Carcinogenesis Advance Access originally published online on January 3, 2008
Carcinogenesis 2008 29(3):536-543; doi:10.1093/carcin/bgm293
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Jun D cooperates with p65 to activate the proximal 
B site of the cyclin D1 promoter: role of PI3K/PDK-1
Institut National de la Santé et de la Recherche Médicale (INSERM) U697, Université Paris 7 Denis Diderot, 75010 Paris, France
1 INSERM U773, Centre de Recherche Bichat Beaujon CRB3, Université Paris 7 Denis Diderot, site Bichat, Paris, France
2 INSERM U591, Hôpital Necker, 75015 Paris, France
3 Ciberehd and Institute of Biomedicine, University of Leon, 24071 Leon, Spain
* To whom correspondence should be addressed. Unité INSERM U697, Pavillon Bazin, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France. Tel: +33 1 53 72 20 71; Fax: +33 1 53 72 20 51; Email: bernuau{at}stlouis.inserm.fr
Nuclear factor kappaB (NF-
B) 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 B2 site of the CCND1 promoter in reporter assays, in a manner strictly dependent on the presence of NF-B 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 B2 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 B2 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-B and JunD in the transcriptional regulation of CCND1 by serum.
Abbreviations: ALLN, N-acetyl-leucyl-leucyl-norleucinal; AP-1, activator protein 1; CCND1, cyclin D1; ChIP, chromatin immunoprecipitation; DTT, dithiothreitol; HDAC, histone deacetylase; IHH, immortalized human hepatocyte; NF-B, nuclear factor kappaB; PCR, polymerase chain reaction; PDK-1, phosphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; RSV, rous sarcoma virus
Received September 24, 2007; revised December 14, 2007; accepted December 15, 2007.