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Using murine AtT20 pituitary cells transfected with a rat pro-opiomelanocortin (POMC) promoter (-706/+64) linked to the luciferase reporter, we showed leukemia inhibitory factor (LIF) to strongly potentiate corticotropin-releasing hormone (CRH) induction of POMC gene expression. We therefore tested mechanisms for molecular interactions between LIF and CRH. Although LIF and CRH synergized to induce an 8-fold induction of POMC transcription, CRH alone (but not LIF) induced cAMP response element-binding protein phosphorylation (5-fold) or an increase of c-fos mRNA levels (>100-fold), suggesting that these pathways are not implicated in LIF transcriptional synergistic effects. Using a DNase I footprint assay, POMC promoter regions protected by AtT20 cell nuclear extracts were identified (-121/-109, and -143/-134, and -173/-160). The protected -173/-160 element fused to a heterologous promoter conferred LIF-CRH synergy (6.5-fold induction of POMC) and formed a specific complex with AtT20 cell nuclear extracts. This complex was supershifted by an anti-phosphoserine antibody, and a serine/threonine kinase inhibitor also altered both this complex and LIF-CRH transcriptional synergy on the POMC promoter-luciferase reporter construct, indicating that these events depend on post-translational serine phosphorylations. LIF-CRH synergy on POMC transcription is therefore mediated at least in part by -173/-160 sequences conferring confluent transcriptional activity of both peptides.

Original publication

DOI

10.1074/jbc.272.16.10551

Type

Journal article

Journal

J Biol Chem

Publication Date

18/04/1997

Volume

272

Pages

10551 - 10557

Keywords

Animals, Base Sequence, Cell Line, Corticotropin-Releasing Hormone, DNA Footprinting, Deoxyribonuclease I, Drug Synergism, Genes, Reporter, Genes, fos, Growth Inhibitors, Interleukin-6, Leukemia Inhibitory Factor, Luciferases, Lymphokines, Mice, Oligodeoxyribonucleotides, Pituitary Gland, Pituitary Neoplasms, Pro-Opiomelanocortin, Promoter Regions, Genetic, Proto-Oncogene Proteins c-fos, Rats, Recombinant Fusion Proteins, Transcription, Genetic, Transfection