Ann. N.Y. Acad. Sci.

ISSN 0077-8923

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S
Issue: Skeletal Biology and Medicine

CITED2 mechanoregulation of matrix metalloproteinases

Hui B. Sun
Leni and Peter W. May Department of Orthopaedics, Mount Sinai School of Medicine, New York, New York, USA Address for correspondence: Hui B. Sun, PhD, One Gustave L. Levy Place, Box 1188, Department of Orthopaedics, Annenberg Building 2066, New York, NY 10029. Herb.Sun@mssm.edu

Joint tissues are exquisitely sensitive to their mechanical environment. Indeed, mechanical loading may be the most important external factor regulating the development and long-term maintenance of joint tissues. Moderate mechanical loading maintains the integrity of articular cartilage, and under these conditions of homeostasis, matrix metalloproteinases (MMPs) are expressed at modest levels. However, both disuse and overuse can result in altered likely high levels of MMP expression, leading to cartilage degradation. The transcriptional regulator ED-rich tail 2 (CITED2) is expressed in chondrocytes in a load-dependent manner but in a pattern inversely related to that of MMPs. CITED2 mediates the moderate load-induced suppression of MMPs, possibly by competing with Ets-1, a known MMP transactivator, for binding to the co-activator p300, suggesting a mechanism for transcriptional suppression by CITED2. These ndings suggest that CITED2 may act as a mechanosensitive molecular switch regulating cartilage matrix breakdown. This regulatory pathway could be exploited clinically to limit pathologic cartilage degradation. Keywords: CITED2; matrix metalloproteinases; mechanical loading; Ets-1; p300; mechanoregulation

Introduction Mechanical force has long been appreciated as a regulator of musculoskeletal tissues and may be the most important single environmental factor responsible for joint homeostasis. The transmission of mechanical loads requires the participation of multiple joint components, including bone, muscle, articular cartilage, and ligaments/tendons, and it has become apparent that these and other joint tissues (e.g., synovium) are sensitive to the magnitude, duration, and nature of mechanical stimuli. Longstanding clinical and experimental evidence has established that both mechanical overloading and complete unloading are detrimental to bone,1,2 cartilage,3,4 and the synovium.5 By contrast, normal physiological loading does not harm cartilage and helps to prevent degradative changes in cartilage due to arthritis.6,7 These signal transduction pathways could be used in the development of therapeutic approaches designed to limit pathologic cartilage degradation but to do so will require greater understanding of the molecular mechanisms underlying the chondroprotective effects of moderate
doi: 10.1111/j.1749-6632.2009.05305.x

mechanical loading. In this article, I will rst review the current studies on the role of mechanical loading in matrix metalloproteinase (MMP) regulation and propose that cAMP-responsive element-binding protein (CBP)/p300-interacting transactivator with ED-rich tail 2 (CITED2), a transcriptional co-regulator, is a required signaling molecule in the regulation of MMPs. I will then discuss the ability of CITED2 to respond to various extracellular stimuli and highlight its role in transcriptional control through its interaction with transcriptional co-activators. Finally, I discuss our current hypothesis on the role of CITED2 in the mechanoregulation of MMPs. Matrix degradation, metalloproteinases, and a possible mechanosensitive switch Among the many cellular activities reported to be subject to mechanical regulation are the expression and activation of MMPs in vivo and in vitro.8 The MMPs, of which 26 have currently been identied, are a family of structurally related zinc endopeptidases that differ in substrate specicity but whose

