sample 4 versus sample 1),respectively, compared with control untreated cells.

Moreover, the roles of PI 3-kinase in fucoidan regulation ofproIL-1 protein and IL-1 secretion were examined. In cellspreincubated with wortmannin for 60 min followed by exposureto fucoidan for an additional 8 h, proIL-1 protein in cell lysateswas detected by Western blotting analysis. Results showed that

preincubation of wortmannin in fucoidan-treated cells in-creases proIL-1 production significantly (Fig. 12A, sample 4)compared with fucoidan-treated cells or wortmannin-treatedcells (Fig. 12A, sample 2 or sample 3) or compared with controluntreated cells (Fig. 12A, sample 1). In addition, the effect ofwortmannin on fucoidan-induced mature IL-1 secretion de-tected by ELISA was examined. There was a 5-fold higher IL-1secretion (20 pg/ml) detected in cells pretreated with wortman-nin prior to incubation with fucoidan (Fig. 12B, sample 4)compared with cells (4 pg/ml) incubated only with fucoidan(Fig. 12B, sample 2); as expected, no IL-1 was in the controlcells (Fig. 12B, sample 1). Without fucoidan, wortmanninslightly increased IL-1 secretion in cells (Fig. 12B, sample 3versus sample 1).

Fucoidan Stimulates Tyrosine Phosphorylation of NRPTKand pp60Src and Induces the Association (Complex Formation)between pp60Src and Hsp90 in J774A.1 Cells—Although thereis no functional PTK motif found in cytoplasmic domain of MSR(9, 41–43), ligation of fucoidan to MSR induces tyrosine phos-phorylation of various proteins in human THP-1 macrophages(1) and in J774A.1 cells, implying that MSR is a PTK-linkedreceptor. We hypothesize that a NRPTK such as pp60Src in-

FIG. 7. Time course of fucoidan-activated p38 activity. Panel A,analysis of p38 activity in fucoidan-treated J774A.1 cells. The p38-induced phosphorylation of ATF-2 was measured by quantitative im-munoblotting with phospho-ATF-2 (Thr-71) antibody and the Photo-tope-HRP Western detection system. Briefly, whole cell lysates (200 mgof protein) at various times were incubated with 20 ml of resuspendedimmobilized phospho-p38 MAP kinase (Thr-180/Tyr-182) monoclonalantibody at 4 °C for 24 h. The immunoprecipitated active (phosphoryl-ated) p38 was centrifuged at 4 °C for 10 min, the pellet was washedtwice with 13 lysis buffer and 13 kinase buffer, respectively, andresuspended in 13 kinase buffer. The kinase reactions were performedin the presence of 200 mM cold ATP and 2 mg of ATF-2 fusion protein at30 °C for 30 min. The phosphorylation of ATF-2 at Thr-71 was meas-ured by Western blotting of nonradioactive labeled samples using phos-pho-ATF-2 (Thr-71) antibody plus the Phototope-HRP Western detec-tion system via chemiluminescence. The detailed method is describedunder “Experimental Procedures” or in NEB instruction manual. PanelB, histograms represent quantification by PhosphorImager of phospho-ATF-2 (Thr-71) for fucoidan-stimulated p38 activity in each samplewith using ImageQuaNT software from Molecular Dynamics. All dataof relative activity are expressed in comparison with untreated cells (t 50; activity of control cells defined as 1). Similar experiments wererepeated four times, and a representative result is shown.

FIG. 8. Effect of p38 inhibitor SB203580 on fucoidan-inducedp38 activity and on fucoidan-induced proIL-1 protein expres-sion. Panel A, effect of the inhibitor of p38, SB203580, on fucoidan-induced p38 activity. J774A.1 cells were pretreated with various con-centrations (0.1, 1.0, and 10 mM) of SB203580 followed by incubationwith 25 mg/ml fucoidan for additional times as in Fig. 7. After theindicated treatments, samples were subjected to analysis of p38 activityvia phosphorylation of ATF-2, and the representative histograms are asdescribed in Fig. 7. All data of p38 relative activity are expressed as acomparison with untreated cells (t 5 0; activity of control cells definedas 1). Experiments were repeated, and a representative of three similarresults is shown. Panel B, effect of the p38 inhibitor SB203580 onfucoidan-induced proIL-1 protein. After pretreatment or no pretreat-ment with 0.1, 1.0, or 10 mM SB203580 for 60 min, cells were dividedinto two groups, fucoidan-untreated (samples 1, 3, 5, and 7) and fu-coidan-treated (samples 2, 4, 6, and 8). For fucoidan treatment cellswere incubated with 25 mg/ml fucoidan for an additional 8 h. Aftertreatments, samples were subjected to Western blot analysis of proIL-1protein as described. Both proIL-1 and a-tubulin are as indicated.Results are representative of three similar experiments.

