title: Hypoxia-inducible factor 1 transactivates the human leptin gene promoter.
abstract: Increased placental leptin has been demonstrated in preeclampsia, a pregnancy disorder associated with placental hypoxia. This suggests that leptin gene expression is enhanced in response to oxygen deficiency in this organ. In support of this hypothesis, we have previously shown that hypoxia activates the leptin promoter in trophoblast-derived BeWo cells. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric HIF-1alpha/HIF-1beta complex that regulates the transcription of hypoxia-responsive genes. To test whether this factor is involved in hypoxia-induced leptin promoter activation, BeWo cells were transiently transfected with a HIF-1alpha expression vector. Exogenous HIF-1alpha markedly increased luciferase reporter activity driven by the leptin promoter when HIF-1beta was co-expressed in the same cells. This effect was similar to that elicited by CoCl2, an agent known to stabilize endogenous HIF-1alpha. These data suggest that HIF-1alpha/HIF-1beta dimers are involved in the effect of CoCl2 to activate the leptin promoter. To confirm the implication of HIF-1, the cells were transfected with a dominant negative form of HIF-1alpha producing transcriptionally inactive HIF-1beta/HIF-1alpha dimers. This mutant HIF-1alpha protein abolished CoCl2 activation of the leptin promoter, providing direct evidence that the effect of CoCl2 is mediated by endogenous HIF-1alpha. Deletion analysis and site-specific mutagenesis demonstrated that a HIF-1 consensus binding site (HRE) spanning -120 to -116 bp relative to the start site was required for CoCl2 and exogenous HIF-1alpha induction of leptin promoter activity. Electrophoretic mobility shift assays performed with in vitro-translated HIF-1alpha and HIF-1beta proteins demonstrated binding to this HRE and not to mutated sequences only when both subunits were used together. These data demonstrate that leptin is a new hypoxia-inducible gene, which is stimulated in a placental cell line through HIF-1 interaction with a consensus HRE site located at -116 in the proximal promoter.
authors.affiliation: INSERM U 465, Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, 15 Rue de l'Ecole de Médecine, 75006 Paris, France.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Grosfeld
authors.forename: Jocelyne
authors.initials: J
authors.lastname: Andre
authors.forename: Sylvie
authors.initials: S
authors.lastname: Hauguel-De Mouzon
authors.forename: Edurne
authors.initials: E
authors.lastname: Berra
authors.forename: Jacques
authors.initials: J
authors.lastname: Pouyssegur
authors.forename: Michele
authors.initials: M
authors.lastname: Guerre-Millo
mesh_terms: D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D015687:Cell Hypoxia; D002474:Cell-Free System; D002822:Choriocarcinoma; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020738:Leptin / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011817:Rabbits; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; D012156:Reticulocytes / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: C497396:ARNT protein, human; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020738:Leptin; D009687:Nuclear Proteins; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator