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Published ahead of print on February 4, 2005, doi:10.1165/rcmb.2004-0314OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 395-403, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0314OC

Role of Hypoxia-Inducible Factor-1{alpha} in Hypoxia-Induced Apoptosis of Primary Alveolar Epithelial Type II Cells

Stefanie Krick, Bastian G. Eul, Jörg Hänze, Rajkumar Savai, Friedrich Grimminger, Werner Seeger and Frank Rose

Department of Internal Medicine/Pulmonary and Critical Care Medicine, Justus-Liebig-University, Giessen, Germany

Correspondence and requests for reprints should be addressed to Stefanie Krick, Department of Internal Medicine II, Klinikstrasse 36, D-35392 Giessen, Germany. E-mail: Stefanie.Krick{at}innere.med.uni-giessen.de


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Hypoxia affects alveolar homeostasis and may induce epithelial injury, which has been implicated in lung diseases such as fibrosis. The underlying cellular and molecular mechanisms are, however, largely unknown. Primary rat alveolar epithelial type II cells (ATII) exposed to graded hypoxia for 24 and 48 h caused a dose-dependent induction of cell cycle arrest and suppression of proliferation, which were comparable to the effects of angiotensin II, a potent inducer of ATII cell death. Hypoxia-induced changes in ATII homeostasis are thought to proceed primarily via activation of hypoxia inducible-factor (HIF)-1{alpha}, because hypoxia increased HIF-1{alpha} protein expression, nuclear translocation, and transactivation of its specific DNA binding domain, the hypoxia responsive element (HRE). Under hypoxic conditions, expression of the proapoptotic protein Bnip3L, which belongs to the Bcl 2 family and is known to be one of the HIF-1–dependent target genes, was upregulated. Suppression of HIF-1{alpha} or Bnip-3L with small interfering RNA (siRNA) fully blocked the hypoxia-induced apoptosis and Bnip3L expression. In line with these data, overexpression of HIF-1{alpha} by transient transfection enhanced the hypoxia-induced apoptosis. Thus, we conclude that hypoxia suppresses alveolar epithelial cell proliferation and enhances ATII apoptosis through activation of the HIF-1{alpha}/HRE axis and a mechanism that involves Bnip3L. Targeting HIF-1{alpha} may represent a new strategy that could impede the alveolar denudation that is observed in several lung diseases.

Key Words: alveolar epithelial type II cells • apoptosis • hypoxia • hypoxia-inducible transcription factor 1


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Increasing evidence suggests a role for programmed cell death in the homeostasis of the alveolar epithelium under normal and pathologic conditions. Alveolar epithelial cells undergo apoptosis during normal lung development and maturation, and pathologically in acute lung injury and lung fibrosis (1–4). Both in animal models and in human lung biopsies, the development of lung fibrosis correlates with a reduced ability of alveolar epithelial cells to proliferate and restore alveolar architecture (4). Apoptosis of alveolar epithelial type II cells has consistently been observed in sections of human and murine fibrotic lungs, which exhibited alveolar damage–induced epithelial denudation (3, 5). Thus, apoptosis of type II pneumocytes (ATII) may play an important role in the pathogenesis of lung fibrosis. The molecular triggers and signaling pathways are, however, still not known in detail.

Alveolar hypoxia provokes structural changes in the lung, which lead to chronic fibroproliferation resulting in inflammation and thickening of the alveolar wall (1, 5). In the early phase of hypoxia, destructive exudative changes occur, causing damage to the alveolar lining layer, apoptosis of ATII cells, and lung edema. In contrast, in the later phase of alveolar hypoxia, reactive hyperplasia of ATII cells is predominant (6). Both in cell culture and in vivo, alveolar hypoxia causes suppression of alveolar epithelial sodium and fluid transport by downregulation of the two primary alveolar sodium transport systems: the epithelial sodium channel (ENaC) and the Na,K-ATPase (7–11). The molecular mechanisms of hypoxia sensing and signaling underlying these structural alterations to ATII cells are still not clear; however, cumulative evidence suggests that activation of the hypoxia-inducible transcription factors (HIFs) is a fundamental hypoxia-driven signaling pathway in the lung (12, 13).

Hypoxia-inducible factors are heterodimeric transcription factors composed of a strictly regulated {alpha} subunit and a constitutive ß subunit (HIF-1ß/ARNT) (14, 15). HIF-1{alpha} is constitutively expressed, but undergoes rapid ubiquitination and proteasomal degradation, the rate of which is directly proportional to cellular oxygen tension. Under conditions of hypoxia, this consititutive decay of HIF-1{alpha} is suppressed, allowing it to translocate into the nucleus where it dimerizes with ARNT (16). The resultant heterodimeric effector molecule binds to hypoxia-responsive elements (HRE) located in the promoter and enhancer regions of hypoxia-regulated genes, causing their transactivation. Target genes include those fostering glucose uptake, glycolysis, erythropoeisis, and proliferation (12).

