The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells.

Poncelet AC, de Caestecker MP, Schnaper HW
Kidney Int. 1999 56 (4): 1354-65

PMID: 10504488 · DOI:10.1046/j.1523-1755.1999.00680.x

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.

MeSH Terms (24)

Blotting, Northern Blotting, Western Cells, Cultured Collagen DNA-Binding Proteins Extracellular Matrix Proteins Gene Expression Regulation Genes, Reporter Glomerular Mesangium Humans Luciferases Mutagenesis Phosphorylation Promoter Regions, Genetic RNA, Messenger Serine Signal Transduction Smad2 Protein Smad3 Protein Smad4 Protein Trans-Activators Transcription, Genetic Transfection Transforming Growth Factor beta

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