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A., Schneider C., Rosen N., Hartl F. the first time the function of extracellular Hsp90 in promoting tumor angiogenesis. (8) reported that extracellular Hsp90 can interact with matrix metalloproteinase 2 (MMP-2) and that the impermeable inhibitor of Hsp90 (immobilized geldanamycin) inhibits MMP-2 proteolytic activity, but the ND-646 regulatory molecular mechanism behind this phenomenon is still a mystery. In the present study, to further elucidate the molecular mechanism of extracellular Hsp90 ND-646 function, we have investigated the regulatory mechanism of extracellular Hsp90 on MMP-2 activity. We reveal that extracellullar Hsp90 stabilizes MMP-2 and protects it from processing and subsequent inactivation in tumor cells. The regulatory function of Hsp90 on MMP-2 processing is usually isoform-specific and ATP-independent. The conversation of Hsp90 and MMP-2 is usually mediated by the middle domain name of Hsp90 and the C-terminal hemopexin domain name of MMP-2. Moreover, we further confirm this mechanism in endothelial cells, which can secrete increasing amounts of Hsp90 upon the treatment ND-646 of VEGF. The effects of recombinant human Hsp90 (rHsp90) and the Hsp90 antibody on angiogenesis and were also examined. The result shows that rHsp90 promotes whereas the antibody of Hsp90 suppresses angiogenesis in an MMP-2-dependent manner, suggesting that extracellular Hsp90 is usually a potential therapeutic target for not only tumor metastasis but also tumor angiogenesis. EXPERIMENTAL PROCEDURES Cell Lines and Transfectants Human breast malignancy cell lines MDA-MB-231 and MCF-7 and mouse melanoma cell line B16/F10 were from the American Type Culture Collection. HMEC is usually a human dermal microvascular endothelial cell line (Sciencell) transfected with SV40 large T antigen (13). Human umbilical ND-646 venous endothelial cells (HUVECs) were isolated from human umbilical vein (14). The stable cell line overexpressing MMP-2 was screened by G418 (200 g/ml) from MCF-7 cells transfected with pcDNA3.1-MMP-2. Antibodies Anti-Myc, anti-His, and anti-CD31 antibody (PECAM-1, M-20) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MMP-2 antibody (Ab-7) for Western blotting was from Calbiochem (Darmstadt, Germany). Anti-Hsp90 antibody (9D2) was from Stressgen Bioreagents (Victoria, Canada). Anti-Hsp90 antibody (H90C10) was from Abcam (Cambridge, UK). Anti-FLAG antibody was from Sigma. Monoclonal antibody against Hsp90 (Hsp90 mAb) and Hsp90 (Hsp90 mAb) for endothelial cell transmigration, tube formation, and tumor growth assay was prepared by our laboratory. The specificity and efficiency were confirmed by ELISA. MMP-2 Processing Assay in Vitro This assay was performed according to the previous report (15). Purified rMMP-2 was incubated with the indicated proteins with or without ATP (2 mm) at 37 C for different periods. The incubation buffer contains 40 mm HEPES, 10 mm MgCl2, 20 mm KCl, 5 mm CaCl2, 2 mm and and and and and and and and and was quantified by Gel-Pro Analyzer software. The percentage of MMP-2 activation (activated MMP-2/ProMMP-2) and inactivation processing (MMP-2 30-kDa inactive fragment/total MMP-2 proteins) was analyzed. and noncell system. Because Hsp90 is an ATP-dependent molecular chaperone in the cytosol Mouse monoclonal to TYRO3 (17, 18), we first examined whether the stabilization effect of Hsp90 on MMP-2 is usually ATP-dependent. Purified rProMMP-2 was mixed with PBS, equal molar of Hsp90 or Hsp90 with or without ATP, and then incubated at 37 C for 3 h. The autocatalytic processing products of ProMMP-2 were assayed by Western blotting. It was found that Hsp90 but not Hsp90 can safeguard MMP-2 from autocatalytic processing and inactivation and that ATP exhibited no effect on the stabilization activity of both Hsp90 and Hsp90 (supplemental Fig. S3was calculated according to the intensity of the bands, which was quantified by Gel-Pro Analyzer software. was quantified using the same method as ND-646 that in of Fig. 3top of the panel). of the panel) with MMP-2. and and of Fig. 3and and supplemental Fig. S5and supplemental Fig. S5(Fig. 5and represent S.D. (= 5). value: Student’s test. *, 0.05; **, 0.01; #, 0.05. represent S.D. (= 5). value: Student’s test. *, 0.05; **, 0.01; #, 0.05. represent S.D. (= 5). value: Student’s test. *, 0.05; **, 0.01; #, 0.05. and and supplemental Fig. S6and supplemental Fig. S6and and for 25 days. represent S.D. (= 5). value: Student’s test. *, .