STAT1 and STAT3 mediate thrombin-induced expression of TIMP-1 in human glomerular mesangial cells

Abstract

Thrombin exhibits numerous biological effects on glomerular resident cells, such as cell proliferation, release and synthesis of cytokines and collagen, expressions of metalloproteinases and their inhibitors, especially tissue inhibitor of metalloproteinase-1 (TIMP-1). However, the signaling mechanisms underlying these cellular events have not been fully elucidated. The present study was designed to examine the role of signal transducers and activators of transcription (STAT) in thrombin-induced TIMP-1 expression in human mesangial cells. Cultured human glomerular mesangial cells were incubated with thrombin up to 12 hours. The effects of the antisense of STAT1 and antisense of STAT3 on stimulated TIMP-1 mRNA levels and DNA-binding activities of both STAT1 and STAT3 were determined using Northern blot, electrophoretic mobility shift assay (EMSA), and supershift assay. Cultured human mesangial cells constitutively expressed TIMP-1, and thrombin induced TIMP-1 gene transcription in a time- and dose-dependent manner. Hirudin, a specific inhibitor of thrombin, could block thrombin-induced TIMP-1 expression. Thrombin also induced STAT-DNA binding activity in a similar time- and dose-dependent manner. In order to examine the role of STAT in thrombin-induced TIMP-1 expression, STAT1 and STAT3 antisense oligonucleotides were used. EMSA showed that STAT1 and STAT3 antisense oligonucleotides could inhibit both thrombin-induced STAT-DNA binding activities and TIMP-1 mRNA expression; the supershift assay showed that the SIF band consisted of STAT1 and STAT3 proteins. Both STAT1 and STAT3 may be involved, at least in part, in thrombin-induced expression of the TIMP-1 gene in cultured human mesangial cells.