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Am J Physiol Lung Cell Mol Physiol 290: L880-L889, 2006. First published December 22, 2005; doi:10.1152/ajplung.00213.2005
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Regulated expression of hypoxia-inducible factors during postnatal and postpneumonectomy lung growth

Quiyang Zhang, Orson W. Moe, Joseph A. Garcia, and Connie C. W. Hsia

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 16 May 2005 ; accepted in final form 19 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously found increased expression of erythropoietin receptor (EPO-R) in peripheral dog lung during postnatal and postpneumonectomy (PNX) lung growth. To study the upstream regulation of EPO-R, we analyzed the expression of hypoxia-inducible factors (HIF)-1{alpha}, -2{alpha}, and -3{alpha} during postnatal lung growth in immature and mature (2.5 and 12 mo old, respectively) dogs and during compensatory lung growth 3 wk and 10 mo after right PNX. Relative to their respective controls, HIF-1{alpha} transcript was 52–95% higher in immature lungs and 284% higher in the remaining lung 3 wk post-PNX. HIF-2{alpha} transcript did not change during maturation but was 42% lower 3 wk post-PNX. HIF-3{alpha} transcript was 53–65% lower in both the immature lung and 3 wk post-PNX. Changes were no longer detectable 10 mo post-PNX. No change in HIF transcripts was observed in kidney and liver post-PNX. Consistent with the mRNA changes, HIF-1{alpha} protein was 120 and 196% higher in growing lungs and 3 wk post-PNX relative to their respective controls. Overexpression of HIF-1{alpha} in cultured HEK-293 cells increased endogenous expression of EPO-R protein. These results demonstrate regulated expression of the HIF system and parallel changes in HIF-1{alpha} and EPO-R expression during two types of lung growth. Because the normal growing lung is not hypoxic, the HIF system likely responds to other signals encountered during sustained lung strain.

hypoxia-inducible factors; pneumonectomy; postnatal development; lung growth; ribonucleic acid blot; real-time polymerase chain reaction; immunoblot

WE HAVE PREVIOUSLY SHOWN increased erythropoietin receptor (EPO-R) protein in peripheral dog lung during the following two types of lung growth: postnatal maturation and postpneumonectomy (PNX) compensatory growth (10). These findings provide evidence that the EPO axis regulates oxygen transport via more than one mechanism. In addition to endocrine EPO signaling that stimulates bone marrow erythropoiesis to enhance convective oxygen delivery, paracrine or autocrine EPO signaling in the lung may modulate growth of gas exchange tissue to enhance alveolar oxygen uptake. Because the EPO axis is one of many oxygen-sensitive pathways responsive to the hypoxia-inducible factors (HIF; see Refs. 3 and 33), activation of the HIF system may be responsible for the elevated EPO-R protein level during lung growth.

HIF is a heterodimer composed of {alpha}- and beta-subunits. The beta-subunit, also known as aryl hydrocarbon receptor nuclear translocator (ARNT), is constitutively expressed independently of oxygen availability. The {alpha}-subunit is stabilized by hypoxia, which allows the HIF dimer to interact with transcriptional coactivator proteins (20). HIF-1{alpha} is expressed widely in a hypoxia-dependent manner in the bronchial epithelium, bronchial smooth muscle cells, and alveolar epithelium (39). HIF-2{alpha} and HIF-3{alpha} are two other members of the basic-loop-helix (bHLH) PER/ARNT/SIM (PAS) domain protein superfamily. HIF-2{alpha}, also known as endothelial PAS domain protein-1 or HIF-1{alpha}-like factor, exhibits hypoxic stabilization and ARNT binding similar to HIF-1{alpha} (9) and is expressed predominantly in pneumocytes and in pulmonary vascular endothelial cells (8, 9). HIF-3{alpha} is similar to HIF-1{alpha} and HIF-2{alpha} in the bHLH and PAS domains but lacks the COOH-terminal transactivation domain (14).

