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Am J Physiol Lung Cell Mol Physiol 293: L913-L922, 2007. First published July 27, 2007; doi:10.1152/ajplung.00459.2006
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Exposure of differentiated airway smooth muscle cells to serum stimulates both induction of hypoxia-inducible factor-1{alpha} and airway responsiveness to ACh

Georgia Chachami,1,2 Apostolia Hatziefthimiou,1 Panagiotis Liakos,2 Maria G. Ioannou,3 Georgios K. Koukoulis,3 Sofia Bonanou,2 Paschalis-Adam Molyvdas,1 George Simos,2 and Efrosyni Paraskeva1

Departments of 1Physiology, 2Biochemistry, and 3Pathology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Thessaly, Greece

Submitted 20 November 2006 ; accepted in final form 16 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Airway smooth muscle (ASM) cells are characterized by phenotypic plasticity and can switch between differentiated and proliferative phenotypes. In rabbit tracheal ASM cells that had been differentiated in vitro by serum starvation, readdition of FBS caused initiation of proliferation and induction of nuclear and transcriptionally active hypoxia-inducible factor (HIF)-1{alpha}. In addition, FBS stimulated the induction of HIF-1{alpha} by the hypoxia mimetic cobalt. Treatment with actinomycin D, cycloheximide, the phosphatidylinositol 3-kinase inhibitors LY-294002 and wortmannin or the reactive oxygen species scavenger diphenyleneiodonium inhibited the FBS-dependent induction of HIF-1{alpha}. These data indicate that, in differentiated ASM cells, FBS upregulates HIF-1{alpha} by a transcription-, translation-, phosphatidylinositol 3-kinase-, and reactive oxygen species-dependent mechanism. Interestingly, addition of FBS and cobalt also induced HIF-1{alpha} in organ cultures of rabbit trachea strips and synergistically increased their contractile response to ACh, suggesting that HIF-1{alpha} might be implicated in airway hypercontractility.

cobalt; fetal bovine serum

AIRWAY SMOOTH MUSCLE (ASM) cells of trachea and bronchi are important for O2 homeostasis, since they control the diameter of the airways and regulate the amount of air that reaches the lungs. Smooth muscle cells are not terminally differentiated but retain phenotypic plasticity and switch between contractile-differentiated and synthetic-proliferative phenotypes with unique morphological, biochemical, functional, and gene expression characteristics (21). Inflammation and injury promote acquisition of the proliferative phenotype and cause hyperplasia and hypertrophy of ASM, which in turn result in airway narrowing (24). These phenotypic changes can be reconstituted in vitro in ASM cell cultures, which have been used to study airway remodeling (19, 20). ASM cell proliferation is mainly controlled by the phosphatidylinositol 3-kinase (PI3K) and MAPK pathways (1, 51), which are activated by growth factors present in serum.

In ASM, as in most other cell types, cellular proliferation is closely linked to the availability of O2. In vitro, O2 levels are suggested to modulate ASM cell growth, with moderate hypoxia inducing and severe hypoxia inhibiting cell proliferation (10). At the tissue level, chronic hypoxia results in hyperplasia of bronchial smooth muscle (26), increased sensitivity of ASM to cholinergic agonists (3), and epithelium-dependent attenuation of contractile responses in isolated rat trachea, which is reversed on removal of the epithelium (9). Therefore, an interplay appears to exist between the hypoxia response pathway and physiological functions of ASM cells, including their contractile properties.

The key mediator of the hypoxia response is the hypoxia-inducible factor (HIF-1) (for review see Refs. 39 and 42). HIF-1{alpha}, the regulatory subunit of HIF-1, is controlled by hydroxylation, which in normoxia causes its inactivation and degradation by the proteasome (35). Lack of O2 inhibits hydroxylation and causes stabilization and activation of HIF-1{alpha}. Its subsequent phosphorylation by p42/44 MAPK (37, 40), nuclear accumulation, dimerization with HIF-1beta (aryl hydrocarbon receptor nuclear translocator), and binding to DNA hypoxia response elements (HREs) induce transcription of hypoxia target genes. Hydroxylation of HIF-1{alpha} can also be inhibited under normoxic conditions by heavy metals, such as cobalt (23), or specific inhibitors, such as dimethyloxalylglycine (DMOG) (12, 27). Furthermore, treatment with growth factors or oncogenic transformation can also upregulate HIF-1{alpha} by transcription- and translation-dependent mechanisms (13, 15, 29, 32, 38, 41, 45, 46).

