Cyclooxygenase 2‐dependent prostaglandin E2 modulates cartilage proteoglycan degradation in human osteoarthritis explants

Objective

To examine cyclooxygenase‐2 (COX‐2) enzyme expression, its regulation by interleukin‐1β (IL‐1β), and the role of prostaglandin E₂ (PGE₂) in proteoglycan degradation in human osteoarthritic (OA) cartilage.

Methods

Samples of human OA articular cartilage, meniscus, synovial membrane, and osteophytic fibrocartilage were obtained at knee arthroplasty and cultured ex vivo with or without IL‐1β and COX inhibitors. COX expression was evaluated by immunohistochemistry and Western blot analysis. The enzymatic activity of COX was measured by conversion of arachidonic acid to PGE₂. Cartilage degradation was evaluated by measuring the accumulation of sulfated glycosaminoglycans in the medium.

Results

IL‐1β induced robust expression of COX‐2 and PGE₂ in OA meniscus, synovial membrane, and osteophytic fibrocartilage explants, whereas low levels were produced in OA articular cartilage. IL‐1β also induced cartilage proteoglycan degradation in OA synovial membrane‐cartilage cocultures. Increased proteoglycan degradation corresponded to the induction of COX‐2 protein expression in, and PGE₂ production from, the synovial membrane. Dexamethasone, neutralizing IL‐1β antibody, or the selective COX‐2 inhibitor, SC‐236, attenuated both the IL‐1β‐induced PGE₂ production and cartilage proteoglycan degradation in these cocultures. The addition of PGE₂ reversed the inhibition of proteoglycan degradation caused by SC‐236.

Conclusion

IL‐1β‐induced production of COX‐2 protein and PGE₂ was low in OA articular cartilage compared with that in the other OA tissues examined. IL‐1β‐mediated degradation of cartilage proteoglycans in OA synovial membrane‐cartilage cocultures was blocked by the selective COX‐2 inhibitor, SC‐236, and the effect of SC‐236 was reversed by the addition of exogenous PGE₂. Our data suggest that induction of synovial COX‐2‐produced PGE₂ is one mechanism by which IL‐1β modulates cartilage proteoglycan degradation in OA.

     

Effects of cyclooxygenase-2 on human esophageal squamous cell carcinoma

AIM:To study the relationship between the cyclooxy-genase(COX)-2 gene and the proliferation and apopto-sis of esophageal squamous carcinoma EC109 cells.METHODS:The techniques of RNA interference(RNAi)and cell transfection,as well as the levels of oncogenic-ity in nude mice,were used to study the role of COX-2 in the esophageal squamous carcinoma cell(ESCC)line EC109.Following RNAi and transfection,Western blot-ting analysis was used to determine the expression of the COX-2 protein.The 3-(4,5-dimethylthiazol...     

Expression of ECRG4, a novel esophageal cancer－related gene,downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma

AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR. RESULTS: The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80 %) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing. CONCLUSION: These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.     

S100A4 regulates motility and invasiveness of human esophageal squamous cell carcinoma through modulating the AKT/Slug signal pathway

The involvement of S100A4 in modulating invasiveness of esophageal squamous cell carcinoma (ESCC) cell lines was explored. It was shown that S100A4 expression is positively correlated with the degree of invasiveness in human ESCC cells. The S100A4‐rich EC‐1 cells displayed higher migratory and invasive cell behavior while ET‐1 cells with low S100A4 expression levels displayed lower migratory and invasive cell behavior. S100A4 silencing by small interfering (siRNA) in EC‐1 cells induced E‐cadherin expression, and overexpression of S100A4 in a lowly invasive TE‐1 cells suppressed E‐cadherin expression. It is suggested that S100A4 silencing inhibit invasion via E‐cadherin upregulation, and overexpression of S100A4 promote invasion via E‐cadherin downregulation in ESCC cells. Compared with the vector‐transfected cells, S100A4 silencing in EC‐1 cells showed reduced ability of migration and invasiveness, and overexpression of S100A4 in TE‐1 cells showed increased ability of migration and invasiveness via wound‐healing and Transwell assay, and pseudometastatic model assay. Furthermore, re‐expression of S100A4 could increase the invasive phenotypes in S100A4 siRNA transfected EC‐1 cells, and S100A4 silencing could decrease the invasive phenotypes in S100A4 circular DNA (cDNA) transfected TE‐1 cells. It was found that Slug is downregulated in S100A4 siRNA transfected EC‐1 cells, and Slug is upregulated in S100A4 cDNA transfected TE‐1 cells. It was also discovered S100A4 cDNA induced protein kinase B (AKT) phosphorylation at Serine‐473(phospho‐AKT [p‐AKT]) levels, followed by the Slug upregulation, and S100A4 siRNA decreases the phospho‐AKT levels, followed by the Slug downregulation. The data suggested that S100A4 could regulate migratory and invasive behavior of human ESCC cells through modulating AKT/Slug pathway.     

