Role of hypoxia-inducible factor-1 alpha in the regulation of plasminogen activator activity in rat knee joint chondrocytes.
OBJECTIVE
To examine the effects of hypoxia-inducible factor-1alpha (HIF-1alpha) on the plasminogen activator's (PA) activity and on the expression of components of PA system in articular chondrocytes of rats.


METHODS
Chondrocytes from rat knee joint cartilage were cultured under normoxic, hypoxic, CoCl(2) simulated hypoxic, and interleukin-1beta (IL-1beta)-stimulated conditions. siRNA targeting HIF-1alpha was transfected into cells cultured under hypoxic, simulated hypoxic, and IL-1beta-stimulated conditions to silence HIF-1alpha. 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-1alpha binds to the hypoxia response element (HRE) of target genes.


RESULTS
The enhancement of HIF-1alpha by CoCl(2) resulted in a decrease of PA activity, and the silence of HIF-1alpha 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-1alpha. CHIP assay further confirmed that the induction of PAI-1 involved the binding of HIF-1alpha to the PAI-1 promoter, while the enhancement or silencing of HIF-1alpha did not affect the expression of urokinase type PA (uPA), tissue type PA (tPA) or uPA receptor (uPAR). Additionally, IL-1beta stimulated both HIF-1alpha and PAI-1 in articular chondrocytes, and the IL-1beta-mediated induction of PAI-1 was inhibited partly by HIF-1alpha silencing.


CONCLUSIONS
HIF-1alpha may inhibit the PA activity through stimulating the expression of PAI-1 in normal articular chondrocytes. The inhibition of HIF-1alpha in the PA activity of articular chondrocytes probably plays an important role in the maintenance of articular cartilage matrix.
Zhu
G
G
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China.
Tang
Y
Y
Liang
X
X
Zheng
M
M
Yang
J
J
Zhou
H
H
Li
L
L
Qin
T
T
D000818:Animals; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism*; D007719:Knee Joint / Q000502:physiology*; D010960:Plasminogen Activators / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051793:Hypoxia-Inducible Factor 1; D012333:RNA, Messenger; D010960:Plasminogen Activators