X Chen1, Q He, W Liu, Q Xu, Y Ye, B Fu, L Yu. 1. Department of Nephrology, General Hospital of Chinese PLA, Beijing 100853, China. xmchen@public.bta.net.cn
Abstract
OBJECTIVE: To evaluate activator protein-1 (AP-1) mediated mechanisms in thrombin-induced qlasminogen activator inhibitor-1 (PAI-1) expression in cultured human glomerular mesangial cells (MCs). METHODS: Electrophoretic mobility shift assay (EMSA) was employed to assess AP-1 DNA-binding activity, and Western blot hybridization was used for quantification of c-fos and c-jun, two subunits of AP-1 dimers. PAI-1 activity and mRNA expression were analysed by the fibrin plate assay and Northern hybridization, respectively. RESULTS: Thrombin concentration enhanced PAI-1 activity in the supernatant and stimulated PAI-1 mRNA expression in cultured MCs. PAI-1 activity was blocked by hirudin, a specific inhibitor of thrombin. Further study demonstrated that thrombin promoted AP-1 DNA-binding activity but exerted little effect on c-fos or c-jun. Curcumin (AP-1 inhibitor), staurosporine (PKC inhibitor), and genistein (PTK inhibitor) all reduced AP-1-mediated PAI-1 mRNA expression induced by thrombin in cultured MCs. CONCLUSION: The present study indicates that in cultured human MCs, thrombin stimulates PAI-1 expression through an AP-1 signal pathway, which may be mediated by PKC and PTK.
OBJECTIVE: To evaluate activator protein-1 (AP-1) mediated mechanisms in thrombin-induced qlasminogen activator inhibitor-1 (PAI-1) expression in cultured human glomerular mesangial cells (MCs). METHODS: Electrophoretic mobility shift assay (EMSA) was employed to assess AP-1 DNA-binding activity, and Western blot hybridization was used for quantification of c-fos and c-jun, two subunits of AP-1 dimers. PAI-1 activity and mRNA expression were analysed by the fibrin plate assay and Northern hybridization, respectively. RESULTS:Thrombin concentration enhanced PAI-1 activity in the supernatant and stimulated PAI-1 mRNA expression in cultured MCs. PAI-1 activity was blocked by hirudin, a specific inhibitor of thrombin. Further study demonstrated that thrombin promoted AP-1 DNA-binding activity but exerted little effect on c-fos or c-jun. Curcumin (AP-1 inhibitor), staurosporine (PKC inhibitor), and genistein (PTK inhibitor) all reduced AP-1-mediated PAI-1 mRNA expression induced by thrombin in cultured MCs. CONCLUSION: The present study indicates that in cultured human MCs, thrombin stimulates PAI-1 expression through an AP-1 signal pathway, which may be mediated by PKC and PTK.