Expression In Human Chondrocytes Research Articles

ALK1 Opposes ALK5/Smad3 Signaling and Expression of Extracellular Matrix Components in Human Chondrocytes

TGF-beta is a multifunctional regulator of chondrocyte proliferation, differentiation, and extracellular matrix production. Dysregulation of TGF-beta action has been implicated in cartilage diseases such as osteoarthritis. TGF-beta signaling is transduced through a pair of transmembrane serine/threonine kinases, known as the type I (ALK5) and type II receptors. However, recent studies on endothelial cells have identified ALK1 as a second type I TGF-beta receptor and have shown that ALK1 and ALK5 have opposing functions in these cells. Here we examined ALK1 expression and its regulation of TGF-beta signaling and responses in human chondrocytes. ALK1 expression in human chondrocytes was examined by RT-PCR and Western blot. The ability of ALK1 to form complexes with other TGF-beta receptors was determined by affinity labeling/immunoprecipitation and by immunoprecipitation followed by Western blot. The effect of ALK1 on TGF-beta1-induced signaling and responses was determined by varying ALK1 expression levels and measuring transcriptional activity using promoter/luciferase assays, Smad1/5 and Smad3 phosphorylation, and expression of type II collagen, PAI-1, and fibronectin. Our results indicate that ALK1 is expressed in human chondrocytes and that it is a component of the TGF-beta receptor system, associating with ALK5, type II TGF-beta receptor, endoglin, and betaglycan. Furthermore, we show that both ALK1 and ALK5 are needed for TGF-beta-induced phosphorylation of intracellular mediators Smad1/5, whereas only ALK5 is essential for TGF-beta1-induced phosphorylation of Smad3. In addition, our results show that ALK1 inhibits, whereas ALK5 potentiates, TGF-beta-induced Smad3-driven transcriptional activity and the expression of PAI-1, fibronectin, and type II collagen in chondrocytes. Our results suggest that ALK1 and ALK5 display opposing functions in human chondrocytes, implicating an essential role for ALK1 in the regulation of TGF-beta signaling and function in these cells.

Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes

Transforming growth factor beta (TGF-β1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-α-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-β1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-β1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-β1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-β induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-β. TIMP-3 promoter deletion analysis revealed that − 828 deletion retains the original promoter activity while − 333 and − 167 deletions display somewhat reduced activity suggesting that most of the TGF-β-responsive, cis-acting elements are found in the − 333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the − 940 and − 333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-β-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

Stromal Cell-Derived Factor-1 Induces Matrix Metalloprotease-13 Expression in Human Chondrocytes

The production of chemokine stromal cell-derived factor (SDF)-1 is significantly higher in synovial fluid of patients with osteoarthritis and rheumatoid arthritis. Matrix metalloproteinase (MMP)-13 may contribute to the breakdown of articular cartilage during arthritis. Here, we found that SDF-1alpha increased the secretion of MMP-13 in cultured human chondrocytes, as shown by reverse transcriptase-polymerase chain reaction, Western blot, and zymographic analysis. SDF-1alpha also increased the surface expression of CXCR4 receptor in human chondrocytes. CXCR4-neutralizing antibody, CXCR4-specific inhibitor [1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)], or small interfering RNA against CXCR4 inhibited the SDF-1alpha-induced increase of MMP-13 expression. The transcriptional regulation of MMP-13 by SDF-1alpha was mediated by phosphorylation of extracellular signal-regulated kinases (ERK) and activation of the activator protein (AP)-1 components of c-Fos and c-Jun. The binding of c-Fos and c-Jun to the activator protein (AP-1) element on the MMP-13 promoter and the increase in luciferase activity was enhanced by SDF-1alpha. Cotransfection with dominant-negative mutant of ERK2 or c-Fos and c-Jun antisense oligonucleotide inhibited the potentiating action of SDF-1alpha on MMP-13 promoter activity. Taken together, our results provide evidence that SDF-1alpha acts through CXCR4 to activate ERK and the downstream transcription factors (c-Fos and c-Jun), resulting in the activation of AP-1 on the MMP-13 promoter and contributing cartilage destruction during arthritis.