Elevated plasma prostaglandins and acetylated histone in monocytes in Type 1 diabetes patients

Aims/hypothesis Inflammation is implicated in diabetes and cyclooxygenase (COX) is involved in vascular inflammatory processes, participating in both atherosclerosis and thrombosis. The aims were to determine whether levels of monocyte COX and plasma COX metabolites are increased in Type 1 diabetic patients and to determine whether these could be linked to histone hyperacetylation. Materials and methods Monocytes from 19 Type 1 diabetic and 39 non‐diabetic control subjects were probed for COX and acetylated histone H4 proteins by immunoblotting. Plasma COX metabolite levels [thromboxane B₂ (TXB₂) and prostaglandin E₂ (PGE₂)] were determined by enzyme immunoassay. Results Monocyte COX‐2 expression was significantly up‐regulated (1.3‐fold) in diabetic relative to the non‐diabetic control subjects and plasma PGE₂ was markedly elevated (2.7‐fold). In diabetic subjects, monocyte acetylated histone H4 levels were significantly elevated; sub‐group analysis indicated that the increased histone acetylation was found only in the complication‐free group. Conclusions Results support increased inflammatory activity in Type 1 diabetes that involves COX‐2 and increased prostaglandin production, which may predispose patients to cardiovascular events. The observation of elevated histone acetylation only in complication‐free diabetic subjects suggests that this may be a protective mechanism. This merits further investigation as histone hyperacetylation has been associated with reduced expression of factors involved in vascular injury and remodelling.     

5-Aza-CdR对人食管癌细胞株中T-cadherin基因表达和细胞增殖的影响

背景：T-cadherin在肿瘤的发生中可能扮演肿瘤抑制基因的角色．在食管癌等多种肿瘤中的表达明显下降。目的：探讨去甲基化制剂5-氮杂-2’-脱氧胞苷（5-Aza-CdR）对人食管癌细胞株EC109中T—cadherin基因表达和细胞增殖的影响。方法：常规培养人食管癌细胞株EC109．并分为5μmol／L5-Aza-CdR组和对照组。以甲基化特异性PCR（MSP）法检测T-cadherin基因启动子区甲基化状态．RT-PCR和蛋白质印迹法分别检测T-cadherinmRNA和蛋白表达,MTr法检测EC109细胞增殖。结果：对照组EC109细胞中T-cadhefin基因启动子区呈异常甲基化状态．5-Aza-CdR干预可逆转甲基化状态。与对照组相比,5-Aza-CdR组T-cadherin基因mRNA和蛋白表达均显著增高（P〈0．01）．细胞增殖明显受到抑制。结论：去甲基化制剂5-Aza-CdR通过逆转食管癌细胞中T-cadherin基因启动子区甲基化状态来增强其表达．并抑制肿瘤细胞增殖．     

Bradykinin potentiates cytokine‐induced prostaglandin biosynthesis in osteoblasts by enhanced expression of cyclooxygenase 2, resulting in increased RANKL expression

Objective

Bradykinin (BK) stimulates bone resorption in vitro and synergistically potentiates interleukin‐1 (IL‐1)–induced bone resorption and prostaglandin (PG) formation, suggesting that kinins are important in inflammation‐induced bone loss. The present study was undertaken to study 1) the role of the kinin B1 and B2 receptors in the synergistic interaction with IL‐1 and tumor necrosis factor α (TNFα), 2) the molecular mechanisms involved in synergistic enhancement of PG formation, and 3) the effects of kinins on cytokine‐induced expression of RANKL, RANK, and osteoprotegerin (OPG) (the latter being crucial molecules in osteoclast differentiation).

Methods

Formation of PGs, expression of enzymes involved in arachidonic acid metabolism, and expression of RANKL, RANK, and OPG were assessed in the human osteoblastic cell line MG‐63 and in mouse calvarial bones. The role of NF‐κB and MAP kinases was studied using pharmacologic inhibitors.

Results

PGE₂ formation and cyclooxygenase 2 (COX‐2) protein expression were induced by IL‐1β and potentiated by kinins with affinity for the B1 or B2 receptors, resulting in PGE₂‐dependent enhancement of RANKL. The enhancements of PGE₂ formation and COX‐2 were markedly decreased by inhibition of p38 and JNK MAP kinases, whereas inhibition of NF‐κB resulted in abolishment of the PGE₂ response with only slight inhibition of COX‐2.

Conclusion

Kinin B1 and B2 receptors synergistically potentiate IL‐1– and TNFα‐induced PG biosynthesis in osteoblasts by a mechanism involving increased levels of COX‐2, resulting in increased RANKL. The synergistic stimulation is dependent on NF‐κB and MAP kinases. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including that in rheumatoid arthritis.