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-1α/HIF-1β 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-1α expression vector. Exogenous HIF-1α markedly increased luciferase reporter activity driven by the leptin promoter when HIF-1β was co-expressed in the same cells. This effect was similar to that elicited by CoCl2, an agent known to stabilize endogenous HIF-1α. These data suggest that HIF-1α/HIF-1β 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-1α producing transcriptionally inactive HIF-1β/HIF-1α dimers. This mutant HIF-1α protein abolished CoCl2 activation of the leptin promoter, providing direct evidence that the effect of CoCl2 is mediated by endogenous HIF-1α. 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-1α induction of leptin promoter activity. Electrophoretic mobility shift assays performed with in vitro-translated HIF-1α and HIF-1β 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. 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-1α/HIF-1β 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-1α expression vector. Exogenous HIF-1α markedly increased luciferase reporter activity driven by the leptin promoter when HIF-1β was co-expressed in the same cells. This effect was similar to that elicited by CoCl2, an agent known to stabilize endogenous HIF-1α. These data suggest that HIF-1α/HIF-1β 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-1α producing transcriptionally inactive HIF-1β/HIF-1α dimers. This mutant HIF-1α protein abolished CoCl2 activation of the leptin promoter, providing direct evidence that the effect of CoCl2 is mediated by endogenous HIF-1α. 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-1α induction of leptin promoter activity. Electrophoretic mobility shift assays performed with in vitro-translated HIF-1α and HIF-1β 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. hypoxia-inducible factor 1 hypoxia response element wild-type Leptin, originally identified as a satiety factor secreted by adipose tissue, is also produced by the placenta in humans (1Ahima R.S. Flier J.S. Annu. Rev. Physiol. 2000; 62: 413-437Crossref PubMed Scopus (1486) Google Scholar). Placental leptin mRNA (2Mise H. Sagawa N. Matsumoto T. Yura S. Nanno H. Itoh H. Mori T. Masuzaki H. Hosoda K. Ogawa Y. Nakao K. J. Clin. Endocrinol. Metab. 1998; 83: 3225-3229Crossref PubMed Scopus (261) Google Scholar) and protein (3Hauguel De-Mouzon S. Grosfeld A. Lepercq J. Turban S. Andre J. Cauzac M. Guerre-Millo M. Diabetes. 2001; 50 Suppl. 2: A4Google Scholar) are markedly increased in preeclampsia, a disorder associated with maternal hypertension, reduction in placental blood flow, and placental hypoxia (4Redman C.W. Placenta. 1991; 12: 301-308Crossref PubMed Scopus (506) Google Scholar). These observations have led to the proposal that the leptin gene could be induced by hypoxia. In support of this hypothesis, we have previously shown that gene expression and leptin release were increased in trophoblast-derived BeWo cells in response to various conditions of natural or chemical hypoxia. Moreover, the human leptin promoter was activated by hypoxia in these cells (5Grosfeld A. Turban S. Andre J. Cauzac M. Challier J.C. Hauguel-de Mouzon S. Guerre-Millo M. FEBS Lett. 2001; 502: 122-126Crossref PubMed Scopus (75) Google Scholar). Hypoxia-inducible factor 1 (HIF-1)1 is a transcription factor of major importance in the cellular response to oxygen deficiency. HIF-1 comprises HIF-1α and HIF-1β subunits, which both belong to the basic-loop-helix-PAS protein family (for review, see Ref.6Brahimi-Horn C. Berra E. Pouyssegur J. Trends Cell Biol. 2001; 11: S32-S36Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The HIF-1β subunit is constitutively expressed. By contrast, HIF-1α is maintained at a low level in normoxic cells through proteasomal degradation of the protein. The von Hippel-Lindau tumor supressor protein is a component of the complex that targets HIF-1α for polyubiquitination and degradation (7Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4117) Google Scholar). Two recent observations indicate that von Hippel-Lindau protein binds to HIF-1α when a proline residue at codon 564 is hydroxylated (8Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3873) Google Scholar, 9Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4432) Google Scholar). Hydroxylation of HIF-1α is controlled by a Fe2+-dependent hydroxylase activity that is inhibited by decreased