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-1 28725

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

volves MSR ligand-mediated signal transductions. Preincuba-tion of J774A.1 cells with specific inhibitors of pp60Src, e.g.PP1 or PP2, was followed by exposure to fucoidan. First, celllysates were immunoprecipitated with monoclonal anti-pp60Src IgG, and immunocomplexes were recovered via incu-bation with protein A/G plus agarose. Immunoprecipitateswere separated by SDS-polyacrylamide gel electrophoresis,then immunoblotted with monoclonal anti-phosphotyrosineIgG. As seen in Fig. 13A, the 60-kDa phosphotyrosyl proteinwas immunoreactive with anti-pp60Src IgG in fucoidan-treated cells (sample 2) and less reaction in PP2-preincubatedcells (sample 4). In contrast, there was no difference of tyrosinephosphotyrosine between PP1-preincubated cells and controluntreated cells (samples 3 and 1). These data indicate thatligation of fucoidan to MSR in J774A.1 cells leads to tyrosinephosphorylation of pp60Src, but PP1 and PP2 inhibit tyrosinephosphorylation.

HB inhibits fucoidan-stimulated protein tyrosine phospho-rylation (1), suggesting that unidentified PTK involve fu-coidan-mediated signals such as stress-related JNK and p38activity. It is known that one of the HB cellular targets isHsp90 (26), which is expressed under certain stress conditions.GA and HB inhibit signal transduction proteins includingNRPTK and pp60Src. Pharmacologically, GA and HB bind in aspecific manner to Hsp90 and inhibit pp60SrczHsp90 hetero-complex formation (26–28). Here in J774A.1 cells preincubatedwith GA and HB as well as PP1 and PP2 followed by exposureto fucoidan, we evaluated further the association (interaction)of pp60Src and Hsp90 by performing immunoprecipitation withanti-Hsp90 IgG and probing the resultant blots with anti-pp60Src IgG (Fig. 13B). Whereas pp60Src co-precipitated withHsp90 in fucoidan-treated cells (sample 2), interaction between

FIG. 10. Effect of Rac1 transfection on fucoidan-stimulatedJNK and p38 activity as well as effect of Rac1 transfection onfucoidan-induced proIL-1 protein expression in J774A.1 cells. Inpanels A, B, and C, cells were transiently transfected with DN-Rac1 orwith the empty expression vector as control, as described under “Ex-perimental Procedures.” Panel A, effect of DN-Rac1 transfection onreduction of fucoidan-stimulated JNK activity. After a 24-h transfec-tion, cells were treated with or without fucoidan for 120 min, and theJNK activity of the treated cells was analyzed as described above. PanelB, effect of DN-Rac1 transfection on reduction of fucoidan-stimulatedp38 activity. After a 24-h transfection, cells were treated with or with-out fucoidan for 240 min, and p38 activity of the treated cells wasanalyzed as described above. Panel C, effect of DN-Rac1 transfection onreduction of fucoidan-induced proIL-1 protein expression. After a 24-,48-, and 72-h transfection, cells were treated with fucoidan for 8 h, andwhole cell lysates were analyzed by Western blot with anti-IL-1 anti-serum, as described under “Experimental Procedures.” The proIL-1 (34kDa) and a-tubulin are indicated as arrows on the right. Panel D,transfection of CA-Rac1 increases fucoidan-induced proIL-1 proteinexpression. Two groups of cells were prepared and used for the exper-iments. Sample 1 contained cells stably transfected with the CA-Rac1construct; sample 2 was made up of cells transfected by the emptyexpression vector (i.e. control). Cells were treated with or withoutfucoidan for 4, 12, and 24 h as indicated; whole cell lysates wereanalyzed by Western blot with anti-IL-1 antiserum. ProIL-1 is as indi-cated. All experiments in panels A–D were repeated, and a represent-ative of three similar results (n 5 3) is shown.

FIG. 9. Time course of fucoidan-stimulated PAK activity inJ774A.1 cells. Panel A, activity of PAK in fucoidan-treated J774A.1cells. Samples were examined in a radioactive immunocomplex kinaseassay method, using myelin basic protein (MBP) as substrate with anadditional 1 ml of [g-32P]ATP (5 mCi, 330 mM) and incubated at 30 °C for15 min. Equal numbers of cells were treated with fucoidan at each timepoint as indicated. Details are described under “Experimental Proce-dures.” Panel B, histograms representing fucoidan-stimulated PAK ac-tivity are quantified by phosphorimaging of [g-32P]ATP of each samplevia using ImageQuaNT software from Molecular Dynamics. All data ofrelative activity are expressed as comparison with untreated cells (t 50; activity of control cells is 1). Similar experiments were repeated fourtimes, and a representative result is shown.