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major actions include the breakdown of many extracellular matrix (ECM) constituents and the posttranslational activation of proenzymes by proteolytic cleavage. The MMP family consists of the collagenases, (MMPs 1, 8, and 13) that degrade collagen types I, II, and III; the gelatinases (MMPs 2 and 9) that target denatured collagen; the stromelysins (MMPs 3, 7, 10, and 11) that degrade several ECM proteins and are involved in proenzyme posttranslational activation; the membrane-type MMPs (MTMMP 14); and a diverse subgroup including MMPs 12, 20, and 23.8,9 MMP regulation occurs at several levels: gene transcription, latent proenzyme synthesis, proenzyme posttranslational activation, and inhibition of active MMPs.10 Extensive studies of tissue and cell responses to a range of loading types and magnitudes in vivo and in vitro suggest that a moderate level of mechanical load is required to maintain normal tissue function, and that under these conditions of mechanical homeostasis, MMP expression is modest and responsive to regulation. On the other hand, unusually high or low levels of mechanical loading, particularly when sustained over prolonged periods, can result in high levels of MMP activity that are not effectively regulated. In vivo, such conditions can eventually lead to a breakdown in the mechanical integrity of the tissue itself. Animal models have clearly demonstrated the effects of various types and magnitudes of joint loading on tissue integrity and have shown that degeneration of articular cartilage is directly associated with abnormal biomechanical function and increased MMP activity. Excessive running in an in vivo rat model induced osteoarthritic changes accompanied by an increase of MMP-3 activity.11 Altered joint loading, caused by knee destabilization, resulted in cartilage destruction and an increase of MMP-13.12 Similarly, in cell or tissue culture model systems, chondrocytes and synoviocytes respond to a range of loading conditions through diverse metabolic responses, including MMP synthesis. In particular, mechanical unloading and intermediate shearing of synoviocytes resulted in increased production of MMPs, while gentle shearing suppressed these catabolic changes.5,13 Anticatabolic effects of mechanical loading were similarly observed in chondrocytes. Decreases of MMP-2 were also demonstrated in human osteoarthritic chondrocytes subject to intermittent hydrostatic pressure,14

a loading mode more commonly experienced than shearing by chondrocytes in vivo. Moderate mechanical loading was also demonstrated to have an anti-inammatory effect. Cyclic strain inhibited the expression of MMP-1 and MMP-13 induced by proinammatory cytokines interleukin-1 (IL-1) or tumor necrosis factor (TNF)- in rheumatoid arthritis synoviocytes.15 A study in periodontal ligament cells found that high magnitudes of tensile strain upregulated the expression of proinammatory genes, including IL-1, IL-6, IL-8, MMP-1, MMP-3, and MMP-9, while low magnitudes of tensile strain inhibited the transcription of several proinammatory genes, suggesting that low levels of strain play an anti-inammatory role via direct inhibition of the nuclear factor (NF)-B pathway.16 Similar anti-inammatory responses induced by mechanical stimuli have been reported in other cultured cells, including osteoblast-like cells and smooth muscle cells (Table 1), indicating that loading-induced anticatabolic effects are common to many tissues and may be part of a fundamental cellular homeostatic mechanism.8 Yet while accumulating evidence suggests that mechanical load within a moderate/physiological range is a potent anticatabolic factor, little is known about its underlying molecular mechanisms, especially at the transcriptional level. In particular, the identity of a putative mechanosensitive signaling molecule regulating MMP downregulation remains to be established. CITED2, a transcriptional co-regulator, has been shown to be inducible by various stimuli, including cytokines, serum growth factors, lipopolysaccharide, and hypoxia.17,18 Interestingly, a recent study demonstrated that CITED2 responds to moderate loading and its expression is inversely correlated to the expression of MMPs, such as MMP-1 and MMP13 in chondrocytes.19 As the study further suggested, load-induced CITED2 proteins may compete with MMP transactivator Ets-1 for limiting amounts of co-factor p300, allowing CITED2 to play a negative regulatory role in MMP transcription, providing a possible regulatory mechanism for load-driven MMP downregulation. CITED2 in transcriptional control CITED2 (also known as MRG1 or p35srj) encodes a 28-kDa nuclear protein and was identied as a gene present in a variety of cell types and

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CITED2 in matrix metalloproteinases

