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CITED2 in matrix metalloproteinases Sun

major actions include the breakdown of many ex-tracellularmatrix (ECM) constituents and the post-translational activation of proenzymes by prote-olytic cleavage. The MMP family consists of thecollagenases, (MMPs 1, 8, and 13) that degrade col-lagentypesI,II,andIII;thegelatinases(MMPs2and9) that target denatured collagen; the stromelysins(MMPs 3, 7, 10, and 11) that degrade several ECMproteins andare involved in proenzymeposttransla-tional activation; the membrane-type MMPs (MT-MMP14);anda diverse subgroup includingMMPs12, 20, and 23.8, 9 MMP regulation occurs at severallevels: gene transcription, latent proenzyme synthe-sis, proenzyme posttranslational activation, and in-hibition of active MMPs. 10

Extensive studies of tissue and cell responses toa range of loading types and magnitudes in vivo and in vitro suggest that a moderate level of me-chanical load is required to maintain normal tissuefunction, and that under these conditions of me-chanical homeostasis, MMP expression is modestand responsive to regulation. On the other hand,unusually high or low levels of mechanical loading,particularly when sustained over prolonged peri-ods, can result in high levels of MMP activity thatarenoteffectively regulated. Invivo, suchconditionscan eventually lead to a breakdown in the mechan-ical integrity of the tissue itself.

Animal models have clearly demonstrated the ef-fects of various types and magnitudes of joint load-ing on tissue integrity and have shown that degener-ation of articular cartilage is directly associated withabnormal biomechanical function and increasedMMP activity. Excessive running in an in vivo ratmodel induced osteoarthritic changes accompaniedby an increase of MMP-3 activity. 11 Altered joint

loading, caused by knee destabilization, resulted incartilage destruction and an increase of MMP-13. 12

Similarly, in cell or tissue culture model systems,chondrocytes and synoviocytes respond to a rangeof loading conditions through diverse metabolicresponses, including MMP synthesis. In particu-lar, mechanical unloading and intermediate shear-ing of synoviocytes resulted in increased produc-tion of MMPs, while gentle shearing suppressedthese catabolic changes. 5, 13 Anticatabolic effects of mechanical loading were similarly observed inchondrocytes. Decreases of MMP-2 were alsodemonstrated in human osteoarthritic chondro-cytes subject to intermittent hydrostatic pressure, 14

a loading mode more commonly experienced thanshearing by chondrocytes in vivo.

Moderate mechanical loading was also demon-strated to have an anti-inammatory effect. Cyclicstrain inhibited the expression of MMP-1 andMMP-13 induced by proinammatory cytokinesinterleukin-1 (IL-1 ) or tumor necrosis factor(TNF)- in rheumatoid arthritis synoviocytes. 15 Astudy in periodontal ligament cells found that highmagnitudes of tensile strain upregulated the expres-sion of proinammatorygenes, including IL-1, IL-6,IL-8, MMP-1,MMP-3, andMMP-9, while lowmag-nitudes of tensile strain inhibited the transcriptionof several proinammatory genes, suggesting thatlow levels of strain play an anti-inammatory role

via direct inhibition of the nuclear factor (NF)- Bpathway. 16 Similar anti-inammatory responses in-duced by mechanical stimuli have been reported inother cultured cells, including osteoblast-like cellsand smooth muscle cells (Table 1), indicating thatloading-induced anticatabolic effects are commonto many tissues and may be part of a fundamen-tal cellular homeostatic mechanism. 8 Yet while ac-cumulating evidence suggests that mechanical loadwithin a moderate/physiological range is a potentanticatabolic factor, little is known about its under-lying molecular mechanisms, especially at the tran-scriptional level. In particular, the identity of a puta-tive mechanosensitive signaling molecule regulatingMMP downregulation remains to be established.

CITED2, a transcriptional co-regulator, has beenshown to be inducible by various stimuli, includingcytokines, serum growth factors, lipopolysaccha-ride, and hypoxia. 17, 18 Interestingly, a recent study demonstrated that CITED2 responds to moderateloading and its expression is inversely correlated to

theexpression of MMPs, such as MMP-1 andMMP-13 inchondrocytes. 19 Asthestudy furthersuggested,load-induced CITED2 proteins may compete withMMP transactivator Ets-1 for limiting amounts of co-factor p300, allowing CITED2 to play a nega-tive regulatory role in MMP transcription, provid-inga possible regulatorymechanismfor load-drivenMMP downregulation.