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-128726

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

pp60Src and Hsp90 was greatly decreased in cells preincubatedwith HB (sample 3) or GA (sample 4). Interestingly, there wasa similar complex formation between pp60Src and Hsp90 in thecells pretreated with PP1 and PP2 (samples 5 and 6), indicatingthat inhibition of Src kinase activity does not interfere thespecific association. Second, in the converse experiments, celllysates were immunoprecipitated with monoclonal anti-pp60Src IgG and immunoblotted with monoclonal anti-Hsp90IgG. Results similar to those Fig. 13B were observed; the 90-kDa protein was immunoreactive with anti-Hsp90 IgG in fu-coidan-treated cells, but there was no association in GA- orHB-preincubated cells (data not shown).

Roles of Tyrosine Phosphorylation of pp60Src and of Associ-ation between pp60Src and Hsp90 in the Regulation of Fu-coidan-induced ProIL-1 Protein Expression—We investigatedfurther the biological significance of fucoidan-stimulated tyro-sine phosphorylation of pp60Src and the association of pp60Srcwith Hsp90 in regulating proIL-1 protein. Results of Westernblotting cell lysates with anti-IL-1 antiserum show that fu-coidan-induced proIL-1 protein expression can be completelyblocked in cells pretreated with inhibitors of the pp60SrczHsp90 complex (i.e. GA or HB) (Fig. 14, samples 3 and 4 versussample 2) in a dose-response study (data not shown). In con-trast, fucoidan-induced proIL-1 protein can be partially re-duced by inhibitors of pp60Src (PP1 or PP2) (Fig. 14, samples 5and 6 versus sample 2); relatively, PP1 exerts more inhibitoryeffect than that of PP2 (Fig. 14, sample 5 versus sample 6).Similarly, there was no detectable IL-1 secretion in cells pre-treated with these inhibitors (data not shown).

DISCUSSION

In atherosclerotic lesions, activated macrophages via overex-pressed scavenger receptors aberrantly taking up OxLDL arealso the main source of secretion of inflammatory cytokinesTNF (2, 17–19) and IL-1 (17, 19). Yet it is unclear whetherthere is a relationship between receptor ligand binding andstimulation of cytokine expression. Fucoidan, a polyanionicpolysaccharide, has been used as an effective competitor forOxLDL or modified LDL in studies of receptor binding (9, 10).Moreover, fucoidan stimulates production of proteases (1, 9, 24)

and cytokines in macrophages (9, 16, and this paper); however,there is no molecular mechanism to date for fucoidan-mediatedreactions. Using ELISA, we demonstrate here that 24-h fu-coidan induces about 50-fold more TNF production thanOxLDL does in J774A.1 cells. Based on the time course of TNFproduction (Fig. 1, A versus B), there are different patterns andmechanisms for TNF induction by the two ligands, althoughTNF preexists in untreated cells. In addition, only the rela-tively lower concentration of OxLDL (5 mg/ml) induced TNFbecause there was no induction of TNF at a higher concentra-tion of OxLDL (50 mg/ml) (data not shown), comparable to theprevious demonstration (21). Using RT-PCR for TNF mRNA,there was no apparent difference of TNF message among cellstreated with fucoidan and OxLDL and untreated cells. The factthat there was increased TNF production but no alteration ofTNF message under fucoidan stimulation indicates that fu-coidan regulation of TNF expression is likely at a post-tran-scriptional level.

Neither IL-1 nor proIL-1 preexists in quiescent J774A.1cells. Upon fucoidan stimulation, IL-1 can be detected byELISA after 6–8 h; IL-1 secretion is consistent with the se-quential times for synthesis of proIL-1 mRNA and proIL-1protein under stimulation for 2 and 4 h, respectively. The ICE

FIG. 11. Effect of the PI 3-kinase inhibitor wortmannin onfucoidan-induced JNK and p38 activity in J774A.1 cells. Panel A,effect of wortmannin on fucoidan-stimulated JNK activity. Cells werepretreated with 25 mM wortmannin for 1 h prior to stimulating with 25mg/ml fucoidan for an additional 120 min followed by nonradioactivemethod for JNK assay as described previously. Panel B, effect of wort-mannin on fucoidan-stimulated p38 activity. Cells were pretreated with25 mM wortmannin for 1 h prior to stimulating with 25 mg/ml fucoidanfor an additional 240 min followed by a nonradioactive p38 kinase assayas described previously. All data of relative JNK and p38 activity areexpressed as a comparison with untreated cells (t 5 0; activity of controlcells is 1). Similar results were obtained in four separate experiments.

