Blockade of the Effects of TGF-β1 on Mesangial Cells by Overexpression of Smad7

ABSTRACT. Smad7, a protein induced by transforming growth factor–β1 (TGF-β1) in many target cells, inhibits TGF-β1 signal transduction and is thought to mediate an intracellular negative feedback response that limits TGF-β1 effects. It is possible that overexpression of Smad7 could block specified effects of TGF-β1 on mesangial cells, a TGF-β target in glomerular disease. Smad7 mRNA was induced by TGF-β1 within 1 h in a concentration-dependent manner in a transformed mouse mesangial cell (MMC) line. Uptake of 14C-spermidine from the medium by MMC and the transcriptional activity of a segment of the human collagen pro-α2 type 1 chain (COL1A2) promoter fused to a luciferase reporter gene were used as indices of TGF-β1. Treatment with TGF-β1 increased 14C-spermidine uptake rate in a time-, concentration-, and temperature-dependent manner. For example, exposure to 1 ng/ml TGF-β1 for 15 h increased uptake approximately twofold, a response that was attenuated by cycloheximide. Transfection of Smad7 expression vector into MMC abrogated both TGF-β1-dependent stimulation of spermidine uptake and COL1A2 promoter activity. It is concluded that: (1) TGF-β1 induces Smad7 in MMC; (2) 14C-spermidine uptake is a convenient quantitative index of TGF-β1 effect in these cells; and (3) overexpression of Smad7 is a highly effective method of blocking at least some mesangial cell effects of TGF-β1 that may warrant evaluation in vivo in experimental glomerular disease.