In the alveolar epithelium, it has been demonstrated that HIF-1{alpha} and HIF-2{alpha} subtypes are both present, but their hypoxic regulation differs markedly (17). Although hypoxic induction of HIF-2{alpha} seems to be independent from the cellular environment, the regulation of HIF-1{alpha} is more complex. Previous findings indicate that acute hypoxia leads to a strong induction of HIF-1{alpha} in A549 cells, as well as in the ferret lung, whereas mild hypoxia and chronic hypoxia did not show any upregulation of HIF-1{alpha} (18). Furthermore, recent studies have demonstrated feedback inhibition of HIF-1{alpha} by HIF-2{alpha} during chronic hypoxia, which is indicative of a transregulation mechanism between different HIF subtypes (19). Whether this is important in the adaptation of primary alveolar epithelial type II cells requires further investigation.

In the present study, performed in primary rat alveolar epithelial type II cells, we document a significant induction of apoptosis and suppression of proliferation during hypoxia. This hypoxia-induced effect was comparable to that provoked by angiotensin II, a potent inducer of ATII cell apoptosis, and may be linked to the development of lung fibrosis (20–23). The hypoxic signal transduction included HIF-1{alpha} stabilization, nuclear translocation, and HRE-dependent gene activation with the proapoptotic protein Bnip3L as a major target gene. Using RNA interference targeting HIF-1{alpha} and Bnip-3L, this hypoxic response was strongly suppressed. Transient transfection with HIF-1{alpha} cDNA enhanced the hypoxia-induced apoptosis. This hypoxia-induced cell death may impact upon alveolar epithelium homeostasis under conditions of chronic alveolar oxygen deprivation, with involvement of the HIF-1 signaling pathway and Bnip3L as major downstream effector.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents and Materials
Fluorescein isothiocyanate (FITC)-conjugated annexin-V, Alexa 568–conjugated annexin-V, and propidium iodide (PI) were purchased from Boehringer Mannheim (Mannheim, Germany). The HIF-1{alpha} antibody was kindly provided by Dr. Gassman (15). Male CD 18 Sprague-Dawley rats (180–200 g) were purchased from Charles River (Sulzfeld/Main, Germany). Affinity-purified polyclonal rabbit antibody against Bnip3L and the potent irreversible pancaspase inhibitor Boc-Asp(Ome) Fluoromethyl Ketone (Boc-D-FMK) were obtained from Sigma (St. Louis, MO). The FITC-conjugated goat anti-rabbit IgG was purchased from DAKO (Glostrup, Denmark). SiRNA targeting Bnip3L was purchased from Qiagen (Hilden, Germany).

Isolation of ATII Cells
ATII cells were isolated as described previously (24). Briefly, inflated and perfused lungs from pathogen-free male CD 18 Sprague-Dawley rats were lavaged and filled to capacity with a solution of elastase (30 U/ml) and trypsin (0.05 mg/ml). Lungs were minced and free cells were separated from lung tissue by sequential filtration through sterile gauze, 100 µm, and 10 µm nylon mesh. "Panning" of the resultant cell suspension was performed on rat IgG–coated plates. Nonadherent ATII cells were harvested after 1 h and resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetal calf serum. The yield of epithelial type II cells from each rat was in the range of 30–50 x 106 cells. The percentage of type II cells was 96 ± 2% as assessed by modified Papanicolaou, tannic acid, and alkaline phosphatase staining. Contaminating cells included alveolar macrophages (< 2% in all experiments), and neutrophils (< 2%). The ATII cell viability, as assessed by 5-carboxyfluorescein diacetate (CFDA) loading and trypan blue exclusion, was consistently above 96%. In transfection experiments, the human lung adenocarcinoma cell line A549 was obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM containing 3.7 g/liter bicarbonate and 1.6 g/liter glucose (F12/DMEM) medium supplemented with 10% (vol/vol) fetal bovine serum, glutamine, vitamins, nonessential amino acids, and penicillin/streptomycin. Cells were seeded in 12-well chambers and 100-mm diameter petri dishes at density of 500,000/cm2, and all experiments were conducted at subconfluent cell densities of 70–80% in serum-free F12/DMEM. Test reagents were diluted in DMEM.

Hypoxia Treatment
Culturing of cells under hypoxic conditions was performed in a chamber equilibrated with a water saturated gas mixture of 0 or 1% (vol/vol) oxygen, 10% carbon dioxide, and rest nitrogen at 37°C. Culturing of control cells under normoxic conditions was performed using an incubator saturated with water under normal atmospheric conditions, supplemented with 10% carbon dioxide at 37°C. Cells were seeded at high density and used for experiments 24 h after isolation to prevent transdifferentiation into type I cells. Additionally, the medium was supplemented only as a thin layer to decrease the diffusion distance of the ambient gas.

In additional experiments, cells were exposed to mild hypoxia (5%, 10%) above over a 24-h period. PO2 levels assayed in culture medium were 0, 10, 40, 75, and 150 mm Hg for 0, 1, 5, 10, and 21% O2, respectively. For the analysis of the HIF-1{alpha} system, we used 1% O2 because these are optimal conditions for the activation of HIF-1{alpha} (13).