Requirement of phosphatidylinositol 3-kinase/Akt signaling pathway for regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-β in human chondrocytes

Transforming growth factor beta (TGF-β1) induces cartilage extracellular matrix synthesis and tissue inhibitor of metalloproteinases-3 (TIMP-3), an important natural inhibitor of matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme, which are implicated in cartilage degradation and joint inflammation. This study tested the hypothesis that Akt/protein kinase B signaling pathway could mediate TGF-β1 induction of TIMP-3 in human articular chondrocytes. TGF-β activated phosphorylation of Akt in a delayed and sustained fashion that correlated with TIMP-3 mRNA induction. Phosphatidylinositol kinase (PI3K) inhibitors, Wortmannin and LY294002 and Akt inhibitor (NL-71-101) significantly inhibited TGF-β-induced Akt phosphorylation, TIMP-3 expression, TIMP-3 promoter (− 940 to + 376)-driven luciferase activity and Sp1 transcription factor binding. PI3K p85, Akt and Sp1 small interfering RNA (siRNA)-driven knockdown of the respective gene products significantly suppressed TGF-β-induced TIMP-3 gene expression. TGF-β-stimulated phosphorylation of p70S6 Kinase and TIMP-3 protein induction was inhibited by rapamycin. Thus TGF-β induces TIMP-3 gene expression in human chondrocytes partly through PI3K/Akt pathway and Sp1 transcription factor and by translational mechanisms via mammalian target of rapamycin (mTOR) signaling. TGF-β induction of pro-survival Akt cascade and TIMP-3 may be related to strengthening of cartilage extracellular matrix, increased chondrocyte viability and maintenance of joint tissue integrity.

P38MAPK mediates IL-1-induced down-regulation of aggrecan gene expression in human chondrocytes

The pleiotropic cytokine interleukin 1 (IL-1) is considered to be the principal inducer of mediators of cartilage degradation in both, osteoarthritis (OA) and rheumatoid arthritis (RA). IL-1 activates numerous signaling pathways involved in cartilage destruction and dedifferentiation of chondrocytes. In this study, we analyzed expression and functional effects of IL-1 in human chondrocytes. We found an IL-1-induced reduction in the expression of the cartilage specific proteoglycan aggrecan as an indicator for the IL-1-mediated dedifferentiation of chondrocytes. To block the IL-1-induced signaling pathways specifically, we incubated human chondrocytes and cartilage explants with IL-1 in the presence of different signal transduction inhibitors and analyzed their effect on aggrecan mRNA expression and IL-6 secretion. IL-6 has been found to act synergistically in the IL-1-induced suppression of the proteoglycan synthesis in chondrocytes. Our results led to the identification of p38MAPK and/or PI3K/JNK as being crucial for IL-1-induced IL-6 secretion by chondrocytes. IL-1-induced down-regulation of aggrecan expression was found to be mediated by p38MAPK and/or ERK1/2. The identification and characterization of these signaling pathways will enable us to develop new modulation strategies for therapeutic use in inflammatory joint diseases.

Catabolic stress induces features of chondrocyte senescence through overexpression of caveolin 1: Possible involvement of caveolin 1–induced down‐regulation of articular chondrocytes in the pathogenesis of osteoarthritis

