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Biomedical subjects

Gary S Firestein

Publications and source records attributed to Gary S Firestein.

At least 37 records · Page 2Linked to original sources
PubMed2005

Regulation of p38 MAPK by MAPK kinases 3 and 6 in fibroblast-like synoviocytes.

The p38 MAPK signal transduction pathway is a key regulator of IL-1 and TNF-alpha production in rheumatoid arthritis. Previous studies demonstrated that upstream MAPK kinases (MKK3 and MKK6) that regulate p38 are activated in rheumatoid arthritis synovium. However, their functional relevance in fibroblast-like synoviocytes (FLS) has not been determined. To investigate the relative contribution of MKK3 and MKK6 to p38 activation, the effect of dominant-negative (DN) MKK3 and MKK6 constructs on cultured FLS was evaluated. Cultured FLS were stimulated with medium or IL-1beta, and immunoblotting was performed. In some experiments, cells were lysed and immunoprecipitated with anti-p38 Ab, followed by in vitro kinase assay with [gamma-(32)P]ATP and GST-activating transcription factor-2 as substrate. IL-1beta rapidly induced p38 phosphorylation in cells transfected with empty vector (pcDNA3.1), but was inhibited by 25% in cells expressing DN MKK3 or DN MKK6. Cotransfection with both DN plasmids decreased phospho-p38 by almost 75%. In vitro kinase assays on IL-1-stimulated FLS also showed that the combination of DN MKK3 and DN MKK6 markedly decreased kinase activity compared with empty vector or the individual DN plasmids. Furthermore, IL-1beta-induced IL-8, IL-6, and matrix metalloproteinase-3 protein production was significantly inhibited in DN MKK3/DN MKK6-transfected cells. The constructs had no effect on the respective mediator mRNA levels. These data demonstrate that MKK3 and MKK6 make individual contributions to p38 activation in FLS after cytokine stimulation, but that both must be blocked for maximum inhibition.

Arthritis, Rheumatoid↗
PubMed2005

Fibroblast-like synoviocytes derived from patients with rheumatoid arthritis show the imprint of synovial tissue heterogeneity: evidence of a link between an increased myofibroblast-like phenotype and high-inflammation synovitis.

OBJECTIVE: Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast-like synoviocyte (FLS) cultured from RA synovial tissues. METHODS: Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real-time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues. RESULTS: Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high-inflammation synovial tissues revealed increased expression of a transforming growth factor beta/activin A-inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin-like growth factor 2/insulin-like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low-inflammation synovial tissues. The molecular feature that defines the myofibroblast-like phenotype was reflected as an increased proportion of myofibroblast-like cells in the heterogeneous FLS population. Myofibroblast-like cells were also found upon immunohistochemical analysis of synovial tissue. CONCLUSION: Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.

Arthritis, Rheumatoid↗
PubMed2005

Regulation of arthritis by p53: critical role of adaptive immunity.

OBJECTIVE: The p53 tumor-suppressor protein is expressed in rheumatoid arthritis synovium, and loss of p53 function through somatic mutation can occur in longstanding disease. Previous studies demonstrated that p53 is protective in murine collagen-induced arthritis (CIA). To determine if adaptive immune responses or synovial effector functions are responsible for this effect, passive models of arthritis were studied in p53 wild-type and knockout mice. METHODS: Models of passive CIA, passive K/BxN serum transfer arthritis, and active CIA were induced in DBA/1 p53(-/-) or p53(+) mice. Hind paws were evaluated for histologic evidence of inflammation and joint destruction. Synovial interleukin-6 and matrix metalloproteinases 3 and 13 gene expression was analyzed by real-time quantitative polymerase chain reaction. To evaluate T cell function in p53(-/-) mice, draining lymph node (LN) cells from mice immunized with type II collagen (CII) were evaluated in vitro. RESULTS: Increased disease severity in p53(-/-) mice was confirmed in the standard CIA model. However, clinical arthritis, joint destruction, and synovial gene expression in the passive CIA and K/BxN serum transfer arthritis models were similar in p53(-/-) and p53(+) mice. To determine if the p53 effect was related to T cell function, LN cells from CII-immunized mice were isolated and stimulated with antigen in vitro. CII-stimulated T cell proliferation and interferon-gamma production were significantly higher in p53(-/-) mice. An independent assessment of Th1 function using the cutaneous delayed-type hypersensitivity model confirmed that p53(-/-) mice have enhanced T cell responses in vivo. CONCLUSION: Adaptive immune responses, rather than antibody-mediated responses, in p53(-/-) mice account for increased disease severity in the active CIA model.