Little is known about the regulation of HIF-1 in ASM cells and the role of HIF-1{alpha} in ASM cell physiology. In our previous study, we showed that exposure to cobalt, a hypoxia mimetic transition metal implicated in occupational asthma (1a), induced HIF-1{alpha} expression in proliferative ASM cells of rabbit trachea (8). To obtain information more relevant to the in vivo situation, which normally concerns differentiated ASM cells, we extended our studies to ASM cells that had been differentiated in vitro by serum deprivation. More specifically, we addressed the following questions. 1) Does serum stimulation of differentiated ASM cells affect HIF-1 induction and, if so, by what mechanism? 2) How does the induction of HIF-1 correlate with the ASM cell phenotype and the contractile properties of isolated rabbit trachea?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. Unless otherwise stated, chemicals were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). DMEM-Ham's F-12 medium (DMEM-F-12) with L-glutamine, FBS, penicillin-streptomycin, and trypsin was purchased from GIBCO-Invitrogen (Paisley, Scotland, UK).

ASM cell isolation and culture. ASM cells were isolated from rabbit trachea as previously described (8). Briefly, epithelium was removed from tracheal muscle, which was then dissected with scissors from cartilage and washed in low-Ca2+ Krebs solution (mM: 139 NaCl, 5.4 KCl, 1.47 MgSO4, 11 glucose, 1.47 KH2PO4, 2.8 Na2HPO4, 1.4 NaHCO3, and 0.2 CaCl2). Myocytes were isolated enzymatically. Specifically, tracheal smooth muscle was digested in 2 ml of low-Ca2+ Krebs solution containing 0.25% BSA, 2 mg/ml collagenase I, and 10 U/ml elastase IV for 30 min at 37°C with vigorous shaking, washed twice in low-Ca2+ Krebs solution, centrifuged (1,000 g for 10 min), and subsequently incubated for 60 min in low-Ca2+ Krebs solution containing 0.25% BSA, 1 mg/ml collagenase I, and 20 U/ml elastase IV. Dispersed myocytes were washed twice in DMEM-F-12 containing L-glutamine, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, plated in 75-cm2 flasks in the same medium, and grown at 37°C in a humidified incubator under 5% CO2. Cells were subcultured with 0.2% trypsin and plated on 10-cm tissue culture dishes. Experiments were performed with cells at passages 2–3. The smooth muscle origin of the cells was confirmed by immunofluorescence with the monoclonal antibody A104 (Sigma-Aldrich Chemie) against smooth muscle {alpha}-actin (44).

For in vitro differentiation, cells were grown to 80–100% confluence and then incubated for 3 days in medium without FBS, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and ITS (5 µg/ml insulin, 5 µg/ml transferin, and 5 ng/ml selenium).

To estimate the homogeneity of the ASM cells after serum deprivation, we assessed the percentage of differentiated cells from fields of independent experiments by comparing the number of cells that immunostained intensely for smooth muscle-specific myosin heavy chain (SM-MHC) with the total number of cells visualized by staining of the cell nuclei with 4,6-diamidino-2-phenylindole.

Treatment of ASM cells. In vitro differentiated ASM cells were stimulated with 100 µM CoCl2, 10% FBS, or CoCl2 + FBS for the indicated times. Cells were pretreated with 0.5 µg/ml actinomycin D, 50 µM LY-294002, or 500 nM wortmannin for 15 min, 50 nM rapamycin for 30 min, 50 µM PD-98059 for 1 h, and 30 µM diphenyleneiodonium (DPI) for 2 h before CoCl2 or serum addition. Cycloheximide (10 µg/ml) was added after 4 h of CoCl2, FBS, or DMOG (1 mM) induction, and cells were harvested at the indicated times.

Immunofluorescence. Cells grown on coverslips were fixed with 3% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. Coverslips were then incubated with an anti-HIF-1{alpha} mouse monoclonal antibody (1:200 dilution), an anti-smooth muscle {alpha}-actin mouse monoclonal antibody (1:400 dilution), or an anti-SM-MHC mouse monoclonal antibody (1:100 dilution; Sigma-Aldrich Chemie), washed, and incubated further with FITC-coupled goat anti-mouse IgG (Amersham). Cell nuclei were stained with 4,6-diamidino-2-phenylindole. The fluorescent signal was analyzed with an Optiphot-2 microscope and a UFX-DX camera system (Nikon).

Flow cytometric analysis of cellular DNA content. Cells were detached from the tissue culture dish by trypsinization and fixed overnight in 70% ice-cold ethanol. Cells were washed with PBS, resuspended at a density of 106 cells/ml in staining solution (10 pg/ml propidium iodide and 100 pg/ml RNase A) for 45 min in the dark, and then immediately analyzed on a flow cytometer (EPICS, Beckman Coulter).

Cell transfection and reporter gene assays. ASM cells seeded in 12-well plates were cultured for 2 days in serum-free medium and then transiently cotransfected with 0.5 µg of the hypoxia-responsive luciferase reporter plasmid pGL3–5HRE-VEGF (kindly provided by Dr. A. Giaccia) (43) and 0.5 µg of the constitutive beta-galactosidase expression plasmid CMV-lacZ (kindly provided by Dr. A. Kretsovali) (33) using the FuGENE 6 transfection reagent (Roche, Manheim, Germany). At 24 h after transfection, 100 µM CoCl2, 10% FBS, or CoCl2 + FBS was added, and cells were further incubated for 18 h before lysis. Luciferase activity in the cell extracts (20 µl) was determined by a chemiluminescence assay kit (Promega, Madison, WI) using a luminometer (model TD20/20, Turner Designs) and was normalized to beta-galactosidase activity. Values are means ± SE. Comparisons between two groups of experiments were performed using the unpaired t-test and were considered significant when P < 0.05.

