Peroxisome Proliferator-Activated Receptor- -Independent Repression of Collagenase Gene Expression by 2-Cyano-3,12- dioxooleana-1,9-dien-28-oic Acid and Prostaglandin 15-Deoxy- (12,14) J2: A Role for Smad Signaling

Matrix metalloproteinases (MMPs) degrade extracellular matrix components, and overexpression of these enzymes contributes to tissue destruction in arthritis. Of particular importance are the collagenases, MMP-1 and MMP-13, which have high activity against the interstitial collagens in cartilage. In this study, we address the mechanisms of two inhibitors of collagenase gene expression, the synthetic triterpenoid 2-cyano-3,12-dioxooleana1,9-dien-28-oic acid (CDDO) and 15-deoxy(12,14)-prostaglandin J2 (15-dPGJ2). Although both inhibitors are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor(PPAR), a connection between PPARand collagenase gene expression has yet to be established. Here, we test the hypothesis that CDDO and 15-dPGJ2 use PPARto repress MMP gene expression. Our findings with the PPARantagonist 2-[4-[2-[3-(2,4-difluorophenyl)-1-heptylureido]ethyl]rsqb]-phenylsulfanyl]-2-methylpropionic acid (GW9662) and mouse embryonic fibroblasts lacking PPARdemonstrate that CDDO and 15dPGJ2 use PPAR-independent mechanisms to inhibit collagenase gene expression. To address a potential PPAR-independent mechanism leading to the repression of MMPs by CDDO, we tested the effect of CDDO on the transforming growth factor(TGF) signaling pathway. We found that CDDO requires Smads (transcription factors activated by TGF) for the repression of MMP-1. Specifically, MMP-1 is inhibited neither by CDDO in the absence of TGFreceptor-activated Smad3 nor when a negative regulator, Smad7, attenuates TGFsignaling. We conclude that CDDO represses MMP gene expression through a novel PPAR-independent mechanism that requires Smad signaling. Peroxisome proliferator-activated receptor(PPAR) is a nuclear hormone receptor that regulates the expression of genes involved in insulin sensitivity and adipogenesis (Forman et al., 1996). Recent studies have proposed a distinct role for this nuclear receptor as an anti-inflammatory mediator activated by PPARligands (Ricote et al., 1998; Kawahito et al., 2000; Chawla et al., 2001). Although it is clear these ligands act directly through PPARto regulate insulin sensitivity and adipogenesis, it is unclear whether a similar mechanism regulates genes involved in inflammation. PPAR-independent mechanisms have been described for some of these ligands (Straus et al., 2000; Chawla et al., 2001; Ward et al., 2002), indicating diverse roles for PPARin the regulation of different target genes. Thus, it is essential to determine the requirement for PPARin the regulation of multiple target genes and to define receptor-independent mechanisms used by PPARligands. The pathologies of rheumatoid arthritis and osteoarthritis are associated with inflammation that is mediated partly by the action of inflammatory cytokines secreted from macrophages within affected joints (Mengshol et al., 2002). These cytokines [i.e., interleukin-1 (IL-1 ) and tumor necrosis factor(TNF)] induce the expression of matrix metalloproteinases (MMPs) in chondrocytes and synovial fibroblasts This work was supported by the National Institutes of Health Grants AR-26599, CA-77617, and the Department of Defense Grant DoD-991121 (to C.E.B.), NIH Grant CA-78814, The National Foundation for Cancer Research, and Eli Lilly and Co. (to M.B.S.), and NIH Grant T32-AI07363 (to K.S.M.). ABBREVIATIONS: PPAR, peroxisome proliferatoractivated receptor; IL, interleukin; TNF, tumor necrosis factor; MMP, matrix metalloproteinase; 15-dPGJ2, 15-deoxy(12,14) prostaglandin J2; CDDO, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid; TGF, transforming growth factor; DMEM, Dulbecco’s modified Eagle’s medium; MEF, mouse embryonic fibroblast; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPRE, PPARresponse element; RSV, Rous sarcoma virus; RLU, relative luciferase unit; AP, activator protein. 0026-895X/04/6502-309–318$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 2 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2840/1122752 Mol Pharmacol 65:309–318, 2004 Printed in U.S.A. 309 at A PE T Jornals on July 5, 2017 m oharm .aspeurnals.org D ow nladed from (Mengshol et al., 2002). In turn, the enzymatic action of these MMPs contributes to the destruction of extracellular matrix components in bone, cartilage, and tendons (Mengshol et al., 2002). Several studies have suggested that PPARligands may modulate the process of joint destruction, either by reducing the expression of inflammatory cytokines (Ricote et al., 1998; Chawla et al., 2001) or MMPs (Shu et al., 2000; Fahmi et al., 2001, 2002; Sabatini et al., 2002). Furthermore, although not all cells express PPAR, its expression has been noted in chondrocytes and synovial fibroblasts (Bordji et al., 2000; Fahmi et al., 2001, 2002; Sabatini et al., 2002), suggesting that PPARmay have a function within joints. An endogenous ligand for PPAR, the cyclopentone prostaglandin 15-dPGJ2 binds to PPARwith a Ki of 1.2 10 9 M (Kliewer et al., 1995). This prostaglandin