Animals↗
PubMed2005

Immunologic mechanisms in the pathogenesis of rheumatoid arthritis.

Although much is known about the etiology and pathogenesis of rheumatoid arthritis (RA), our understanding of the immune pathways remains incomplete. The observed clinical and pathologic manifestations result from activation of several interrelated immune pathways. Current concepts of RA pathogenesis, supported by animal models, laboratory studies, and clinical observation, have reestablished and revised some of the original views. Early proposals emphasized the importance of autoantibodies and immune complexes in the initiation of RA, suggested a role for T cells in the inflammatory response characteristic of RA, and based disease perpetuation on an imbalance in the cytokine networks. We now recognize that each of these interrelated mechanisms significantly contributes to RA pathogenesis, including T cells that can help initiate and perpetuate the disease. This article reviews the major components and immune pathways involved in RA and briefly discusses the animal models that contribute to our understanding. Although a unified theory of RA pathogenesis may not be possible at this time, a paradigm is presented that considers the immune pathways that contribute to disease progression and joint destruction. These pathways may have important implications for treatment, because their modulation by biologic response modifiers (BRMs) directed toward specific targets provides benefits to patients with RA. BRMs are a new class of therapeutic agents derived from biologically active molecules and designed to modulate specific immune or inflammatory pathways. Although currently approved BRMs still have limitations, choosing an appropriate target, possibly early rather than late in the immune response, might result in new and improved therapies for RA.

Arthritis, Rheumatoid↗
PubMed2005

Common mechanisms in immune-mediated inflammatory disease.

Characterization of the K/BxN mouse model of spontaneous arthritis contributed to the rediscovery of immune complex-mediated inflammation in rheumatoid arthritis (RA). Serum from these animals can transfer joint-specific inflammation to normal mice. Fc receptors, interleukin 1, mast cells, and complement are all essential for the development of arthritis after serum transfer. In RA, additional amplifying factors have been identified, including cytokines and intracellular signaling molecules, such as mitogen-activated protein kinases and nuclear factor kappa B, that perpetuate inflammation. Understanding the autoimmune and inflammatory pathways implicated in disease has led to targeted drug development and improved clinical outcomes.

Animals↗
PubMed2005

Synovial tissue analysis in clinical trials.

Synovial tissue analysis has considerable potential for future randomized controlled trials (RCT). The synovial membrane is the target tissue in treatment strategies of rheumatoid arthritis and other arthropathies. Effective modulation of synovitis is critical when attempting to control symptoms and signs, to prevent joint damage, and to maintain function. In RCT, the systematic evaluation of changes in synovial tissue after commencing treatment enables identification of an early therapeutic effect, using relatively small numbers of patients. This special interest group is working on establishing the evidence to have this endpoint meet the OMERACT filter criteria.

Antirheumatic Agents↗
PubMed2004

Innate immunity contributes to cochlear adaptive immune responses.

Inner ear immune responses mediated by antigen-specific processes are thought to contribute to hearing loss in humans. Systemic activation of innate immunity contributes to immune responses in the central nervous system. We hypothesized that activation of innate immunity can prime the inner ear for adaptive immune responses and exacerbate disease. Mice were systemically immunized with antigen. Three weeks after initial antigen exposure, the antigen was injected intrathecally coincident with systemic injection of lipopolysaccharide (LPS), an activator of innate immunity. Responses were measured by quantifying the leukocyte infiltrate and cochlear IL-1beta expression. LPS stimulation markedly amplified the adaptive immune response to exogenous antigen in the inner ear. These data indicate that the cochlea is activated by systemic events that stimulate innate immunity and when antigen is present in the inner ear, a robust cochlear adaptive response is generated. If true in humans, this implies that priming may be an important component in the development of immune-mediated hearing loss.