Measurement of Cell Proliferation
For assessment of cell number, culture medium was aspirated and cells were incubated in 1 ml trypsin/EDTA at room temperature. After cell detachment, cells were centrifuged for 5 min at 1,200 rpm (25°C) and resuspended in 1 ml DMEM. Counting was performed using a Neubauer hemocytometer (Merck, Haar, Germany). Three wells were counted for each condition.

Detection of Apoptosis
After trypsinization from culture wells, cells were incubated in 50% (vol/vol) fetal calf serum for 15 min to restore membrane integrity and then centrifuged for 5 min at 1,200 rpm. Apoptotic cells were identified by detection of annexin-V binding using FACScan according to the protocol provided by Boehringer Mannheim. To exclude necrotic cells, cells were double-stained with 5 µg/ml PI. As an alternative method to measure apoptosis, we used a TUNEL assay kit (Calbiochem, La Jolla, CA) in a selected number of experiments. In all cases, TUNEL staining and annexin-V binding assays yielded similar results.

Cell Cycle Analysis
Cells (1 x 106) were maintained under hypoxic conditions (1% O2) for 24 h. S-phase cells were labeled with 10 µM bromodeoxyuridine (BrdU) for the last 30 min of treatment under either hypoxic or normoxic conditions. After trypsinization, cells were fixed with ice-cold 70% ethanol for at least 24 h before staining. Double-staining with propidium iodide and FITC-conjugated anti-BrdU antibody (Becton Dickinson, Heidelberg, Germany) was performed according to the manufacturer's protocol (25). Briefly, cells were treated with 2 N hydrochloric acid plus 0.5% Triton X-100 to denature the DNA, washed in 0.1 M sodium tetraborate to neutralize the acid, and then stained with anti-BrdU antibody diluted 1:50 in phosphate-buffered saline (PBS) containing 0.5% Tween 20 and 1% bovine serum albumin (BSA). After anti-BrdU staining, cells were washed again and resuspended in 5 µg/ml PI in PBS. After 1 h on ice, cells were analyzed on a FACScalibur flow cytometer (Becton Dickinson).

HIF-1{alpha} Western Blot Analysis
Western blot analysis of HIF-1{alpha} was performed using a previously described polyclonal HIF-1{alpha} antibody (15). Briefly, cells maintained under hypoxic and normoxic conditions were scraped from cell culture dishes, and cellular protein extracts were prepared by homogenization in an ice-cold buffer (10 mM Tris-HCl pH 6.8, 8 M urea, 10 [vol/vol] % glycerol, 1 [m/vol] % sodium dodecyl sulfate, 5 mM dithiothreitol) and a protease inhibitor cocktail (Sigma-Aldrich, Deisenhofen, Germany). Approximately 20 µg of protein was run per lane on a sodium dodecyl sulfate polyacrylamide gel. After electroblotting to a nylon membrane, HIF-1{alpha}–specific bands were visualized by chemiluminescence (ECL; Amersham, Freiburg, Germany) using a secondary anti-chicken antibody coupled with horseradish peroxidase.

Immuncytochemistry of HIF-1{alpha}
ATII cells were grown on chamber slides and incubated under hypoxic conditions for 24 h. The supernatant was aspirated and cells were fixed immediately in acetone and methanol (1:1). The fixed cells were incubated overnight at 4°C with PBS containing a HIF-1{alpha} monoclonal mouse antibody (BD Biosciences, Heidelberg, Germany) diluted 1:100. Indirect immunofluorescence was obtained by incubation with FITC-conjugated rabbit anti-mouse IgG antibody (DAKO), diluted 1:100 in PBS.

Reporter Gene Assay
A dual reporter gene assay for studying HIF-1{alpha}–dependent gene regulation was performed as previously described (26). In the first vector, a firefly luciferase reporter gene was controlled by a three-tandem repeat of the HRE from the erythropoietin gene, coupled to a thymidine-kinase (TK) promoter. In the second reporter vector, a Renilla luciferase gene was controlled by the TK promoter without HRE. Both vectors were co-transfected at a molar ratio of 3:1 (firefly/Renilla) with Lipofectamine (Invitrogen, San Diego, CA). Chemiluminescence of firefly and Renilla luciferase was measured independently in cell extracts with a bioluminometer. While firefly luciferase activity represents the specific HRE-dependent induction, the Renilla luciferase served as a reference value for transfection effiency.

Transfection of A549 Cells with HIF-1{alpha} cDNA
Because transient transfection of primary isolated ATII cells remained largely inefficient, we used the stable alveolar epithelial cell line A549 in our experiments. Cells were seeded on six-well dishes with 1.5 x 105 cells/well and transfected 24 h later using Effectene transfection reagent (Qiagen). Cells were maintained under hypoxic (1% O2) conditions for an additional 24 h before analysis. Control cells were maintained under normoxic conditions. A cDNA fragment containing the complete coding region of HIF-1{alpha} was prepared by reverse transcription-PCR from RNA extracts of human lung tissue and inserted into Bam HI/Xba I sites of the eukaryotic expression plasmid pMG (Invitrogen). Integrity of the plasmid was verified by automated capillary sequencing (ABIPrism310). The activity of HIF-1 was assessed by a reporter assay sensitive to HRE activity. Control cells were transfected with empty pMG plasmid at a similar concentration. To assess transfection efficiency, cells were transfected with 1 µg PMG/HIF-1{alpha} and 1 µg pEGFP, which constitutively expresses the green fluorescent protein (GFP) from a cytomegalovirus (CMV) promoter. The GFP-positive cells were quantified and indicated a transfection efficiency of 30%. Analysis of apoptosis was performed on GFP-positive cells and, for this condition, HIF-1{alpha} function was assessed by additionally co-transfecting the HIF-1{alpha}–responsive luciferase reporter construct. As control, we transfected the empty vector pMG, which showed an average percentage of apoptotic cells of 4 ± 1%, which was normalized as 100%.