Articular chondrocyte senescence is responsible, at least in part, for the increased incidence of osteoarthritis (OA) with increased age. Recently, it was suggested that caveolin 1, a 21-24-kd membrane protein, participates in premature cellular senescence. Caveolin 1 is the principal structural component of caveolae, vesicular invaginations of the plasma membrane. This study was undertaken to investigate whether the catabolic factors oxidative stress and interleukin-1beta (IL-1beta) induce features of premature senescence of articular chondrocytes through up-regulation of caveolin 1 expression. Caveolin 1 expression was investigated in human OA cartilage by real-time polymerase chain reaction and in rat OA cartilage by immunohistologic analysis. We studied whether IL-1beta and H2O2 induce caveolin 1 expression in OA chondrocytes and analyzed the relationship between cellular senescent phenotypes and caveolin 1 expression in human chondrocytes. In human and rat OA articular cartilage, caveolin 1 positivity was associated with cartilage degeneration. Both IL-1beta and H2O2 up-regulated caveolin 1 messenger RNA and protein levels, and both treatments induced marked expression of senescent phenotypes: altered cellular morphology, cell growth arrest, telomere erosion, and specific senescence-associated beta-galactosidase activity. Caveolin 1 overexpression induced p38 MAPK activation and impaired the ability of chondrocytes to produce type II collagen and aggrecan. In contrast, down-regulation of caveolin 1 with antisense oligonucleotide significantly inhibited the features of chondrocyte senescence induced by catabolic factors. Caveolin 1 induction and stresses with both IL-1beta and H2O2 up-regulated p53 and p21 and down-regulated phosphorylated retinoblastoma (Rb), suggesting that the p53/p21/Rb phosphorylation pathway, as well as prolonged p38 MAPK activation, may mediate the features of chondrocyte senescence induced by stress. Our findings suggest that IL-1beta and oxidative stress induce features of premature senescence in OA chondrocytes, mediated, at least in part, by stress-induced caveolin 1 expression. This indicates that caveolin 1 plays a role in the pathogenesis of OA via promotion of chondrocyte down-regulation.

Retinoic acid and oncostatin M combine to promote cartilage degradation via matrix metalloproteinase-13 expression in bovine but not human chondrocytes

Retinoic acid (RetA) and oncostatin M (OSM) have both been shown to mediate potent effects with respect to extracellular matrix integrity. This study assesses the effects of a RetA + OSM combination on cartilage catabolism. Animal and human cartilage samples were used to assess the ability of RetA + OSM to promote the release of collagen and proteoglycan fragments, which was determined by measuring glycosaminoglycan and hydroxyproline, respectively. Total collagenolytic and tissue inhibitor of metalloproteinases (TIMP) inhibitory activities were determined by bioassay, whilst gene expression of matrix metalloproteinases (MMPs) and TIMP-1 were determined by northern blotting. Immunohistochemistry was used to assess the presence of MMP-1 and -13 in resorbing cartilage explants. Both agents alone induced proteoglycan release from bovine cartilage, whilst RetA-induced collagen release was variable. Reproducible and synergistic collagenolysis was observed with RetA + OSM, which appeared to be due to MMP-13. Similar collagen release was observed from porcine cartilage. Conversely, no collagen release was seen with human articular cartilage. In primary human chondrocytes, RetA + OSM failed to induce MMP-1 or -13 but caused a significant increase in TIMP-1 expression. These novel observations show that the combination of RetA + OSM has profound effects on cartilage matrix turnover, but these effects are species-specific. A better understanding of the mechanism by which this combination differentially regulates MMP and TIMP expression in human chondrocytes could provide valuable insight into new therapeutic strategies aimed at the prevention of cartilage destruction.

Inhibitors of Mitogen‐Activated Protein KinasesDownregulate COX‐2 Expression in Human Chondrocytes

Inducible prostaglandin synthase (cyclooxygenase-2, COX-2) is expressed in rheumatoid and osteoarthritic cartilage and produces high amounts of proinflammatory prostanoids in the joint. In the present study we investigated the effects of the inhibitors of mitogen-activated protein kinase (MAPK) pathways Erk1/2, p38, and JNK on COX-2 expression and prostaglandin E2 (PGE2) production in human chondrocytes. Proinflammatory cytokine IL-1β caused a transient activation of Erk1/2, p38, and JNK in immortalized human T/C28a2 chondrocytes and that was followed by enhanced COX-2 expression and PGE2 production. PD98059 (an inhibitor of Erk1/2 pathway) suppressed IL-1-induced COX-2 expression and PGE2 production in a dose-dependent manner, and seemed to have an inhibitory effect on COX-2 activity. SB203580 (an inhibitor of p38 pathway) but not its negative control compound SB202474 inhibited COX-2 protein and mRNA expression and subsequent PGE2 synthesis at micromolar drug concentrations. SP600125 (a recently developed JNK inhibitor) but not its negative control compound N1-methyl-1,9-pyrazolanthrone downregulated COX-2 expression and PGE2 formation in a dose-dependent manner. SP600125 did not downregulate IL-1-induced COX-2 mRNA expression when measured 2 h after addition of IL-1β but suppressed mRNA levels in the later time points suggesting post-transcriptional regulation. Our results suggest that activation of Erk1/2, p38, and JNK pathways belongs to the signaling cascades that mediate the upregulation of COX-2 expression and PGE2 production in human chondrocytes exposed to proinflammatory cytokine IL-1β.