CITED2 in transcriptional control

CITED2 (also known as MRG1 or p35srj) en-codes a 28-kDa nuclear protein and was identi-ed as a gene present in a variety of cell types and

430 Ann. N.Y. Acad. Sci. 1192 (2010) 429436 c 2010 New York Academy of Sciences.

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Sun CITED2 in matrix metalloproteinases

Table 1. Moderate mechanical loading downregulation of matrix metalloproteinases (MMPs) in skeletal tissue

MMPs Type of mechanical loading Cell type Reference

MMPs 1, 3 Pressure-induced strain Human normal and OA

chondrocytes

Millward-Sadler et al. 200036

MMPs 1, 3, 13 Mechanical shear Human MH7A RA-FLS Sun et al. 200113

MMP-13 Oscillatory shear Human MH7A RA-FLS Sun et al. 20015

MMPs 1, 13 Cyclic strain Human MH7A RA-FLS Sun et al. 200215

MMPs 1, 13 Fluid ow shear Human C28/I2 chondrocytes Yokota et al. 200319

MMPs 2, 9 IHP Human OA chondrocytes Trindade et al. 200414

MMP-1 Continuous passive motion Rabbit chondrocytes frommeniscal brocartilage

Ferretti et al. 200537

MMPs 1, 2, 8, 9, 13 Strain+ ow MC3T3-E1 osteoblasts Tanaka et al. 200538

MMP-13 Intermittent hydrostaticpressure

Rat tenocytes Sun et al. 200839

MMPs 3, 9, 13 Immobilization and motionloading

In vivo rat chondrocytes (Leong et al. unpublishedobservations)

MMP-9 Uniaxial stretch Smooth muscle cells Asanuma et al. 200340

OA, osteoarthritis; IHP, intermittent hydrostatic pressure; RA-FLS, rheumatoid arthritis-broblast-like synoviocytes.

responsive to several stimuli, including cytokines,serum, and lipopolysaccharide. 18 During early de-velopment CITED2 is widely expressed in both em-bryonic and extraembryonic cells and is involved in

regulation of cell proliferation and embryonic de-velopment. 20 Loss of CITED2 was shown to resultin senescence of cultured broblasts, 21 while stud-ies with CITED2 / animals revealed roles in many different developmental processes, including the es-tablishmentof leftrightbody axis anddevelopmentof cardiac, neural, liver, and lung tissues. 20, 22 Inter-estingly, CITED2 has been suggested as an interven-tional approach to suppress colon cancer becauseectopic expression was found to arrest colon cancergrowth and invasion by downregulation of MMP-13.23 However, as with many transcriptionally activemolecules, dysregulation of CITED2 could lead tooncogenic cellular changes. 18

CITED2 is a founding member of the CITEDfamily of transcriptional co-regulators. Its mem-bers have two conserved regions within their codingsequences. The conserved 32-amino acid sequence(CR2) shared by all CITED members is located atthe carboxyl terminus and is necessary and suf-cient for binding cAMP-response-element-binding

protein (CBP) and co-activator p300. In CR2, theLPEL motif is critical for CITED2 binding to therst cysteinehistidine-rich (CH1) region of p300

(Fig. 1A).17, 24 As a co-transcriptional regulator,CITED2 does not possess intrinsic DNA-bindingactivity. Instead it functions as a context-dependenttranscriptional co-activator or repressor via inter-

actions with other proteins.17, 25

It has been shownthat CITED2 binds directly to DNA-binding tran-scription factors, such as LIM/homeobox protein(Lhx2), transcription factor AP2 (TFAP2), peroxi-some proliferator-activated receptors (PPAR andPPAR ), and SMAD 2 and, through the recruit-ment of CBP/p300, acts as a positive regulator of transcription. On the other hand, CITED2 can alsonegatively regulate the activation of target genes by competing with transcription factors for binding tofactors like p300. Such a mechanismwas establishedin hypoxia response where CITED2 downregulatedhypoxia inducible factor (HIF)-1 -triggered trans-activation by competing with HIF-1 for binding top300 via the CH1 domain. 17

That CITED2 is able to exert a wide range of reg-ulatory roles in numerous cellular processes, suchas cell proliferation and embryonic development,is mainly from the ubiquity of p300 as a tran-scriptional co-regulator. 17 By binding to p300 viathe CH1 domain, CITED2 may interact, and likely

compete, with a multitude of transcription factors,including HIF-1 , Ets-1, NF- B, retinoid X recep-tor (RXR ), signal transducer and activator of

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Figure 1. Structural domain and motif of CITED2 protein ( A ) and Sp1, Sp3, nuclear factor (NF)- B, signaltransducerand activator of transcription (STAT), and Ets-1 transcription factor binding sites on the human CITED2promoter region ( B ). LEPL is a four amino acid sequence on the CITED2 protein essential for binding to p300.