FIG. 12. Effect of the PI 3-kinase inhibitor wortmannin onfucoidan-induced proIL-1 expression and IL-1 secretion inJ774A.1 cells. Panel A, effect of wortmannin on fucoidan-inducedproIL-1 expression. After incubated with or without 25 mM wortmanninfor 1 h, the cells were divided into two groups, fucoidan-untreated(samples 1 and 3) and fucoidan-treated (samples 2 and 4). For fucoidan-treated groups cells were incubated with 25 mg/ml fucoidan for anadditional 8 h. After treatments, the samples were subjected to Westernblot analysis of proIL-1 as described above. This experiment is repre-sentative of three similar experiments. Panel B, effect of wortmannin onfucoidan-induced IL-1 secretion. Cells were treated as described inpanel A. After the indicated treatments, concentrated conditioned me-dia were collected at 8 h. The IL-1 contents in tested samples wereassayed by an IL-1-specific ELISA as described above. The data arereported as representative of four separate experiments (n 5 4).

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-1 28727

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

activity of fucoidan-treated cells peaks around 6 h and simul-taneously IL-1 secretion increases, which reflects that activeICE hydrolyzes proIL-1 into IL-1 as in various cells (31, 32),although the reason for continuous increasing IL-1 needs fur-ther study. Our results indicate that there are complicatedmechanisms, likely at transcriptional, post-transcriptional,and post-translational levels for fucoidan induction of proIL-1protein and IL-1 secretion in macrophages. Surprisingly, thereis no detectable expression of proIL-1/IL-1 in the cell incubatedwith OxLDL. One of explanations for the results is that bindingof different ligands to the conserved lysine clusters of thecollagen-like domain in MSR (43–45) may trigger on different

signal transductions (44, 45).2 For the first time we demon-strate that different MSR ligands transduce diverse signalingand further lead to different biochemical reactions such ascytokine production.

Ligation of MSR induces signal transduction pathways inmacrophages (1, 16, 46, 47); for example, binding of fucoidan toMSR in human monocytic macrophage THP-1 cells inducedPKC signaling-mediated IL-1 secretion (16) and also stimu-lated protein tyrosine phosphorylation including phospholipaseC-g1, PI 3-kinase, and PKC activity leading to up-regulatedurokinase-type plasminogen activator expression (1). To focusfurther on molecular mechanism by which fucoidan mediatessignaling cascades including MAPK in regulation of proIL-1/IL-1 expression, we utilized specific pharmacological antago-nists such as calphostin C, PD98059, wortmannin, curcumin,and SB203580, which inhibit the phosphorylation of PKC (1,48), MEK1 (49), PI 3-kinase (50), JNK (51, 52), and p38 (53),respectively. Because PKC and MEK1 are involved in the in-duction of ERK activity (54), and ligation of fucoidan inducesERK activity, we examined the roles of PKC and MEK1 infucoidan regulation of proIL-1/IL-1. Initially, both calphostin Cand PD98059 inhibit fucoidan-stimulated ERK activity, butthey fail to block fucoidan-induced proIL-1 protein and IL-1secretion. Results indicate that the fucoidan-induced PKC/MEK1/ERK pathway is not involved in regulating proIL-1/IL-1expression, although we could not rule out the possibility ofcross-talking between MEK kinase members and ERK. Simi-larly, using pharmacological inhibitors and genetic dominantmutants, we established a fucoidan-mediated signaling rela-tionship among JNK and p38 activity and regulation of proIL-1protein expression. In preliminary studies, we determinedwhether OxLDL or fucoidan induce cytokines in fully differen-tiated human macrophages. Peripheral blood monocytes wereallowed to differentiate in culture at indicated times, and con-ditioned medium and cell lysates were collected and prepared.Exposure of monocyte-derived macrophages to OxLDL or fu-coidan increased TNF secretion as detected by ELISAs; butonly fucoidan induced proIL-1/IL-1 expression. Moreover, fu-coidan activated ERK, JNK, and p38 in human macrophages

