017 INHIBITORY INTERACTIONS BETWEEN HMGB1 AND HEME OXYGENASE-1 PATHWAYS IN OSTEOARTHRITIC SYNOVIOCYTES

Abstract

Purpose: Extracellular high mobility group box 1 (HMGB1) has been demonstrated to participate in inflammatory processes in joint diseases. Activation of osteoarthritic synoviocytes by proinflammatory cytokines results in HMGB1 translocation and release. We have shown recently a potential modulation of HMGB1 by heme oxygenase-1 (HO-1). In this study, we have examined the interactions between both pathways in osteoarthritic synoviocytes. Methods: Synovial tissue samples were obtained from 15 osteoarthritic patients undergoing total knee joint replacement. Synoviocytes were obtained by digestion with collagenase and cultured until third passage. Cells were treated with human recombinant HMGB1 (15 ng/ml) in the presence or absence of IL-1β (100 U/ml). HO-1 was induced by cobalt protoporphyrin IX (CoPP). Lentiviral HO-1.flag vector was also used for HO-1 overexpression. HO-1 gene silencing was achieved by using a specific siRNA. Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. HMGB1 translocation into the cytoplasm was studied by immunofluorescence. Results: Treatment of synoviocytes with HMGB1 down-regulated HO-1. This effect was potentiated by IL-1β and accompanied by a significant increase in matrix metalloproteinase (MMP)-1 and MMP-3 gene expression and activity. Conversely, induction of HO-1 by CoPP in the presence of IL-1 β led to reduced expression of both protein and mRNA of HMGB1. These effects were accompanied by a significant reduction in gene expression of MMP-1 and MMP-3, and MMP activity. The consequences of HO-1 induction were counteracted by HO-1 gene silencing. Interestingly, we observed a marked reduction in the translocation of HMGB1 from the nucleus into the cytoplasm. Transfection with lentiviral HO-1.flag vector confirmed the inhibitory effect of HO-1 on HMGB1 translocation. Conclusions:We have provided direct evidence that HO-1 inhibits the translocation of HMGB1 into the cytoplasm. Our data indicate a reciprocal negative regulation between HO-1 and HMGB1 pathways in osteoarthritic synoviocytes.