Other than oxygen tension, nitric oxide, growth factors, and mechanical strain can modulate HIF-1{alpha} expression and function (6, 7); these same factors are also involved in regulating lung growth. Silencing HIF-1{alpha} mRNA retards lung cell proliferation in vitro (13). Targeted gene knockout of HIF-1{alpha} (22) and HIF-2{alpha} (8) results in embryonic lethality or neonatal respiratory distress. In addition, alveolar hypoxia enhances postnatal lung development in dogs (23) and compensatory lung growth in post-PNX rats (32), indirectly suggesting that hypoxia regulatory pathways may also mediate lung growth. Expression of HIF-2{alpha} correlates with that of vascular endothelial growth factor (VEGF) and VEGF receptors in newborn rat lungs during normal development or hyperoxic injury (17). The level of HIF-3{alpha} mRNA increases with moderate hypoxia (15) and may oppose the action of HIF-1{alpha} (14). However, the expression pattern of HIF-1{alpha}, -2{alpha}, and -3{alpha} in vivo during lung growth and maturation has not been established.

We hypothesized that the elevated autocrine/paracrine EPO-R expression during lung growth (10) is accompanied by upregulation of its upstream transcriptional regulators HIF-1{alpha}, -2{alpha}, and -3{alpha}. We measured the mRNA levels of HIF-1{alpha}, -2{alpha}, and -3{alpha} by RNA blot and real-time RT-PCR in the lungs of growing (immature, 2.5-mo old) and mature (12 mo old) dogs and in dogs that underwent right PNX or sham PNX at 2.5 mo of age and followed for 3 wk or 10 mo. We demonstrated that HIF-1{alpha} mRNA and protein expression is higher in the actively growing lungs of young animals than in mature lungs and that these changes are further accentuated during post-PNX compensatory lung growth. In separate in vitro experiments, we overexpressed human HIF-1{alpha} in HEK-293 cells and measured EPO-R protein in these cells after transfection to directly demonstrate the linkage between HIF transactivation and EPO-R gene expression.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animal surgery and tissue collection. The Institutional Animal Care and Use Committee approved the animal protocols. To examine postnatal growth, lung tissue was obtained during thoracotomy under general anesthesia from peripheral and central locations within the right upper lobe of normal foxhounds at 2.5 or 12 mo of age. To examine post-PNX lung growth, litter- and sex-matched foxhounds (2.5 mo of age) underwent either right PNX or right thoracotomy without PNX (sham). Under isoflurane anesthesia, the right lung was exposed via a lateral thoracotomy through the fifth intercostal space. Pre-PNX tissue samples were obtained from the right upper lobe. The lobar arteries and veins were ligated individually and cut between ligatures. The right main stem bronchus was stapled and cut. The stump was immersed under saline to check for leaks. After hemostasis was ensured, the thorax was closed in layers. Residual air within the thorax was aspirated. Sham animals underwent right thoracotomy without lung resection. At 3 wk or 10 mo after surgery, corresponding to ~3.5 and 12 mo of age, respectively, animals were deeply anesthetized with intravenous pentobarbital sodium and mechanically ventilated. Via a left thoracotomy, the left upper lobe was removed. Post-PNX tissue samples were taken from the peripheral and central regions of the lobe, rinsed with saline, flash-frozen in liquid nitrogen, and stored at –70°C for later analysis.

cDNA cloning and sequencing. Initial fragments of canine dog HIF-1{alpha}, -2{alpha}, and -3{alpha} cDNA were amplified by conventional RT-PCR. The primers were based on published human sequences for HIF-1{alpha} (GenBank accession no. NM_001530) and HIF-2{alpha} (BC051338 [GenBank] ), respectively, and primers for HIF-3{alpha} were based on a rat sequence (NM_022528 [GenBank] ; Table 1). The PCR fragments were cloned into pGEM-T Easy Vector (Promega, Madison, WI); after sequence verification, the inserts were excised and used as probes in RNA blot analysis.


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  		Table 1. Oligonucleotide primers

 
Probe preparation and RNA blot. Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction and DNA-free DNase Treatment and Removal Reagents (Ambion, Austin, TX) to remove genomic DNA. Aliquots of total RNA (20 µg) were fractionated by agarose gel electrophoresis, transferred to a nylon membrane by capillary action, and cross-linked to the membrane by ultraviolet irradiation. The cDNA probes for HIF-1{alpha}, HIF-2{alpha}, and HIF-3{alpha} were generated by the Rediprime II random prime labeling system (Amersham Biosciences, Piscataway, NJ). Nylon membranes were incubated in Quick-Hyb (Stratagene, La Jolla, CA) containing [32P]cDNA probe (>109 counts·min–1·µg cDNA–1) at 68°C for 2 h. The blots were washed in 2x saline-sodium citrate (SSC, 1x saline-sodium citrate is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and 0.1% SDS three times for 15 min each at room temperature, two times in 0.2x SSC for 10 min at 68°C, and subjected to autoradiography, and signals were quantified by densitometry. For control of loading, the membranes were stripped and rehybridized with a mouse 18S rRNA probe. Because of the very weak signal intensity, the RNA blot for HIF-3{alpha} was not quantified.