oxygen. This mechanism accounts for HIF-1α stabilization in hypoxic cells, allowing nuclear translocation and dimerization with HIF-1β. Stabilization of HIF-1α is also induced by chelating or substituting Fe2+ with desferrioxamine and cobalt chloride (CoCl2), respectively. This provides a molecular mechanism accounting for the ability of these agents to mimic the effect of hypoxia in experimental cell systems. The present study was designed to test whether HIF-1 is involved in hypoxia-induced activation of the human leptin gene promoter in the placental BeWo cells. To investigate this, the transcriptional activity of HIF-1 was manipulated by overexpressing the wild-type or dominant negative form of HIF-1α. Our data provide evidence that induction of leptin promoter activity by hypoxia is mediated by HIF-1, through a HIF-1 consensus binding site (HRE) located at −116 in the proximal promoter. This study adds the leptin gene to the list of hypoxia-inducible genes regulated by this transcription factor. The human choriocarcinoma cell line BeWo was obtained through American Type Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum and antibiotics in a humidified ambient atmosphere with 5% CO2 at 37 °C. The 5′-deleted constructs containing various lengths of the human leptin promoter sequences upstream of the luciferase reporter gene have been described previously (5Grosfeld A. Turban S. Andre J. Cauzac M. Challier J.C. Hauguel-de Mouzon S. Guerre-Millo M. FEBS Lett. 2001; 502: 122-126Crossref PubMed Scopus (75) Google Scholar). Promoter fragments are designated according to their length in bp (p(bp)luc), relative to the transcription start site described in Ref. 10Miller S.G., De Vos P. Guerre-Millo M. Wong K. Hermann T. Staels B. Briggs M.R. Auwerx J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5507-5511Crossref PubMed Scopus (145) Google Scholar. Expression vectors encoding wild-type HIF-1α (pcDNA3-HA-HIF-1α), a dominant negative form of HIF-1α (pcDNA3-HA-DN-HIF-1α), or wild-type HIF-1 β (pcDNA3-HA-HIF-1β) have been described in Refs. 11Richard D.E. Berra E. Pouyssegur J. J. Biol. Chem. 2000; 275: 26765-26771Abstract Full Text Full Text PDF PubMed Google Scholar and 12Richard D.E. Berra E. Gothie E. Roux D. Pouysségur J. J. Biol. Chem. 1999; 274: 32631-32637Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar. A sequence contained within 0.146 kb of the leptin promoter sequence in the p(146)luc construct was mutated by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Two distinct mutations were generated. The motif 5′-GCACGTCG-3′ spanning −121 to −114 was replaced by 5′-GCTTAATG-3′ or by 5′-GCAAAACG-3′. The generated plasmids were designated p(146)lucmut1 and p(146)lucmut2, respectively. Each mutation was verified by direct sequencing, and two plasmid preparations isolated from distinct clones were tested in transfection. LipofectAMINE (Invitrogen)-mediated transfection of BeWo cells was performed as described previously (5Grosfeld A. Turban S. Andre J. Cauzac M. Challier J.C. Hauguel-de Mouzon S. Guerre-Millo M. FEBS Lett. 2001; 502: 122-126Crossref PubMed Scopus (75) Google Scholar). Briefly, 1 day before transfection, cells were plated into 35-mm 6-well dishes and transfected with 500 ng/well luciferase reporter construct and 60 ng/well pRSV-β-galactosidase expression vector to normalize for transfection efficiency. In some experiments, expression vectors encoding HIF-1β, HIF-1α wild-type, or HIF-1α mutant cDNA were co-transfected with the reporter constructs, as indicated in the figure legends. The total amount of transfected DNA was kept constant by adding the appropriate amount of pcDNA3 empty vector. Five h after transfection, the medium was changed, and the cells were cultured for 24 h in serum-free medium with or without addition of 100 μm CoCl2(Sigma). Transfections were performed at least in triplicate. In each experiment, individual data were calculated as the mean of triplicates and expressed as the ratio of luciferase to β galactosidase activity measured in the same cell lysate, as described previously (5Grosfeld A. Turban S. Andre J. Cauzac M. Challier J.C. Hauguel-de Mouzon S. Guerre-Millo M. FEBS Lett. 2001; 502: 122-126Crossref PubMed Scopus (75) Google Scholar). HIF-1α and HIF-1β proteins were expressed by in vitro translation in rabbit reticulocyte lysates (Promega, Madison, WI). pcDNA3 empty vector, pcDNA3-HA-HIF-1α, or pcDNA3-HA-HIF-1β was used as template. Electrophoretic mobility shift assays were performed using a double-stranded probe encompassing a sequence contained within the first 0.146 kb of the human leptin gene promoter. The sense strand sequence of the wild-type (wt) oligonucleotide is 5′-GCTAGCAGCCGCCCGGCACGTCGCTACCCTGAGGGGCG-3′. Two oligonucleotides containing distinct mutations of the underlined sequence in the wt probe, 5′-GCTAGCAGCCGCCCGGCTTAATGCTACCCTGAGGGGCG-3′ for mutant 1 (mut1) and 5′-GCTAGCAGCCGCCCGGCAAAACGCTACCCTGAGGGGCG-3′ for mutant 2 (mut2) were also used as probes in electrophoretic mobility shift assays. 