Up-regulation of microsomal prostaglandin E synthase 1 in osteoarthritic human cartilage: critical roles of the ERK-1/2 and p38 signaling pathways.

Microsomal prostaglandin E synthase 1 (mPGES-1) is the final enzyme of the cascade that produces prostaglandin E(2) (PGE(2)), a key actor in arthritis. To study mPGES-1 synthesis in human cartilage and its regulation by interleukin-1beta (IL-1beta), we used human cartilage and an immortalized human chondrocyte cell line. Furthermore, we investigated the signaling pathways involved in mPGES-1 expression. We used real-time quantitative reverse transcription-polymerase chain reaction, Northern blotting, and Western blotting to measure mPGES-1 messenger RNA (mRNA) and protein expression in human chondrocytes. PGE(2) production was measured by enzyme-linked immunosorbent assay. Cartilage specimens from osteoarthritis (OA) patients contained far greater amounts of mPGES-1 and cyclooxygenase 2 (COX-2) mRNA than did normal cartilage. Incubation with IL-1beta markedly increased mPGES-1 mRNA and protein in a dose-dependent and time-dependent manner, in parallel with an increase in PGE(2) levels. Both PD98059, an ERK pathway inhibitor, and SB203580, a p38alpha/beta MAPK inhibitor, abolished the increases in mPGES-1 mRNA and protein in response to IL-1beta. The specific p38alpha MAPK inhibitor SC906 suppressed IL-1beta-induced COX-2 expression but not IL-1beta-induced mPGES-1 expression, suggesting preferential involvement of p38beta MAPK in IL-1beta-induced mPGES-1 expression. This study is the first to show that mPGES-1 is stimulated in human chondrocytes by the proinflammatory cytokine IL-1beta via activation of both ERK-1/2 and p38 MAPK in an isoform-specific manner. We postulate that mPGES-1 may be a novel target for OA therapy.

Inhibitory Effects of Insulin-like Growth Factor-1 and Osteogenic Protein-1 on Fibronectin Fragment- and Interleukin-1β-stimulated Matrix Metalloproteinase-13 Expression in Human Chondrocytes

Matrix metalloproteinase-13 (collagenase-3), a member of the family of matrix metalloproteinases (MMPs), plays a major pathological role in the cartilage destruction of arthritis. A dramatic up-regulation of MMP-13 by inflammatory cytokines such as interleukin (IL)-1beta or by fibronectin fragments has been observed in chondrocytes. In this study, we investigated the inhibitory effects of insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) on the expression of MMP-13, which was induced by fibronectin fragment or IL-1beta in human immortalized or human primary chondrocytes. IGF-1 and OP-1 each significantly reduced the basal level as well as fibronectin fragment- or IL-1beta-stimulated transcription of the MMP-13 gene in a dose-dependent fashion with the corresponding decreases in the protein level of MMP-13. The most prominent suppressive effect was observed by the combination of IGF-1 and OP-1, which decreased the basal promoter activity by 60% and almost completely abrogated the fibronectin fragment-stimulated MMP-13 promoter activity. OP-1 was found to enhance mRNA levels of IGF-1 and the IGF-1 receptor, the latter of which appeared to be responsible for the combined effect of IGF-1 and OP-1. The suppressive effect of IGF-1 and OP-1 on MMP-13 expression was due in part to down-regulation of the expression of pro-inflammatory cytokines and the activity of their intermediate molecules, including NF-kappaB and AP-1 factors. We propose that IGF-1 and OP-1 could be key physiological regulators of MMP-13 gene expression and that the combination of IGF-1 and OP-1 may be useful in controlling the excess catabolic activity in arthritis.

