RNA Interference Targeting Thrombospondin 1 Suppresses the Activation of Latent Transforming Growth Factor-b1 in Human Renal Tubular Epithelial Cell

Abstract: Thrombospondin 1 (TSP1) is a major endogenous activator for transforming growth factor-b1 (TGF-b1)， which plays an critical role on the development of renal tubulointerstitial fibrosis. The purpose of the study was to determine whether small interference RNAs (siRNA) targeting the thrombospondin-1 could be used to suppress the over-activation of TGF-b1 induced by angiotensin II in human renal tubular epithelial cells. TSP1 specific siRNA designed from the human gene sequence was transfected into cultured human renal tubular epithelial cells (HK-2). The protein and transcript levels of the TSP1 and TGF-b1 were determined by Western bloting and RT-PCR. The co-localization of TSP1 and TGF-b1 was observed by flow-cytometry. Western blotting was performed to measure the level of Smad2 and phsophorylated-Smad2. The secreting extracellular matrix such as fibronectin and PAI-1 was examined by ELISA. TGF-b1 bioactivity was determined by ELISA. siRNA targeting TSP1 was successfully transferred into HK-2 cells and markedly inhibited de novo synthesis of TSP1 in a dose dependent manner. This effect was accompanied by decreased activation TGF-b1 but the total level of TGF-b1 remained unaffected， siRNA additionally inhibited TGF-b1-dependent Smad-signaling pathway， markedly suppressed accumulation of fibronectin as well as transcription of TGF-b1 target gene PAI-1. TSP1 is the major endogenous activator of TGF-b1， TSP1 specific siRNA were efficacious in vitro knocking down the expression of TSP1 and further suppressing TGF-b1 activation.
    

CSTR: 32200.14.cjcb.2006.02.0018