Quantitative real-time RT-PCR. Real-time RT-PCR was performed for HIF-1{alpha}, -2{alpha}, and -3{alpha} using the respective primers shown in Table 1. The primers for HIFs were based on partial dog cDNA sequences cloned in this study, and the for the reference gene cyclophilin A primers were based on a published dog sequence (AF243140 [GenBank] ; see Ref. 16). These primers were designed using Primer Express (Applied BioSystems, Foster City, CA), and manufactured by Integrated DNA Technologies (Coralville, IA). The cDNA was generated from 200 ng DNase I-treated total RNA in a 20-µl reaction using a Taqman Reverse Transcription Kit (Applied BioSystems). Amplification was carried out using an ABI Prism 7000 Sequence Detector (Applied BioSystems), with one cycle of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The reaction was performed in triplicate for each sample. Each reaction had a final volume of 20 µl, containing 1x SYBR Green Universal PCR Master Mix, 200 nM each of forward primer and reverse primers, and 5 ng cDNA. Pooled cDNA from four normal adult dog lungs was used as a calibration standard for different runs.

Analysis of RNA blot and PCR data. For RNA blot, three to four replicate assays were prepared; each replicate used a separate tissue sample obtained from a different location of the lobe to compare immature with mature (n = 6/group) or sham with PNX (n = 4/group) animals. The autoradiographic intensity of target mRNA bands was normalized to that of the corresponding 18S rRNA and then expressed as a ratio of the mean signal intensity of the respective control group (mature or sham) obtained on the same blot. The normalized ratios from replicate assays in each animal were averaged.

For real-time PCR, at least three replicate assays were performed; each replicate used a separate RNA sample from each lung (immature and mature, n = 6/group; sham and 3 wk post-PNX, n = 6/group; and sham and 10 mo post-PNX, n = 5/group). The relative quantity of mRNA with respect to the standard was calculated using standard equations (Applied BioSystems):

Formula
where {Delta}CT = mean threshold cycle(CT; HIF) – mean CT(cyclophilin), and {Delta}{Delta}CT = {Delta}CT(sample) – {Delta}CT(standard).

Individual results were expressed as a ratio to the mean value in the respective control group (mature or sham); replicate results from each animal were averaged and then compared among groups by one-way ANOVA (3 wk post-PNX vs. sham and pre-PNX, 10 mo post-PNX vs. sham) or unpaired t-test (immature vs. mature). Differences were considered significant at P < 0.05.

Cell culture and in vitro transfection. To explore whether there is a link between HIF-1{alpha} upregulation and increased EPO-R expression, we cultured human embryonic kidney (HEK)-293 cells in 5% CO2 and 95% air at 37°C in DMEM (GIBCO, Rockville, MD) containing FBS (10% vol/vol), L-glutamine (4 mM), and penicillin (100 µg/ml)-streptomycin (100 IU/ml). Before transfection (1 day), cells were transferred to six-well plates and incubated with the same medium without antibiotics. Next, the cells were transfected with a human HIF-1{alpha} expression vector construct; the HIF-1{alpha} expression vector contained inactivated prolyl and asparagyl hydroxylation sites generated by site-directed mutagenesis (37). Control cells were transfected with random DNA. Transfection was performed using Lipofetamine 2000 (Invitrogen, Carlsbad, CA) at a cell density of 75% confluence. After transfection (8 h), the medium was aspirated and replaced with medium containing serum and antibiotics, and the cells were incubated for another 24, 36, or 56 h. Next, the cells were rinsed two times with cold PBS and lysed with radioimmunoprecipitation assay (RIPA) buffer [50 mmol/l Tris·HCl at pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Triton X-100, 0.5% (wt/vol) deoxycholic acid, and 0.1% SDS] supplemented with the protease inhibitor cocktail (1 tablet/10 ml; Roche Diagnostics, Indianapolis, IN) on ice. The cell lysates were incubated at 4°C with a continuous rocking motion for 30 min and centrifuged (16,000 g) for 20 min. The supernatant was collected for immunoblot (see below).