The triterpenoid CDDO inhibits expression of matrix metalloproteinase-1, matrix metalloproteinase-13 and Bcl-3 in primary human chondrocytes.

A synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), has been reported to have anti-inflammatory properties and to decrease the interleukin-1 (IL-1)-induced expression of matrix metalloproteinase-1 (MMP-1) and MMP-13. We have shown previously that IL-1 induces expression of the inhibitor of NF-kappaB (IkappaB) family member Bcl-3, and that this contributes to MMP-1 expression. To quantify the effects of CDDO on IL-1-induced MMP-1, MMP-13 and Bcl-3 expression, we stimulated the chondrosarcoma cell line SW-1353 and human primary chondrocytes with IL-1, in the presence or absence of CDDO. Harvested RNA was subjected to quantitative real-time reverse-transcriptase polymerase chain reaction. In SW-1353 cells, 300 nM CDDO significantly decreased the induction of MMP-1 and MMP-13 by IL-1. In human primary chondrocytes, 300 nM CDDO inhibited the induction of these genes by IL-1 to an even greater extent. In both cell types, inhibition of MMP-1 required 24 hours of pretreatment with CDDO, whereas MMP-13 could be inhibited when CDDO and IL-1 were added simultaneously to culture. In human primary chondrocytes, IL-1-induced Bcl-3 expression was inhibited when cells were pretreated with CDDO. To determine whether the inhibitory effect of CDDO on MMP worked through inhibition of Bcl-3 gene expression, SW-1353 cells stably transfected with a Bcl-3 expression plasmid were treated with IL-1 and/or CDDO, and MMP gene expression was assayed. Overexpression of Bcl-3 increased MMP-1, but not MMP-13, mRNA levels. Furthermore, overexpressed Bcl-3 could sustain the CDDO-dependent inhibition of IL-1-induced MMP-1 expression. Our data demonstrate that CDDO inhibits IL-1-induced MMP-1 and MMP-13 expression in human chondrocytes. CDDO also inhibits the expression of Bcl-3, an IL-1-responsive gene that preferentially contributes to MMP-1 gene expression.

The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin-1 and oncostatin M: a time-course study using real-time quantitative reverse transcription-polymerase chain reaction.

Previous studies have reported elevated levels of interleukin-1 (IL-1) and oncostatin M (OSM) in rheumatoid joints, as well as the synergistic degradation of human articular cartilage by this cytokine combination. The present study was undertaken to investigate the ability of IL-1 and OSM to modulate gene expression of matrix metalloproteinase (MMP), ADAM, and ADAM-TS (ADAM with thrombospondin motifs) family members in human chondrocytes. T/C28a4 human chondrocytes were stimulated for 2-48 hours with IL-1 and/or OSM. Total RNA was harvested, reverse transcribed, and assessed by real-time polymerase chain reaction for the expression of various MMP, ADAM, and ADAM-TS messenger RNAs (mRNA). Results were normalized to 18S ribosomal RNA. IL-1 and OSM synergized to markedly induce the expression of the collagenases MMP-1, MMP-8, and MMP-13 as well as MMP-3, an activator of proMMPs. Expression of mRNA for MMPs 1, 3, and 13 was induced early, whereas that of MMP-8 mRNA occurred late. Gene expression of MMP-14, an MMP that degrades collagen and activates proMMP-13, was elevated by this combination. IL-1 and OSM also synergized to induce gene expression of the aggrecanase ADAM-TS4, but not ADAM-TS5. These data indicate that the potent cartilage-degrading properties of the combination of IL-1 and OSM are potentially mediated by a synergistic induction of the aggrecan-degrading enzyme ADAM-TS4 and the collagen-degrading enzymes MMP-1, MMP-8, MMP-13, and MMP-14, although differences in the magnitude of response and in the time course of induction were observed. A role for MMPs 3 and 14 in the activation of proMMPs may also be implicated.