2 H.-Y. Hsu, S.-L. Chiu, M.-H. Wen, K.-Y. Chen, and K.-F. Hua,unpublished data.

FIG. 13. Fucoidan stimulates tyrosine phosphorylation ofpp60Src and induces the association (complex formation) be-tween pp60Src and Hsp90. Panel A, fucoidan stimulates tyrosinephosphorylation of pp60Src. J774A.1 cells were preincubated with in-hibitors of pp60Src, 1 mM PP1 and 1 mM PP2, respectively, followed byexposure to fucoidan. Various cell lysates were immunoprecipitated (IP)with monoclonal anti-pp60Src IgG at room temperature for 4 h, andimmunocomplexes were recovered via incubation with protein A/G plusagarose at 4 °C for 24 h. Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted (IB) with mono-clonal anti-phosphotyrosine IgG. Visualization of protein tyrosine phos-phorylation on each immunoblot was performed with Renaissance®

Western blot chemiluminescence reagent, PerkinElmer Life Sciences,as described (1). Similar results were obtained in three separate exper-iments. Panel B, fucoidan induces the association (complex formation)between pp60Src and Hsp90. Cells were preincubated with 5 mM GAand 1 mM HB as well as inhibitors of Src, 1 mM PP1 and 1 mM PP2,followed by exposure to fucoidan. Various cell lysates were immunopre-cipitated with monoclonal anti-Hsp90 IgG at room temperature for 4 h,and immunocomplexes were recovered via incubation with protein A/Gplus agarose at 4 °C for 24 h. Immunoprecipitates were separated bySDS-polyacrylamide gel electrophoresis and immunoblotted with mono-clonal pp60Src IgG. Protein visualization on each immunoblot wasperformed with Renaissance® Western blot chemiluminescence reagentas described. The positions of pp60Src and the IgG heavy chain (IgG(H))are indicated. Molecular mass markers are indicated in kDa. Similarresults were obtained in three separate experiments.

FIG. 14. Roles of tyrosine phosphorylation of pp60Src as wellas of association between pp60Src and Hsp90 in the regulationof fucoidan-induced proIL-1 protein expression. Initially, a dose-response study (concentration of GA and HB from 1 to 15 mM) for theeffect of GA and HB on fucoidan-induced proIL-1 protein was performed(data not shown), and the effective concentration was determined as 1mM GA and 1 mM HB. Preincubation of J774A.1 cells with 5 mM GA and1 mM HB as well as with inhibitors of Src, 1 mM PP1 and 1 mM PP2, for30 min, was followed by exposure of cells to 25 mg/ml fucoidan. ProIL-1protein expression in various cell lysates was analyzed by Western blotwith anti-IL-1 antiserum, as in Fig. 2. The experiment is a represent-ative of three similar experiments (n 5 3).

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-128728

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

examined by protein kinase assays, similar to those in J774A.1cells.

Upstream signaling molecules, Rac1 and Cdc42, activatingJNK activity have been reported (55, 56), and likely p38 activ-ity is activated in a similar way (57). The PAK family of proteinkinase has been recognized as one of the main targets to inter-act with the GTPases of Rac1/Cdc42 and generating down-stream signaling networks (58). We demonstrate that fucoidanquickly stimulates PAK activity followed by increasing theactivity of JNK and p38, as well as induction of proIL-1 protein.The current results of fucoidan-induced PAK, JNK, and p38activity are comparable to the recent demonstration of Rac1activation of PAK-mediated signals (59), leading to induction ofactivity of JNK (34) and p38 (57), respectively. Upon fucoidanstimulation, DN-Rac1-transfected cells express lower activityof JNK and p38 (Figs. 4B and 10A) as well as less proIL-1protein production (Fig. 10C) than those of empty expressionvector-transfected control cells. Moreover, fucoidan induceshigher JNK and p38 activity2 and more proIL-1 protein pro-duction (Fig. 10D) in CA-Rac1-stably transfected J774A.1 cellsthan those of control cells. Together, the results of using vari-ous Rac1 genetic mutants support that the levels of Rac1 andPAK are at the upstream of JNK and p38. In addition, wedemonstrate the important roles in fucoidan-mediated signal-ing pathways of PTK/Rac1/PAK/JNK and of PTK/Rac1/PAK/p38 in regulating proIL-1 expression. Furthermore, inhibitorsof PI 3-kinase, wortmannin and LY294002 (data not shown)up-regulate JNK and p38 activity as well as increase proIL-1protein and superinduce IL-1 secretion, suggesting that undernormal conditions, endogenous PI 3-kinase and/or PI 3-kinase-related downstream signals inhibit fucoidan-stimulated JNKand p38 activity and further suppress fucoidan-induced proIL-1/IL-1 expression.

