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

Anne Christine Poncelet*, Mark P. De Caestecker, H. William Schnaper

*Corresponding author for this work

Research output: Contribution to journalArticle

135 Scopus citations

Abstract

Background. Transforming growth factor-β (TGF-β) 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-β in the extracellular matrix accumulation, we sought to characterize the role of SMAD proteins in mediating TGF-β1 responses in a more physiological system using nontransformed human mesangial cells. Methods. Endogenous SMAD expression and its modulation by TGF-β1 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-β1 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-β1 beginning at 5 minutes, with maximal phosphorylation at 15 minutes, and decreasing phosphorylation by 2 hours. Smad2/3 and Smad4 coimmunoprecipitate only after TGF-β1 treatment. The activity of a transiently transfected, TGF-β-responsive construct, p3TP-Lux, was stimulated 3.6-fold by TGF-β1. Overexpressed wild-type Smad3 increased basal luciferase activity, which was further stimulated by TGF-β1. A dominant negative mutant form of Smad3 lacking the C-terminal serine phosphoacceptor sites (Smad3A) inhibited TGF-β1-induced luciferase activity. TGF-β1 also increased the activation of an α2(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-β1 in human mesangial cells. The SMAD pathway is functional in these cells and appears to be involved in TGF-β1-induced type I collagen gene transcription. These findings raise the possibility that SMAD signaling plays a role in glomerular matrix accumulation.

Original languageEnglish (US)
Pages (from-to)1354-1365
Number of pages12
JournalKidney international
Volume56
Issue number4
DOIs
StatePublished - Jan 1 1999

Keywords

  • Extracellular matrix
  • Glomerulosclerosis
  • Luciferase
  • Serine phosphoacceptor sites
  • Type I collagen

ASJC Scopus subject areas

  • Nephrology

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