A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development.
Journal: 1997/May - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 9113979
Abstract:
We have isolated and characterized a cDNA for a novel Per-Arnt/AhR-Sim basic helix-loop-helix (bHLH-PAS) factor that interacts with the Ah receptor nuclear translocator (Arnt), and its predicted amino acid sequence exhibits significant similarity to the hypoxia-inducible factor 1alpha (HIF1alpha) and Drosophila trachealess (dTrh) gene product. The HIF1alpha-like factor (HLF) encoded by the isolated cDNA bound the hypoxia-response element (HRE) found in enhancers of genes for erythropoietin, vascular endothelial growth factor (VEGF), and various glycolytic enzymes, and activated transcription of a reporter gene harboring the HRE. Although transcription-activating properties of HLF were very similar to those reported for HIF1alpha, their expression patterns were quite different between the two factors; HLF mRNA was most abundantly expressed in lung, followed by heart, liver, and other various organs under normoxic conditions, whereas HIF1alpha mRNA was ubiquitously expressed at much lower levels. In lung development around parturition, HLF mRNA expression was markedly enhanced, whereas that of HIF1alpha mRNA remained apparently unchanged at a much lower level. Moreover, HLF mRNA expression was closely correlated with that of VEGF mRNA. Whole mount in situ hybridization experiments demonstrated that HLF mRNA was expressed in vascular endothelial cells at the middle stages (9.5 and 10.5 days postcoitus) of mouse embryo development, where HIF1alpha mRNA was almost undetectable. The high expression level of HLF mRNA in the O2 delivery system of developing embryos and adult organs suggests that in a normoxic state, HLF regulates gene expression of VEGF, various glycolytic enzymes, and others driven by the HRE sequence, and may be involved in development of blood vessels and the tubular system of lung.
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Proc Natl Acad Sci U S A 94(9): 4273-4278

A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1α regulates the <em>VEGF</em> expression and is potentially involved in lung and vascular development

Department of Chemistry, Graduate School of Science, Tohoku University, Sendai 980–77, Japan; Toneyama Institute for Tuberculosis Research, Osaka City University Medical School, Osaka 545, Japan; and Genome Research Group, National Institute of Radiological Sciences, Inage, Chiba 263, Japan
To whom reprint requests should be addressed.
Minor J. Coon, University of Michigan School of Medicine, Ann Arbor, MI
Received 1996 Dec 26; Accepted 1997 Feb 14.

Abstract

We have isolated and characterized a cDNA for a novel Per-Arnt/AhR-Sim basic helix–loop–helix (bHLH-PAS) factor that interacts with the Ah receptor nuclear translocator (Arnt), and its predicted amino acid sequence exhibits significant similarity to the hypoxia-inducible factor 1α (HIF1α) and Drosophila trachealess (dTrh) gene product. The HIF1α-like factor (HLF) encoded by the isolated cDNA bound the hypoxia-response element (HRE) found in enhancers of genes for erythropoietin, vascular endothelial growth factor (VEGF), and various glycolytic enzymes, and activated transcription of a reporter gene harboring the HRE. Although transcription-activating properties of HLF were very similar to those reported for HIF1α, their expression patterns were quite different between the two factors; HLF mRNA was most abundantly expressed in lung, followed by heart, liver, and other various organs under normoxic conditions, whereas HIF1α mRNA was ubiquitously expressed at much lower levels. In lung development around parturition, HLF mRNA expression was markedly enhanced, whereas that of HIF1α mRNA remained apparently unchanged at a much lower level. Moreover, HLF mRNA expression was closely correlated with that of VEGF mRNA. Whole mount in situ hybridization experiments demonstrated that HLF mRNA was expressed in vascular endothelial cells at the middle stages (9.5 and 10.5 days postcoitus) of mouse embryo development, where HIF1α mRNA was almost undetectable. The high expression level of HLF mRNA in the O2 delivery system of developing embryos and adult organs suggests that in a normoxic state, HLF regulates gene expression of VEGF, various glycolytic enzymes, and others driven by the HRE sequence, and may be involved in development of blood vessels and the tubular system of lung.

Keywords: vasculogenesis, tubular morphogenesis, trachealess
Abstract

Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells that has been shown to be expressed in many tumor cell lines and vascular smooth muscle (1, 2) and is important not only for vasculogenesis and angiogenesis, but also for the maintenance of existing blood vessels (3). VEGF gene expression is induced by environmental stresses such as hypoxia, anemia, myocardial ischemia, and tumor progression to initiate subsequent angiogenesis and neovascularization (46).