Total cellular protein extraction and Western blot analysis. Cells were lysed in 20 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1 mM DTT, and 100 µg/ml PMSF and incubated on ice for 15 min. The cell lysate was obtained by centrifugation at 10,000 g for 20 min at 4°C. Total cell extracts (40 µg) were resolved by 8% SDS-PAGE and analyzed by Western blot using an anti-HIF-1{alpha} mouse monoclonal antibody (1:500 dilution; BD Transduction Laboratories, BD Biosciences, San Diego, CA), an anti-phosphorylated Akt (Ser473) rabbit polyclonal antibody (1:1,000 dilution; Cell Signalling, Danvers, MA), or an anti-beta-actin mouse monoclonal antibody (1:5,000 dilution; Sigma-Aldrich Chemie). Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (Amersham) or anti-rabbit IgG (Cell Signalling) and analyzed by enhanced chemiluminescence (Amersham). Densitometric analysis of the bands was performed with the Scion Image program to determine the HIF-1{alpha} expression levels.

Trachea contraction studies. Adult rabbits were killed by intravenous administration of thiopentone sodium (Abbot), and exothoracic tracheal tissue was removed and placed in Krebs solution (mM: 110.9 NaCl, 5.9 KCl, 1.1 MgCl2, 2 CaCl2, 1.2 NaH2PO4, 25 NaHCO3, and 9.6 glucose, pH 7.4 at 37°C) gassed with 95% O2-5% CO2. Tracheal rings (2 mm wide) were dissected, and strips were cut longitudinally through the cartilage, opposite the smooth muscle layer. Epithelium was removed from the strips, which were incubated in DMEM-F-12 containing L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and ITS in the presence or absence of 100 µM CoCl2, 10% FBS, or CoCl2 + FBS. Each strip was placed with the superfused luminal side up in a water-jacketed horizontal organ bath. One end of the tracheal strip was fixed on the bottom of the organ bath and the other to a force displacement transducer. The entire strip was continuously perfused with oxygenated Krebs solution at 37°C. The volume of the organ bath was ~5 ml, and the perfusion rate was 4 ml/min. The strips were stretched manually to 1 g of resting tension and allowed to equilibrate for ≥60 min. Contractions were induced by 80 mM KCl (the KCl solution had the same composition as normal Krebs solution with equimolar substitution of NaCl with KCl), 10–8 M endothelin-1, or 10–7–10–3 M ACh, which was added cumulatively to the organ bath. Changes in tension were recorded on a force displacement transducer (Grass FT03C, Astro Med) and displayed via an oscillograph recorder (model 7400, Grass). Contraction experiments with tracheal strips under control conditions or treated with CoCl2, FBS, or CoCl2 + FBS were performed in parallel. Values in trachea contraction studies are expressed as tension in grams per tissue cross section (in mm2), as previously described (22); n represents the number of experiments. Values are means ± SE. Comparisons between two groups of experiments were performed using the unpaired t-test and were considered significant when P < 0.05.

Immunohistochemistry. Immunostaining was performed on formalin-fixed paraffin-embedded tissue sections of rabbit tracheal strips treated as described above after microwave antigen retrieval in 10 mM citrate buffer, pH 6.0, with use of a DAKO Envision Plus kit and diaminobenzidine color development. The primary antibody for HIF-1{alpha} was obtained from BD Biosciences (1:20 dilution), the primary antibody for cyclin D1 from Neomarkers (1:50 dilution), and the primary antibody for MIB-1 (Ki-67) from DAKO (1:80 dilution).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Stimulation by FBS increases protein levels and activity of HIF-1{alpha} in ASM cells differentiated in vitro. To differentiate rabbit tracheal ASM cells in vitro, we maintained confluent primary cultures in serum-free medium for 3–10 days. ASM cells in culture comprise a heterogeneous population. Prolonged serum deprivation is expected to increase the size of the subpopulation with the contractile phenotype characterized by elongated shape (19, 31). This change in shape was evident in our ASM cell system 3 days after serum starvation, as shown by indirect immunofluorescence using an antibody against smooth muscle {alpha}-actin (Fig. 1A, a and b). In addition, 3 days of serum starvation resulted in a significant increase of cells that immunostained intensely for the contractile phenotype marker SM-MHC (Fig. 1A, c and d). Concomitant with these phenotypic changes, the cell cycle of serum-starved ASM cells was arrested in the G1 phase as measured by flow cytometry (Fig. 1B, b vs. a).