Our results demonstrate that ligation of fucoidan to MSRinduces multiple protein kinases activity, mitogen-like signals,and stimulation of proIL-1/IL-1 expression in macrophages,comparable to cells treated with certain cytokines (35, 60–63).There is no functional tyrosine kinase motif in the MSR cyto-plasmic domain (9, 41–43); however, engagement of MSR withfucoidan quickly triggers HB-blockable protein tyrosine phos-phorylation (1). Here, using specific inhibitors, we further dem-onstrate for the first time that fucoidan-mediated signals arevia MSR, likely associated with cytosolic tyrosine kinase, e.g.pp60Src in concert with Hsp90; or via other receptor(s), butwhich, if any, of these mediate signal transductions and regu-late proIL-1 protein and IL-1 secretion needs more study. Al-though the physiologic ligands for MSR are currently un-known, in comparing the polysaccharide structure of fucoidanwith the relevant structure of another MSR ligand, lipopolysac-charide (9, 14), fucoidan might exert lipopolysaccharide-likesignals such as activation of MAPKs and induction of IL-1(64).2 Phosphatidylserine, one of the effective competitors forMSR binding (9), translocates from the inner face of the cellplasma membrane to the cell surface after initiating apoptosis.The role for MSR in the phagocytosis and clearance of apoptoticthymocytes has been reported (65), and it is likely that phos-phatidylserine on dying cells becomes an endogenous ligand ofMSR upon apoptosis. Besides, another MSR ligand is advancedglycation end products of protein related to age-enhanced dis-ease processes including atherosclerosis, which are endocyti-cally taken up by scavenger receptors on macrophages (66).Considering the physiological relevance of our observations infucoidan-mediated signals and the potential endogenous li-gand(s) as fucoidan, we speculate that similar transducingsignals might arise upon the ligation of MSR, and the sequen-tial biological impacts need further investigation.

In summary, continuing to expose MSR as a functional sig-naling receptor, we are the first to examine that MSR ligandsinduce several transducing signal cascades differentially, andfurther we systematically to dissect the molecular mechanismsby which fucoidan-mediated signals regulate IL-1. Specifically,we establish signal transduction pathways of PTK(Src)/Rac1/PAK/JNK and PTK(Src)/Rac1/PAK/p38 in the regulation ofproIL-1 protein and IL-1 secretion (Fig. 15). In contrast, al-though fucoidan induces activity of PKC, MEK1, and ERK,there is less role for the pathway of PTK/phospholipase C-g1/PKC/MEK1/ERK in proIL-1/IL-1 regulation (Fig. 15). On theother hand, fucoidan-induced ICE activity results in the alter-native relationship between degradation of proIL-1 protein andincrease of mature IL-1 secretion with time, suggesting thatfucoidan probably mediates transcriptional, post-transcrip-tional, and post-translational regulation of IL-1 expression.Moreover, considering the important role of IL-1 in stimulatinginflammatory abnormality, the model for ligation of MSR-me-diated signal transduction pathways in regulating proIL-1/IL-1expression provides certain biological significance in the devel-opment of vascular-related diseases.

REFERENCES

1. Hsu, H. Y., Hajjar, D. P., Khan, K. M., and Falcone, D. J. (1998) J. Biol. Chem.273, 1240–1246

2. Ross, R. (1993) Nature 362, 801–8093. Sakaguchi, H., Takeya, M., Suzuki, H., Hakamata, H., Kodama, T., Horiuchi,

S., Gordon, S., van der Laan, L. J., Kraal, G., Ishibashi, S., Kitamura, N.,and Takahashi, K. (1998) Lab. Invest. 78, 423–434

4. Goldstein, J. L., Ho, Y. K., Basu, S. K., and Brown, M. S. (1979) Proc. Natl.Acad. Sci. U. S. A. 76, 333–337

5. Naito, M., Suzuki, H., Mori, T., Matsumoto, A., Kodama, T., and Takahashi, K.(1992) Am. J. Pathol. 141, 591–599

6. Parthasarathy, S., Fong, L. G., Otero, D., and Steinberg, D. (1987) Proc. Natl.

FIG. 15. The proposed fucoidan-mediated signal transductionpathways in the regulation of proIL-1 protein expression andIL-1 secretion.

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-1 28729

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Acad. Sci. U. S. A. 84, 537–5407. Sparrow, C. P., Parthasarathy, S., and Steinberg, D. (1989) J. Biol. Chem. 264,

2599–26048. Ottnad, E., Parthasarathy, S., Sambrano, G. R., Ramprasad, M. P.,

Quehenberger, O., Kondratenko, N., Green, S., and Steinberg, D. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 1391–1395

9. Krieger, M., and Herz, J. (1994) Annu. Rev. Biochem. 63, 601–63710. Brown, M. S., and Goldstein, J. L. (1983) Annu. Rev. Biochem. 52, 223–26111. Fraser, I., Hughes, D., and Gordon, S. (1993) Nature 364, 343–34612. El Khoury, J., Thomas, C. A., Loike, J. D., Hickman, S. E., Cao, L., and

Silverstein, S. C. (1994) J. Biol. Chem. 269, 10197–1020013. Resnick, D., Freedman, N. J., Xu, S., and Krieger, M. (1993) J. Biol. Chem.