Adjuvants, Immunologic↗
PubMed2004

Telomerase-transduced osteoarthritic fibroblast-like synoviocyte cell line.

To examine whether the life span of fibroblast-like synoviocytes (FLSs) can be extended and to establish FLS cell lines that preserve the characteristics of primary FLSs, we introduced human catalytic subunit of telomerase (hTERT) gene into human osteoarthritic (OA) FLSs. Two hTERT-transduced clonal cell lines were established and one line, hTERT-OA FLS 13A, was characterized. The hTERT-OA FLS 13A cells have a morphology similar to that of the parental untransduced cells and a population-doubling time similar to that of the parental cells of early passages. While the parental untransduced OA FLSs reached senescence after 100 days in culture, the hTERT-OA FLS 13A cells continued to grow at a population-doubling rate of once in about every 2-3 days. The hTERT-OA 13A cells have so far grown in culture beyond 450 days and maintained the same growth rate. Furthermore, the hTERT-OA FLS 13A cells preserved their sensitivity and response to the treatment with basic calcium phosphate crystals and interleukin-1beta. In conclusion, exogenous expression of telomerase represents a way to extend the life span of human FLSs and telomerase-transduced FLS cells offer a promising tool for gene regulation, cell-based assay, cell transplantation-based gene therapy, and tissue engineering research and development.

Aged↗
PubMed2004

p53 tumor suppressor gene mutations in fibroblast-like synoviocytes from erosion synovium and non-erosion synovium in rheumatoid arthritis.

Abnormalities in the p53 tumor suppressor gene have been detected in rheumatoid arthritis (RA) and could contribute to the pathogenesis of chronic disease. To determine whether synoviocytes from invasive synovium in RA have an increased number of mutations compared with non-erosion synoviocytes, p53 cDNA subclones from fibroblast-like synoviocytes (FLS) derived from erosion and non-erosion sites of the same synovium were examined in patients requiring total joint replacement. Ten erosion FLS lines and nine non-erosion FLS lines were established from nine patients with RA. Exons 5-10 from 209 p53 subclones were sequenced (114 from erosion FLS, 95 from non-erosion FLS). Sixty percent of RA FLS cell lines and 8.6% of the p53 subclones isolated from FLS contained p53 mutations. No significant differences were observed between the erosion and non-erosion FLS with regard to the frequency or type of p53 mutation. The majority of the mutations were missense transition mutations, which are characteristic of oxidative damage. In addition, paired intact RA synovium and cultured FLS from the same joints were evaluated for p53 mutations. Matched synovium and cultured synoviocytes contained p53 mutations, although there was no overlap in the specific mutations identified in the paired samples. Clusters of p53 mutations in subclones were detected in some FLS, including one in codon 249, which is a well-recognized 'hot spot' associated with cancer. Our data are consistent with the hypothesis that p53 mutations are randomly induced by genotoxic exposure in small numbers of RA synoviocytes localized to erosion and non-erosion regions of RA synovium. The determining factor for invasiveness might be proximity to bone or cartilage rather than the presence of a p53 mutation.

Arthritis, Rheumatoid↗
PubMed2004

P53 down-regulates matrix metalloproteinase-1 by targeting the communications between AP-1 and the basal transcription complex.