Immunoblot. Lung tissue (~60 mg wet wt) was minced on ice and transferred to tubes containing RIPA buffer (see above). Tissue fragments were homogenized by Polytron followed by an ultrasonic device. The homogenates were incubated at 4°C for 30 min and cleared by centrifugation (16,000 g for 20 min). The supernatant was transferred to another tube for immunoblot.

Total protein concentration was measured by the Bradford method; aliquots (~150 µg from tissue homogenate and 40 µg from cell lysate) were resolved on 7.5% SDS-PAGE, transferred to polyvinylidene difluoride membranes, blocked in Blotto-Tween solution (5% nonfat dry milk, 0.05% Tween in PBS) for 1 h, and then incubated in Blotto-Tween with the primary antibody for 2 h. The primary antisera were rabbit anti-human HIF-1{alpha} polyclonal antibody (0.4 µg/ml, H-206; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-mouse EPO-R polyclonal antibody (1 µg/ml, C20; Santa Cruz). Labeled protein was visualized by chemiluminescence (ECL-plus; Amersham) and quantified by densitometry. For loading control, the membranes were stripped and re-probed with mouse anti-beta-actin monoclonal antibody (1 µg/ml, A1978; Sigma, St. Louis, MO). Specificity of the anti-HIF-1{alpha} antibody was examined by incubating a duplicate blot with 5X excess blocking peptide (sc-8711P; Santa Cruz).

For studies of dog lung, three assays were performed, each using a separate tissue sample from each animal. The HIF-1{alpha} signal intensity was normalized to that of beta-actin in corresponding samples and expressed as a ratio to the mean signal intensity of the respective control group (mature or sham) obtained on the same blot. Replicate results in a given animal were averaged and compared between groups (PNX vs. sham or immature vs. mature) by unpaired t-test.

For studies on HEK-293 cells, the HIF-1{alpha} and EPO-R protein levels in cells transfected with the human HIF-1{alpha} expression vector were compared with control cells transfected with random DNA in the same blot prepared at different time points (24, 36, and 56 h) posttransfection. The target signal intensity was normalized to that of beta-actin. The entire experiment was repeated three times. Results from treatment and control groups were compared by paired t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of HIF-{alpha} subunit mRNA in normal dog lung. For RNA blot, we used dog-specific, sequence-verified cDNA probes cloned from different regions of each HIF-{alpha} subunit (Fig. 1). Unique transcripts were obtained for HIF-1{alpha} (~4.5 kb) and HIF-2{alpha} (~4.7 kb). Three bands were labeled with the HIF-3{alpha} probe (~1.4, 1.6, and 3.2 kb) but were too faint for accurate quantification. Normalized with respect to cyclophilin A, mRNA abundance in normal dog lungs was greatest for HIF-2{alpha} followed by HIF-1{alpha} and HIF-3{alpha}.


Figure 1
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  		Fig. 1. The hypoxia-inducible factor (HIF)-1{alpha}, -2{alpha}, and -3{alpha} cDNA probes and primers are shown. The structural frame of the nucleotide sequences is outlined according to published cDNA data for human HIF-1{alpha} (NM_001530 [GenBank] ), rat HIF-2{alpha} (BC051338 [GenBank] ), and rat HIF-3{alpha} (NM_022528 [GenBank] ). The primers (upstream, downstream, or both) were designed to span the junctions of exons and introns for use in real-time RT-PCR amplification.

 
Expression of HIF-{alpha} subunit mRNA during postnatal lung growth. By RNA blot, HIF-1{alpha} was significantly (52%) higher in the actively growing lungs of 2- to 3-mo-old animals than in the lungs of 12-mo-old mature animals (P = 0.003; Fig. 2A). By comparison, HIF-2{alpha} was similar between the two age groups (P = 0.189; Fig. 3A). Real-time RT-PCR corroborated these findings: HIF-1{alpha} was 95% higher in the actively growing lungs compared with the mature lungs (P = 0.0007; Fig. 4A), whereas HIF-2{alpha} was not different between groups (P = 0.447; Fig. 4B). By real-time RT-PCR, HIF-3{alpha} was 65% lower in actively growing lungs than in mature lungs (P = 0.0001; Fig. 4C).