Figure 1
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  		Fig. 1. FBS stimulation of differentiated airway smooth muscle (ASM) cells causes induction of hypoxia-inducible factor (HIF)-1{alpha}. A: indirect immunofluoresence using anti-smooth muscle (SM) {alpha}-actin (a and b) or anti-SM-specific myosin heavy chain (SM-MHC) (c and d) antibody. ASM cells were cultured for 3 days in the presence of 10% FBS (a and c) or in the absence of FBS and in the presence of insulin-transferin-selenium (ITS)-containing medium (b and d). B: flow cytometric analysis of proliferative ASM cells (a), differentiated in vitro ASM cells (b), or differentiated in vitro ASM cells incubated with FBS for 4 h (c) or 24 h (d). C and D: time course of HIF-1{alpha} accumulation in differentiated in vitro ASM cells treated with 10% FBS or 100 µM CoCl2. Left: total cell extracts (40 µg) were analyzed by SDS-PAGE and Western blotting using an anti-HIF-1{alpha} antibody and an anti-beta-actin antibody as control for equal loading. Right: mean (n = 3) fold expression of HIF-1{alpha} protein normalized to beta-actin protein expression.

 
To study the effect of serum stimulation on in vitro differentiated ASM cells, we treated primary rabbit tracheal ASM cell cultures as described above and subsequently exposed the cultures to 10% FBS for up to 48 h. Analysis of the cell cycle by flow cytometry showed that cell proliferation resumed 24 h after serum addition (Fig. 1Bd). To investigate HIF-1{alpha} expression under these conditions, we analyzed ASM cell extracts by Western blotting at various time points after FBS addition. HIF-1{alpha} rapidly accumulated: it was detectable 1 h after FBS addition and reached maximal expression after 4 h (Fig. 1C). At 4 h after FBS addition, cell proliferation as monitored by cell cycle progression was not evident (Fig. 1Bc); therefore, HIF-1{alpha} induction is a very early step in the transition of ASM cells to the proliferating phenotype. After 4 h, HIF-1{alpha} levels gradually declined but persisted for up to 48 h (Fig. 1C). A comparable and kinetically similar increase of HIF-1{alpha} protein levels was also observed in the absence of serum stimulation when in vitro differentiated ASM cells were treated with cobalt, a transition metal that is known to mimic hypoxia and upregulate HIF-1{alpha} (Fig. 1D).

Next, we examined the effect of simultaneous exposure to FBS + CoCl2. Addition of FBS stimulated the induction of HIF-1{alpha} by CoCl2 (Fig. 2A, lane 2 vs. lane 4). Quantification of the Western blotting results showed that the effect of FBS + CoCl2 was greater than the sum of the individual treatments (Fig. 2A). Thus FBS was not only able to increase HIF-1{alpha} but could also augment its induction by the hypoxia mimetic cobalt, suggesting a synergistic effect of CoCl2 + FBS on the regulation of HIF-1{alpha}. Indirect immunofluorescence analysis (Fig. 2B) showed that, in all cases of induction, HIF-1{alpha} was localized predominantly inside the nucleus and confirmed that HIF-1{alpha} could be overinduced by CoCl2 + FBS.


Figure 2
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  		Fig. 2. Synergistic induction of HIF-1{alpha} by cobalt and serum in differentiated ASM cells. A: differentiation of ASM cells in vitro by incubation for 3 days in DMEM-Ham's F-12 medium (DMEM-F-12) without FBS supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and ITS and analyzed for expression of HIF-1{alpha} without (lane 1) or with 100 µM CoCl2 (lane 2), 10% FBS (lane 3), or CoCl2 + FBS (lane 4) for 4 h. Left: total cell extracts (40 µg) were analyzed by SDS-PAGE and Western blotting using an anti-HIF-1{alpha} antibody and an anti-beta-actin antibody as control for equal loading. Right: mean fold (n = 3) expression of HIF-1{alpha} protein normalized to beta-actin protein expression. B: immunofluorescence microscopy analysis of differentiated in vitro ASM cells before (a) and after addition of 100 µM CoCl2 (b), 10% FBS (c), or CoCl2 + FBS (d) using an antibody against HIF-1{alpha}. C: differentiated in vitro ASM cells were transiently transfected with pGL3–5HRE-VEGF, and luciferase activity was measured 18 h after induction by 10% FBS, 100 µM CoCl2, or CoCl2 + FBS. Values (means ± SE of 3 separate experiments performed in triplicate) are expressed as fold increase of luciferase activity (normalized to beta-galactosidase activity) in relation to untreated cells. Statistical differences between all responses and control were assessed using unpaired t-tests: *P < 0.05; ***P < 0.001.

 
To test whether FBS-induced HIF-1{alpha} was transcriptionally active, we employed a luciferase reporter assay (Fig. 2C). Differentiated ASM cells were transiently transfected with a plasmid containing the luciferase reporter gene under the control of five tandemly repeated HRE motifs from the VEGF gene and then stimulated for 18 h with CoCl2, FBS, or CoCl2 + FBS. FBS increased reporter activity 3.7-fold over control, showing that the FBS-induced HIF-1{alpha} is transcriptionally active. The CoCl2-induced increase of luciferase activity was higher (10.4-fold over control), indicating that CoCl2 was a more potent inducer of HIF-1 activity than FBS. On treatment of cells with CoCl2 + FBS, the increase in activity was significantly stimulated to 21.6-fold over control (Fig. 2C), confirming the synergistic overinduction of HIF-1{alpha} at the protein level (Fig. 2, A and B).

