缺氧诱导因子-1α在大鼠膝关节软骨细胞中纤溶酶原激活物活性调节中的作用。

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

机构信息

State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China.

出版信息

Osteoarthritis Cartilage. 2009 Nov;17(11):1494-502. doi: 10.1016/j.joca.2009.05.005. Epub 2009 May 19.

DOI:10.1016/j.joca.2009.05.005

PMID:19490965

Abstract

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.

CONCLUSION

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.

摘要

目的

探讨低氧诱导因子-1α（HIF-1α）对关节软骨细胞中纤溶酶原激活物（PA）活性及 PA 系统成分表达的影响。

方法

在常氧、低氧、氯化钴（CoCl2）模拟低氧及白细胞介素-1β（IL-1β）刺激条件下培养大鼠膝关节软骨细胞。将针对 HIF-1α 的 siRNA 转染至低氧、模拟低氧及 IL-1β 刺激条件下培养的细胞中，以沉默 HIF-1α。通过水解发色底物 H-D-Val-Leu-Lys-pNA（S-2251）测定 PA 活性。通过实时定量逆转录聚合酶链反应（RT-PCR）测定 mRNA 水平。通过 Western blot 检测细胞内/基质相关蛋白水平，通过酶联免疫吸附测定（ELISA）检测可溶性蛋白水平。进行染色质免疫沉淀（CHIP）实验以确定 HIF-1α是否与靶基因的低氧反应元件（HRE）结合。

结果

CoCl2 增强 HIF-1α导致 PA 活性降低，而 siRNA 沉默 HIF-1α导致 PA 活性增加。低氧或模拟低氧增加了 PA 抑制剂-1（PAI-1）的 mRNA 和蛋白水平，而 siRNA2 介导的 HIF-1α沉默则逆转了这一作用。CHIP 实验进一步证实，PAI-1 的诱导涉及 HIF-1α与 PAI-1 启动子的结合，而 HIF-1α 的增强或沉默并不影响尿激酶型 PA（uPA）、组织型 PA（tPA）或 uPA 受体（uPAR）的表达。此外，IL-1β刺激关节软骨细胞中的 HIF-1α和 PAI-1，而 HIF-1α 沉默部分抑制了 IL-1β 介导的 PAI-1 诱导。