Figure 2
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  		Fig. 2. RNA blot of HIF-1{alpha} expression. Signal intensity was normalized to 18S rRNA and expressed as a ratio to the average intensity in control animals (mature or sham) within each assay. Four replicate assays were performed. Data are means ± SE. A: developmental lung growth (6 animals/group). HIF-1{alpha} mRNA was elevated significantly in immature compared with mature lungs. B: postpneumonectomy (PNX) lung growth (4 animals/group). HIF-1{alpha} mRNA in the remaining left lung was elevated further 3 wk post-PNX, reaching statistical significance with respect to the left lung of control animals (sham) but not with respect to the normal right lung removed at the time of PNX (pre-PNX).

 

Figure 3
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  		Fig. 3. RNA blot of HIF-2{alpha} expression. Signal intensity was normalized to that of the corresponding 18S rRNA and expressed as a ratio to the average intensity in control animals (mature or sham) within each assay. Three replicate assays were performed; data are means ± SE. A: developmental lung growth (6 animals/group). HIF-2{alpha} mRNA was not different between immature and mature lungs by unpaired t-test B: post-PNX lung growth (4 animals/group). HIF-2{alpha} mRNA was significantly lower in the remaining left lung 3 wk post-PNX compared with the left lung of age- and litter-matched controls (sham) and the normal right lung removed at the time of PNX (pre-PNX) by 1-way ANOVA. NS, not significant.

 

Figure 4
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  		Fig. 4. The abundance of HIF-1{alpha} (A), -2{alpha} (B), and -3{alpha} (C) transcript was quantified by real-time RT-PCR during postnatal lung growth. At least 3 replicate assays were performed using separate tissue samples from immature and mature lungs (6 animals/group). The target signal was normalized by that of the reference gene (cyclophilin) and a standard calibrator (pooled normal adult dog lung tissue) and expressed as a ratio to the average control (mature) value within each assay (left). Data are means ± SE; significance determined by unpaired t-test. Representative amplification plots are also shown for the target genes (middle) and the reference gene (cyclophilin A; right).

 
Expression of HIF-{alpha} subunit mRNA during compensatory lung growth. By RNA blot, HIF-1{alpha} mRNA levels were 41% higher 3 wk after right PNX than in corresponding sham lungs (P = 0.04) and ~33% higher than in normal lung tissue removed at the time of PNX, but the latter difference did not reach statistical significance (P = 0.10; Fig. 2B). On the other hand, post-PNX HIF-2{alpha} mRNA was 41% lower than that in sham lungs (P = 0.02) and 15% lower than in pre-PNX normal lungs (P = 0.04; Fig. 3B).

By real-time RT-PCR at 3 wk post-PNX, HIF-1{alpha} mRNA levels were 284% higher than in corresponding sham lungs (P = 0.001) and 272% higher than in pre-PNX normal lungs (P = 0.001; Fig. 5A). The HIF-2{alpha} transcript was 42% lower than that in sham lungs (P = 0.006) and 30% lower than that in pre-PNX normal lungs (P = 0.04; Fig. 5C). The HIF-3{alpha} transcript was 53% lower than that in sham lungs (P = 0.003) and 97% lower than that of pre-PNX normal lungs (P = 0.001; Fig. 5E). By 10 mo after surgery, the levels of HIF-1{alpha} (Fig. 4B), HIF-2{alpha} (Fig. 4B), and HIF-3{alpha} (Fig. 4F) transcript were no longer different from that in corresponding sham lungs (P > 0.05).


Figure 5
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  		Fig. 5. Relative mRNA abundance of HIF-1{alpha} (A and B), HIF-2{alpha} (C and D), and HIF-3{alpha} (E and F) was quantified by real-time RT-PCR during post-PNX lung growth. At least 3 assays were performed using separate tissue samples from each lung obtained 3 wk (A, C, and E, 6 animals/group) or 10 mo (B, D, and F, 5 animals/group) post-PNX and compared with age-matched controls (sham). The target mRNA signal was normalized to that of the reference gene (cyclophilin A) and a standard calibrator (pooled normal adult dog lung tissue) and expressed as a ratio to the average control value within each assay. Data are means ± SE; significance was determined by 1-way ANOVA (3 wk post-PNX vs. sham and pre-PNX) and unpaired t-test (10 mo post-PNX vs. sham).