suppresses adjuvant-induced arthritis in rats (Kawahito et al., 2000), reduces the expression of inflammatory cytokines involved in arthritis (Ricote et al., 1998; Chawla et al., 2001), and inhibits the expression of MMP-1 and MMP-13 produced by synovial fibroblasts and chondrocytes (Fahmi et al., 2001, 2002). MMP-1 and MMP-13 are chief mediators of joint destruction because these enzymes are able to degrade type II collagen, the most abundant collagen within cartilage (Mengshol et al., 2002). However, some of the anti-inflammatory effects of 15-dPGJ2 have been attributed to PPAR-independent mechanisms (Straus et al., 2000; Chawla et al., 2001; Ward et al., 2002), and the role of PPARin the inhibition of MMP-1 and MMP-13 by 15-dPGJ2 has not been elucidated. We have described another compound with anti-inflammatory activity, the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) (Suh et al., 1999), which inhibits the synthesis of inflammatory mediators inducible nitric-oxide synthase and cyclooxygenase-2 (Suh et al., 1999) and blocks MMP-1 and MMP-13 gene expression in human SW-1353 chondrosarcoma cells (Mix et al., 2001) and primary osteoarthritic chondrocytes (Elliott et al., 2003). Although the chemical structures of CDDO and 15-dPGJ2 are distinct, both compounds contain an , -unsaturated ketone group (Kliewer et al., 1995; Honda et al., 1998), potentially explaining some of their similar effects. Interestingly, CDDO binds to PPARwith a Ki between 10 8 and 10 7 M and, like other PPARligands, mediates adipogenic differentiation by activating PPAR(Wang et al., 2000). CDDO inhibits cell proliferation and induces differentiation of leukemia cells independent of PPAR(Place et al., 2003). Thus, like 15dPGJ2, CDDO exerts its effects through PPAR-dependent and -independent mechanisms. Here too, however, the role of PPARin the inhibition of MMP-1 and MMP-13 expression by CDDO is unclear. Given the potential benefits of using PPARligands as inhibitors of MMP gene expression, addressing the role of PPARin the repression of MMPs by CDDO and 15-dPGJ2 is critical. Many of the cellular activities of CDDO parallel the effects of the pleiotropic growth factor known as transforming growth factor(TGF). For example, TGFcan regulate differentiation, suppress cell proliferation, and, importantly, modulate the expression of MMPs (Massague and Wotton, 2000; Verrecchia and Mauviel, 2002). TGFmediates many of its effects via TGFreceptor-activated transcription factors known as Smads (Massague and Wotton, 2000; Verrecchia and Mauviel, 2002). A recent study indicates that synthetic triterpenoids CDDO and CDDO-imidazolide target the TGFpathway in epithelial and leukemia cells (Suh et al., 2003), thus potentially affecting numerous cellular processes. However, the connection between CDDO and TGFhas not been elucidated in other cell types, and this elucidation is critical given the tissue-specific effects of TGF(Massague and Wotton, 2000; Verrecchia and Mauviel, 2002). Furthermore, the impact of CDDO on TGFsignaling has not been addressed as a mechanism contributing to MMP repression. In this study, we test the hypothesis that PPARmediates the inhibition of MMP-1 and MMP-13 gene expression by 15-dPGJ2 and CDDO. We use SW-1353 human chondrosarcoma cells stimulated with IL-1 as a model for osteoarthritic chondrocytes. Our findings indicate that CDDO and 15dPGJ2 use PPAR-independent mechanisms to inhibit MMP-1 and MMP-13 gene expression. We further characterize the mechanism of CDDO by addressing its effect on the TGFsignaling pathway, and we identify a novel mechanism for CDDO that uses Smad signaling for the repression of MMPs. Materials and Methods Cell Culture. Human SW-1353 chondrosarcoma cells (American Type Culture Collection, Manassas, VA) and wild-type and Smad3 / dermal fibroblasts (gift from Kathleen Flanders, National Cancer Institute) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum and penicillin/streptomycin at 37°C, 5% CO2. PPAR/ and / mouse embryonic fibroblasts (MEFs) (Rosen et al., 2002) were cultured in DMEM containing 10% fetal calf serum and penicillin/streptomycin at 37°C, and 10% CO2. PPARstatus of the MEFs was confirmed by RT-PCR (data not shown). Cells were grown to confluence, washed with Hanks’ balanced salt solution, and placed in serum-free DMEM containing 0.2% lactalbumin hydrolysate for experiments. CDDO was synthesized (Honda et al., 1998) and provided for use by Drs. Tadashi Honda and Gordon Gribble (Dartmouth College, Hanover, NH). As described previously (Mix et al., 2001), cells were treated with 300 nM CDDO for 24 h before the addition of 10 ng/ml IL-1 (Promega, Madison, WI) for an additional 24 h. 15-dPGJ2 at 1 or 5 M (Cayman Chemical, Ann Arbor, MI) was added to cells for 24 h before the addition of IL-1 . Where indicated, 10 M GW9662 (gift from Timothy Willson, GlaxoSmithKline, Uxbridge, Middlesex, UK) was added to cells for 1 h before the CDDO or 15-dPGJ2 pretreatment. Rosiglitazone (gift from Timothy Willson, GlaxoSmithKline) was used at a concentration of 1 M. Where indicated, 10 ng/ml TGF(R&D Systems, Minneapolis, MN) was added to cells simultaneously