We have previously reported that human matrix metalloproteinase-1 (MMP1) is a p53 target gene subject to down-regulation (Sun et al. [1999]: J Biol Chem 274:11535-11540]. In the present study, we demonstrate that the down-regulation of the human -83MMP1 promoter fragment by p53 was abolished when the -72AP-1 site was eliminated and that a GAL4-cJun-mediated but not a GAL4-Elk1-mediated induction of pFR-luci was effectively inhibited by p53 suggesting an AP-1 dependent but AP-1 binding independent mechanism. Results from gel mobility shift assays were consistent with an AP-1 binding independent mechanism. We also demonstrate that both p300 and TATA box binding proteins cooperated with the transcription factor AP-1 to induce the promoter of MMP1; however, p53 only inhibited the p300-mediated induction of the MMP1 promoter and the inhibition was -72AP-1 dependent. Furthermore, the down-regulation of the MMP1 promoter and mRNA by p53 could be reversed by p300 and by a p53 binding p300 fragment that had no coactivator activity. Taken together, these results indicate that p53 down-regulates MMP1 mainly by disrupting the communications between the transactivator AP-1 and the basal transcriptional complex, which are partially mediated by p300. Finally, by using p53 truncated mutant constructs, we demonstrate that both the N-terminal activation domain and the C-terminal oligomerization domains of p53 were required for the down-regulation of MMP1 transcription.

Binding Sites↗
PubMed2004

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis.

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.

Arthritis, Rheumatoid↗
PubMed2004

Retinoblastoma suppression of matrix metalloproteinase 1, but not interleukin-6, through a p38-dependent pathway in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by increased synovial lining cellularity, inflammation, and destruction of cartilage and bone. During the pathogenesis of RA, synovial fibroblasts reenter the cell cycle and multiply in number. RA synovial fibroblasts express high levels of the MAP kinase p38, which may contribute to the production of interleukin-6 (IL-6) and matrix metalloproteinases (MMPs). IL-6 and MMP-1 promote inflammation and joint destruction, respectively. Taken together, these findings indicate that in RA the enhanced cell cycle activity and production of IL-6 and MMP-1 may be linked. Therefore, we sought to determine if the tumor suppressor gene product retinoblastoma (Rb), a negative regulator of cell cycle activity, inhibits IL-6, MMP-1, and p38 in RA synovial fibroblasts. METHODS: RA and non-RA synovial fibroblasts were examined by enzyme-linked immunosorbent assay (ELISA) for the relative expression of inactive hyperphosphorylated Rb (inactive Rb/total Rb). Ectopic Rb expression was mediated by infection with a replication-defective adenovirus that expresses Rb (Ad-Rb). A control replication-defective adenovirus that expresses beta-galactosidase (Ad-beta-gal) was used. Cell cycle activity was determined by flow cytometry. IL-6 and MMP-1 expression was examined by real-time polymerase chain reaction and ELISA. Expression and activation of p38 were determined by kinase assays and ELISA. The activity of p38 was enhanced by infecting RA synovial fibroblasts with a replication-defective adenovirus that expresses a constitutively active form of MAPK kinase 3 (Ad-CA-MKK3), an upstream activator of p38. RESULTS: Quiescent RA, compared with non-RA synovial fibroblasts, displayed a 200% (P < 0.02) increase in the inactive Rb isoform. Proliferating RA synovial fibroblasts exhibited a 60% (P < 0.12) increase in the inactive Rb isoform compared with non-RA synovial fibroblasts. Increased levels of the active Rb isoform inhibited cell cycle progression and suppressed IL-6 and MMP-1 secretion in RA synovial fibroblasts, although the steady-state levels of IL-6 and MMP-1 messenger RNA remained unchanged. However, Rb overexpression had no effect on spontaneous or IL-1beta-induced production of IL-6 or MMP-1 in non-RA synovial fibroblasts. Ectopic Rb expression reduced the activity of p38. Ad-CA-MKK3 infection in RA synovial fibroblasts increased p38 phosphorylation, and MMP-1 but not IL-6 secretion. In contrast, Rb overexpression inhibited Ad-CA-MKK3-mediated phosphorylation of p38 and subsequent increase in MMP-1. CONCLUSION: Rb-mediated suppression of IL-6 and MMP-1 occurs at a posttranscriptional level. However, Ad-Rb reduction of MMP-1 but not IL-6 requires inhibition of the p38 pathway. These results suggest that Rb negatively regulates p38 activation, leading to decreased MMP-1 secretion in RA synovial fibroblasts.