FBS does not stabilize HIF-1{alpha} but, rather, induces its synthesis. HIF-1{alpha} induction by FBS may be due to stimulation of its synthesis or inhibition of protein degradation. To test whether FBS altered HIF-1{alpha} protein stability, we studied the half-life of HIF-1{alpha} on addition of FBS. Differentiated in vitro ASM cells were incubated with FBS for 4 h, cycloheximide was added to inhibit new HIF-1{alpha} protein synthesis, and incubation was continued for 15–60 min. HIF-1{alpha} became undetectable by Western blotting 30 min after cycloheximide addition (Fig. 3A). On the other hand, after induction with CoCl2 for 4 h, HIF-1{alpha} protein levels decreased but remained detectable for 60 min after cycloheximide addition (Fig. 3B). We then used the prolylhydroxylase inhibitor DMOG, which is known to stabilize HIF-1{alpha}. Incubation of differentiated ASM cells with DMOG for 4 h caused an increase in the amount of HIF-1{alpha}, which persisted for 60 min after protein synthesis was blocked by cycloheximide (Fig. 3C). These data show that the half-life of HIF-1{alpha} protein that accumulates in ASM cells after the addition of FBS is much shorter than the half-life of HIF-1{alpha} induced by DMOG or CoCl2. This suggests that FBS, in contrast to cobalt or DMOG, enhances the level of HIF-1{alpha} protein without significantly increasing its stability. Moreover, the differences indicate that FBS and CoCl2 induce HIF-1{alpha} in differentiated ASM cells through different mechanisms.


Figure 3
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  		Fig. 3. Effect of cycloheximide (Chx) and actinomycin D (ActD) on induction of HIF-1{alpha} in differentiated ASM cells. Differentiated in vitro ASM cells were treated with 10% FBS (A), 100 µM CoCl2 (B), or 1 mM dimethyloxylglycine (DMOG, C) for 4 h. Cycloheximide (10 µg/ml) was added, and incubation was continued for 15–60 h in the presence (lanes 2–5) of the inducers. D: cells were pretreated (+) with actinomycin D (0.5 µg/ml) for 15 min and stimulated with 100 µM CoCl2, 10% FBS, or CoCl2 + FBS for 4 h. Right: total cell extracts (40 µg) were analyzed by SDS-PAGE and Western blotting using an anti-HIF-1{alpha} antibody and an anti-beta-actin antibody as control for equal loading. Left: mean (n = 3) fold expression of HIF-1{alpha} protein normalized to beta-actin protein expression.

 
The increase in the synthesis of HIF-1{alpha} induced by FBS may be caused by upregulation of gene transcription and/or enhanced mRNA translation. To investigate whether a transcriptional mechanism is involved, differentiated ASM cells were pretreated for 15 min with actinomycin D and then for 4 h with FBS, CoCl2, or FBS + CoCl2 (Fig. 3D). Inhibition of transcription by actinomycin D abolished the induction of HIF-1{alpha} by FBS, demonstrating the requirement for active transcription (lane 3 vs. lane 6). In contrast, actinomycin D had no significant effect on the induction of HIF-1{alpha} by CoCl2 (lanes 2 and 5) and, in addition, failed to attenuate the stimulatory effect of FBS on the induction of HIF-1{alpha} by cobalt (lanes 4 and 7). These data suggest that induction of HIF-1{alpha} in differentiated ASM cells by FBS differs from hypoxia mimetic induction and requires ongoing DNA transcription.

Induction of HIF-1{alpha} by FBS in differentiated ASM cells involves reactive oxygen species and the PI3K signaling pathway. HIF-1{alpha} mRNA translation is believed to be regulated by the PI3K pathway (8, 29, 30, 45–47), which can be activated by FBS. We therefore investigated the role of the PI3K pathway in the induction of HIF-1{alpha} protein synthesis by FBS. As expected, addition of FBS activated the PI3K pathway, which is demonstrated by the increased levels of phosphorylated Akt, the downstream effector of PI3K (Fig. 4, A and B, lane 3, and Fig. 4D, lane 2). CoCl2, on the other hand, affected Akt phosphorylation to a lesser degree (Fig. 4, A and B, lane 2). Treatment of the cells with the PI3K inhibitors LY-294002 and wortmannin reduced phosphorylated Akt levels and abolished the induction of HIF-1{alpha} by FBS (Fig. 4, A and B, lane 5) but had much less effect on the induction of HIF-1{alpha} by CoCl2 (Fig. 4, A and B, lane 4), suggesting that induction of HIF-1{alpha} by FBS involves the PI3K pathway. To further confirm this finding, we used rapamycin, which inhibits mammalian target of rapamycin (mTOR), an effector downstream from PI3K and Akt. Treatment of cells with rapamycin impaired the induction of HIF-1{alpha} by FBS (Fig. 4C, lane 3 vs. lane 5) but influenced CoCl2-induced HIF-1{alpha} much less (Fig. 4C, lane 2 vs. lane 4).


