A common pro-opiomelanocortin-binding element mediates leukemia inhibitory factor and corticotropin-releasing hormone transcriptional synergy.
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.
Bousquet
C
C
Department of Medicine, Cedars-Sinai Research Institute-UCLA School of Medicine, Los Angeles, California 90048, USA.
Ray
D W
DW
Melmed
S
S
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I; D004357:Drug Synergism; D017930:Genes, Reporter; D016762:Genes, fos / Q000235:genetics; D006131:Growth Inhibitors / Q000494:pharmacology*; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008156:Luciferases / Q000096:biosynthesis; D008222:Lymphokines / Q000494:pharmacology*; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010902:Pituitary Gland; D010911:Pituitary Neoplasms; D011333:Pro-Opiomelanocortin / Q000096:biosynthesis* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D006131:Growth Inhibitors; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins; D011333:Pro-Opiomelanocortin; D003346:Corticotropin-Releasing Hormone; D008156:Luciferases; D003850:Deoxyribonuclease I