c-Fos accelerates hepatocyte conversion to a fibroblastoid phenotype through ERK-mediated upregulation of paxillin-Serine178 phosphorylation.

Transforming growth factor beta (TGF-beta) exerts an important role in the late steps of carcinogenesis by cooperating with Ras to induce cell motility and tumor invasion. The transcription complex AP-1 has been implicated in the regulation of genes involved in motility and invasion, by mechanisms not yet delineated. We utilized a model of immortalized human hepatocytes (IHH) overexpressing c-Fos (IHH-Fos) or not (IHH-C) to investigate the role of c-Fos on cell motility in response to a prolonged treatment with TGF-beta, EGF or a combination of both. Cotreatment with EGF and TGF-beta, but neither cytokine alone, induced the conversion of hepatocytes to a fibroblastoid phenotype and increased their motility in Boyden chambers. EGF/TGF-beta cotreatment induced a higher effect on ERK phosphorylation compared to TGF-beta treatment alone. It also induced an increase in total and phosphorylated Ser(178) paxillin, a protein previously implicated in cell motility. This response was inhibited by two specific MEK inhibitors, indicating the involvement of the ERK pathway in paxillin activation. Overexpression of c-Fos correlated with increased cell scattering and motility, higher levels of ERK activation and phospho Ser(178) paxillin, increased levels of EGF receptor (EGF-R) mRNA and higher EGF-R phosphorylation levels following EGF/TGF-beta cotreatment. Conversely, siRNA-mediated invalidation of c-Fos delayed the appearance of fibroblastoid cells, decreased EGF-R mRNA and downregulated ERK and Ser(178) paxillin phosphorylations, indicating that c-Fos activates hepatocyte motility through an EGF-R/ERK/paxillin pathway. Since c-Fos is frequently overexpressed in hepatocarcinomas, this newly identified mechanism might be involved in the progression of hepatic tumors in vivo.

Jun D cooperates with p65 to activate the proximal kappaB site of the cyclin D1 promoter: role of PI3K/PDK-1.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 are transcription factors involved in the regulation of cell proliferation that play important roles in tumorigenesis. We investigated whether these two factors cooperate for transcriptional regulation of cyclin D1 (CCND1), a gene whose deregulation is critical during carcinogenesis. We demonstrate that overexpression of JunD in human hepatocarcinoma cells strongly activates transcription mediated by the kappaB2 site of the CCND1 promoter in reporter assays, in a manner strictly dependent on the presence of NF-kappaB proteins. Serum stimulation increased the expression of p65, p50, c-Fos, c-Jun and JunD and induced the recruitment of p65, p50 and JunD to the kappaB2 site of the promoter in DNA pull-down assays. Chromatin immunoprecipitation (ChIP) analysis confirmed the serum-induced recruitment of JunD to the promoter in vivo and showed that the presence of JunD was dependent on the presence of p65 and p50, indicating a protein-protein-dependent mechanism of JunD recruitment. Serum-induced activation of protein binding to kappaB2 correlated with high levels of phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylation. Both LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), and overexpression of a dominant-negative form of PDK-1 inhibited the JunD-stimulating effect in reporter assays. LY294002 also prevented the serum-induced recruitment of JunD, but not p65 or p50 to the promoter in ChIP assay. JunD-p65 complexes, identified in vivo by co-immunoprecipitation, were decreased by LY294002 and by small interfering RNA inhibition of PDK-1. Taken together, our data demonstrate a PI3K/PDK-1-dependent functional cooperation of NF-kappaB and JunD in the transcriptional regulation of CCND1 by serum.