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

MMPs MMPs 1, 3 MMPs 1, 3, 13 MMP-13 MMPs 1, 13 MMPs 1, 13 MMPs 2, 9 MMP-1 MMPs 1, 2, 8, 9, 13 MMP-13 MMPs 3, 9, 13 MMP-9

Type of mechanical loading Pressure-induced strain Mechanical shear Oscillatory shear Cyclic strain Fluid ow shear IHP Continuous passive motion Strain+ow Intermittent hydrostatic pressure Immobilization and motion loading Uniaxial stretch

Cell type Human normal and OA chondrocytes Human MH7A RA-FLS Human MH7A RA-FLS Human MH7A RA-FLS Human C28/I2 chondrocytes Human OA chondrocytes Rabbit chondrocytes from meniscal brocartilage MC3T3-E1 osteoblasts Rat tenocytes In vivo rat chondrocytes Smooth muscle cells

Reference Millward-Sadler et al. 200036 Sun et al. 200113 Sun et al. 20015 Sun et al. 200215 Yokota et al. 200319 Trindade et al. 200414 Ferretti et al. 200537 Tanaka et al. 200538 Sun et al. 200839 (Leong et al. unpublished observations) 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 development CITED2 is widely expressed in both embryonic and extraembryonic cells and is involved in regulation of cell proliferation and embryonic development.20 Loss of CITED2 was shown to result in senescence of cultured broblasts,21 while studies with CITED2/ animals revealed roles in many different developmental processes, including the establishment of leftright body axis and development of cardiac, neural, liver, and lung tissues.20,22 Interestingly, CITED2 has been suggested as an interventional approach to suppress colon cancer because ectopic expression was found to arrest colon cancer growth and invasion by downregulation of MMP13.23 However, as with many transcriptionally active molecules, dysregulation of CITED2 could lead to oncogenic cellular changes.18 CITED2 is a founding member of the CITED family of transcriptional co-regulators. Its members have two conserved regions within their coding sequences. The conserved 32-amino acid sequence (CR2) shared by all CITED members is located at the carboxyl terminus and is necessary and sufcient for binding cAMP-response-element-binding protein (CBP) and co-activator p300. In CR2, the LPEL motif is critical for CITED2 binding to the rst cysteinehistidine-rich (CH1) region of p300

(Fig. 1A).17,24 As a co-transcriptional regulator, CITED2 does not possess intrinsic DNA-binding activity. Instead it functions as a context-dependent transcriptional co-activator or repressor via interactions with other proteins.17,25 It has been shown that CITED2 binds directly to DNA-binding transcription factors, such as LIM/homeobox protein (Lhx2), transcription factor AP2 (TFAP2), peroxisome proliferator-activated receptors (PPAR and PPAR ), and SMAD 2 and, through the recruitment of CBP/p300, acts as a positive regulator of transcription. On the other hand, CITED2 can also negatively regulate the activation of target genes by competing with transcription factors for binding to factors like p300. Such a mechanism was established in hypoxia response where CITED2 downregulated hypoxia inducible factor (HIF)-1-triggered transactivation by competing with HIF-1 for binding to p300 via the CH1 domain.17 That CITED2 is able to exert a wide range of regulatory roles in numerous cellular processes, such as cell proliferation and embryonic development, is mainly from the ubiquity of p300 as a transcriptional co-regulator.17 By binding to p300 via the CH1 domain, CITED2 may interact, and likely compete, with a multitude of transcription factors, including HIF-1, Ets-1, NF-B, retinoid X receptor (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, signal transducer and activator of transcription (STAT), and Ets-1 transcription factor binding sites on the human CITED2 promoter 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 in humans.26 Because both CITED2 and these other CH1 domain binding proteins are differentially expressed in various tissue and cell types, CITED2 can act in a tissue- and stimulus-dependent manner. Therefore, the regulatory roles played by CITED2 should be understood through its interaction with

