1 We examined the e ects of endogenous prostaglandin E 2 (PGE 2 ) on the production of interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF) by interleukin-1b (IL-1b)-stimulated human synovial ®broblasts. 2 NS-398 (1 mM), a cyclo-oxygenase-2 (COX-2) inhibitor, inhibited IL-6 and VEGF production (35+4% and 26+2%, respectively) but enhanced M-CSF production (38+4%) by IL-1b (1 ng ml 71 ) in synovial ®broblasts isolated from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Exogenous PGE 2 completely abolished the e ects of NS-398 on the production of each mediator by OA ®broblasts stimulated with IL-1b. 3 8-Bromo cyclic AMP and dibutyryl cyclic AMP, cyclic AMP analogues, mimicked the e ects of PGE 2 on IL-6, M-CSF, and VEGF production by OA ®broblasts. 4 The EP 2 selective receptor agonist ONO-AE1-259 (2 nM) and the EP 4 selective receptor agonist ONO-AE1-329 (2 or 20 nM), but not the EP 1 selective receptor agonist ONO-DI-004 (1 mM) and the EP 3 selective receptor agonist ONO-AE-248 (1 mM), replaced the e ects of PGE 2 on IL-6, M-CSF, and VEGF production by OA and RA ®broblasts stimulated with IL-1b in the presence of NS-398. 5 Both OA and RA ®broblasts expressed mRNA encoding EP 2 and EP 4 but not EP 1 receptors. In addition, up-regulation of EP 2 and EP 4 receptor mRNAs was observed at 3 h after IL-1b treatment. 6 These results suggest that endogenous PGE 2 regulates the production of IL-6, M-CSF, and VEGF by IL-1b-stimulated human synovial ®broblasts through the activation of EP 2 and EP 4 receptors with increase in cyclic AMP.

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Rheumatoid arthritis (RA) is characterized by a chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages, and plasma cells, all of which manifest signs of activation. Recent studies have revealed the essential role of osteoclasts in joint destruction in RA. Src family tyrosine kinases are implicated in various intracellular signaling pathways, including mitogenic response to growth factors in fibroblasts, activation of lymphocytes, and osteoclastic bone resorption. Therefore, inhibiting Src activity can be a good therapeutic strategy to prevent joint inflammation and destruction in RA. We constructed an adenovirus vector carrying the csk gene, which negatively regulates Src family tyrosine kinases. Csk overexpression in cultured rheumatoid synoviocytes remarkably suppressed Src kinase activity and reduced their proliferation rate and IL-6 production. Bone-resorbing activity of osteoclasts was strongly inhibited by Csk overexpression. Furthermore, local injection of the virus into rat ankle joints with adjuvant arthritis not only ameliorated inflammation but suppressed bone destruction. In conclusion, adenovirus-mediated direct transfer of the csk gene is useful in repressing bone destruction and inflammatory reactions, suggesting the involvement of Src family tyrosine kinases in arthritic joint breakdown and demonstrating the feasibility of intervention in the kinases for gene therapy in RA.

Three of four natural compounds, which are caffeic acid, eupatilin and 4'-demethyleupatilin, isolated from Chinese plant, Artbmisia rubripes Nakai selectively inhibited 5lipoxygenase of cultured mastocytoma cells. Half-inhibition doses (1.50) for caffeic acid, eupatilin and 4' -demethyleupatilin were 3.7, 14 and 18 x 10e6 M, respectively. The inhibition by caffeic acid was non-competitive types. Prostaglandin synthase activities were little inhibited by eupatilin and 4'-demethyleupatilin, but rather stimulated by affeic acid. The formation of leukotriene C4 and D4 by mast tumor cells was almost completely suppressed by these compounds at 10m4 M. J-Lipoxygenase Inhibitor leukotriene Caffeic acid Prostaglandin synthease Lipoxygenase

Osteoarthritis (OA) is the most prevalent joint disease and is characterized by pain and functional loss of the joint. However, the pathogenic mechanism of OA remains unclear, and no drug therapy for preventing its progress has been established. To identify genes related to the progress of OA, the gene expression profiles of paired intact and damaged cartilage obtained from OA patients undergoing joint substitution were compared using oligo microarrays. Using functional categorization combined with gene ontology and a statistical analysis, five genes were found to be highly expressed in damaged cartilage (HBEGF, ASUS, CRLF1, LOX, CDA), whereas three genes were highly expressed in intact tissues (CHST2, PTPRD, CPAN6). Among these genes, the upregulated expression of CRLF1 was reconfirmed using real-time PCR, and the in vivo expression of CRLF1 was detected in clusters of chondrocytes and fibrocartilage-like cells in damaged OA cartilages using in situ hybridization. In vitro, the transcriptional level of CRLF1 was positively regulated by TGF-beta1 in the mouse chondrogenic cell line ATDC5. Additionally, the CRLF1/CLC complex promoted the proliferation of ATDC5 cells and suppressed the expression level of aggrecan and type II collagen. Our data suggest that the CRLF1/CLC complex disrupts cartilage homeostasis and promotes the progress of OA by enhancing the proliferation of chondrocytes and suppressing the production of cartilage matrix. A component of the complex, CRLF1, may be useful as a biomarker of OA; and the corresponding receptor is a potential new drug target for OA.