Transforming growth factor-β-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38β mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial–myofibroblast transdifferentiation

A. Sebe (Corresponding Author), Suvi-Katri Leivonen, A. Fintha, A. Masszi, L. Rosivall, V.-M. Kähäri, I. Mucsi

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Background. Transforming growth factor-β (TGFβ)-induced epithelial–myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of α-smooth muscle cell actin (αSMA) expression, a hallmark of myofibroblast formation, induced by TGFβ in renal proximal tubular cells.

Methods. Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The αSMA synthesis was assessed by western blotting or immunofluorescent staining of cellular αSMA. To assess the regulation of the αSMA promoter, tubular cells were transiently transfected with a 785 bp αSMA promoter–luciferase reporter construct and vectors interfering with the Smad pathway.

Results. Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFβ-induced αSMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38β but not of p38α potently inhibited αSMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFβ effect, confirming the role of p38β in the regulation of TGFβ-induced αSMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFβ-induced αSMA synthesis.

Conclusion. TGFβ-induced αSMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial–mesenchymal transdifferentiation.

Original languageEnglish
Pages (from-to)1537 - 1545
Number of pages9
JournalNephrology, Dialysis, Transplantation
Volume23
Issue number5
DOIs
Publication statusPublished - 2008
MoE publication typeA1 Journal article-refereed

Fingerprint

Transforming Growth Factor alpha
p38 Mitogen-Activated Protein Kinases
Smooth Muscle Myocytes
Actins
Transforming Growth Factors
Kidney
Proteins
Receptor-Regulated Smad Proteins
Western Blotting
Phospho-Specific Antibodies
Myofibroblasts
Extracellular Signal-Regulated MAP Kinases
Adenoviridae
Protein Kinases
Fibrosis
Phosphotransferases
Swine
Staining and Labeling

Keywords

  • Alpha-smooth muscle cell actin
  • Epithelial-myofibroblast transdifferentiation
  • p38 nitrogen-activated protein kinase
  • Renal proximal tubular cell
  • Smad proteins

Cite this

@article{b26b325d6d3c49c7b2212b96eb12f56a,
title = "Transforming growth factor-β-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38β mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial–myofibroblast transdifferentiation",
abstract = "Background. Transforming growth factor-β (TGFβ)-induced epithelial–myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of α-smooth muscle cell actin (αSMA) expression, a hallmark of myofibroblast formation, induced by TGFβ in renal proximal tubular cells. Methods. Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The αSMA synthesis was assessed by western blotting or immunofluorescent staining of cellular αSMA. To assess the regulation of the αSMA promoter, tubular cells were transiently transfected with a 785 bp αSMA promoter–luciferase reporter construct and vectors interfering with the Smad pathway. Results. Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFβ-induced αSMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38β but not of p38α potently inhibited αSMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFβ effect, confirming the role of p38β in the regulation of TGFβ-induced αSMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFβ-induced αSMA synthesis. Conclusion. TGFβ-induced αSMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial–mesenchymal transdifferentiation.",
keywords = "Alpha-smooth muscle cell actin, Epithelial-myofibroblast transdifferentiation, p38 nitrogen-activated protein kinase, Renal proximal tubular cell, Smad proteins",
author = "A. Sebe and Suvi-Katri Leivonen and A. Fintha and A. Masszi and L. Rosivall and V.-M. K{\"a}h{\"a}ri and I. Mucsi",
year = "2008",
doi = "10.1093/ndt/gfm789",
language = "English",
volume = "23",
pages = "1537 -- 1545",
journal = "Nephrology, Dialysis, Transplantation",
issn = "0931-0509",
publisher = "Oxford University Press",
number = "5",

}

Transforming growth factor-β-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38β mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial–myofibroblast transdifferentiation. / Sebe, A. (Corresponding Author); Leivonen, Suvi-Katri; Fintha, A.; Masszi, A.; Rosivall, L.; Kähäri, V.-M.; Mucsi, I.

In: Nephrology, Dialysis, Transplantation, Vol. 23, No. 5, 2008, p. 1537 - 1545.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Transforming growth factor-β-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38β mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial–myofibroblast transdifferentiation

AU - Sebe, A.

AU - Leivonen, Suvi-Katri

AU - Fintha, A.

AU - Masszi, A.

AU - Rosivall, L.

AU - Kähäri, V.-M.

AU - Mucsi, I.

PY - 2008

Y1 - 2008

N2 - Background. Transforming growth factor-β (TGFβ)-induced epithelial–myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of α-smooth muscle cell actin (αSMA) expression, a hallmark of myofibroblast formation, induced by TGFβ in renal proximal tubular cells. Methods. Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The αSMA synthesis was assessed by western blotting or immunofluorescent staining of cellular αSMA. To assess the regulation of the αSMA promoter, tubular cells were transiently transfected with a 785 bp αSMA promoter–luciferase reporter construct and vectors interfering with the Smad pathway. Results. Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFβ-induced αSMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38β but not of p38α potently inhibited αSMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFβ effect, confirming the role of p38β in the regulation of TGFβ-induced αSMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFβ-induced αSMA synthesis. Conclusion. TGFβ-induced αSMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial–mesenchymal transdifferentiation.

AB - Background. Transforming growth factor-β (TGFβ)-induced epithelial–myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of α-smooth muscle cell actin (αSMA) expression, a hallmark of myofibroblast formation, induced by TGFβ in renal proximal tubular cells. Methods. Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The αSMA synthesis was assessed by western blotting or immunofluorescent staining of cellular αSMA. To assess the regulation of the αSMA promoter, tubular cells were transiently transfected with a 785 bp αSMA promoter–luciferase reporter construct and vectors interfering with the Smad pathway. Results. Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFβ-induced αSMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38β but not of p38α potently inhibited αSMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFβ effect, confirming the role of p38β in the regulation of TGFβ-induced αSMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFβ-induced αSMA synthesis. Conclusion. TGFβ-induced αSMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial–mesenchymal transdifferentiation.

KW - Alpha-smooth muscle cell actin

KW - Epithelial-myofibroblast transdifferentiation

KW - p38 nitrogen-activated protein kinase

KW - Renal proximal tubular cell

KW - Smad proteins

U2 - 10.1093/ndt/gfm789

DO - 10.1093/ndt/gfm789

M3 - Article

VL - 23

SP - 1537

EP - 1545

JO - Nephrology, Dialysis, Transplantation

JF - Nephrology, Dialysis, Transplantation

SN - 0931-0509

IS - 5

ER -