Recent studies have revealed that the VEGF gene is induced through binding of a protein factor designated hypoxia-inducible factor 1 (HIF1) to the hypoxia response element (HRE) upstream of the VEGF gene (79). HIF1 was originally found as a critical mediator for inducible expression of the erythropoietin (Epo) gene by hypoxia (10), and has been shown to activate transcription of many genes including tyrosine hydroxylase (11), inducible nitric oxide synthase (12), and various glycolytic enzymes (13, 14) in a hypoxia-dependent manner. Recent cDNA cloning has demonstrated that HIF1 is a heterodimer composed of HIF1α and HIF1β (15). The latter is a factor already known as Ah receptor nuclear translocator (Arnt), which functions in association with the Ah receptor (AhR) as a mediator of various biological and toxicological effects of dioxin and other xenobiotics (16). HIF1α is a novel factor comprising a basic helix–loop–helix (bHLH) domain and a Per-Arnt/AhR-Sim (PAS) domain in the N terminus and exhibiting a striking homology with transcription factors encoded by Drosophila sim (dSim) (17), a master gene regulating the CNS midline development, and D. trachealess (dTrh) (18, 19), a regulator of development of the O2 delivery system such as trachea. Interestingly, a basic amino acid sequence immediately prior to the N terminus of the HLH domain, which is known to be the site for DNA recognition, is completely conserved between HIF1α and dTrh (18, 19).

In the course of searching for novel dimerization partners of Arnt, we isolated a cDNA clone encoding a bHLH-PAS protein closely related to HIF1α and dTrh, which we term HLF (HIF1α-like factor). Although HLF exhibits very similar characteristics to HIF1α in the properties of dimerization, DNA-binding, and transcriptional activation, their modes of expression are quite different from each other, in that HLF mRNA is abundantly expressed in a variety of organs including lung, heart, and other organs of adult mice in a normoxic state, whereas HIF1α mRNA is ubiquitous at much lower levels. In lung development around parturition, expression of HLF mRNA was markedly enhanced, while the low levels of HIF1α mRNA remained apparently unchanged. These expression patterns of HLF mRNA were found to closely parallel those of VEGF mRNA. Whole mount in situ hybridization experiments demonstrated that HLF was expressed in vascular endothelial cells at the middle stages of mouse embryo development where HIF1α was almost undetectable. Proposed regulatory functions of HLF include transcription of genes for VEGF and glycolytic enzymes and development of the vascular and pulmonary tubular system.

Yeast strain SFY526 containing lacZ under the control of the GAL1 promoter was cotransfected with the indicated plasmids, and β-galactosidase activities were determined.

Acknowledgments

The authors thank Drs. M. Shibuya (The University of Tokyo, Tokyo) for mflt-1 and mflk-1 cDNA, L. Poellinger (Karolinska Institute, Stockholm) for hHIF1α cDNA, M. Gassmann (University of Zurich–Irchel, Zurich) for mHIF1α cDNA, M. Whitelaw (The University of Adelaide, Adelaide) for critically reading this manuscript, and Mr. H. Abe (The University of Tohoku, Sendai) for help in transfections experiments. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas, for Scientific Research (B); and the International Scientific Research Program from the Ministry of Education, Culture, Sport and Science of Japan; and by a fund from Sankyo. M.E. is a research fellow of the Japan Society for the Promotion of Science.

Acknowledgments

ABBREVIATIONS

VEGFvascular endothelial growth factor
Epoerythropoietin
AhRAh receptor
HIF1αhypoxia-inducible factor
P0postnatal day 0
EMSAelectrophoretic mobility shift assay
HLFHIF1α-like factor
HREhypoxia-response element
ArntAh receptor nuclear translocator
dTrh, Drosophila trachealessdpc, days postcoital
bHLHbasic helix–loop–helix
PASPer-Arnt/AhR-Sim
ABBREVIATIONS

Footnotes

Data deposition: The sequence reported in this paper has been deposited in the DDBJ, EMBL, and GenBank databases (accession no. {"type":"entrez-nucleotide","attrs":{"text":"D89787","term_id":"2117019"}}D89787{"type":"entrez-nucleotide","attrs":{"text":"D89787","term_id":"2117019"}}D89787).

Footnotes

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