Figure 4
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  		Fig. 4. Inhibition of phosphatidylinositol 3-kinase (PI3K) prevents HIF-1{alpha} induction by FBS. Cells were pretreated (+) with 50 µM LY-294002 for 15 min (A), 500 nM wortmannin for 15 min (B), 50 nM rapamycin for 30 min (C), and 30 µM diphenyleneiodonium (DPI) for 2 h (D) before addition of 100 µM CoCl2 or 10% FBS for 4 h. Left: total cell extracts (40 µg) were analyzed by SDS-PAGE and Western blotting using an anti-HIF-1{alpha} antibody, an anti-phosphorylated Akt (Ser473) antibody, and an anti-beta-actin antibody as control for equal loading. Right: mean (n = 3) fold expression of HIF-1{alpha} protein normalized to beta-actin protein expression.

 
The PI3K pathway can be activated by reactive oxygen species (ROS) (28), which are also involved in ASM cell proliferation (5, 6). To test the possible involvement of ROS in the induction of the PI3K pathway and FBS-dependent HIF-1{alpha} synthesis, we used the NAD(P)H oxidase inhibitor DPI. Incubation of FBS-treated cells with DPI blocked Akt phosphorylation and HIF-1{alpha} synthesis (Fig. 4D, lanes 2 and 3). Another cell signaling pathway involved in cell proliferation and HIF-1{alpha} upregulation is the MAPK pathway (1, 2, 51). Although addition of FBS to differentiated ASM cells also led to activation of this pathway, treatment with the MAPK pathway inhibitor PD-98059 did not influence induction of HIF-1{alpha} (data not shown).

Induction of HIF-1{alpha} does not correlate with expression of SM-MHC in differentiated in vitro ASM cells. As shown above, modulation of in vitro differentiated ASM cells with FBS results in HIF-1{alpha} induction and reinitiation of cell proliferation. Proliferating ASM cells normally lose their contractile phenotype (21). To test whether expression of HIF-1{alpha} in our system is accompanied by loss of contractility, we used indirect immunofluorescence to examine whether HIF-1{alpha}-inducing conditions affected the expression of the contractile marker SM-MHC. Incubation of in vitro differentiated ASM cells with CoCl2 or FBS for 24 h did not significantly change the number of cells with strong SM-MHC staining (Fig. 5). The same was also true with CoCl2 + FBS, which, as shown above, leads to overinduction of HIF-1{alpha}.


Figure 5
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  		Fig. 5. Treatment of differentiated ASM cells with CoCl2 and FBS does not affect SM-MHC expression. Immunofluorescence microscopic images show differentiated in vitro ASM cells before and after addition of 100 µM CoCl2, 10% FBS, or CoCl2 + FBS for 24 h using an antibody against SM-MHC.

 
Incubation of tracheal strips with CoCl2 and FBS causes induction of HIF-1{alpha} and synergistically increases their responsiveness to ACh. To correlate induction of HIF-1{alpha} to the function of ASM in the context of intact tissue, we measured the contractility of epithelium-denuded tracheal strips after 24 h of organ culture under HIF-1{alpha}-inducing conditions. The presence of FBS or CoCl2 in the culture medium enhanced the responsiveness of tracheal strips to increasing concentrations of ACh, but this effect was not statistically significant (Fig. 6A). In contrast, incubation in the presence of FBS + CoCl2, which leads to overinduction of HIF-1{alpha} in cultured differentiated ASM cells, resulted in a significant increase in ACh-evoked contractions (Fig. 6A). In control experiments, a short (30-min) exposure to CoCl2, FBS, or CoCl2 + FBS did not alter the contractility of tracheal strips (Fig. 6B), precluding the possibility that the enhanced responsiveness to FBS + CoCl2, shown in Fig. 6A, is due to a direct and acute effect of FBS or cobalt on the contractile machinery. Furthermore, this effect is specific for ACh-evoked contractions, since culture of tracheal strips for 24 h with CoCl2, FBS, or CoCl2 + FBS did not affect significantly their responsiveness to 80 mM KCl (Fig. 6C) or 10–8 M endothelin-1 (Fig. 6D) (50).


