Transcriptional Activation of the Human Leptin Gene in Response to Hypoxia: INVOLVEMENT OF HYPOXIA-INDUCIBLE FACTOR 1

Grazia Ambrosini,Anjali K Nath,M Rocıáo Sierra-Honigmann,Jaime Flores-Riveros

doi:10.1074/jbc.m205172200

Abstract

In addition to having a major role in energy homeostasis, leptin is emerging as a pleiotropic cytokine with multiple physiological effector functions. The recently discovered proangiogenic activity of leptin suggested the hypothesis that its production might be regulated by hypoxia, as are other angiogenic factors. To examine this proposal, the expression of leptin protein and mRNA was measured and found to be markedly up-regulated in response to ambient or chemical hypoxia (upon exposure to desferrioxamine or cobalt chloride), an effect that requires intact RNA synthesis, suggesting a transcriptional mechanism. Transient transfection of cultured cells with deletion constructs of the leptin gene promoter linked to a reporter gene revealed a functional hypoxia response element (HRE) located at position -116 within the proximal upstream region. This putative HRE harbors a characteristic 5'-RCGTG-3' core motif, a hallmark of hypoxia-sensitive genes and recognized by the hypoxia-inducible factor 1 (HIF1), which consists of a HIF1alpha/HIFbeta heterodimer. Constructs harboring this -116/HRE supported reporter gene expression in response to hypoxia but not when mutated. Expression of HIF1alpha cDNA in normoxic cells mimicked hypoxia-induced reporter gene expression in cells cotransfected with the wild type leptin -116/HRE construct but not with the mutant. Gel shift assays with a (32)P-labeled leptin promoter -116/HRE probe and nuclear extracts from hypoxia-treated cells indicated binding of the HIF1alpha/beta heterodimer, which was blocked with an excess of unlabeled -116/HRE probe or a HIF1-binding probe from the erythropoietin gene enhancer. Taken together, these observations demonstrate that the leptin gene is actively engaged by hypoxia through a transcriptional pathway commonly utilized by hypoxia-sensitive genes.

Highlights

In addition to having a major role in energy homeostasis, leptin is emerging as a pleiotropic cytokine with multiple physiological effector functions
We examine the activity of the leptin gene promoter linked to a reporter gene in transiently transfected cells and demonstrate the existence of a functional hypoxia response element (HRE), which is required for hypoxic induction and is transactivated by hypoxia-inducible factor 1 (HIF1)␣
We show for the first time that the leptin gene is transcriptionally activated in response to hypoxia through a mechanism that involves binding of the heterodimer HIF1␣/␤ to a functional HRE site located within the proximal promoter region

Summary

The abbreviations used are

Erythropoietin; C/EBP, CCAAT/ enhancer-binding protein; DFO, desferrioxamine; EMSA, electrophoretic mobility shift assay; HIF1, hypoxia-inducible factor 1; HRE, hypoxia response element; hSDF, human skin dermal fibroblasts; LUC, luciferase; RT-PCR, reverse transcription-PCR. We have recently reported that the hormone leptin exhibits robust angiogenic activity when assayed by in vitro and in vivo experiments [20] This unexpected biological activity of leptin is in contrast with the prevailing view of its function, which portrays leptin as a key regulator of body weight primarily through its extensively documented central nervous systemmediated effects on food intake and energy expenditure [21]. We show that the HIF1␣/␤ heterodimer binds to this leptin promoter HRE upon exposure to hypoxia, and we demonstrate that HRE mutations that disable HIF1␣/␤ binding eliminate leptin promoter activation Taken together, these observations demonstrate that the leptin gene is actively engaged by hypoxia through mechanisms that are common to other hypoxiainducible genes, consistent with the concept of leptin as a bona fide angiogenic factor

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION