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Objective. To compare induction of the aggrecanases (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15) by interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣) in chondrocyte-like OUMS-27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.Methods. OUMS-27 cells were stimulated for different periods of time and with various concentrations of IL-1␤ and/or TNF␣. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL-1␤ and/or TNF␣. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real-time polymerase chain reaction and Northern blotting. ADAMTS-9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL-1␤-stimulated OUMS-27 cells was investigated.Results. IL-1␤ increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL-1␤ stimulation was greater in chondrocytes than in fibroblasts. The combination of IL-1␤ and TNF␣ had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS-9 protein was also induced in IL-1␤-stimulated OUMS-27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 upregulation in OUMS-27 cells.Conclusion. ADAMTS9 is an IL-1␤-and TNF␣-inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS-9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS-9 may have a pathologic role in arthritis.

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is a member of the matrix metalloproteinase family. We have previously reported that ADAMTS1 was strongly expressed in myocardial infarction. In this study, we investigated whether hypoxia induced ADAMTS1 and investigated its regulatory mechanism. In hypoxia, the expression level of ADAMTS1 mRNA and protein rapidly increased in endothelial cells, but not in other cell types. Interestingly, the induction of ADAMTS1 by hypoxia was transient, whereas vascular endothelial growth factor induction by hypoxia in human umbilical vein endothelial cells (HUVEC) increased in a time-dependent manner. CoCl 2 , a transition metal that mimics hypoxia, induced ADAMTS1 in HUVEC. The phosphatidylinositol 3-kinase inhibitor LY294002 dose-dependently inhibited the increase of ADAMTS1 mRNA expression in hypoxia. We characterized the promoter region of ADAMTS1, and the secreted luciferase assay system demonstrated that hypoxia induced luciferase secretion in the culture medium 4.6-fold in HUVEC. In the promoter region of ADAMTS1, we found at least three putative hypoxiainducible factor (HIF) binding sites, and the chromatin immunoprecipitation assay revealed HIF-1 binding to HIF binding sites in the promoter region of ADAMTS1 under hypoxia. Recombinant ADAMTS1 protein promoted the migration of HUVEC under hypoxic conditions. In summary, we found that ADAMTS1 is transiently induced by hypoxia in endothelial cells, and its transcription is mediated by HIF-1 binding. Our data indicate that ADAMTS1 is a novel acute hypoxia-inducible gene.ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is a newly discovered matrix metalloproteinase with multiple domains, including propeptide, metalloproteinase, disintegrin, and spacer region domains, and domains containing thrombospondin type I motifs (1). The ADAMTS family now contains 19 members. One of the best characterized biological functions of ADAMTS is proteolytic activity against extracellular matrix proteins, including proteoglycans (2, 3). In arthritic cartilage, aggrecan breakdown, mediated by members of ADAMTS, not matrix metalloproteinases, is one of the features of the progressive stage. ADAMTS5 (aggrecanase-2) null mice show protection against experimental arthritis (4, 5).ADAMTS1 was originally cloned from a murine colon carcinoma cell line (6). Recently, it has been reported that a polymorphism of ADAMTS1 is closely associated with the occurrence of ischemic heart disease (7). In the cardiovascular system, ADAMTS1 plays roles in atherosclerosis by degrading versican (8) and TFPI-2 (tissue factor pathway inhibitor-2) (9). We have previously reported that ADAMTS1 was induced in the infarcted heart (10); however, the regulatory mechanism of ADAMTS1 in ischemia is unknown.Low oxygen tension (hypoxia) affects endothelial cells in a number of ways, including the transcriptionally regulated expression of vasoactive substances and matrix proteins (11). One mechanism of the regulation of gene expre...

Thrombospondin-1 (TSP-1) is a multifunctional, rapid-turnover matricellular protein. Recent studies demonstrated that TSP-1 has a role in regulating inflammatory reactions. Myocardial infarction (MI) is associated with an inflammatory response, ultimately leading to healing and scar formation. In particular, an enhanced inflammatory reaction and a massive accumulation of monocytes/macrophages is seen with reperfusion after MI. To examine the role of TSP-1 in MI, we isolated rat TSP-1 complementary DNA (cDNA) and analyzed the level and distribution of the mRNA expression. In infarcted rat hearts, TSP-1 mRNA increased markedly at 6 and 12 hrs after coronary artery ligation (27.97 +/- 3.40-fold and 22.77 +/- 1.83-fold, respectively, compared with sham-operated hearts). Western blot analysis revealed that TSP-1 protein was transiently induced in the infarcted heart. Using in situ hybridization analysis, TSP-1 mRNA signals were observed in the infiltrating cells at the border area of infarction. We then examined the effect of ischemia/reperfusion (I/R) on TSP-1 mRNA induction in the rats with infarcted hearts. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that I/R enhanced the TSP-1 mRNA expression approximately 4-fold, as compared with the level in the permanently ligated heart. Finally, we examined the effect of TSP-1 on proinflammatory cytokine release in mononuclear cells. The releases of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) from human mononuclear cells were enhanced by TSP-1 in a dose-dependent manner. Thus, the immediate and marked increase of TSP-1 expression suggests that TSP-1 has an inflammatory-associated role in MI.

Abbreviations: bp, base pairs; hr, hours; kbp, kilo base pairs; min, minutes; RNA, ribonucleic acid; sec, seconds Toeda et al, Versican expression in myocardial infarction 2 AbstractVersican, a large chondroitin sulfate proteoglycan, plays a role in conditions such as wound healing and tissue remodeling.To test the hypothesis that versican is transiently expressed and plays a role in the infarcted heart, we examined its expression in a rat model of myocardial infarction.Northern blot analysis demonstrated increased expression of versican mRNA. Quantitative real-time RT-PCR analysis revealed that versican mRNA began to increase as early as 6 hours and reached its maximal level 2 days after coronary artery ligation. Versican mRNA then gradually decreased, while the mRNA of decorin, another small proteoglycan, increased thereafter. gene by monocytes in the infarcted heart. We suggest that upregulation of versican in the infarcted myocardium may have a role in the inflammatory reaction, which mediates subsequent chemotaxis in the infarcted heart.