Hyaluronan does not affect cytokine and chemokine expression in osteoarthritic chondrocytes and synoviocytes

Objective Many studies have evidenced the clinical efficacy of hyaluronan (HA) in the treatment of osteoarthritis (OA). However, human and animal studies have described proinflammatory effects of HA on cells not involved in OA. We therefore investigated whether different molecular weight HA preparations can affect proinflammatory cytokine (IL1β and TNFα) or chemokine (IL8, MCP-1 and RANTES) expression in human chondrocytes and synoviocytes isolated from OA patients.Design Human chondrocytes and synoviocytes were cultured in vitro in the presence or absence of three different purified HA pharmaceutical preparations (1×106Kd, 5×105Kd and 6.5×104Kd) and assessed for the production of proinflammatory cytokines and chemokines and their mRNA expression.Results In basal conditions, both chondrocytes and synoviocytes produce only MCP-1 and IL8, along with low quantities of IL1β and TNFα, but not RANTES. IL8 production was generally about 100 times higher in chondrocytes than in synoviocytes, while MCP-1 was roughly twice as high in synoviocytes than in chondrocytes. At the mRNA level, expression of IL1β, TNFα, IL8, MCP-1 and RANTES did not change in the presence of the three HA preparations either in synoviocytes or in chondrocytes with respect to basal condition. None of the three different HA preparations significantly affected production of IL8 or MCP-1.ConclusionsThese data demonstrate that preparations of HA of the same origin but with different MWs do not induce proinflammatory cytokines and chemokines expressed by chondrocytes and synoviocytes that are either directly or indirectly involved in OA progression.

FrzB-2: a human secreted frizzled-related protein with a potential role in chondrocyte apoptosis

Objective To characterize a novel secreted frizzled-related protein (SFRP) and determine its tissue distribution at the mRNA and protein level.Methods The FrzB-2 gene was identified by expressed sequence tag (EST) analysis of human tissue-derived libraries. Tissue distribution of FrzB-2 mRNA was determined by Northern blot analysis and in situ hybridization. FrzB-2 protein reactivity was localized in human OA articular cartilage by immunocytochemistry, using a polyclonal antibody against a peptide sequence unique to FrzB-2. Apoptosis was detected in articular cartilage sections using Tunel staining.Results ESTs corresponding to FrzB-2 were found in osteoblast, chondrosarcoma, osteosarcoma, osteoclastoma and synovial fibroblast libraries. FrzB-2 mRNA is expressed in a number of tissues and cell types including bone-related cells and tissues such as primary human osteoblasts and osteoclastoma. In situ hybridization studies showed strong FrzB-2 mRNA expression in human chondrocytes in human osteoarthritic (OA) cartilage but negligible levels in normal cartilage chondrocytes. The FrzB-2 cDNA encodes a secreted 40 kDa protein consisting of 346 amino acids. FrzB-2 is 92.5% identical to the rat orthologue, DDC-4, which has been shown to be associated with physiological apoptosis. FrzB-2 protein was selectively detected in human OA articular cartilage by immunocytochemistry, using a polyclonal antibody. Consistent with its potential role in apoptosis, positive FrzB-2 staining and Tunel positive nuclei staining were detected in chondrocyte clones in sections of human OA cartilage.Conclusion These data suggest that FrzB-2 may play a role in apoptosis and that the expression of this protein may be important in the pathogenesis of human OA.

Cytokine control of interstitial collagenase and collagenase-3 gene expression in human chondrocytes

Cytokine control of interstitial collagenase and collagenase-3 gene expression in human chondrocytes

Cytokine Control of Interstitial Collagenase and Collagenase-3 Gene Expression in Human Chondrocytes

Human collagenase-3 expression, previously seen only in a breast tumor tissue, is here shown to be expressed in primary human chondrocytes derived from the joint tissue and in transformed human chondrocytes. Its mRNA is inducible by the inflammatory cytokines interleukin-1beta plus tumor necrosis factor-alpha, but not by phorbol 12-myristate 13-acetate and only slightly by the growth factors platelet-derived growth factor and epidermal growth factor. Human synovial fibroblasts, another prominent cell type in the joint tissue, do not produce collagenase-3 message. Expression of the murine collagenase, which is possibly the counterpart of human collagenase-3, is induced by interleukin-1beta plus tumor necrosis factor-alpha, and its full induction requires the presence of the transcription factor, c-FOS. This family of transcription factors also plays a role in induction of human collagenase-3, since it binds to the AP-1 site of this matrix metalloproteinase.