 
Expression of HIF-{alpha} subunit mRNA in kidney and liver after PNX. By real-time RT-PCR, the mRNA levels of HIF-1{alpha} (Fig. 6A), -2{alpha} (Fig. 6B), and -3{alpha} (Fig. 6C) in the kidney and liver did not differ between animals studied 3 wk post-PNX and corresponding sham controls (P > 0.05). Thus the changes in mRNA expression of HIF-{alpha} subunits in response to PNX were specific to the lung.


Figure 6
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  		Fig. 6. Relative mRNA abundance of HIF-1{alpha} (A), HIF-2{alpha} (B), and HIF-3{alpha} (C) measured by real-time RT-PCR in the kidney and liver 3 wk post-PNX did not differ from that in the corresponding controls (sham). Triplicate assays were performed using separate tissue samples from each animal (6/group). The target mRNA was normalized to that of the reference gene (cyclophilin A) and a standard calibrator (pooled normal adult dog lung tissue) and expressed as a ratio to the average control (sham) value within each assay. Data are means ± SE; significance was determined by the unpaired t-test.

 
Expression of HIF-1{alpha} protein during lung growth. In the normal dog lung, we detected a major band of HIF-1{alpha} protein, ~70 kDa, that was specifically blocked by a blocking peptide (Fig. 7A). In the immature lung, the HIF-1{alpha} protein level was 120% higher than in the mature lung (P = 0.0001; Fig. 7B). In the remaining lung, 3 wk post-PNX HIF-1{alpha} protein was 196% higher than in the control lung from sham animals (P = 0.001; Fig. 7C).


Figure 7
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  		Fig. 7. Immunoblot of HIF-1{alpha}. Triplicate blots were performed using separate tissue samples from each lung. Signal intensity was normalized to that of beta-actin and expressed as a ratio to the average intensity in control animals (mature or sham) within an assay. Data are means ± SE; significance was determined by unpaired t-test. A: specificity of HIF-1{alpha} immunolabeling was demonstrated using 5x excess of a blocking peptide. B: developmental lung growth (5 animals/group). HIF-1{alpha} protein level was significantly higher in immature than in mature lungs. C: post-PNX lung growth (5 animals/group). HIF-1{alpha} protein in the remaining lung was further elevated 3 wk post-PNX compared with the same lung from matched controls (sham).

 
Effect of HIF-1{alpha} overexpression on EPO-R production. In cultured HEK-239 cells, we observed enhanced expression of HIF-1{alpha} protein 24, 36, and 56 h after transfection with the HIF-1{alpha} expression construct compared with control cells transfected with random DNA (Fig. 8); differences were statistically significant at 36 h (5.4-fold, P = 0.01) and 56 h (5.0-fold, P = 0.02). Concurrently, EPO-R protein level was also elevated in HIF-1{alpha}-transfected cells at 36 and 56 h (1.9- and 1.4-fold, respectively, P < 0.05; Fig. 8).


Figure 8
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  		Fig. 8. Overexpression of HIF-1{alpha} increased erythropoietin receptor (EPO-R) expression in HEK-293 cells. Cultured cells were transfected with either a HIF-1{alpha} expression vector (HIF) or random DNA [control (con)]. At 24, 36, and 56 h after transfection, total cell lysates were immunolabeled with anti-HIF-1{alpha} or anti-EPO-R. Signal intensity was normalized to that of the corresponding beta-actin. The experiment was repeated 3 times. Data are means ± SD; significance was determined by paired t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study was undertaken to determine if the previously reported upregulation of EPO-R protein during postnatal and compensatory lung growth (10) was associated with upregulation of its transcriptional factors, the HIFs. We document, for the first time, the in vivo upregulation of HIF-{alpha} subunits during these two types of lung growth. The mRNA of HIF-1{alpha} was 95% higher in actively growing lungs than in mature lungs and further increased by 284% during compensatory growth 3 wk after right PNX when cell proliferation in the remaining lung was markedly activated (10). HIF-2{alpha} mRNA was not altered during postnatal lung growth and was 42% lower 3 wk post-PNX compared with sham controls. HIF-3{alpha} mRNA was 65% lower in postnatal growing lungs than in mature lungs and further reduced (by 53%) 3 wk post-PNX. By 10 mo after PNX, the mRNA of all HIF-{alpha} subunits had completely returned to control levels corresponding to normalized growth markers in the remaining lung (10). The differential response of various HIF-{alpha} subunits to PNX was specific to the lung; no effect was observed in the kidney or liver. Consistent with the elevated transcripts, HIF-1{alpha} protein was also elevated in normal growing lungs, and the elevation was further accentuated during compensatory lung growth. In addition, in vitro overexpression of HIF-1{alpha} enhanced endogenous EPO-R production in cultured HEK-293 cells, supporting a direct link between HIF-1{alpha} activation and EPO-R production. We conclude that the differential regulated expression of HIF subunits could modulate in vivo expression of EPO-R during normal and compensatory lung growth.

HIF-1{alpha} expression. Mammalian bHLH-PAS proteins such as HIF-1{alpha} show considerable homology to Trachealess, the protein that regulates tracheal development in Drosophila (21, 38). The HIF-1{alpha} subunit is responsive not only to a low oxygen tension but also to androgens (27), epidermal growth factor (40), insulin and interleukin-1 (34), and mechanical strain (6, 24). The downstream target genes of HIF-1{alpha} are involved in erythropoiesis, cell proliferation and survival, anti-apoptosis, angiogenesis, vascular remodeling, and vascular tone (1, 5, 12). Blocking HIF-1{alpha} protein synthesis markedly downregulates gene expression of heme oxygenase-I, phosphoglycerate kinase, and VEGF (13) that subserve a wide range of metabolic functions. A high level of HIF-1{alpha} is present in aggressively proliferating tumor cells in association with neoangiogenesis (25). Interference of HIF-1{alpha} mRNA synthesis retards lung cell proliferation in vitro (13). Targeted disruption of HIF-1{alpha} (22) is embryonically lethal, associated with widespread vascularization defects. In light of its diverse action, it is not surprising that HIF-1{alpha} is involved in postnatal organ growth and development, even though this aspect has not been investigated extensively.

Although severe ambient hypoxia (1% O2) suppresses markers of cell differentiation in human fetal lung explants (2), a more modest level (3% O2) enhances epithelial branching morphogenesis in cultured fetal mouse lung (35). Chronic alveolar hypoxia stimulates postnatal lung development in dogs (23) and guinea pigs (19) as well as compensatory lung growth in post-PNX rats (32). In post-PNX dogs, alveolar-arterial oxygen tension gradient is normal at rest but becomes abnormally elevated upon exercise (18). One might expect the intermittent and modest alveolar hypoxia after PNX to increase HIF-1{alpha} expression, leading to enhanced vascularization. However, organ hypoxia/ischemia typically stabilizes HIF-1{alpha} protein without increasing HIF-1{alpha} mRNA expression (31). Our finding of more abundant HIF-1{alpha} mRNA and protein reflects the contribution of transcriptional induction of HIF-1{alpha} in the growing lung. Because the maturing postnatal lung is not overtly hypoxic, elevated HIF-1{alpha} steady-state transcript levels in these lungs suggest the contribution of other signal(s) to HIF-1{alpha} activation, possibly including persistent mechanical lung strain imposed by the enlarging rib cage as well as hormones and growth factors associated with lung development.

HIF-2{alpha} expression. Although HIF-1{alpha} shows marked changes in oxygen-dependent protein degradation in cultured cells, HIF-2{alpha} is less dynamically regulated by hypoxia (30). In developing lungs, HIF-2{alpha} gene expression is temporally and spatially associated with that of hypoxia-induced mitogenic factor, a potent hypoxia-induced cytokine involved in pulmonary vasoconstriction, proliferation, and angiogenesis (36). During fetal lung maturation, HIF-2{alpha} may also regulate VEGF synthesis or the activity of VEGF receptor-2 (8). The expression of HIF-2{alpha} correlates with that of VEGF and VEGF receptors (VEGFR-1 and -2) in newborn rat lungs during normal development or hyperoxic injury (17). Although chronic upregulation of HIF-1{alpha} leads to active vascular remodeling and increased pulmonary arterial pressure (26), downregulation of HIF-2{alpha} ameliorates the pulmonary hypertension and right ventricular hypertrophy induced by chronic hypoxia (4). Mice with heterozygous deficiency of HIF-2{alpha} show reduced pulmonary levels of endothelin-1 and plasma catecholamine compared with wild-type mice. In our study, HIF-2{alpha} mRNA was reduced significantly after PNX but not during postnatal growth; the reduction may mitigate the post-PNX increase in pulmonary vascular resistance and the associated vascular remodeling.

