Role of Smad4 on TGF-beta-induced extracellular matrix stimulation in mesangial cells

Kidney Int. 2003 Jun;63(6):2000-9. doi: 10.1046/j.1523-1755.2003.00009.x.

Abstract

Background: The best characterized signaling pathway employed by transforming growth factor-beta (TGF-beta) is the Smad pathway; however, its role in matrix production in mesangial cells is unclear. We focused on Smad4, as Smad4 is essential for the activation of Smad-dependent target genes.

Methods: To investigate the function of Smad4 in extracellular matrix (ECM) production, we generated several stably transfected mesangial cell lines (MMC) that have a deletion in the linker region (Smad4 Delta M4: Delta 275-322) or have a deletion in MH1 of Smad4 (Smad4N4: Delta 1-136). The ECM genes, alpha1 type I collagen (COL1A1), plasminogen activator inhibitor-1 (PAI-1) and fibronectin (FN) were assessed in wild-type mesangial cells and stably transfected Smad4-DN cell lines in the absence and presence of TGF-beta.

Results: As compared to wild-type MMC that had a 10.8-fold stimulation of TGF-beta-induced p3TP-Lux activity, MMC stably transfected with Smad4 Delta M4 and Smad4N4 had only a 2.0-fold and 1.3-fold stimulation, respectively, indicating that they had dominant-negative effects on TGF-beta signaling. Basal and TGF-beta-induced COL1A1 expression in Smad4 dominant-negative cells were dramatically reduced to very low levels. The early (2 hours) TGF-beta-induced PAI-1 mRNA expression was inhibited; however, the sustained (24 to 48 hours) TGF-beta-induced expression was not affected in Smad4 dominant-negative cells. For FN, TGF-beta-induced expression was maintained in Smad4-dominant negative cells.

Conclusion: These results indicate that Smad4 is essential for basal and TGF-beta-induced COL1A1 expression, and contributes to the early, but not sustained TGF-beta-induced PAI-1 expression in mesangial cells. However, TGF-beta-induced FN expression is independent of Smad4. In conclusion, Smad4 has a discriminate effect in mediating specific ECM molecules stimulated by TGF-beta in mesangial cells.

Publication types

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

MeSH terms

  • Animals
  • Anti-Bacterial Agents
  • Cell Line
  • Collagen Type I / genetics
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism*
  • Drug Resistance
  • Extracellular Matrix / metabolism
  • Fibronectins / genetics
  • Gene Expression / drug effects
  • Gene Expression / physiology
  • Gentamicins
  • Glomerular Mesangium / cytology
  • Glomerular Mesangium / metabolism*
  • Mice
  • Oligopeptides
  • Peptides
  • Plasminogen Activator Inhibitor 1 / genetics
  • Smad4 Protein
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism*
  • Transfection
  • Transforming Growth Factor beta / pharmacology*

Substances

  • Anti-Bacterial Agents
  • Collagen Type I
  • DNA-Binding Proteins
  • Fibronectins
  • Gentamicins
  • Oligopeptides
  • Peptides
  • Plasminogen Activator Inhibitor 1
  • Smad4 Protein
  • Smad4 protein, mouse
  • Trans-Activators
  • Transforming Growth Factor beta
  • collagen type I, alpha 1 chain
  • FLAG peptide