Adenoviridae↗
PubMed2004

Expression and activation of mitogen-activated protein kinase kinases-3 and -6 in rheumatoid arthritis.

The p38 mitogen-activated protein (MAP) kinase signal transduction pathway regulates the production of interleukin-1 and tumor necrosis factor-alpha. p38 kinase inhibitors are effective in animal models of arthritis and are currently being developed in rheumatoid arthritis (RA). However, little is known about the upstream kinases that control the activation of p38 in RA synovium. In vitro studies previously identified the MAP kinase kinases (MAPKKs) MKK3 and MKK6 as the primary regulators of p38 phosphorylation and activation. To investigate a potential role for MKK3 and MKK6 in RA, we evaluated their expression and regulation in RA synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that MKK3 and MKK6 are expressed in RA and osteoarthritis (OA) synovium. Digital image analysis showed no significant differences between OA and RA with regard to expression or distribution. However, phosphorylated MKK3/6 expression was significantly higher in RA synovium and was localized to the sublining mononuclear cells and the intimal lining. Actin-normalized Western blot analysis of synovial tissue lysates confirmed the increased expression of phosphorylated MKK3/6 in RA. Western blot analysis demonstrated constitutive expression of MKK3 and MKK6 in RA and OA FLS. Phospho-MKK3 levels were low in medium-treated FLS, but were rapidly increased by interleukin-1 and tumor necrosis factor-alpha, although phospho-MKK6 levels only modestly increased. p38 co-immunoprecipitated with MKK3 and MKK6 from cytokine-stimulated FLS and the complex phosphorylated activating transcription factor-2 in an in vitro kinase assay. These data are the first documentation of MKK3 and MKK6 activation in human inflammatory disease. By forming a complex with p38 in synovial tissue and FLS, these kinases can potentially be targeted to regulate the production of proinflammatory cytokine production in inflamed synovium.

Arthritis, Rheumatoid↗
PubMed2004

Rheumatoid arthritis: regulation of synovial inflammation.

Rheumatoid arthritis (RA) is a systemic, inflammatory autoimmune disorder that presents as a symmetric polyarthritis associated with swelling and pain in multiple joints, often initially occurring in the joints of the hands and feet. Articular inflammation causes activation and proliferation of the synovial lining, expression of inflammatory cytokines, chemokine-mediated recruitment of additional inflammatory cells, as well as B cell activation with autoantibody production. A vicious cycle of altered cytokine and signal transduction pathways and inhibition of programmed cell death contribute to synoviocyte and osteoclast mediated cartilage and bone destruction. A combination of targeted interventions at various stages in the pathogenesis of RA will likely be required to control symptoms in certain patients with this complex and potentially disabling disease. The regulation of rheumatoid synovial inflammation will be reviewed, followed by a brief summary of the therapeutic implications of these advances, including strategies targeting key cytokines, signal transduction molecules, co-stimulatory molecules, B cells, chemokines, and adhesion molecules.

Arthritis, Rheumatoid↗
PubMed2004

Signal transduction in rheumatoid arthritis.

