TY - JOUR
T1 - Role of Smad4 on TGF-β-induced extracellular matrix stimulation in mesangial cells
AU - Tsuchida, Ken Ichi
AU - Zhu, Yanqing
AU - Siva, Senthuran
AU - Dunn, Stephen R.
AU - Sharma, Kumar
N1 - Funding Information:
We appreciate generous provision of the 3′ Flag-tagged Smad4N4 and Smad4ΔM4 by Dr. M. P. de Caestecker and the pcDNA3.1-LacZ by Dr. S. Sasaki. This work was supported by a fellowship grant to K.T. by the Juvenile Diabetes Research Foundation International and by the National Institutes of Health (grants NIDDK R01-DK 53867 and K08-DK02308 to K.S.). We also appreciate the critical review of the manuscript by Dr. M. P. de Caestecker.
PY - 2003/6/1
Y1 - 2003/6/1
N2 - Background. The best characterized signaling pathway employed by transforming growth factor-β (TGF-β) 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 (Smad4ΔM4: Δ275-322) or have a deletion in MH1 of Smad4 (Smad4N4: Δ1-136). The ECM genes, α1 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-β. Results. As compared to wild-type MMC that had a 10.8-fold stimulation of TGF-β-induced p3TP-Lux activity, MMC stably transfected with Smad4ΔM4 and Smad4N4 had only a 2.0-fold and 1.3-fold stimulation, respectively, indicating that they had dominant-negative effects on TGF-β signaling. Basal and TGF-β-induced COL1A1 expression in Smad4 dominant-negative cells were dramatically reduced to very low levels. The early (2 hours) TGF-β-induced PAI-1 mRNA expression was inhibited; however, the sustained (24 to 48 hours) TGF-β-induced expression was not affected in Smad4 dominant-negative cells. For FN, TGF-β-induced expression was maintained in Smad4-dominant negative cells. Conclusion. These results indicate that Smad4 is essential for basal and TGF-β-induced COL1A1 expression, and contributes to the early, but not sustained TGF-β-induced PAI-1 expression in mesangial cells. However, TGF-β-induced FN expression is independent of Smad4. In conclusion, Smad4 has a discriminate effect in mediating specific ECM molecules stimulated by TGF-β in mesangial cells.
AB - Background. The best characterized signaling pathway employed by transforming growth factor-β (TGF-β) 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 (Smad4ΔM4: Δ275-322) or have a deletion in MH1 of Smad4 (Smad4N4: Δ1-136). The ECM genes, α1 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-β. Results. As compared to wild-type MMC that had a 10.8-fold stimulation of TGF-β-induced p3TP-Lux activity, MMC stably transfected with Smad4ΔM4 and Smad4N4 had only a 2.0-fold and 1.3-fold stimulation, respectively, indicating that they had dominant-negative effects on TGF-β signaling. Basal and TGF-β-induced COL1A1 expression in Smad4 dominant-negative cells were dramatically reduced to very low levels. The early (2 hours) TGF-β-induced PAI-1 mRNA expression was inhibited; however, the sustained (24 to 48 hours) TGF-β-induced expression was not affected in Smad4 dominant-negative cells. For FN, TGF-β-induced expression was maintained in Smad4-dominant negative cells. Conclusion. These results indicate that Smad4 is essential for basal and TGF-β-induced COL1A1 expression, and contributes to the early, but not sustained TGF-β-induced PAI-1 expression in mesangial cells. However, TGF-β-induced FN expression is independent of Smad4. In conclusion, Smad4 has a discriminate effect in mediating specific ECM molecules stimulated by TGF-β in mesangial cells.
KW - Diabetic nephropathy
KW - Fibronectin
KW - Kidney fibrosis
KW - PAI-1
KW - Signaling
KW - TGF-β
KW - Type I collagen
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U2 - 10.1046/j.1523-1755.2003.00009.x
DO - 10.1046/j.1523-1755.2003.00009.x
M3 - Article
C2 - 12753287
AN - SCOPUS:0037513430
SN - 0085-2538
VL - 63
SP - 2000
EP - 2009
JO - Kidney international
JF - Kidney international
IS - 6
ER -