Treatment of Primary ATII Cells with siRNA
It has recently been shown that introduction into mammalian cells of double-stranded oligoribonucleotides, also called siRNA, triggers the degradation of the endogenous mRNA to which the siRNA hybridizes. This mechanism is highly sequence specific and facilitated knock-down of endogenous levels of the target protein (27, 28). Using the HUSAR software package (Heidelberg Unix Sequence Analysis resources), an siRNA motif according to the AA-N19 rule was selected by applying the program to the human HIF-1{alpha} cDNA sequence (GenBank Accession No. U22431). A putative target sequence was identified at a position 146 bases downstream of the start codon from HIF-1{alpha}. The forward and reverse RNA strands (5'-UGU GAG UUC GCA UCU UGA U DTDT-3') and (5'-AUC AAG AUG CGA ACU CAC A DTDT-3') with two 50 deoxy-thymidine overhangs were commercially synthesized (Biospring, Frankfurt, Germany) and annealed at a final concentration of each 20 µM by heating at 95°C for 1 min and incubating at 37°C for 1 h in annealing buffer (20 mM potassium acetate, 6 mM Hepes–OH, pH 7.4, and 0.4 mM magnesium acetate). Transfection of siRNA was performed at a concentration of 100 nM using Oligofectamine or Lipofectamine 2,000 (Invitrogen) as described previously (26). As a control for HIF-1{alpha}–siRNA we used either a corresponding random siRNA sequence (control-siRNA: 5'-UAC ACC GUU AGC AGA CAC C DTDT-3') or in the case of luciferase reporter gene assay also luciferase siRNA (5'-CUU ACG CUG AGU ACU UCG A DTDT-3') targeting firefly luciferase (27). The siRNA targeting Bnip-3L was purchased from Qiagen, which provided validation by qRT-PCR.

Immuncytochemistry of Bnip3L Protein
ATII cells were grown on chamber slides and transfected with siHIF-1{alpha} or siRan as control. After another 24 h under hypoxic or normoxic conditions, cells were fixed in acetone/methanol (1:1). After blocking unspecific binding sites with 3% (m/vol) BSA, fixed cells were incubated for 60 min in PBS containing 0.1% (m/vol) BSA and an anti-human Bnip3L polyclonal rabbit antibody diluted 1:100. Indirect immunofluorescence was obtained by incubation with FITC-conjugated goat anti-rabbit IgG antibody (DAKO), diluted 1:100 in PBS. The chamber slides were mounted on glass slides and subjected to microscopic analysis.

Data Handling and Statistical Analysis
The data are given as mean ± SEM with n = 3–6. ANOVA with Scheffe's post-test was used to test for significant differences between the different groups; a P value of less than 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Hypoxia Induces Apoptosis and Inhibits Growth of ATII Cells
Annexin-V and PI staining were assessed by FACScan (10,000 counted cells per sample). Hypoxia (1% O2) increased the basal apoptotic rate of ATII cells from 5.9% to 9.7% (P < 0.005; Figure 1A). As a positive control, ATII cells were incubated with angiotensin (Ang) II (10 nM), which enhanced apoptosis up to 9.5 ± 0.04%. There was no significant change in necrosis due to hypoxia or Ang II. To investigate the effect of oxygen concentration on the apoptotic rate of ATII cells, we exposed cells to different O2 concentrations (0, 1, 5, 10%) for 24 h. Our results demonstrate a clear dose dependence of hypoxia-induced apoptosis, increasing significantly upon exposure to 10, 5, and 1% O2. Maximum was reached after a 24-h exposure to anoxia (0% O2; 37 ± 8%; P < 0,001; Figure 1B).



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  		Figure 1. Hypoxia strongly induces apoptosis in primary ATII cells in a dose-dependent manner. (A) Hypoxia increased levels of apoptosis as demonstrated by flow cytometric annexin-V staining. Cells in the lower right quadrant represent annexin-V–positive cells that are undergoing apoptosis; cells in the upper right quadrant are double-stained with annexin-V and PI, indicating necrosis. The summarized data in the histogram demonstrate the hypoxia-induced effect on apoptosis as compared with application of 10 nM Ang II. (B) Effect of different oxygen concentrations (0, 1, 5, 10, and 21% O2, incubation time 24 h) on apoptosis of ATII cells. Exposure to 10% O2 already showed a marked increase of apoptosis. This effect was further increased by lowering the oxygen concentration to 5%, 1%, or anoxia (0%) (**P < 0.01, ***P < 0.001 as compared with normoxic control cells).

