Activation of PPARs α, β/δ, and γ Impairs TGF-β1-Induced Collagens' Production and Modulates the TIMP-1/MMPs Balance in Three-Dimensional Cultured Chondrocytes

Background and Purpose. We investigated the potency of Peroxisome Proliferators-Activated Receptors (PPARs) α, β/δ, and γ agonists to modulate Transforming Growth Factor-β1 (TGF-β1-) induced collagen production or changes in Tissue Inhibitor of Matrix Metalloproteinase- (TIMP-) 1/Matrix Metalloproteinase (MMP) balance in rat chondrocytes embedded in alginate beads. Experimental Approach. Collagen production was evaluated by quantitative Sirius red staining, while TIMP-1 protein levels and global MMP (-1, -2, -3, -7, and -9) or specific MMP-13 activities were measured by ELISA and fluorigenic assays in culture media, respectively. Levels of mRNA for type II collagen, TIMP-1, and MMP-3 & 13 were quantified by real-time PCR. Key Results. TGF-β1 increased collagen deposition and type II collagen mRNA levels, while inducing TIMP-1 mRNA and protein expression. In contrast, it decreased global MMP or specific MMP-13 activities, while decreasing MMP-3 or MMP-13 mRNA levels. PPAR agonists reduced most of the effects of TGF-β1 on changes in collagen metabolism and TIMP-1/MMP balance in rat in a PPAR-dependent manner, excepted for Wy14643 on MMP activities. Conclusions and Implications. PPAR agonists reduce TGF-β1-modulated ECM turnover and inhibit chondrocyte activities crucial for collagen biosynthesis, and display a different inhibitory profile depending on selectivity for PPAR isotypes

All-trans retinoic acid suppresses interleukin-6 expression in interleukin-1-stimulated synovial fibroblasts by inhibition of ERK1/2 pathway independently of RAR activation

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Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes

ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-β1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-β1. Chondrocytes were exposed to 10 ng/mL of TGF-β1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-β1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-β1. TGF-β1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-β1-induced ePPi generation. Induction of Ank by TGF-β1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-β1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCδ inhibitor). These data suggest a regulatory role for calcium in TGF-β1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-β1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed to affect the inducing effect of TGF-β1 on Ank mRNA level. These data show that TGF-β1 increases ePPi levels, mainly by the induction of the Ank gene, which requires activation of Ras, Raf-1, ERK, and Ca2+-dependent PKC pathways in chondrocytes

Xylosyltransferase-I Regulates Glycosaminoglycan Synthesis during the Pathogenic Process of Human Osteoarthritis

Loss of glycosaminoglycan (GAG) chains of proteoglycans (PGs) is an early event of osteoarthritis (OA) resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I) that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as “normal”, “late-stage” or adjacent to “late-stage”. The results showed that GAG synthesis and content increased in cartilage from areas flanking OA lesions compared to cartilage from macroscopically “normal” unaffected regions, while decreased in “late-stage” OA cartilage lesions. This increase in anabolic state was associated with a marked upregulation of XT-I expression and activity in cartilage “next to lesion” while a decrease in the “late-stage” OA cartilage. Importantly, XT-I inhibition by shRNA or forced-expression with a pCMV-XT-I construct correlated with the modulation of GAG anabolism in human cartilage explants. The observation that XT-I gene expression was down-regulated by IL-1β and up-regulated by TGF-β1 indicates that these cytokines may play a role in regulating GAG content in human OA. Noteworthy, expression of IL-1β receptor (IL-1R1) was down-regulated whereas that of TGF-β1 was up-regulated in early OA cartilage. Theses observations may account for upregulation of XT-I and sustained GAG synthesis prior to the development of cartilage lesions during the pathogenic process of OA

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E(2 )synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δ(12,14)prostaglandin J(2)

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E(2 )synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF(1α )and PGE(2 )in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF(1α )and PGE(2 )peaked 24 hours after stimulation with IL-1β; the induction of PGE(2 )was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ(12,14)prostaglandin J(2 )(15d-PGJ(2)) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE(2 )level than on 6-keto-PGF(1α )level (-90% versus -66% at 10 μM). A high dose of 15d-PGJ(2 )partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 μM. Inhibitory effects of 10 μM 15d-PGJ(2 )were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM(® )analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE(2 )production, whereas 15d-PGJ(2 )inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE(2 )synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ(2 )strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ(2 )occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ(2)

Impact of the Exposome on the Epigenome in Inflammatory Bowel Disease Patients and Animal Models

peer reviewedInflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract that encompass two main phenotypes, namely Crohn’s disease and ulcerative colitis. These conditions occur in genetically predisposed individuals in response to environmental factors. Epigenetics, acting by DNA methylation, post-translational histones modifications or by non-coding RNAs, could explain how the exposome (or all environmental influences over the life course, from conception to death) could influence the gene expression to contribute to intestinal inflammation. We performed a scoping search using Medline to identify all the elements of the exposome that may play a role in intestinal inflammation through epigenetic modifications, as well as the underlying mechanisms. The environmental factors epigenetically influencing the occurrence of intestinal inflammation are the maternal lifestyle (mainly diet, the occurrence of infection during pregnancy and smoking); breastfeeding; microbiota; diet (including a low-fiber diet, high-fat diet and deficiency in micronutrients); smoking habits, vitamin D and drugs (e.g., IBD treatments, antibiotics and probiotics). Influenced by both microbiota and diet, short-chain fatty acids are gut microbiota-derived metabolites resulting from the anaerobic fermentation of non-digestible dietary fibers, playing an epigenetically mediated role in the integrity of the epithelial barrier and in the defense against invading microorganisms. Although the impact of some environmental factors has been identified, the exposome-induced epimutations in IBD remain a largely underexplored field. How these environmental exposures induce epigenetic modifications (in terms of duration, frequency and the timing at which they occur) and how other environmental factors associated with IBD modulate epigenetics deserve to be further investigated