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.
Toualbi-Abed
Kahina
K
Institut National de la Santé et de la Recherche Médicale (INSERM) U697, Université Paris 7 Denis Diderot, 75010 Paris, France.
Daniel
Fanny
F
Güller
Meryem C
MC
Legrand
Agnès
A
Mauriz
Jose-Luis
JL
Mauviel
Alain
A
Bernuau
Dominique
D
D001483:Base Sequence; D001665:Binding Sites; D001769:Blood; D047369:Chromatin Immunoprecipitation; D002867:Chromones / Q000494:pharmacology; D019938:Cyclin D1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D016755:Proto-Oncogene Proteins c-jun / Q000502:physiology*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D051996:Transcription Factor RelA / Q000378:metabolism / Q000502:physiology*
D002867:Chromones; D019938:Cyclin D1; D017931:DNA Primers; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D016755:Proto-Oncogene Proteins c-jun; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D051996:Transcription Factor RelA; C498143:RELA protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one