 
Hypoxic Exposure Leads to Growth Arrest of ATII Cells
Cell proliferation was assessed by counting cell number. Freshly isolated cells were plated at high density to prevent differentiation into type I cells (29). Exposure to 1% O2 for 24 h decreased the cell number, similar to the decrease provoked by 10 nM Ang II (–35 ± 3% and –31 ± 4%; P < 0.01; Figure 2A).



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  		Figure 2. Hypoxia decreases proliferation and induces cell cycle arrest in ATII cells. (A) Assessment of proliferation by counting cell number after hypoxic or normoxic treatment for 24 h. Cells were initially seeded with 500,000 cells per well. Incubation with Ang II (10 nM), maintained under normoxic conditions is shown as an internal control. (B) Analysis of cell cycle progression by staining with propidium iodide and Anti-BrdU. Original FACS dot blots show cells in G0,1 phase and in S phase (as indicated). The summarized histogram demonstrates the percentage of cells in G0/G1 and S phase. Data represent means + SEM of five independent experiments each; **P < 0.01 as compared with normoxic controls.

 
Cell cycle progression was evaluated by double-staining with anti-BrdU and PI. Flow-cytometric analysis revealed that both hypoxia and Ang II induced cell cycle arrest by a significant increase of cells in G0/G1 phase and a decrease of cells in the S phase (P < 0.01; Figure 2B).

Inhibition of Caspases Prevents Hypoxia-Induced Apoptosis and the Decrease of Cell Number
Before exposure to 24 h of hypoxia (1% O2) or anoxia (0% O2), cells were pre-incubated with Boc-D-FMK (30 µM), a potent irreversible pancaspase inhibitor (30, 31). Our results showed a significant inhibition of apoptosis by BOC-FMK at 1 and 0% O2 (P < 0.05 and P < 0.01, respectively; Figure 3A). In line with these results, hypoxic cells treated with BOC-FMK showed no decrease of cell number compared with their hypoxic control cells (P < 0.01; Figure 3B).



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  		Figure 3. Inhibition of the hypoxia-induced effects by the pancaspase inhibitor BOC-FMK. (A) Preincubation with BOC-FMK (30 µM) attenuated the hypoxia-induced apoptosis at 1 and 0% oxygen. (B) Assessment of cell number after 24 h of normoxia or hypoxia with increasing concentrations of BOC-FMK, compared with untreated control cells. Cells were initially seeded with 500,000 cells per well. Data represent means ± SEM of five independent experiments each; *P < 0.05, **P < 0.01.

 
Comparison of Primary ATII and A549 Cells: Hypoxia-Induced Apoptosis and Upregulation of HIF-1{alpha} Protein
Because the A549 lung adenocarcinoma cells are used as a substitute for primary type II cells, we compared the effect of hypoxia on apoptosis and HIF-1{alpha} protein levels between these two cell types. To investigate whether exposure to hypoxic conditions influenced the development of apoptosis, we exposed both cell types to 1% O2 for 24 and 48 h. Our results indicated that there was an increase in apoptosis after 24 h (+70 ± 7% in ATII cells and +50 ± 7% in A549 cells). After 48 h under hypoxic conditions, ATII cell apoptosis was still elevated in both cell types (Figure 4A). Control cells maintained under normoxic conditions showed no change of basic apoptotic levels over the 48 h.



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  		Figure 4. Comparison between ATII cells and A549 cells concerning time dependence of apoptosis (flowcytometry), expression levels of HIF-1{alpha} protein, and its nuclear translocation. (A) Hypoxia increased levels of apoptosis after 24 and 48 h of hypoxia, compared with normoxic controls. (B) Assessment of HIF-1{alpha} by immunocytochemistry showing nuclear fluorescence after 24 h under hypoxic conditions in both cell types. This fluorescent signal was still detectable after 48 h under hypoxic conditions, but was absent in the normoxic controls. (C) Effects of hypoxia (24 h) on nuclear abundance of HIF-1{alpha} as assessed by immunoblotting, compared with normoxic conditions. Data represent means ± SEM of five independent experiments each; **P < 0.01, ***P < 0.001.

 
The amount and localization of HIF-1{alpha} protein was assessed by immunocytochemistry and Western blot. After 24 h under hypoxic conditions, a significant increase in nuclear-localized HIF-1{alpha} protein was observed in both cell types, compared with control cells maintained under normoxic conditions. This nuclear localization was still observed after 48 h under hypoxic conditions, but then with lower intensity (Figure 4B). Detection of HIF-1{alpha} protein by Western blot confirmed these results, because protein expression was markedly increased (~ 27-fold) after 24 h (Figure 4C, P < 0.05).

Overexpression of HIF-1{alpha} in A549 Cells
To achieve a high transfection efficiency, we employed A549 alveolar epithelial cells. Control cells were transfected with the empty vector pMG. The HIF-1{alpha} protein was not detected in transfected cells maintained under normoxic conditions, whereas transfected cells exposed to a hypoxic environment increased nuclear HIF-1{alpha} protein levels when compared with the hypoxic control cells (Figure 5A).