32P-labeled oligonucleotides were generated by 5′ end labeling using T4 polynucleotide kinase (New England Biolabs, Hitchin, UK) with [γ-32P]ATP (50 μCi) and gel-purified. Reticulocyte lysates were incubated at room temperature in a 20-μl reaction containing 20 mmTris-HCl, pH 7.5, 50 mm KCl, 1 mmMgCl2, 0.5 mm EDTA, 5 mmdithiothreitol, 5% glycerol (v/v), and 250 ng of poly(dI-dC). One ng of 32P-labeled oligonucleotide (∼50,000 cpm/ng) was added 5 min later. Binding was then allowed to proceed for 15 min at room temperature. For competition experiments, a 5-, 10-, and 50-fold molar excess of unlabeled oligonucleotide, either wild-type or mutated, were added simultaneously with the labeled probe. DNA-protein complexes were resolved on native 5% polyacrylamide gels in 0.5× TBE (45 mm Tris-HCl, pH 8.3, 45 mm boric acid, and 1 mm EDTA). The gels were then dried and analyzed by autoradiography. Statistical analysis was performed using Student's t test for paired data, with significance defined as p < 0.05. An initial series of experiments was conducted to test whether increasing HIF-1 transcriptional activity would activate the leptin gene promoter in BeWo cells. Because transcriptional activation by HIF-1 depends on the amounts of HIF-1α available for heterodimerization with the constitutively expressed partner HIF-1β, we increased the cellular level of HIF-1α by the use of the pcDNA3-HA-HIF-1α expression vector. Previous experiments have shown that transient transfection of a fibroblast cell line with this plasmid allowed the detection of HIF-1α protein under normoxia (12Richard D.E. Berra E. Gothie E. Roux D. Pouysségur J. J. Biol. Chem. 1999; 274: 32631-32637Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar). The effect of HIF-1α overexpression on the luciferase activity driven by 1.872 kb of the human leptin promoter was tested. As shown in Fig.1, overexpression of HIF-1α moderately increased luciferase activity (by 2-fold) compared with cells expressing the reporter only. However, when cells were co-transfected with HIF-1α and HIF-1β, reporter gene expression increased markedly compared with cells transfected with either subunit individually. CoCl2 treatment induced a stimulatory effect on leptin promoter activity, which was very similar to that elicited by HIF-1α overexpression in the absence or presence of added HIF-1β. Moreover, when cells overexpressing HIF-1α were treated with CoCl2, luciferase activity did not double, indicating that the effects of CoCl2 and HIF-1α were not fully additive. These data support the implication of HIF-1α/HIF-1β dimers in the transactivation of the leptin gene promoter. Because overexpression of HIF-1β markedly enhanced luciferase activity in the presence of HIF-1α and/or CoCl2, it is likely that low endogenous HIF-1β levels restrict HIF-1 transcriptional activity in these experimental conditions. Thus, in the next series of experiments, the cells were systematically transfected with the HIF-1β expression vector. To determine the promoter region mediating activation by exogenous HIF-1α or CoCl2, BeWo cells were transfected with reporter constructs containing various lengths of the leptin gene promoter region. For each construct, the fold increase in luciferase activity elicited by either HIF-1α overexpression, CoCl2treatment, or a combination of both was determined over non-stimulated cells. As mentioned above, HIF-1β was routinely transfected, whatever the experimental condition. A similar pattern of reporter gene expression was observed for several constructs containing up to 0.146 kb of the leptin gene 5′-flanking region (Fig.2). Both HIF-1α and CoCl2individually stimulated luciferase activity by 5–7-fold. The effect of the two stimuli in combination was always greater than that elicited by HIF-1α or CoCl2 alone. However, when combined, these effects were never fully additive. In contrast