Figure 6
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  		Fig. 6. Incubation of ASM with CoCl2 and FBS increases responsiveness of rabbit tracheal strips to ACh. A: dose-response curve of rabbit tracheal strips to ACh after 24 h of incubation in DMEM-F-12 containing 10% FBS, 100 µM CoCl2, or CoCl2 + FBS. B: dose-response curve of rabbit tracheal strips to ACh after 30 min of incubation in DMEM-F-12 containing 10% FBS, 100 µM CoCl2, or CoCl2 + FBS. C: response of rabbit tracheal strips to 80 mM KCl after 24 h of incubation in DMEM-F-12 containing 10% FBS, 100 µM CoCl2, or CoCl2 + FBS. D: response of rabbit tracheal strips to 10–8 M endothelin-1 after 24 h of incubation in DMEM-F-12 containing 10% FBS, 100 µM CoCl2, or CoCl2 + FBS. Values are means ± SE; n represents number of independent experiments. Statistical differences between all responses and control were assessed using unpaired t-tests: *P < 0.05; **P < 0.001.

 
As shown above for cultured in vitro differentiated ASM cells, induction of HIF-1{alpha} is linked to cell proliferation, which should normally lead to a reduction, rather than an increase, of contractile function. To address this paradox, we used immunohistochemistry to analyze the correlation between HIF-1{alpha} expression and proliferation in smooth muscle cells of intact trachea treated with CoCl2, FBS, or CoCl2 + FBS. Induction of nuclear HIF-1{alpha} could be readily detected in smooth muscle cells of tracheas exposed to CoCl2, FBS, CoCl2 + FBS for 24 h, whereas tracheas incubated in culture medium alone were negative for HIF-1{alpha} expression (Fig. 7). However, immunohistochemistry of the same tissues with antibodies against the proliferation markers cyclin D1 (14) and MIB-1 (Ki-67) (7) failed to detect these proteins in tracheal strips treated with CoCl2, FBS, or CoCl2 + FBS for 24 h (data not shown), suggesting that, under these ex vivo conditions, HIF-1{alpha} expression is established in the absence of any signs of cellular proliferation. This is in accordance with our observation that induction of HIF-1{alpha} by FBS in cultured in vitro differentiated ASM cells is an early event that precedes progression of the cells to the S phase of the cell cycle (Fig. 1). Taken together, these data indicate that overinduction of HIF-1 specifically increases the response of the trachea to ACh before detectable cellular proliferation.


Figure 7
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  		Fig. 7. Induction of HIF-1{alpha} in ASM of rabbit tracheal strips. Rabbit tracheal strips were incubated for 24 h in DMEM-F-12 containing 10% FBS, 100 µM CoCl2, or CoCl2 + FBS using an antibody against HIF-1{alpha} and subjected to immunohistochemical analysis. Arrows, representative nuclei expressing HIF-1{alpha}.

 

   DISCUSSION
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 MATERIALS AND METHODS
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In the present study, we used previously established protocols (19, 31) to differentiate in vitro primary rabbit tracheal ASM cells. This was achieved by maintaining confluent ASM cell cultures in serum-free ITS-containing medium for ≥3 days. This treatment caused arrest of the cell cycle, as well as a significant increase in the subpopulation of cells with elongated cell shape and high expression of the contractile marker SM-MHC. Using these cultures as an in vitro model of differentiated ASM cells, we subsequently investigated the effect of serum stimulation on induction of HIF-1{alpha}, a transcription factor that, apart from its role in the hypoxic response, is highly involved in many other cell processes, including proliferation, migration, and cytoskeletal organization (42).

Exposure of these in vitro differentiated ASM cells to FBS caused a rapid induction of nuclear and transcriptionally active HIF-1{alpha}, which was followed by reinitiation of cell cycle progression. Moreover, addition of FBS significantly enhanced the effect of the hypoxia mimetic CoCl2, suggesting that FBS and CoCl2 act synergistically to upregulate HIF-1{alpha}. Our results with inhibitors of transcription (actinomycin D), translation (cycloheximide), and the PI3K pathway (LY-294002, wortmannin, and rapamycin) showed that FBS induced HIF-1{alpha} by increasing its synthesis and suggested the involvement of transcriptional and translational mechanisms. Activation of the PI3K pathway and mTOR has indeed been proposed as a general mechanism regulating HIF-1{alpha} mRNA translation (4, 49). We have also shown that inhibition of NAD(P)H oxidase impairs FBS-dependent HIF-1{alpha} induction and stimulation of the PI3K pathway, suggesting the involvement of ROS in the activation of PI3K, as previously proposed (28). Similar mechanisms have been suggested to operate during induction of HIF-1{alpha} in vascular smooth muscle cells by FBS, or several growth factors, including ANG II, thrombin, fibroblast growth factor, and platelet-derived growth factor (38, 41). The FBS component responsible for HIF-1{alpha} induction in our case is unknown, and its identification would require extensive further investigation.