PURPOSE: Signal transduction pathways are the intracellular mechanism by which cells respond and adapt to environmental stress. Understanding the critical networks in diseases like rheumatoid arthritis can potentially identify novel therapeutic targets. RECENT FINDINGS: Dissecting the complex pathways involved in rheumatoid synovitis, including mitogen-activated protein kinases, NF-kB, tumor suppressors, Janus kinases, the signal transducer and activator of transcription, suppressors of cytokine stimulation, and toll-like receptors may lead to new approaches to inflammatory arthritis. For instance, targeting NF-kB via IkB kinase 2 with specific inhibitors may block an array of proinflammatory cytokines that contribute to synovitis. Inhibition of Janus kinases and p38 could block metalloproteinase expression and protect the extracellular matrix. Overexpression of suppressors of cytokine stimulation and inhibition of signal transducer and activator of transcription are additional approaches that have demonstrated efficacy in animal models of arthritis. Tumor suppressor proteins and cell cycle inhibitors represent additional targets with unexpected anti-inflammatory activities. Recent evidence also suggests that targeting toll-like receptors may regulate cytokine expression in rheumatoid arthritis. SUMMARY: Multiple signal transduction pathways have been implicated in rheumatoid arthritis, and preclinical models have confirmed the therapeutic potential of small molecule inhibitors. Orally bioavailable inhibitors of the mitogen-activated protein kinase and NF-kB pathways have been designed and are currently being evaluated. Many other pathways could be targeted and offer new therapeutic options for rheumatoid arthritis.

Arthritis, Rheumatoid↗
PubMed2004

The T cell cometh: interplay between adaptive immunity and cytokine networks in rheumatoid arthritis.

The etiology of autoimmunity in humans remains poorly defined, and animal models provide a unique opportunity to study potential autoimmune mechanisms. A novel model of autoimmune inflammatory arthritis results from a point mutation in the zeta-associated-protein of 70 kDa (ZAP-70), which causes abnormal thymic T cell selection and survival of autoreactive clones. Although the resulting clinical and pathologic abnormalities are clearly T cell-dependent, macrophage and fibroblast cytokines such as IL-1 and TNF-alpha are required for full expression of the disease. The studies of Hata et al. raise the intriguing possibility that traditional proinflammatory cytokine networks represent common effector mechanisms in inflammatory joint diseases such as rheumatoid arthritis. Hence, effective therapeutic interventions can target either unique etiologic pathways related to adaptive immune responses or shared terminal mechanisms.

Animals↗
PubMed2004

Wnt1 inducible signaling pathway protein-3 regulation and microsatellite structure in arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) synovial tissue expresses several embryonic gene families, including wingless (wnt) and their receptors, frizzled (fz). The Wnt proteins, including Wnt-1, activate the Wnt inducible signaling pathway proteins (WISP), which are members of the CCN family that regulate cell growth and differentiation. WISP3 is of particular interest because it contains a microsatellite region in its coding region that is susceptible to frameshift mutations and leads to a truncated protein. To investigate the contribution of WISP3 to synovial inflammation, we evaluated its expression and regulation in arthritis. METHODS: mRNA and protein expression of WISP3 were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. For mutation analysis, PCR product amplified from genomic DNA of synovial tissue and cultured fibroblast-like synoviocytes (FLS) was subcloned and sequenced. RESULTS: WISP3 mRNA is expressed in synovial tissue, but is 11-fold higher in RA than osteoarthritis (OA) or normal samples. Surprisingly, WISP3 protein levels are similar in RA, OA, and normal synovium samples. Immunohistochemistry of synovial tissue reveals that WISP3 protein is located primarily in the synovial intimal lining. WISP3 mRNA expression is also 6-fold higher in RA FLS compared with OA FLS and 50-fold higher in RA than in normal FLS. When RA FLS are stimulated with interleukin 1 or tumor necrosis factor-a, WISP3 mRNA is significantly increased. The cytokines also increase WISP3 mRNA in OA FLS, but the maximal level in stimulated OA FLS is still less than medium-treated RA FLS. Mutation analysis in the coding region microsatellite of the WISP3 gene in RA and OA synovium and FLS shows a limited number of insertion and deletion mutations. CONCLUSION: WISP3 gene expression is higher in RA synovium and FLS compared with OA and normal synovial tissue and is further induced by proinflammatory cytokines in vitro. Protein levels are not increased, indicating discoordinate regulation of WISP3 protein and mRNA. Although functionally relevant mutations were observed in genomic DNA, they were noted in both OA and RA samples.

Arthritis, Rheumatoid↗