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  		Figure 5. Overexpression of HIF-1{alpha} by transient transfection of A549 cells in hypoxia led to a further increase of HIF-1{alpha} expression, HRE activation, and induction of apoptosis. (A) Detection of HIF-1{alpha} by immunocytochemistry after transfection with HIF-1{alpha} cDNA or the empty vector pMG. Overexpression of HIF-1{alpha} enhanced the hypoxia-induced fluorescence signal compared with control cells. Normoxic transfection did not show any marked increase of fluorescence. Assessment of nuclear abundance of HIF-1{alpha} was performed by immunoblotting and compared with pMG-transfected cells. Hypoxia and HIF-1{alpha} both upregulated HIF-1{alpha} protein, whereas HIF-1{alpha} transfection under normoxia had no visible effect. (B) Reporter gene assay was performed to show HIF-1{alpha}–induced activation of the HRE. The histogram represents mean values ± SEM of five independent experiments each (*P < 0.05 as compared with normoxic controls). (C) Annexin-V staining and flow cytometry identified apoptotic cells. Necrotic cells were excluded by double-staining with propidium iodide. Both hypoxia and transfection with HIF-1{alpha} cDNA significantly induced apoptosis of ATII cells (five independent experiments each; **P < 0.01).

 
Because HIF-1{alpha} overexpression under normoxic condition showed no nuclear accumulation, we tested whether transient transfection with HIF-1{alpha} cDNA under normoxia induces activation of its DNA binding site, the HRE, which is more sensitive than Western blot technique. A549 cells were co-transfected with a luciferase HRE reporter plasmid. In these experiments, the HRE was coupled to the thymidine-kinase minimal promoter (TK-MP). This construct exhibits specific HIF-1–dependent gene induction (26). Incubation for 24 h under hypoxic conditions induced activation of the HRE (203 ± 48%; P < 0.05), compared with control cells maintained under normoxic conditions. Transient transfection with HIF-1{alpha} cDNA further enhanced the hypoxia-induced HRE activation (P < 0.001; Figure 5B).

Transfection of HIF-1{alpha} Induces Apoptosis in A549 Cells
Approximately 4 ± 1% of cells transfected with the empty vector pMG exhibited an apoptotic phenotype, which was normalized to 100%. Transfection with HIF-1{alpha} cDNA significantly increased annexin-V staining in cells maintained under normoxic conditions (42 ± 14%, P < 0.01). Combining transfection and hypoxia further elevated the level of apoptosis (65 ± 2%, P < 0.005; Figure 5C). These data fit very well to the results of the reporter gene assay.

Transfection of Primary ATII Cells with siRNA Targeting HIF-1{alpha}
To analyze the role of endogenous HIF-1{alpha} in hypoxia-induced apoptosis of primary ATII cells, we transfected cells with siRNA targeting HIF-1{alpha} according to previous experiments on A549 cells (26). As a control, we used a random siRNA(siRan). We employed a control siRNA, labeled with FITC, for assessment of transfection efficiency, which was ~ 30% of type II cells staining positive for FITC (Figure 6A). Then, to assess the potency of the siRNA to block HIF-1{alpha} protein expression, we performed immunocytochemistry for HIF-1{alpha} protein. HIF-1{alpha} staining was abolished in ATII cells treated with siHIF-1{alpha}, whereas cells treated with random siRNA were still HIF-1{alpha}–positive after exposure to hypoxic conditions (Figure 6B). Immunostaining of HIF-1{alpha} was corrobated by Western blot analysis using cellular extracts. Cells transfected with siRan showed the hypoxia-induced upregulation of HIF-1{alpha} protein, whereas transfection with siRNA targeting HIF-1{alpha} lowered this hypoxic increase (Figure 6C).



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  		Figure 6. Transfection of ATII cells with siRNA targeting HIF-1{alpha}. (A) Assessment of transfection efficiency of siRNA by using a control siRNA labeled with FITC. (a and b) ATII cells transfected with SiFITC are shown under light (a and c) and fluorescence microscope (b and d) in two different magnifications (x10, x20). (B) Assessment of nuclear abundance of HIF-1{alpha} by immunostaining. ATII cells, transfected with siHIF-1{alpha}, showed no HIF-1{alpha}–positive fluorescent signal, which contrasts to control cells, transfected with random siRNA. (C) Western blot of hypoxia-induced HIF-1{alpha} expression (cytosolic fractions) after 24 h. The histogram represents relative intensity of the bands in the Western blot. Data indicate mean values ± SEM (n = 5); *P < 0.05 for direct comparison between groups. ß-Actin levels were used as control for equal loading.

