Potentiation of Smad Transactivation by Jun Proteins during a Combined Treatment with Epidermal Growth Factor and Transforming Growth Factor-β in Rat Hepatocytes: ROLE OF PHOSPHATIDYLINOSITOL 3-KINASE-INDUCED AP-1 ACTIVATION

Philippe Péron,Mohamed Rahmani,Yvrick Zagar,Anne-Marie Durand-Schneider,Bernard Lardeux,Dominique Bernuau

doi:10.1074/jbc.m005919200

Philippe Péron, Mohamed Rahmani + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m005919200

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Abstract

Cross-talk between Smad and mitogen-activated protein kinase pathways has been described recently, and evidence for Smad cooperation with AP-1 is emerging. Here we report that epidermal growth factor (EGF) potentializes transforming growth factor beta (TGF-beta)-induced Smad3 transactivation in rat hepatocytes, an effect abrogated by TAM-67, a dominant negative mutant of AP-1. Antisense transfection experiments indicated that c-Jun and JunB were involved in the synergistic effect, and endogenous c-Jun physically associated with Smad3 during a combined EGF/TGF-beta treatment. We next investigated which signaling pathway transduced by EGF was responsible for the Jun-induced synergism. Whereas inhibition of JNK had no effect, inhibition of the phosphatidylinositol-3' kinase (PI3-kinase) pathway by LY294002 or by expression of a dominant negative mutant of PI3-kinase reduced EGF/TGF-beta-induced Smad3 transcriptional activity. Transfection of an activated Ras with a mutation enabling the activation of the PI3-kinase pathway alone mimicked the EGF/TGF-beta potentiation of Smad3 transactivation, and TAM-67 abolished this effect, suggesting that the PI3-kinase pathway stimulates Smad3 via AP-1 stimulation. The EGF/TGF-beta-induced activation of Smad3 correlated with PI3-kinase and p38-dependent but not JNK-dependent phosphorylation of c-Jun. Since potentiation of a Smad-binding element-driven gene was also induced by EGF/TGF-beta treatment, this novel mechanism of Jun/Smad cooperation might be crucial for diversifying TGF-beta responses.

Highlights

Cross-talk between Smad and mitogen-activated protein kinase pathways has been described recently, and evidence for Smad cooperation with AP-1 is emerging
epidermal growth factor (EGF) Synergizes with TGF-␤ for Smad 3 Transactivation—To determine whether EGF and TGF-␤ signalizations cross-talk in hepatocytes, we investigated the transcriptional activity of Smad proteins by using a single Gal-hybrid system, which makes use of an expression vector for Smad3-Gal4 fusion protein and a reporter plasmid containing five concatemerized Gal4-binding sites (Ti5xGal4)
These results indicated that whereas the EGF signaling pathway alone weakly activates Smad3 transcriptional activity in hepatocytes, it potently synergizes with Smads once they have been activated by TGF-␤

Summary

EXPERIMENTAL PROCEDURES

Nuclear extracts (5–20 ␮g of protein) were incubated in binding buffer (20 mM Hepes, pH 7.9, 5 mM MgCl2, 4 mM dithiothreitol, 20% glycerol, 0.1 mM PMSF, 5 mM benzamidine, 2 mM levamisole, 0.1 ␮g/ml aprotinin, 0.1 ␮g/ml bestatin) containing 2 ␮g of poly(dI-dC) and 32P-labeled double-stranded probe (3 ϫ 104 cpm) for 20 min at 4 °C. For Luc assays, cells were washed with chilled PBS and lysed for 15 min on ice with lysis buffer (25 mM Tris/HPO4, pH 7.8, 8 mM MgCl2, 1% Triton X-100, 1% bovine serum albumin, 15% glycerol, 1 mM EDTA, and 1 mM dithiothreitol), and the lysates were centrifuged 10 min at 13,000 rpm and stored at Ϫ20 °C. Statistical Tests—We used a Student’s t test and the one-way analysis of variance followed by the least significant difference test

RESULTS

Smads and Jun Cooperation in Rat Hepatocytes

DISCUSSION