these function-associated proteins. For example, as a hypoxia-response gene, CITED2 expression can be upregulated in cardiac tissue, and the increased CITED2 protein can repress HIF-1-mediated transcription by competing with HIF-1 protein p300 binding.17 Because CITED2 acts through competition with other 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 numerous other transcription factors from diverse signaling pathways that are present in a cell under given circumstances.27 Although the CH1 domain exhibits diverse binding properties and has more than one binding surface, some transactivation domain binding surfaces overlap.24 Competition for a common binding site is the structural basis for the negative regulation by CITED2 of certain transactivating targets, like HIF-1.24 Despite a considerable excess of total p300 over CITED2, the number of free CH1 sites would likely be much smaller as a result of occupancy by so many competing binding partners. Thus, the ability of variations in the CITED2 level to affect the proportion of available functional CH1 sites in vivo and thus modulate its binding competitors function would depend on both the amounts of CITED2 and its competitors, and their relative afnities. It has been suggested that nearly all cellular CITED2 complexes bind physically with p300/CBP, indicating that it binds with high afnity.17 The specicity of the CITED2CH1 interaction suggests that it may function to prevent titration of cellular p300 by a competitor, such as HIF-1. Because CITED2 is ubiquitously expressed and regulated by a wide range of stimuli, its effects vary considerably depending on the nature of the signal and of the cell type. For example, in chondrocytes, transforming growth factor (TGF)- induces CITED2 expression, which is consistent with the known role of TGF- in downregulating MMP-1 in those cells.19 However, in MDA-MB-231 breast cancer cells, TGF- downregulates CITED2 at the posttranslational level.28 Similarly, upregulation of CITED2 under moderate ow shear in chondrocytes leads to downregulation of MMP-1 and MMP-13, but CITED2 knockdown in SW480 colon cancer cells results in a mild downregulation of MMP-1.23 While CITED2 interacts with a wide range of transcriptional regulators, its own expression is responsive to multiple stimuli. The ability of CITED2 to respond to a multitude of stimuli may be explained by the presence of regulatory elements, such as the binding sites for various transcription factors (e.g., Sp1, STAT, and NF-B) in the CITED2 promoter (Fig. 1B).29 The Sp1 transcription factorbinding site has previously been identied to be responsive to shear stress and promote gene expression.30 The deletion and site-directed mutations of

the Ets-1 and Sp1 site upstream of the start codon are critical for CITED2 expression in broblasts. Gel mobility shift and supershift assays performed with Rat1 nuclear extracts identied nucleoprotein complexes binding to the Ets-1 site and the Sp1 site on CITED2 promoter DNA. In Drosophila SL2 cells, which lack the Sp and Ets family of transcription factors, expression of Sp1, Sp3, and Ets-1 or Elf-1 functionally stimulated CITED2 promoter activity in a synergistic manner.31 These results suggest that multiple transcription factors acting in synergy are responsible for the induction of CITED2. Role of CITED2 in mechanotransduction and matrix metalloproteinase downregulation CITED2 was considered a likely mediator of mechanically induced MMP suppression because it was known to antagonize transcriptional regulators, like Ets-1, which have several binding sites within MMP promoter regions.32 Yokota et al. used an immortalized human chondrocyte cell line, C28/I2,33 to investigate whether CITED2 was responsive to mechanical stimuli and, if so, could mediate shearmediated downregulation of MMP-1 and MMP13.19 CITED2 expression at the mRNA and protein levels was found to be inducible by moderate ow shear. Expression was maximal at 5 dyn/cm2 , and basal levels of expression were found at 0, 10, and 20 dyn/cm2 . In contrast, mRNA and protein expression and enzyme activities of MMP-1 and MMP13 were upregulated at 0, 10, and 20 dyn/cm2 but were suppressed at 5 dyn/cm2 .19 Consistent with these 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 rat mandibular osteotomy model. Furthermore, overexpression of CITED2 in osteoblasts inhibited the expression and activity of MMPs 2, 3, 9, and 13.34 Taken together, these data provide evidence that CITED2 is a mechanical stimuli-responsive gene, and its inverse relationship with MMPs suggests it may play a regulatory role in load-driven MMP downregulation. To determine the relationship between loadinduced CITED2 expression and MMP downregulation, loss-of-function and gain-of-function approaches were used. Transfecting antisense CITED2 plasmids into chondrocytes abolished the

