ABSTRACT:We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF-1a)-dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl 2 , which induces HIF-1a, to simulate hypoxia, or transfected with siRNAs targeting HIF-1a (si-HIF-1a) and HSP70 (si-HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF-1a under hypoxia or simulated hypoxia, but not in the presence of si-HIF-1a. Hypoxia-induced overexpression of ECM genes was significantly suppressed by si-HIF-1a or si-HSP70. Cell viability positively correlated with hypoxia, but transfection with si-HIF-1a or si-HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside-treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si-HIF-1a or si-HSP70 almost completely blocked these effects. These findings indicated that HIF-1a-induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF1a may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. Keywords: hypoxia-inducible factor-1a; heat shock protein 70; hypoxia; extracellular matrix; apoptosis Articular cartilage is an avascular tissue that derives its nutrition and oxygen supply via diffusion from synovial fluid and subchondral bone. It has been estimated that chondrocytes at the articular surface are exposed to approximately 6-10% oxygen (O 2 ), whereas chondrocytes in the deepest layers of the articular cartilage are exposed to only 1-5% O 2 . 1 Several studies have shown that chondrocytes can survive hypoxic conditions by adjusting their metabolism. 2,3 Oxygen is an important modulator of gene expression, with hypoxia-inducible factor (HIF) being the primary regulatory factor responding to variations in O 2 levels. 4,5 The transcription factor HIF-1alpha (HIF-1a) is important in maintaining proper cellular functions under hypoxic conditions. 5 In addition, HIF1a is essential for chondrogenesis, including chondrocyte growth arrest, survival, maturation, and apoptosis. 6 HIF-1a also promotes the synthesis of relevant extracellular matrix (ECM) components. 2 However, these HIF-1a-dependent anabolic pathways in chondrocytes remain poorly understood.Among the genes regulated via the HIF-1a pathway under hypoxic conditions are the highly conserved heat shock proteins (HSPs), which act as cellular chaperones for proteins misfolded by cellular stress. 7,8 For example, hypoxic conditions in the growth plate of tibial dyschondroplasia increases HIF-1a expression, which, in turn, induces increased levels of heat shock protein 70 (HSP70). 9 We also reported that HSP70 overexpression promotes the metabolic activity of chondrocyte...

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

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