 
SiHIF-1{alpha} Abolished the Hypoxia-Induced Apoptosis and Antiproliferation of ATII Cells
Analysis of apoptosis revealed that siHIF-1{alpha} significantly attenuated the anoxia-induced increase in apoptosis, in contrast to siRan, which exhibited no effect (P < 0.001; Figure 7A). Furthermore, we assessed the effect of siHIF-1{alpha} on the hypoxia-mediated effect on cell number. In line with the apoptosis, inhibition of HIF-1{alpha} expression blocked the hypoxia-mediated decrease of cell number (P < 0.005; Figure 7B). Furthermore, cell cycle analysis was performed with the transfected ATII cells. In line with the latter results, inhibition of HIF-1{alpha} by siRNA technique prevented the hypoxia-induced cell cycle arrest and showed an increase of cells in the S phase compared with cells transfected with siRan (P < 0.005; Figure 7C).



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  		Figure 7. SiRNA targeting HIF-1{alpha} suppresses the hypoxia-induced effects of ATII cells. (A) Annexin-V staining indicates that anoxia-induced apoptosis was fully blocked and even suppressed below normoxic baseline by siHIF-1{alpha} intervention (five independent experiments each; means ± SEM; ***P < 0.01, {dagger}{dagger}{dagger}P < 0.005). (B) Transfection with siHIF-1{alpha} significantly inhibited the hypoxia-induced decrease of cell number (five independent experiments each; means ± SEM; ***P < 0.005, {dagger}{dagger}P < 0.01). (C) Analysis of cell cycle progression by staining with PI and Anti-BrdU. Original FACS dot blots show cells in S phase (as indicated). The summarized histogram demonstrates the percentage of cells in G0/G1 and S phase. Data represent means + SEM of five independent experiments each; ***P < 0.01 as compared with control cells, transfected with random siRNA.

 
SiHIF-1{alpha} Abolished the Hypoxia-Induced Apoptosis by Downregulation of the Proapoptotic Protein Bnip3L
To determine the signaling pathway of HIF-1{alpha}–induced apoptosis, we looked for a factor known to be essentially involved in promoting apoptosis and belonging to the group of HIF-1 target genes. After 24 h of exposure to hypoxic conditions, a clear upregulation of the HIF-1 dependent Bnip3L protein expression was detected by immunocytochemistry (Figure 8A, b). The Bnip3L belongs to the Bcl2 family and is known to promote apoptosis after release from mitochondria. In line with these data, the hypoxia-induced upregulation of Bnip3L could be suppressed by transfection of ATII cells either with siHIF-1{alpha} or siBnip-3L, compared with control cells which were transfected with random siRNA (Figure 8A, d and f).



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  		Figure 8. SiRNA targeting HIF-1{alpha} suppresses the hypoxia-induced effects of ATII cells. (A) Assessment of Bnip3L protein by immunocytochemistry showed a hypoxia-induced increase of fluorescence (b). Transient transfection with siHIF-1{alpha} fully blocked the signal (d). This inhibition of Bnip-3L protein expression under hypoxia was also achieved by transient transfection of ATII cells with siRNA targeting Bnip-3L (f). a, c, and e show normoxic control cells. (B) Annexin-V/PI staining identified apoptotic and necrotic cells. ATII cells were either transfected with siBnip-3L or siRan before a 24-h exposure to anoxia (five independent experiments each; means ± SEM; ***P < 0.01, {dagger}{dagger}{dagger}P < 0.005). (C) Assessment of cell number using ATII cells transfected with siRNA targeting Bnip-3L. Data represent means + SEM of five independent experiments each; ***P < 0.005, {dagger}{dagger}P < 0.01 as compared with control cells transfected with random siRNA.

 
SiRNA Targeting Bnip3L Prevents Hypoxia-Induced Apoptosis of ATII Cells
Using RNA-interfering technique targeting Bnip-3L, we wanted to show the linkage between the hypoxia-induced apoptosis and the upregulation of Bnip-3L. Cells transiently transfected with siBnip-3L showed no hypoxic upregulation of Bnip-3L protein any more (Figure 8A, f), which indicated effectiveness of transfection. Assessment of apoptosis revealed a significant inhibition of anoxia-induced apoptosis compared with cells transfected with random siRNA (P < 0.005, Figure 8B). In line with these results, hypoxia-induced decrease of cell number was also significantly suppressed by transfection with siBnip-3L (P < 0.01, Figure 8C).


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Few studies exist that analyze the response of primary ATII cells to hypoxia (7, 9, 32, 33). In this study, we present several new aspects relevant to type II pneumocyte biology. The alveolar epithelium, as assessed by in vitro analysis of primary rat alveolar epithelial type II cells, responds to hypoxia with enhanced apoptosis, suppression of cell proliferation, and cell cycle arrest in a time- and dose-dependent manner. This antiproliferative effect of hypoxia (1% O2) is comparable to that provoked by the strong alveolar epithelial cell apoptosis–inducing agent angiotensin II. Exposure to anoxia further enhanced ATII apoptosis, which is in line with previous studies (34). HIF-1{alpha}–dependent signaling is shown to be largely responsible for this alveolar epithelial response to hypoxia.