to all other deleted constructs, the p(116)luc reporter vector was unresponsive to HIF-1α and/or CoCl2. This analysis revealed that the first 146 bp of the leptin promoter harbor a sequence responsive to CoCl2 and exogenous HIF-1α, which is missing or disrupted in the p(116)luc construct. To directly evaluate the involvement of HIF-1 in mediating the effect of CoCl2 on this region of the leptin promoter, BeWo cells were co-transfected with the p(146)luc construct and a dominant negative form of HIF-1α (HIF-1α DN). This mutant HIF-1α heterodimerizes with HIF-1β but lacks a DNA binding domain, thereby producing transcriptionally inactive dimers (11Richard D.E. Berra E. Pouyssegur J. J. Biol. Chem. 2000; 275: 26765-26771Abstract Full Text Full Text PDF PubMed Google Scholar). As shown in Fig.3, HIF-1α DN fully inhibited the stimulatory effect of exogenous HIF-1α on leptin promoter activity, demonstrating the efficiency of this dominant negative form of HIF-1α. HIF-1α DN also totally abolished the stimulatory effect of CoCl2 on luciferase activity in cells transfected with the p(146)luc construct. By contrast, the reporter activity arising from the p(116)luc construct was not significantly affected. These data provide direct evidence that endogenous HIF-1α is required for CoCl2-induced activation of the leptin promoter through a region extending 146 bp upstream of the transcription start site. Sequence analysis revealed the presence of a 5′-RCGTG-3′ HIF-1 binding consensus sequence (HRE) within the 0.146-kb promoter fragment. This putative HRE, located between −120 and −116 on the noncoding strand, is disrupted in the p(116)luc construct. To assess its functional importance, the sequence was mutated in the p(146)luc reporter vector. Constructs containing two distinct mutated fragments, p(146)lucmut1 and p(146)lucmut2, were transfected in BeWo cells, and their capacity to respond to CoCl2 treatment and HIF-1α overexpression was tested. As shown in Fig. 4, the luciferase activity produced by both mutated leptin promoter fragments was not increased by these stimuli. This supports the hypothesis that this HRE consensus sequence is required for induction of leptin promoter activity. To test whether the HRE identified within the proximal region of the human leptin promoter binds the HIF-1 complex of HIF-1α and HIF-1β, the two subunits were vitro by using the reticulocyte Electrophoretic mobility shift assays were performed with the proteins obtained in or reticulocyte As shown in Fig. a complex with when and lysates were incubated with the labeled probe containing the complex was when mutated oligonucleotides were used as the probe 5 In the binding of HIF-1α/HIF-1β to the wild-type probe was by competition with an excess of unlabeled probe, but not with each of the two mutated oligonucleotides 5 These data demonstrate that HIF-1 binds to the consensus HRE present within the proximal region of the leptin gene promoter. have shown previously that leptin gene expression is increased by hypoxia in a trophoblast-derived cell line (5Grosfeld A. Turban S. Andre J. Cauzac M. Challier J.C. Hauguel-de Mouzon S. Guerre-Millo M. FEBS Lett. 2001; 502: 122-126Crossref PubMed Scopus (75) Google Scholar). The present study provides functional evidence that HIF-1 this effect a element located at −116 in the human leptin promoter. This is on obtained in experiments the cellular level of HIF-1α was to or HIF-1 transcriptional activity. with the implication of HIF-1, exogenous overexpression of HIF-1α in the BeWo cells markedly activated the leptin promoter. Moreover, a similar amount of was produced by CoCl2 support to the that stabilization of endogenous HIF-1α by this agent leptin promoter The evidence for the implication of HIF-1 from the use of a dominant negative form of HIF-1α, which totally abolished the effect of CoCl2. This that CoCl2-induced leptin promoter activity is driven by increased endogenous HIF-1α to the activation of Sequence analysis the presence of several putative within the first 1.872 kb of the human leptin promoter. have previously observed that two containing and kb of the promoter and to hypoxia (5Grosfeld A. Turban S. Andre J. Cauzac M. Challier J.C. Hauguel-de Mouzon S. Guerre-Millo M. FEBS Lett. 2001; 502: 122-126Crossref PubMed Scopus (75) Google Scholar). These data to that a HRE located at kb in the promoter could the effect of hypoxia. 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