Hsp90 beta inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes

Abstract

Background: Hsp90 beta is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90 beta in key aspects of OA pathogenesis. Methods: Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1 beta or TNF-alpha) and nitric oxide donors (NOC-12 or SNP). For Hsp90 beta inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90 beta in stimulated cells. Results: Hsp90 beta was found to be increased by proinflammatory cytokines. Inhibition of Hsp90 beta by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1 beta in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90 beta. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes. Conclusions: The present results show how Hsp90 beta modulates NO production and NO-mediated cellular death in human OA chondrocytes.