The hypoxia-inducible transcription factor subtype HIF-1{alpha} was found to be upregulated in hypoxic ATII cells and exhibited nuclear translocation. This represents a typical feature of this transcription factor, which has no relevant transcriptional or translational regulation in response to hypoxia, but is largely regulated by proteasomal degradation (13, 16). An HRE-dependent reporter gene assay demonstrated that the hypoxia-induced accumulation of HIF-1{alpha} was accompanied by enhanced HRE-dependent signaling. To definitely link the hypoxia-induced growth inhibition to the activation of HIF-1{alpha}, (1) A549 cells (an alveolar epithelial cell line) were transiently transfected with HIF-1{alpha} cDNA, and (2) HIF-1{alpha} expression was blocked in primary ATII cells using siRNA targeting HIF-1{alpha}. Our results indicate that HIF-1{alpha} overexpression significantly enhanced the growth inhibitory and proapoptotic response to hypoxia, whereas application of siHIF-1{alpha} markedly attenuated these features under hypoxic conditions. This strongly suggests that hypoxia sensing and signaling via the HIF/HRE axis is largely responsible for the proapoptotic and antiproliferative response of ATII cells to hypoxia. Surprisingly, Western blot analysis showed no nuclear signal of HIF-1{alpha} overexpression at normoxic conditions, in contrast to the marked activation of HRE-dependent signaling and the increase of apoptosis. An explanation for this is that the reporter gene assay is a lot more sensitive than Western blot analysis, especially when using nuclear extracts. We also observed a weak signal by HIF-1{alpha} immunocytochemistry due to normoxic overexpression (Figure 5A). Furthermore, others have also shown that overexpression of HIF-1{alpha} under normoxia can mimic the hypoxia-induced effects (35). Berra and coworkers found out that HIF-1{alpha} entry into the nucleus is not, as proposed, a key event that controls its stability (36). By using cellular extracts of ATII cells, we observed already a slight signal under normoxia, which increased significantly under hypoxia (Figure 6C), referring to HIF-1{alpha} being expressed at baseline levels under normoxia in the cytosol (18).

The fact that hypoxia can induce apoptosis has already been shown for different cell types (37, 38), but the molecular signaling pathways have not yet been elucidated. Previous studies suggest that the proapoptotic tumor suppressor gene p53 is stabilized under hypoxic conditions and interacts with HIF-1{alpha}, leading to the transcription of p53 target genes such as p21 (causing cell cycle arrest) and to the induction of apoptosis (39). Carmeliet and colleagues could show that tumors of HIF-1{alpha}–/–ES cells grew more rapidly due to a lower rate of apoptosis (39). Further analyzing the effector pathways of HIF-1–induced apoptosis, we found out that the proapoptotic Bnip3L was upregulated under hypoxia in ATII cells and could be blocked by transfection with siHIF-1{alpha}. This supports previous studies that demonstrated that hypoxia-inducible expression of Bnip-3L was HIF-dependent and caused apoptosis in neuroblastoma cells (40). The response of Bnip-3L to hypoxia in human cell lines was further characterized, and Bnip-3L was overexpressed in human tumors. These data implicate Bnip-3L as an important mediator of the HIF-1 signaling pathway leading to cell death (41).

Our data illustrate that overexpression of HIF-1{alpha} increased apoptosis in cells maintained under normoxic conditions, supporting observations that demonstrated that HIF-1 was expressed in tissue under normoxic conditions (18).

The readiness of the alveolar epithelial HIF-1{alpha}/HRE axis to react to environmental oxygen changes may also be relevant for other oxygen-dependent features of this cell type, such as the cationic transport system or the surfactant secretory machinery (7, 8, 32, 41). Moreover, it is of major interest whether hypoxia-induced apoptosis of ATII cells may play a role in fibroproliferative lung disorders. This is particularly relevant in view of the hypothesis that ATII cells inhibit proliferation of interstitial fibroblasts by direct interaction, thereby "controlling" this cell population and preventing progressive fibrosis (42–45). Recently, it has been demonstrated that enhanced oxidative stress may lead to damage and/or apoptosis of alveolar epithelial cells, with redox-sensitive effector pathways being involved, resulting in increased angiotensin II levels and fostering of apoptosis (22, 45–48). This is of interest because the response to hypoxia observed in our experiments was as strong as treatment with Ang II in promoting proapoptotic and antiproliferative changes in the alveolar epithelial type II cells (4, 21–23, 49).

In conclusion, ATII cells were shown to respond to hypoxia with enhanced apoptosis and a reduced rate of proliferation, largely mediated by the HIF-1{alpha}/HRE axis, with activation of Bnip3L. This may have a significant impact on lung homeostasis under hypoxic conditions, and would be relevant in pathophysiologic sequelae such as pulmonary fibrosis.


   Acknowledgments
 
The authors are grateful to Gabriele Dahlem and Jessica Lange for their technical assistance.


   Footnotes
 
Conflict of Interest Statement: S.K. has no declared conflicts of interest; B.G.E. has no declared conflicts of interest; J.H. has no declared conflicts of interest; R.S. has no declared conflicts of interest; F.G. has no declared conflicts of interest; W.S. has no declared conflicts of interest; and F.R. has no declared conflicts of interest.

Received in original form October 5, 2004

Received in final form January 31, 2005


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 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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