268, 3538–354514. Hampton, R. Y., Golenbock, D. T., Penman, M., Krieger, M., and Raetz, C. R.

(1991) Nature 352, 342–34415. Suzuki, H., Kurihara, Y., Takeya, M., Kamada, N., Kataoka, M., Jishage, K.,

Ueda, O., Sakaguchi, H., Higashi, T., Suzuki, T., Takashima, Y., Kawabe,Y., Cynshi, O., Wada, Y., Honda, M., Kurihara, H., Aburatani, H., Doi, T.,Matsumoto, A., Azuma, S., Noda, T., Toyoda, Y., Itakura, H., Yazaki, Y.,and Kodama, T. (1997) Nature 386, 292–296

16. Palkama, T. (1991) Immunology 74, 432–43817. Ross, R. (1999) N. Engl. J. Med. 340, 115–12618. Schreyer, S. A., Peschon, J. J., and LeBoeuf, R. C. (1996) J. Biol. Chem. 271,

26174–2617819. Tipping, P. G., and Hancock, W. W. (1993) Am. J. Pathol. 142, 1721–172820. Hamilton, T. A., Major, J. A., and Chisolm, G. M. (1995) J. Clin. Invest. 95,

2020–202721. Jovinge, S., Ares, M. P., Kallin, B., and Nilsson, J. (1996) Arterioscler. Thromb.

Vasc. Biol. 16, 1573–157922. Hsu, H. Y., Nicholson, A. C., and Hajjar, D. P. (1996) J. Biol. Chem. 271,

7767–777323. Moulton, K. S., Wu, H., Barnett, J., Parthasarathy, S., and Glass, C. K. (1992)

Proc. Natl. Acad. Sci. U. S. A. 89, 8102–810624. Falcone, D. J., McCaffrey, T. A., and Vergilio, J. A. (1991) J. Biol. Chem. 266,

22726–2273225. Falcone, D. J., and Ferenc, M. J. (1988) J. Cell. Physiol. 135, 387–39626. Pratt, W. B. (1997) Annu. Rev. Pharmacol. Toxicol. 37, 297–32627. Xu, Y., Singer, M. A., and Lindquist, S. (1999) Proc. Natl. Acad. Sci. U. S. A.

96, 109–11428. Whitesell, L., Mimnaugh, E. G., De Costa, B., Myers, C. E., and Neckers, L. M.

(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8324–832829. Mallat, Z., Ohan, J., Leseche, G., and Tedgui, A. (1997) Circulation 96,

424–42830. Geng, Y. J., and Libby, P. (1995) Am. J. Pathol. 147, 251–26631. Cerretti, D. P., Kozlosky, C. J., Mosley, B., Nelson, N., Van Ness, K.,

Greenstreet, T. A., March, C. J., Kronheim, S. R., Druck, T., and Canniz-zaro, L. A. (1992) Science 256, 97–100

32. Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D.,Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., and Aunins,J. (1992) Nature 356, 768–774

33. Li, Y. S., Shyy, J. Y., Li, S., Lee, J., Su, B., Karin, M., and Chien, S. (1996) Mol.Cell. Biol. 16, 5947–5954

34. Bagrodia, S., Derijard, B., Davis, R. J., and Cerione, R. A. (1995) J. Biol. Chem.270, 27995–27998

35. Hsu, H. Y., and Twu, Y. C. (2000) J. Biol. Chem. 275, 41035–4104836. Gille, H., Kortenjann, M., Thomae, O., Moomaw, C., Slaughter, C., Cobb,

M. H., and Shaw, P. E. (1995) EMBO J. 14, 951–962

37. Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., andDavis, R. J. (1994) Cell 76, 1025–1037

38. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad,M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156–160

39. Livingstone, C., Patel, G., and Jones, N. (1995) EMBO J. 14, 1785–179740. Erickson, A. K., Payne, D. M., Martino, P. A., Rossomando, A. J., Shabanowitz,

J., Weber, M. J., Hunt, D. F., and Sturgill, T. W. (1990) J. Biol. Chem. 265,19728–19735

