Abstract
Bile duct epithelial cells (BDECs) contribute to liver fibrosis by expressing αVβ6 integrin, a critical activator of latent transforming growth factor β (TGF-β). β6 integrin (Itgβ6) mRNA induction and αVβ6 integrin expression in BDECs are partially TGF-β-dependent. However, the signaling pathways required for TGF-β-dependent Itgβ6 mRNA induction in BDECs are not known. We tested the hypothesis that the p38 mitogen-activated protein kinase (MAPK) signaling pathway contributes to TGF-β1 induction of Itgβ6 mRNA by activating SMAD and activator protein 1 (AP-1) transcription factors. Pretreatment of transformed human BDECs (MMNK-1 cells) with two different p38 MAPK inhibitors, but not a control compound, inhibited TGF-β1 induction of Itgβ6 mRNA. Inhibition of p38 also reduced TGF-β1 activation of a SMAD-dependent reporter construct. Expression of a dominant-negative SMAD3 (SMAD3ΔC) significantly reduced TGF-β1-induced Itgβ6 mRNA expression. Expression of JunB mRNA, but not other AP-1 proteins, increased in TGF-β1-treated MMNK-1 cells, and induction of JunB expression was p38-dependent. Consistent with a requirement for de novo induction of JunB protein, cycloheximide pretreatment inhibited TGF-β1 induction of Itgβ6 mRNA. Expression of a dominant-negative AP-1 mutant (TAM67) also inhibited TGF-β1 induction of Itgβ6 mRNA. Overall, the results suggest that p38 contributes to TGF-β1-induced Itgβ6 mRNA expression in MMNK-1 cells by regulating activation of both SMAD and AP-1 transcription factors.
Footnotes
This work was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grant R01-ES017537] (to J.P.L.); the Center of Biomedical Research Excellence [Grant P20 RR021940]; and the Molecular Biology Core and the Histology Core, which were supported by the Center of Biomedical Research Excellence grant. B.P.S. was supported in part by the National Institutes of Health National Institute of Environmental Health Sciences [Training Grant T32ES007079].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.177337.
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ABBREVIATIONS:
- BDEC
- bile duct epithelial cell
- TGF-β
- transforming growth factor β
- Itgβ6
- β6 integrin
- MAPK
- mitogen-activated protein kinase
- AP-1
- activator protein 1
- qPCR
- quantitative polymerase chain reaction
- DMSO
- dimethyl sulfoxide
- PBS
- phosphate-buffered saline
- BSA
- bovine serum albumin
- TBST
- 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4
- siRNA
- small interfering RNA
- ATF-2
- activating transcription factor 2
- CMV
- cytomegalovirus
- SB203580
- 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine
- SB202190
- 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]phenol
- SB202474
- 4-ethyl-2(p-methoxyphenyl)-5-(4′-pyridyl)-1H-imidazole.
- Received November 16, 2010.
- Accepted February 7, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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