A C Poncelet1, M P de Caestecker, H W Schnaper. 1. Department of Pediatrics and Children's Memorial Institute for Education and Research, Northwestern University Medical School, Chicago, Illinois, USA. anne-c@nwu.edu
Abstract
BACKGROUND: Transforming growth factor-beta (TGF-beta) signals through a unique set of intracellular proteins, called SMADs, that have been characterized mainly in transient overexpression systems. Because several models of glomerulosclerosis suggest a role for TGF-beta in the extracellular matrix accumulation, we sought to characterize the role of SMAD proteins in mediating TGF-beta1 responses in a more physiological system using nontransformed human mesangial cells. METHODS: Endogenous SMAD expression and its modulation by TGF-beta1 were evaluated by Western and Northern blot analyses. Phosphorylation of Smad2 and Smad3 was determined by both phospholabeling and immunoblot. SMAD function and its role in type I collagen transcription were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. RESULTS: Cultured human mesangial cells express Smad2, Smad3, and Smad4 proteins. TGF-beta1 down-regulated Smad3 mRNA and protein expression, respectively, after 4 and 24 hours of treatment, whereas Smad2 and Smad4 were less affected. Both Smad2 and Smad3 were phosphorylated in response to TGF-beta1 beginning at 5 minutes, with maximal phosphorylation at 15 minutes, and decreasing phosphorylation by 2 hours. Smad2/3 and Smad4 coimmunoprecipitate only after TGF-beta1 treatment. The activity of a transiently transfected, TGF-beta-responsive construct, p3TP-Lux, was stimulated 3.6-fold by TGF-beta1. Overexpressed wild-type Smad3 increased basal luciferase activity, which was further stimulated by TGF-beta1. A dominant negative mutant form of Smad3 lacking the C-terminal serine phosphoacceptor sites (Smad3A) inhibited TGF-beta1-induced luciferase activity. TGF-beta1 also increased the activation of an alpha2(I) collagen promoter-luciferase reporter construct transfected into mesangial cells. This activation was inhibited by cotransfection with the Smad3A mutant. CONCLUSIONS: Smad2, Smad3, and Smad4 are present and activated by TGF-beta1 in human mesangial cells. The SMAD pathway is functional in these cells and appears to be involved in TGF-beta1-induced type I collagen gene transcription. These findings raise the possibility that SMAD signaling plays a role in glomerular matrix accumulation.
BACKGROUND:Transforming growth factor-beta (TGF-beta) signals through a unique set of intracellular proteins, called SMADs, that have been characterized mainly in transient overexpression systems. Because several models of glomerulosclerosis suggest a role for TGF-beta in the extracellular matrix accumulation, we sought to characterize the role of SMAD proteins in mediating TGF-beta1 responses in a more physiological system using nontransformed human mesangial cells. METHODS: Endogenous SMAD expression and its modulation by TGF-beta1 were evaluated by Western and Northern blot analyses. Phosphorylation of Smad2 and Smad3 was determined by both phospholabeling and immunoblot. SMAD function and its role in type I collagen transcription were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. RESULTS: Cultured human mesangial cells express Smad2, Smad3, and Smad4 proteins. TGF-beta1 down-regulated Smad3 mRNA and protein expression, respectively, after 4 and 24 hours of treatment, whereas Smad2 and Smad4 were less affected. Both Smad2 and Smad3 were phosphorylated in response to TGF-beta1 beginning at 5 minutes, with maximal phosphorylation at 15 minutes, and decreasing phosphorylation by 2 hours. Smad2/3 and Smad4 coimmunoprecipitate only after TGF-beta1 treatment. The activity of a transiently transfected, TGF-beta-responsive construct, p3TP-Lux, was stimulated 3.6-fold by TGF-beta1. Overexpressed wild-type Smad3 increased basal luciferase activity, which was further stimulated by TGF-beta1. A dominant negative mutant form of Smad3 lacking the C-terminal serine phosphoacceptor sites (Smad3A) inhibited TGF-beta1-induced luciferase activity. TGF-beta1 also increased the activation of an alpha2(I) collagen promoter-luciferase reporter construct transfected into mesangial cells. This activation was inhibited by cotransfection with the Smad3A mutant. CONCLUSIONS:Smad2, Smad3, and Smad4 are present and activated by TGF-beta1 in human mesangial cells. The SMAD pathway is functional in these cells and appears to be involved in TGF-beta1-induced type I collagen gene transcription. These findings raise the possibility that SMAD signaling plays a role in glomerular matrix accumulation.
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