Regulation of Collagen Gene Expression by Prostaglandins and Interleukin-1beta in Cultured Chondrocytes and Fibroblasts.

To compare the modulatory effects of different prostaglandins on collagen gene expression in human chondrocytes, PGE(2), PGE(1), misoprostol (PGE(1) analog), and PGF(2alpha) (10, 50 and 100 ng ml(minus sign1)) were added to human chondrocytes with or without interleukin-1beta (IL-1beta) in the presence of indomethacin to inhibit endogenous prostaglandin synthesis and the effects evaluated on chondrocyte morphology, collagen synthesis, and procollagen mRNA levels. The effects of prostaglandins on the expression of collagen gene regulatory sequences were examined using transient transfection assays of reporter gene constructs in human chondrocytes and BALB/c3T3 fibroblasts, PGE(1), misoprostol, and PGF(2alpha), similar to PGE(2), inhibited type I collagen gene expression in fibroblasts and promoted type II collagen gene expression in chondrocytes. PGE(2), the major inflammatory prostaglandin produced by IL-1-activated chondrocytes and fibroblasts, and PGF(2alpha) were somewhat more potent than the anti-inflammatory prostaglandins PGE(1) and misoprostol in counteracting the IL-1-induced suppression of type II collagen gene expression by chondrocytes and stimulation of type I collagen gene expression by fibroblasts. Rather than promoting degradation of the cartilage matrix in joint diseases, prostaglandins may be somewhat protective, suppressing fibrosis, and maintaining or promoting appropriate cartilage repair.

Transcriptional suppression by interleukin-1 and interferon-gamma of type II collagen gene expression in human chondrocytes.

Type II collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) are capable of decreasing the levels of alpha 1(II) procollagen mRNA and suppressing the synthesis of type II collagen in cultured human chondrocytes. Data reported here show that these effects of IL-1 and IFN-gamma on the expression of the human type II collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three types of experimental evidence: (1) in nuclear run-off assays, preincubation of chondrocytes with either IL-1 or IFN-gamma decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) indicated that the suppression of alpha 1(II) procollagen mRNA by IL-1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT-B/4.0) containing 4.0 kb of 5'-flanking sequences of COL2A1 (-577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT-B/4.0 was observed in human chondrocytes incubated with an insulin-containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT-B/4.0 in these cells was inhibited by either IL-1 or IFN-gamma. Furthermore, expression of pCAT-B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL-1 or IFN-gamma is mediated primarily at the transcriptional level.

Synergistic Regulation of Collagen Gene Expression in Human Chondrocytes by Tumor Necrosis Factor‐α and Interleukin‐1βa

Annals of the New York Academy of SciencesVolume 580, Issue 1 p. 536-539 Synergistic Regulation of Collagen Gene Expression in Human Chondrocytes by Tumor Necrosis Factor-α and Interleukin-1βa MARY B. GOLDRING, MARY B. GOLDRING Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this authorJAMES BIRKHEAD, JAMES BIRKHEAD Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this authorLINDA J. SANDELL, LINDA J. SANDELL Departments of Orthopaedics and Biochemistry University of Washington Seattle, Washington 98195Search for more papers by this authorSTEPHEN M. KRANE, STEPHEN M. KRANE Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this author MARY B. GOLDRING, MARY B. GOLDRING Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this authorJAMES BIRKHEAD, JAMES BIRKHEAD Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this authorLINDA J. SANDELL, LINDA J. SANDELL Departments of Orthopaedics and Biochemistry University of Washington Seattle, Washington 98195Search for more papers by this authorSTEPHEN M. KRANE, STEPHEN M. KRANE Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this author First published: February 1990 https://doi.org/10.1111/j.1749-6632.1990.tb17983.xCitations: 16 a This work was supported by Grants AR-34390 and AR-03564 from the National Institutes of Health. This is Publication Number 1051 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume580, Issue1Structure, Molecular Biology, and Pathology of CollagenFebruary 1990Pages 536-539 RelatedInformation