transcription 2 (STAT2), murine double minute 2(MDM2), and p53, a tumor suppressor gene inhumans. 26 Because both CITED2 and these otherCH1 domain binding proteins are differentially ex-pressed in various tissue and cell types, CITED2 canact in a tissue- and stimulus-dependent manner.Therefore, the regulatory roles played by CITED2should be understood through its interaction with

these function-associated proteins. For example, asa hypoxia-response gene, CITED2 expression canbe upregulated in cardiac tissue, and the increasedCITED2protein canrepress HIF-1 -mediated tran-scription by competing with HIF-1 protein p300binding. 17

Because CITED2 acts through competition withother factors for p300 at the CH1 binding site,

Figure 2. CITED2 mediates load-induced matrix metalloproteinase (MMP) downregulation. CITED2, induced by mechanical loading, competes with MMP-1 transactivator Ets-1 for binding to limiting amounts of p300. MAPK,mitogen-activated protein kinase.


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its effectiveness will be determined not only by the amount of p300 but also by the numerousother transcription factors from diverse signalingpathways that are present in a cell under givencircumstances. 27 Although the CH1 domain ex-hibits diverse binding properties and has more thanone binding surface, some transactivation domainbinding surfaces overlap. 24 Competition for a com-mon binding site is the structural basis for the nega-tive regulation by CITED2 of certain transactivatingtargets, like HIF-1 .24 Despite a considerable ex-cess of total p300 over CITED2, the number of freeCH1 sites would likely be much smaller as a resultof occupancy by so many competing binding part-ners. Thus, the ability of variations in the CITED2

level to affect the proportion of available functionalCH1 sites in vivo and thus modulate its bindingcompetitors function would depend on both theamounts of CITED2 and its competitors, and theirrelative afnities. It has been suggested that nearly all cellular CITED2 complexes bind physically withp300/CBP, indicating that it binds with high afn-ity.17 The specicity of the CITED2CH1 interac-tionsuggeststhatitmayfunctiontopreventtitrationof cellular p300 by a competitor, such as HIF-1 .

Because CITED2 is ubiquitously expressed andregulated by a wide range of stimuli, its effects vary considerably depending on the nature of the sig-nal and of the cell type. For example, in chondro-cytes, transforming growth factor (TGF)- inducesCITED2 expression, which is consistent with theknown role of TGF- in downregulating MMP-1in those cells.19 However, in MDA-MB-231 breastcancer cells, TGF- downregulates CITED2 at theposttranslational level. 28 Similarly, upregulation of CITED2undermoderateowshearinchondrocytes

leads to downregulation of MMP-1 and MMP-13,but CITED2 knockdown in SW480 colon cancercells results in a mild downregulation of MMP-1. 23

While CITED2 interacts with a wide range of transcriptional regulators, its own expression is re-sponsive to multiple stimuli. The ability of CITED2to respond to a multitude of stimuli may be ex-plained by the presence of regulatory elements, suchas the binding sites for various transcription fac-tors (e.g., Sp1, STAT, and NF- B) in the CITED2promoter (Fig. 1B). 29 The Sp1 transcription factor-binding site has previously been identied to beresponsive to shear stress and promote gene expres-sion.30 The deletion and site-directed mutations of

the Ets-1 and Sp1 site upstream of the start codonare critical forCITED2expression in broblasts. Gelmobility shift and supershift assays performed withRat1 nuclear extracts identied nucleoprotein com-plexes binding to the Ets-1 site and the Sp1 site onCITED2 promoter DNA. In Drosophila SL2 cells,which lack the Sp and Ets family of transcriptionfactors, expression of Sp1, Sp3, and Ets-1 or Elf-1functionally stimulated CITED2 promoter activity in a synergistic manner. 31 These results suggest thatmultiple transcription factors acting in synergy areresponsible for the induction of CITED2.

Role of CITED2 in mechanotransductionand matrix metalloproteinasedownregulation

CITED2 was considered a likely mediator of me-chanically induced MMPsuppressionbecause it wasknown to antagonize transcriptional regulators, likeEts-1, which have several binding sites within MMPpromoter regions. 32 Yokota et al. used an immor-talized human chondrocyte cell line, C28/I2, 33 toinvestigate whether CITED2 was responsive to me-chanical stimuli and, if so, could mediate shear-mediated downregulation of MMP-1 and MMP-13.19 CITED2 expression at the mRNA and proteinlevels was found to be inducible by moderate ow shear. Expression was maximal at 5 dyn/cm 2, andbasal levels of expression were found at 0, 10, and20 dyn/cm 2. In contrast, mRNA and protein expres-sion and enzyme activities of MMP-1 and MMP-13 were upregulated at 0, 10, and 20 dyn/cm 2 butwere suppressed at 5 dyn/cm 2.19 Consistent withthese ndings, CITED2 expression was recently found to be inversely related to the expression of

MMPs 2, 3, 9, and 13 in fractured bone in a ratmandibular osteotomy model. Furthermore, over-expression of CITED2 in osteoblasts inhibited theexpression and activity of MMPs 2, 3, 9, and 13. 34

Taken together, these data provide evidence thatCITED2 is a mechanical stimuli-responsive gene,and its inverse relationship with MMPs suggestsit may play a regulatory role in load-driven MMPdownregulation.

