Role of hypoxia-inducible factor-1 alpha in the regulation of plasminogen activator activity in rat knee joint chondrocytes

SummaryObjectiveTo examine the effects of hypoxia-inducible factor-1α (HIF-1α) on the plasminogen activator's (PA) activity and on the expression of components of PA system in articular chondrocytes of rats.MethodsChondrocytes from rat knee joint cartilage were cultured under normoxic, hypoxic, CoCl2 simulated hypoxic, and interleukin-1β (IL-1β)-stimulated conditions. siRNA targeting HIF-1α was transfected into cells cultured under hypoxic, simulated hypoxic, and IL-1β-stimulated conditions to silence HIF-1α. PA activity was determined by the hydrolysis of the chromogenic substrate H-D-Val-Leu-Lys-pNA (S-2251). The mRNA levels were measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The intracellular/matrix-associate protein levels were detected by Western blot and the soluble protein levels were measured by enzyme linked immunosorbent assay (ELISA). Chromatin immunoprecipitation (CHIP) assay was performed to determine whether HIF-1α binds to the hypoxia response element (HRE) of target genes.ResultsThe enhancement of HIF-1α by CoCl2 resulted in a decrease of PA activity, and the silence of HIF-1α by siRNA led to an increase of PA activity. The PA inhibitor-1 (PAI-1) mRNA and protein were increased by hypoxia or simulated hypoxia, which was reversed by the siRNA2-mediated silencing of HIF-1α. CHIP assay further confirmed that the induction of PAI-1 involved the binding of HIF-1α to the PAI-1 promoter, while the enhancement or silencing of HIF-1α did not affect the expression of urokinase type PA (uPA), tissue type PA (tPA) or uPA receptor (uPAR). Additionally, IL-1β stimulated both HIF-1α and PAI-1 in articular chondrocytes, and the IL-1β-mediated induction of PAI-1 was inhibited partly by HIF-1α silencing.ConclusionHIF-1α may inhibit the PA activity through stimulating the expression of PAI-1 in normal articular chondrocytes. The inhibition of HIF-1α in the PA activity of articular chondrocytes probably plays an important role in the maintenance of articular cartilage matrix.