41. Ashkenas, J., Penman, M., Vasile, E., Acton, S., Freeman, M., and Krieger, M.(1993) J. Lipid Res. 34, 983–1000

42. Matsumoto, A., Naito, M., Itakura, H., Ikemoto, S., Asaoka, H., Hayakawa, I.,Kanamori, H., Aburatani, H., Takaku, F., and Suzuki, H. (1990) Proc. Natl.Acad. Sci. U. S. A. 87, 9133–9137

43. Kodama, T., Freeman, M., Rohrer, L., Zabrecky, J., Matsudaira, P., andKrieger, M. (1990) Nature 343, 531–535

44. Acton, S., Resnick, D., Freeman, M., Ekkel, Y., Ashkenas, J., and Krieger, M.(1993) J. Biol. Chem. 268, 3530–3537

45. Doi, T., Higashino, K., Kurihara, Y., Wada, Y., Miyazaki, T., Nakamura, H.,Uesugi, S., Imanishi, T., Kawabe, Y., and Itakura, H. (1993) J. Biol. Chem.268, 2126–2133

46. Fong, L. G., and Le, D. (1999) J. Biol. Chem. 274, 36808–3681647. Fong, L. G. (1996) J. Lipid Res. 37, 574–58748. Kobayashi, E., Nakano, H., Morimoto, M., and Tamaoki, T. (1989) Biochem.

Biophys. Res. Commun. 159, 548–55349. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995)

J. Biol. Chem. 270, 27489–2749450. Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem.

269, 3563–356751. Lin, J. K., Chen, Y. C., Huang, Y. T., and Lin-Shiau, S. Y. (1997) J. Cell.

Biochem. Suppl. 28–29, 39–4852. Kakar, S. S., and Roy, D. (1994) Cancer Lett. 87, 85–8953. Hazzalin, C. A., Cano, E., Cuenda, A., Barratt, M. J., Cohen, P., and

Mahadevan, L. C. (1996) Curr. Biol. 6, 1028–103154. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841–85255. Coso, O. A., Chiariello, M., Yu, J. C., Teramoto, H., Crespo, P., Xu, N., Miki, T.,

and Gutkind, J. S. (1995) Cell 81, 1137–114656. Minden, A., Lin, A., Claret, F. X., Abo, A., and Karin, M. (1995) Cell 81,

1147–115757. Zhang, S., Han, J., Sells, M. A., Chernoff, J., Knaus, U. G., Ulevitch, R. J., and

Bokoch, G. M. (1995) J. Biol. Chem. 270, 23934–2393658. Manser, E., Chong, C., Zhao, Z. S., Leung, T., Michael, G., Hall, C., and Lim,

L. (1995) J. Biol. Chem. 270, 25070–2507859. Bagrodia, S., Taylor, S. J., Creasy, C. L., Chernoff, J., and Cerione, R. A. (1995)

J. Biol. Chem. 270, 22731–2273760. Chan, E. D., Winston, B. W., Uh, S. T., Wynes, M. W., Rose, D. M., and Riches,

D. W. (1999) J. Immunol. 162, 415–42261. Goebeler, M., Kilian, K., Gillitzer, R., Kunz, M., Yoshimura, T., Brocker, E. B.,

Rapp, U. R., and Ludwig, S. (1999) Blood 93, 857–86562. Kumar, A., Middleton, A., Chambers, T. C., and Mehta, K. D. (1998) J. Biol.

Chem. 273, 15742–1574863. Natoli, G., Costanzo, A., Moretti, F., Fulco, M., Balsano, C., and Levrero, M.

(1997) J. Biol. Chem. 272, 26079–2608264. Zhang, Y., and Rom, W. N. (1993) Mol. Cell. Biol. 13, 3831–383765. Platt, N., Suzuki, H., Kurihara, Y., Kodama, T., and Gordon, S. (1996) Proc.

Natl. Acad. Sci. U. S. A. 93, 12456–1246066. Sano, H., Nagai, R., Matsumoto, K., and Horiuchi, S. (1999) Mech. Aging Dev.

107, 333–346

MSR Ligand-mediated MAPK in Regulation of ProIL-1/IL-128730

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Hsien-Yeh Hsu, Show-Lan Chiu, Meng-Hsuan Wen, Kuo-Yen Chen and Kuo-Feng HuaDifferential Modulation of Protein Kinase Signaling Pathways

Ligands of Macrophage Scavenger Receptor Induce Cytokine Expression via

doi: 10.1074/jbc.M011117200 originally published online June 4, 20012001, 276:28719-28730.J. Biol. Chem. 

  10.1074/jbc.M011117200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/276/31/28719.full.html#ref-list-1

This article cites 66 references, 37 of which can be accessed free at

by guest on April 10, 2019

http://ww

w.jbc.org/

Dow

nloaded from