Transforming growth factor-beta-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38beta mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial-myofibroblast transdifferentiation.
BACKGROUND
Transforming growth factor-beta (TGFbeta)-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 alpha-smooth muscle cell actin (alphaSMA) expression, a hallmark of myofibroblast formation, induced by TGFbeta 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 alphaSMA synthesis was assessed by western blotting or immunofluorescent staining of cellular alphaSMA. To assess the regulation of the alphaSMA promoter, tubular cells were transiently transfected with a 785 bp alphaSMA 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 TGFbeta-induced alphaSMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38beta but not of p38alpha potently inhibited alphaSMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFbeta effect, confirming the role of p38beta in the regulation of TGFbeta-induced alphaSMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFbeta-induced alphaSMA synthesis.


CONCLUSIONS
TGFbeta-induced alphaSMA 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.
Sebe
Attila
A
1st Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, 2/A Koranyi S. u, Budapest, H-1083, Hungary.
Leivonen
Suvi-Katri
SK
Fintha
Attila
A
Masszi
Andras
A
Rosivall
Laszlo
L
Kähäri
Veli-Matti
VM
Mucsi
Istvan
I
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D054338:Cell Transdifferentiation; D017931:DNA Primers / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D018374:LLC-PK1 Cells; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048290:Mitogen-Activated Protein Kinase 11 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D051785:Smad Proteins / Q000378:metabolism; D013552:Swine; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
D000199:Actins; D017931:DNA Primers; D005419:Flavonoids; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011994:Recombinant Proteins; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; D019950:Mitogen-Activated Protein Kinase 1; D048290:Mitogen-Activated Protein Kinase 11; D048052:Mitogen-Activated Protein Kinase 3; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one