Article Figures & Data
Figures
- FIG. 1.
NEPAS is a bHLH/PAS domain protein. (A) RT-PCR analysis of IPAS mRNA expression in the lung. RNA was purified from wild-type lung at various developmental stages: E15.5, E17.5, P8, P15, adult mouse, and hypoxia-treated adult mouse (A-H48; 6% O2 for 48 h). The primers used are indicated with arrows (a to d). The 151-bp band (primer set a-b) was the same size as HIF-3α, whereas the 424-bp band (primer set c-d) was an unexpected size. (B) Amino acid sequence of NEPAS. Underlined amino acids are different from those of HIF-3α. (C) Comparison of NEPAS, IPAS, and HIF-3α showing each splicing pattern (NEPAS, thick line; IPAS, dashed line; HIF-3α, thin line) and the primary structures. (D) GAL4 two-hybrid assay in 293T cells under normoxic conditions. Note that NEPAS interacts with Arnt (left). NEPAS inhibits reporter gene activation by HIFs under normoxic conditions (white bars) or after exposure to CoCl2 (black bars) (middle). The NEPAS-Arnt heterodimer has a weak transactivation activity under normoxic conditions (white bars) or after exposure to CoCl2 (black bars) (right). Data are presented as induction (n-fold) relative to cells transfected with reporter plasmid alone, defined as 1. Note the difference in magnitude of transactivation in the middle and right panels. The error bars represent means ± standard deviations.
- FIG. 2.
Targeted disruption of NEPAS/HIF-3α gene. (A) RT-PCR analysis of NEPAS and HIF-3α expression in various mouse tissues at different stages. Br, brain; H, heart; Lu, lung; Li, liver; K, kidney. As an internal control, 18S rRNA was used. (B) NEPAS/HIF-3α targeting construct. Top, map of the murine NEPAS gene; middle, structure of the NEPAS targeting construct containing a neomycin resistance gene (Neo), a green fluorescent protein gene (GFP), and a diphtheria toxin gene (DT); bottom, structure of the targeted NEPAS/HIF-3α allele that results in insertion of the Neo and GFP genes within the bHLH domain. The black bar represents the hybridization probe used for Southern blot analysis. (C) Southern blot analysis of SpeI-digested genomic DNA from ES cell clones (left) and mouse tails (right). The 10.5-kb band corresponds to the wild-type NEPAS allele/HIF-3α, whereas the 12.2-kb band corresponds to the homologous recombined allele. (D) Macroscopic examination of E8.5 and E13.5 embryos by fluorescent field and E13.5 embryos by bright-field microscopy. The genotypes of each image were either wild type (+/+) or NEPAS/HIF-3α−/− (−/−). Note that GFP was expressed in the entire NEPAS/HIF-3α−/− embryonic body and was strongly expressed in vessels (E8.5, arrowheads) and heart (E13.5, arrowheads). Bars, 1 mm (E13.5) and 0.5 mm (E8.5).
- FIG. 3.
Impaired heart and lung development in NEPAS/HIF-3α−/− mice. (A) Hematoxylin-eosin staining of sections from wild-type mice (top) and NEPAS/HIF-3α−/− mice (bottom) at E17.5, P8, and adult stage showing enlargement of the right sides of NEPAS/HIF-3α−/− hearts. Bars, 0.1 mm (E17.5) and 1 mm (P8 and adult). (B) Histograms of RV and LV dimensions and RV blood pressure obtained by echo analysis of seven or eight mice (mean ± standard deviation). **, P < 0.01. (C) Immunostaining of adult wild-type and NEPAS/HIF-3α−/− hearts with antidesmin and anti-CD31 antibodies. Black staining represents positive signals. Note the irregular striated muscle fibers and large number of microcapillaries in the myocardium of the NEPAS/HIF-3α−/− heart. Bars, 100 μm (CD31) and 50 μm (desmin). (D) Hematoxylin-eosin (H.E.)-stained P2 (top) and P5 (bottom) lung sections of wild-type (left) and NEPAS/HIF-3α−/− (right) mice. Bar, 100 μm. (E) Hematoxylin-eosin (H.E.)-stained and CD31-immunostained P15 (top four panels) and adult (bottom four panels) lung sections. Note the incomplete alveolar spaces surrounded by CD31-positive endothelial cells (black staining) in NEPAS/HIF-3α−/− lung at both P15 and the adult stage. Bars, 100 μm (hematoxylin-eosin staining) and 50 μm (CD31). (F) Victoria blue-van Gieson staining for the detection of elastic fibers surrounding vessels in NEPAS/HIF-3α−/− lung at P8. Elastic fibers were stained blue and classified as SEF-containing vessels (left), PEF-containing vessels (middle), or fully MEF-containing vessels (right). Bar, 10 μm. (G) Comparison of the frequency of SEF (white bars), PEF (crosshatched bars), and MEF (black bars) in P8 and adult lungs between wild-type and NEPAS/HIF-3α−/− mice. Note that the number of vessels <30 μm in diameter, classified as MEF, per 100 alveoli in both P8 and adult NEPAS/HIF-3α−/− lungs increased significantly (P < 0.01).