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loading-mediated downregulation of MMP-1, suggesting that CITED2 is required by load-driven MMP downregulation. Overexpression of CITED2 in chondrocytes reduced the basal level of MMP1 expression under regular culture conditions and protected cells from IL-1-induced upregulation of MMP-1 and MMP-13.19 To elucidate the mechanism of MMP downregulation by CITED2, we carried out a series of experiments in our labs and used co-immunoprecipitation with p300-specic antibody to identify potential regulatory proteins in p300-binding protein complexes. This approach was based on the understanding that CITED2 is not a DNA-binding protein and its role in MMP regulation has to be based on its interaction with DNA-binding transcription factors of 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 interact with p300 and Ets-1 is a DNA-binding protein that can transactivate MMPs with p300 as a co-factor.32 C28/I2 chondrocytes were treated with moderate shear at 5 dyn/cm2 or high intensity shear at 20 dyn/cm2 . Nuclear extracts were prepared and incubated with antibody specic for p300 to immunoprecipitate p300 protein complexes. Western blotting identied equal amounts of p300 in control and treated extracts. In control cells, no p300 Ets-1 or p300CITED2 complexes were detectable. In contrast, p300CITED2 complexes were identied in cells exposed to ow shear at 5 dyn/cm2 , and p300ETS-1 complexes were detected in cells under 20 dyn/cm2 .19 Our ndings support the hypothesis that CITED2 may mediate the mechanical load-induced downregulation of MMP by competing with MMP transactivator Ets-1 for limiting amounts of co-factor p300 protein, as illustrated in Figure 2. As a CITED2-mediated target in MMP downregulation from moderate mechanical loading, Ets1 proteins constitute a highly conserved family of transcription factors that share a unique DNAbinding domain, the Ets domain.32 The Ets domain specically recognizes DNA sequences that contain a GGAA/T core element.35 Ets-1 is a transcription factor that binds to MMP-1 and other MMP promoter regions.32 The Ets domain also serves as the p300 CH1-binding domain, recruiting p300 as a co-factor in the transactivation of MMPs. The Ets-1 activation 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-1 to activate transcription and is also necessary for the interaction of Ets-1 with p300.32 Although CITED2 has only been demonstrated to mediate the load-driven downregulation of MMP1 and MMP-13, an MMP promoter analysis has identied Ets-1-binding sites at the promoter regions of many MMPs, including MMPs 1, 2, 3, 8, 9, and 13 (unpublished observations), suggesting that CITED2 may mediate a general MMP mechanoregulatory mechanism. In addition, many transcriptional regulators can bind to p300 via the p300 CH1 domain, implying the competitive mechanism by which CITED2 regulates MMP expression may extend to other CH1-binding transcriptional regulators. 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 driving MMP downregulation. Understanding the mechanisms by which mechanical loads regulate MMP expression is a critical step toward the development of chondroprotective therapies. The nding that CITED2 expression is mechanically responsive and inversely correlated with MMP, coupled with the identication that CITED2 acts via the p300 Ets-1 pathway to regulate MMP transcription in chondrocytes, claries molecular targets that may have signicant clinical potential for the prevention and treatment of joint disease. Acknowledgment This work was supported by National Institutes of Health grants AR47628 and AR52743 to H.B.S. The author thanks Daniel J. Leong (graduate student) and Drs. Robert J. Majeska and Li Sun for helpful critique and Dr. Zhengzhe Li for promoter analysis. Conict of interest The author declares no conicts of interest. References
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