To determine the relationship between load-induced CITED2 expression and MMP down-regulation, loss-of-function and gain-of-functionapproaches were used. Transfecting antisenseCITED2 plasmids into chondrocytes abolished the


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loading-mediated downregulation of MMP-1, sug-gesting that CITED2 is required by load-drivenMMP downregulation. Overexpression of CITED2in chondrocytes reduced the basal level of MMP-1 expression under regular culture conditions andprotected cells from IL-1 -induced upregulation of MMP-1 and MMP-13. 19

To elucidate the mechanism of MMP downregu-lation by CITED2, we carried out a series of experi-ments inourlabs andused co-immunoprecipitationwith p300-specic antibody to identify potentialregulatory proteins in p300-binding protein com-plexes. This approach wasbased on the understand-ing that CITED2 is not a DNA-binding protein andits role in MMP regulation has to be based on its in-

teraction with DNA-binding transcription factorsof MMPs, either directly or via their co-regulators,such as CBP/p300. Ets-1 was chosen as a primary target because CITED2 and Ets-1 are known to in-teract with p300 and Ets-1 is a DNA-binding pro-tein that can transactivate MMPs with p300 as aco-factor. 32 C28/I2 chondrocytes were treated withmoderate shear at 5 dyn/cm 2 or high intensity shearat 20 dyn/cm 2. Nuclear extracts were prepared andincubated with antibody specic for p300 to im-munoprecipitate p300 protein complexes. Westernblotting identied equal amounts of p300 in con-trol and treated extracts. In control cells, no p300Ets-1 or p300CITED2 complexes were detectable.In contrast, p300CITED2 complexes were identi-ed in cells exposed to ow shear at 5 dyn/cm 2,and p300ETS-1 complexes were detected in cellsunder 20 dyn/cm 2.19 Our ndings support the hy-pothesis that CITED2 may mediate the mechani-cal load-induced downregulation of MMP by com-peting with MMP transactivator Ets-1 for limiting

amounts of co-factor p300 protein, as illustrated inFigure 2.As a CITED2-mediated target in MMP down-

regulation from moderate mechanical loading, Ets-1 proteins constitute a highly conserved family of transcription factors that share a unique DNA-binding domain, the Ets domain. 32 The Ets domainspecically recognizes DNA sequences that containa GGAA/T core element. 35 Ets-1 is a transcriptionfactor that binds to MMP-1 and other MMP pro-moter regions. 32 The Ets domain also serves as thep300 CH1-binding domain, recruiting p300 as aco-factor in the transactivation of MMPs. The Ets-1activation domain or C domain is located between

amino acids 130242 and contains a high content of acidic residues. The C domain is essential for Ets-1to activate transcription and is also necessary for theinteraction of Ets-1 with p300. 32

Although CITED2hasonly beendemonstratedtomediate the load-driven downregulation of MMP-1 and MMP-13, an MMP promoter analysis hasidentied Ets-1-binding sites at the promoter re-gions of many MMPs, including MMPs 1, 2, 3,8, 9, and 13 (unpublished observations), suggest-ing that CITED2 may mediate a general MMPmechanoregulatory mechanism. In addition, many transcriptional regulators can bind to p300 via thep300 CH1 domain, implying the competitive mech-anism by which CITED2 regulates MMP expression

may extend to other CH1-binding transcriptionalregulators.

Summary

MMPs constitute the most inuential family of proteolytic enzymes in joint tissues. Moderate/physiological mechanical load has been recently identied as a potent anticatabolic factor drivingMMP downregulation. Understanding the mech-anisms by which mechanical loads regulate MMPexpression is a critical step toward the develop-ment of chondroprotective therapies. The ndingthat CITED2 expression is mechanically responsiveand inversely correlated with MMP, coupled withthe identication that CITED2 acts via the p300Ets-1 pathway to regulate MMP transcription inchondrocytes, claries molecular targets that may have signicant clinical potential for the preventionand treatment of joint disease.

Acknowledgment

This work was supported by National Institutes of Health grants AR47628 and AR52743 to H.B.S. Theauthor thanks Daniel J. Leong (graduate student)and Drs. Robert J. Majeska and Li Sun for helpfulcritique and Dr. Zhengzhe Li for promoter analysis.

Conict of interest

The author declares no conicts of interest.

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