- FIG. 4.
Coexpression of NEPAS and HIF-2α. (A) Immunostaining of NEPAS/HIF-3α−/− (−/−) and wild-type (+/+) lungs at P15 using anti-HIF-2α, anti-CD31, and anti-GFP antibodies. Normal rabbit IgG was used as a negative control (top left). Positive immunostaining signals are brown. Anti-GFP antibody staining of wild-type mouse lung is similar to that of the normal IgG control (data not shown). Note the colocalization of HIF-2α and GFP expression in vascular endothelium. V, vessel; T, trachea. (B) Immunostaining of NEPAS/HIF-3α−/− and wild-type lungs at P15 using anti-SP-D and anti-GFP antibodies. SP-D is a marker of type II alveolar cells. Note the colocalization of SP-D and GFP expression in alveolar epithelium (arrowheads). Bar, 50 μm.
- FIG. 5.
Elevated ET-1 expression in NEPAS/HIF-3α−/− lung endothelial cells. (A) FACS sorting of lung endothelial cells from wild-type (left) and NEPAS/HIF-3α−/− (right) mice at P8. Lung cells in the rectangles designated R1 and R2 (wild type) and R3, R4, and R5 (NEPAS/HIF-3α−/− mice) were harvested for RT-PCR analyses. R1, CD31+/GFP−; R2, CD31−/GFP−; R3, CD31+/GFP+; R4, CD31−/GFP+; R5, CD31+/GFP−. APC, allophycocyanin. (B) RT-PCR analyses of the mRNA expression of HIF-2α, HIF-1α, VEGF164, Flk-1, ET-1, and PDGF-β in R1 to R5 fractions. Data are presented relative to the internal control 18S rRNA expression levels. The R1 data were set to 1. (C) Expression of ET-1 protein examined by ELISA in lung endothelial cells of wild-type and NEPAS/HIF-3α−/− mice. ET-1 expression was analyzed under normoxic conditions (N) or in the presence of CoCl2 (Co2+). *, P < 0.05.
- FIG. 6.
RT-PCR analysis of NEPAS mRNA expression in lung endothelial cells at P8. (A) FACS-sorted lung endothelial cells from wild-type (+/+) and NEPAS/HIF-3α−/− (−/−) mice at P8 were immortalized and examined for the expression of NEPAS or HIF-3α. (B) RT-PCR analysis of HIF-3α mRNA expression utilizing primer sets different from those used in panel A. Note that wild-type lung endothelial cells at P8 barely expressed HIF-3α, while NEPAS was abundantly expressed. WT, wild type.
- FIG. 7.
Enhanced ET-1 expression in NEPAS/HIF-3α−/− lung endothelial cells under normoxic conditions. (A) RT-PCR analysis of ET-1 (black bars) and VEGF (white bars) expression in wild-type and NEPAS/HIF-3α−/− lung-derived endothelial cells under normoxic conditions (N) and after exposure to CoCl2 (H). (B) Transfection of NEPAS into NEPAS/HIF-3α−/− cells revealing significant suppression of ET-1 expression. (C) ChIP assay using anti-HIF-1α and anti-HIF-2α antibodies on wild-type and NEPAS/HIF-3α−/− lung endothelial cells under normoxic conditions (N) or in the presence of CoCl2 (H). Normal mouse IgG or rabbit IgG was used as a negative control. (D) Transfection of Arnt, HIF-1α, or HIF-2α into lung endothelial cells revealing induction of ET-1 (black bars) but not VEGF (white bars) expression. (E) Western blot analysis of wild-type and NEPAS/HIF-3α−/− cells showing the expression of HIF-2α (top), HIF-1α (middle), and control lamin B (bottom). (F) Model illustrating the effect of NEPAS on ET-1 regulation during embryonic and neonatal stages.