Induction of HIF-1{alpha} in differentiated ASM cells by CoCl2, in contrast to FBS, affected HIF-1{alpha} protein half-life and was insensitive to transcription and PI3K inhibitors. However, we previously showed that, in proliferating ASM cells maintained in the presence of FBS, cobalt induced HIF-1{alpha} predominantly by a transcription-independent but PI3K- and translation-dependent mechanism (8). As shown in the present study, addition of FBS in differentiated ASM cells stimulates the PI3K pathway. Probably, the basal PI3K activity in serum-deprived ASM cells is not sufficient to promote synthesis of HIF-1{alpha}, and cobalt mainly acts by increasing HIF-1{alpha} protein stability through inhibition of HIF-1{alpha} prolylhydroxylases (23). The observation that two different pathways can mediate the effect of cobalt, depending on the proliferation state of ASM cells, also explains why induction of HIF-1{alpha} is much stronger and synergistic when cobalt is used in the presence of FBS. In this case, activation of the PI3K pathway on addition of FBS may "sensitize" the cells to hypoxia mimetic stimuli, such as cobalt, and allow overinduction of HIF-1{alpha}. This "indirect" effect of FBS on the hypoxia mimetic activity of cobalt can also explain the insensitivity of the synergistic induction to the transcription inhibitor actinomysin D.

The upregulation of HIF-1{alpha} on a proliferative stimulus in differentiated ASM cells may indicate a role for this factor in their physiological functions, such as the control of airway diameter. To address this issue, we applied the HIF-1{alpha}-inducing conditions, which have been established in our in vitro model, to epithelium-denuded tracheal strips and measured their effect on ASM responsiveness to contractile stimuli. Our data show that CoCl2 + FBS enhanced significantly the responsiveness of ASM to ACh, but not KCl or endothelin-1. The specificity of the effect suggests that the change in responsiveness is not due to a general change of muscle phenotype during the treatment. Furthermore, as shown by tissue immunohistochemistry, the same treatment did not significantly affect ASM proliferation but led to induction of nuclear HIF-1{alpha}. Therefore, the changes in contractility cannot be due to an increase in ASM mass, which would be insignificant during the 24-h treatment, but correlate well with expression of HIF-1{alpha}. This is in agreement with our in vitro results, which showed that HIF-1{alpha} induction is an early event that precedes detectable cell proliferation and does not affect the expression of the contractile machinery component SM-MHC. Indeed, in previous studies, incubation with FBS for ≥3 days was required before proliferation in epithelium-intact airways was observed (16). Also, in this case, the long treatment resulted in a reduction of general responsiveness to contractile agonists (16, 36).

Overall, our observations support the notion that HIF-1{alpha} plays a role in the regulation of ASM cell contractile properties without affecting the contractile machinery. Which mechanism may then be targeted by HIF-1? The increase of responsiveness under HIF-1{alpha}-inducing conditions is specific for ACh, but not KCl or endothelin-1. ACh, KCl, and endothelin-1 evoke ASM contraction through distinct pathways. KCl induces contraction by depolarizing the cell membrane and causing Ca2+ entry via voltage-dependent Ca2+ channels. The contractile effect of endothelin-1 on mature rabbit airways has been attributed mainly to the mobilization of extracellular Ca2+, at least in part via voltage-dependent Ca2+ channels (18). ACh, on the other hand, induces contraction by causing release of Ca2+ from intracellular stores as well as Ca2+ influx from the extracellular space (34). The selective effect of HIF-1{alpha} on ACh-mediated contractions suggests that either of these two processes may be affected by HIF-1 through regulated expression of genes involved in Ca2+ handling. As shown previously in cardiac myocytes, HIF-1 is required for the expression of SERCA2, a sarco(endo)plasmic reticulum Ca2+ pump gene (25). Moreover, HIF-1 has been shown to upregulate the expression of isoforms of canonical transient receptor potential channels and increase basal intracellular Ca2+ concentration in pulmonary artery smooth muscle cells (48). Similar mechanisms, the delineation of which requires further investigation, may be operating in tracheal ASM cells upon HIF-1 induction. Regardless of the exact mechanism(s) through which HIF-1 increases the ASM response to ACh, HIF-1 could play an important role in pathological conditions such as asthma and chronic obstructive pulmonary disease, which are characterized by altered ACh and muscarinic receptor activity (11, 17).

In summary, our results show that transition of in vitro differentiated ASM cells to the proliferative phenotype by addition of FBS is characterized by early induction of HIF-1{alpha} and sensitization to the hypoxia mimetic CoCl2. HIF-1{alpha} induction occurs via a transcription- and translation-dependent mechanism involving the PI3K-mTOR pathway. Moreover, culture of epithelium-denuded tracheal strips under HIF-1-overinducing conditions enhances their contractile response to ACh, suggesting that HIF-1 may be involved in the regulation of airway tone.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by a grant funded by the European Social Fund and National Resources, EPEAEKII, PYTHAGORAS, from the Greek Ministry of National Education and Religious Affairs (to G. Simos).


   ACKNOWLEDGMENTS
 
The authors thank I. Makadassis for technical assistance, Prof. A. Germenis for the use of the flow cytometer, and the Laboratory of Biology for the use of their fluorescence microscope.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Paraskeva or G. Simos, Papakiriazi 22, 41222 Larissa, Greece (e-mail: fparaskeva{at}med.uth.gr or simos{at}med.uth.gr)

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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 REFERENCES
 

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