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

Kidney Int. 1999 Oct;56(4):1354-65. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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


  • DNA-Binding Proteins
  • Extracellular Matrix Proteins
  • RNA, Messenger
  • SMAD2 protein, human
  • SMAD3 protein, human
  • SMAD4 protein, human
  • Smad2 Protein
  • Smad3 Protein
  • Smad4 Protein
  • Trans-Activators
  • Transforming Growth Factor beta
  • Serine
  • Collagen
  • Luciferases