HIF-3{alpha} expression. The level of HIF-3{alpha} mRNA increases with the duration of moderate hypoxia (15). The limited available literature suggests that HIF-3{alpha} and its splice variants oppose or inhibit the action of HIF-1{alpha} (14, 28, 29). Because of its lack of NH2-terminal and/or COOH-terminal transactivation domain, HIF-3{alpha} and its splice variants may compete with HIF-1{alpha} for association with the beta-subunit, thus forming an abortive complex that is unable to bind to the HIF-responsive element of target genes (14) or directly interact with HIF-1{alpha} (28). In our study, HIF-3{alpha} gene expression changed in the opposite direction from that of HIF-1{alpha} during lung growth, consistent with an inhibitory interaction between HIF-1{alpha} and -3{alpha}.

Comparison of developmental and compensatory lung growth. In the growing remaining lung after PNX, vigorous cellular proliferation and tissue growth are marked by further increased expression of proliferating cell nuclear antigen above the already elevated level observed in age-matched growing animals relative to adult animals (11). In these post-PNX animals, we showed that changes in expression of epidermal growth factor, its receptor, and surfactant proteins-A and -D during compensatory lung growth diverged from that during developmental lung growth (11). Subsequently, we reported that EPO-R protein is higher (by ~60%) in the actively growing postnatal dog lung and the increase is further accentuated (by another 60%) during post-PNX compensatory growth (10). Although all soluble fractions of EPO-R increased during postnatal lung growth, only one of three soluble fractions increased during post-PNX lung growth, suggesting different EPO-R processing during different types of lung growth. In the present study, the higher HIF-1{alpha} and lower HIF-3{alpha} mRNA levels in normal growing lungs were further accentuated after PNX. However, HIF-2{alpha} mRNA level was unchanged in the normal growing lung but significantly lower in the post-PNX lung, suggesting differences in signaling among HIF subunits during different types of lung growth. Thus, in multiple biochemical pathways that have been examined so far, the post-PNX response reproduces some but not all aspects of the developmental response, indicating differential involvement of distinct mediators or regulatory pathways during different types of lung growth.

In summary, these results implicate the HIF system as a potential regulator of lung growth. Postnatal lung growth is associated with elevated HIF-1{alpha} and decreased HIF-3{alpha} mRNA levels; the lower HIF-3{alpha} mRNA probably reflects suppression of a negative regulator to permit full upregulation of HIF-1{alpha} activity. Post-PNX compensation is associated with a further increase in HIF-1{alpha} mRNA and reductions in HIF-2{alpha} and -3{alpha} mRNA in the remaining lung. A selective lowering of HIF-2{alpha} after PNX but not during normal maturation may ameliorate the post-PNX increase in pulmonary vascular resistance and the associated pulmonary vascular remodeling. The protein expression of HIF-1{alpha} paralleled that of its mRNA expression, consistent with activated HIF-1{alpha} production during lung growth. In addition, in vitro overexpression of HIF-1{alpha} enhanced endogenous EPO-R production in cell culture, supporting a link between HIF-1{alpha} activation and EPO-R expression. We conclude that the previously observed EPO-R upregulation in the growing lung (10) is associated with activation of an upstream transcription regulator, HIF-1{alpha}, whereas other HIF subunits (2{alpha} and 3{alpha}) also modulate the response.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institutes of Health Grants RO1 HL-40070, HL-54060, HL-45716, HL-62873, DK-48482, and DK-20543 and by the Department of Veterans Affairs Research Service.


   ACKNOWLEDGMENTS
 
We thank Dr. Aaron S. Estrera for surgical expertise, Heather L. Stanley, Richard T. Hogg, Debbie C. Hogg, and Jue Yang for animal care and technical assistance, and the staff of the Animal Resources Center for veterinary assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. C. W. Hsia, Pulmonary and Critical Care Medicine, Dept. of Internal Medicine, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9034

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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