Articular cartilage is a unique tissue in that it is avascular with its nutrition and oxygen supply being dependent on the diffusion of solutes through the synovial fluid and to and from the subchondral bone. The oxygen levels in articular cartilage, therefore, are assumed to be low. Oxygen is an important modulator of gene expression and this regulation occurs largely through the activation of the transcriptional complex hypoxia-inducible factor-1 (HIF-1). However, little is known about how articular cartilage regulates genes in response to O(2)tension and whether this regulation occurs through HIF-1.The aim of this study was to investigate the expression profile of HIF-1alpha in normal and osteoarthritic (OA) chondrocytes under normoxic and hypoxic conditions, and in response to treatment with tumor necrosis factor alpha (TNFalpha).Articular chondrocytes from human normal and OA knee cartilage were isolated and cultured in suspension under normoxic (21% O(2)) or hypoxic conditions (1% O(2)). Chondrocytes were also treated with TNF-alpha under normoxic conditions. Nuclear extracts and total RNA were prepared and HIF-1alpha protein and mRNA levels were assayed by immunoblotting and Northern hybridization. Localization of HIF-1alpha by immunofluorescence was performed on frozen sections of cartilage tissue by confocal microscopy.HIF-1alpha expression was detectable in human normal and OA chondrocytes and cartilage by Northern analysis, immunoblotting and immunofluorescence under normoxic conditions. Culture of OA or normal chondrocytes under hypoxic conditions for up to 16h resulted in a modest stabilization and/or increase of HIF-1alpha expression. Treatment of articular chondrocytes with TNFalpha resulted in an increase in HIF-1alpha protein steady state levels under normoxic conditions. The increase in HIF-1alpha expression induced by TNFalpha was partially blocked by pretreatment of the chondrocytes with inhibitors of NFkappaB or p38 MAP kinase. We also observed the expression of HIF-2alpha mRNA in human chondrocytes.HIF-1alpha is expressed in human normal and OA articular chondrocytes cultured under normoxic conditions. HIF-1alpha can be further induced or stabilized in articular chondrocytes by hypoxia or by treatment with TNFalpha. The relatively high constitutive expression of HIF-1alpha by chondrocytes may be an important adaptation to survival in the avascular-hypoxic environment of cartilage. Modulation of HIF-1alpha levels by TNF-alpha may have important implications for chondrocyte metabolism during degenerative joint disease. In addition, we detected for the first time the expression of HIF-2alpha mRNA in chondrocytes.
