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Comparative Study
. 1997 Apr 29;94(9):4273-8.
doi: 10.1073/pnas.94.9.4273.

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

M Ema  1 , S Taya, N Yokotani, K Sogawa, Y Matsuda, Y Fujii-Kuriyama
Affiliations
Comparative Study

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

M Ema et al. Proc Natl Acad Sci U S A. .

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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Figures

Figure 1
Figure 1
Comparison of primary structures of mouse HLF and HIF1α gene products. (A) Identical amino acid (∗) and conservative substitutions (.) are indicated. Dashes indicate deletions to maximize the sequence similarity. Basic helix–loop–helix region and direct repeats of PAS domain are boxed. The entire PAS domain is enclosed in brackets. (B) Comparison of the basic amino acid sequence of Arnt (33), dSim (17), AhR (34, 35), HIF1α (15), HLF, and dTrh (18, 19). Amino acids identical to those of HLF are boxed.
Figure 2
Figure 2
Chromosomal localization of HLF gene on mouse R-banded chromosomes. The hybridization signals are indicated by an arrow. The metaphase spreads were hybridized as described (22) and photographed with Nikon B-2A (A) and UV-2A (B) filters. R-banded and G-banded patterns are demonstrated in (A) and (B), respectively.
Figure 3
Figure 3
Coimmunoprecipitation, EMSA, and transient transfection experiments. (A) Interaction of HLF or HIF1α with Arnt was investigated by coimmunoprecipitation assay using extracts from Sf9 cells transfected with baculovirus-carrying Arnt cDNA and 35S-labeled HLF or HIF1α proteins synthesized in the in vitro reticulocyte translation system as described. Lanes 1 and 5, input proteins. (B) EMSA experiment using the Epo HRE oligonucleotide. Combinations of the reaction components are indicated above the blot. (C) Transient transfection experiment in c4 cells. Combinations of effector and reporter plasmids and their amounts (μg) are indicated beneath the chart. (D) Effects of hypoxic treatment on the transcription of the reporter gene in Hep3B cells. Combinations of effector and reporter plasmids used for transfection and their amounts (μg) are indicated below the chart. Cells were grown under normoxic (N, 21% O2) and hypoxic (H, 1% O2) conditions.
Figure 4
Figure 4
RNA blot analysis using HIF1α and HLF cDNA probes. poly(A)-RNAs (3 μg) prepared from various tissues of mice, as described, were electrophoresed in a 0.8% agarose gel containing 2.2 M formaldehyde and then transferred to a nylon membrane. The membrane was probed with 32P-labeled mHIF1α cDNA (A), mHLF cDNA (B), mVEGF (C), and hG3PDH (D). Tissues from which poly(A)-RNAs were prepared are indicated across the top.
Figure 5
Figure 5
mRNA expression of HIF1α, HLF, and VEGF in the process of lung development. (A–C) RNA blot analysis was performed by using total RNAs (15 μg) from the lungs of embryos of 13.5 dpc (E13.5 dpc) to adult mouse with probes for HIF1α (A), HLF (B), and VEGF (C). (D) Ethidium bromide-staining of RNA electrophoresed on agarose gel. (E–G) In situ hybridization analysis of mouse lung of the postnatal day 0 (P0) by the cRNA probes for HIF1α (E), HLF (F), and VEGF (G). Coloring reaction was done for 1 day for F and 3 days for E and G. Arrowheads indicate the expression of alveolar epithelial cells.
Figure 6
Figure 6
Transcriptional regulation of the VEGF gene by a heterodimer of HLF and Arnt. (A) Schematic representation of the VEGF gene promoter region and reporter gene, and summary of the transfection assay. The VEGF gene promoter and the HRE-deleted promoter regions were PCR-amplified and ligated to the firefly luciferase reporter gene as described. The VEGF HRE sequences were tandemly ligated to the promoter of the heterologous SV40 promoter. Plasmid DNAs of a reporter (2 μg), effectors (1 μg), and β-galactosidase expressing pENL (1 μg) were cotransfected, and expressed luciferase and β-galactosidase activities were determined. (B) EMSA experiment using the HRE oligonucleotide of the VEGF gene. Combinations of various proteins components and competitor DNA are indicated above the blot.
Figure 7
Figure 7
Whole-mount in situ hybridization on mouse embryos using HLF, HIF1α, and flt-1 cRNA probe. (A) Lateral view of 9.5-dpc embryo hybridized with HIF1α cRNA probe. Hybridization signal was seen weakly in the vascular system. ∗, nonspecific signal in the otic vesicle. (B) Lateral view of 9.5-dpc embryo hybridized with HLF cRNA probe. Hybridization signal was observed clearly in the vascular system. F, position of cross-section in F. (C) Lateral view of 9.5-dpc embryo hybridized with flt-1 cRNA probe. (D) Lateral view of 10.5-dpc embryo hybridized with HLF cRNA probe. E, photograph of higher magnification (×45) in E. G and H, position of the cross-section in G and H. (E) Photograph of higher magnification (×45) of dorsal region of 10.5-dpc embryo. Arrows indicate the signals in the intersomitic plexus. (F) Transverse section of 9.5-dpc embryo hybridized with HLF cRNA. Note that HLF mRNA was expressed in the endothelial cells of the dorsal aorta. (G) Transverse section of 10.5-dpc embryo. Arrowheads indicate the signals in the intersomitic plexus. (H) Transverse section of the 10.5-dpc embryo. An arrowhead indicates the signal of the endothelial cells of the sprouting blood vessels. da, dorsal aorta: nt, neural tube: o, optic vesicle; sg, sympathetic ganglion.
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