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


CONCLUSIONS
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.
Tsuchida
Ken-ichi
K
Dorrance Hamilton Laboratories, Division of Nephrology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Zhu
Yanqing
Y
Siva
Senthuran
S
Dunn
Stephen R
SR
Sharma
Kumar
K
D000818:Animals; D000900:Anti-Bacterial Agents; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004351:Drug Resistance; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005839:Gentamicins; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009842:Oligopeptides; D010455:Peptides; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004268:DNA-Binding Proteins; D005353:Fibronectins; D005839:Gentamicins; D009842:Oligopeptides; D010455:Peptides; D017395:Plasminogen Activator Inhibitor 1; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C085706:FLAG peptide