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Published ahead of print on July 15, 2004, doi:10.1165/rcmb.2003-0426OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 544-551, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0426OC

Cobalt Induces Hypoxia-Inducible Factor-1{alpha} Expression in Airway Smooth Muscle Cells by a Reactive Oxygen Species– and PI3K-Dependent Mechanism

Georgia Chachami, George Simos, Apostolia Hatziefthimiou, Sophia Bonanou, Paschalis-Adam Molyvdas and Efrosyni Paraskeva

Laboratory of Physiology and Laboratory of Biochemistry, Department of Medicine, School of Health Sciences, University of Thessaly, Larissa, Greece

Address correspondence to: Efrosyni Paraskeva, Laboratory of Physiology, Department of Medicine, School of Health Sciences, University of Thessaly, Papakiriazi 22, 41222 Larissa, Greece. E-mail: fparaskeva{at}med.uth.gr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cobalt can mimic hypoxia and has been implicated as a cause of lung defects. However, the effect of cobalt on airway smooth muscle (ASM) cells has not been analyzed in detail. In this article, we use primary cultures of ASM cells from rabbit trachea and show that exposure to cobalt chloride causes a rapid increase of the intracellular levels of hypoxia-inducible factor–1{alpha}, which is detected predominantly inside the nucleus. With the use of specific inhibitors, we demonstrate that induction of hypoxia-inducible factor–1{alpha} by cobalt depends on active protein synthesis but not transcription. Furthermore, wortmannin, LY294002, and N-acetyl-L-cysteine inhibit the effect of cobalt, suggesting that it involves the phosphatidylinositol 3 kinase pathway and production of reactive oxygen species. Interestingly, cobalt chloride attenuates the contractile response of rabbit airways induced by potassium chloride, but not by acetylcholine, suggesting a link between the cellular response to hypoxic stimuli and the contractile properties of ASM cells.

Abbreviations: airway smooth muscle, ASM • acetylcholine, Ach • bovine serum albumin, BSA • cobalt chloride, CoCl2 • enhanced version of the green fluorescent protein, GFP • hypoxia-inducible factor-1, HIF-1 • N-acetyl-L-cysteine, NAC • phosphatidylinositol 3-kinase, PI3K • reactive oxygen species, ROS • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE • smooth muscle, SM • von Hippel-Lindau, VHL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cobalt is essential for the formation of vitamin B12. The general population is exposed to low levels of cobalt, found mainly in food and at lower levels in air and water. However, exposure to high levels of cobalt in the occupational environment can affect, among others, the lungs. More specifically, cobalt has been implicated as a cause of occupational asthma (1). Moreover, cobalt has been shown to mimic the effects of hypoxia at the cellular level (2).

Reduction in the normal levels of tissue O2 partial pressure, if severe or prolonged, can result in cell death. Therefore, adaptive mechanisms operate during hypoxia to achieve maintenance of cellular and tissue function. The respiratory tract, in particular, has evolved homeostatic responses to correct imbalances between ventilation and perfusion and thus ensure survival. Acute hypoxia results in constriction of pulmonary arteries, increased pulmonary arterial pressure, and a redistribution of blood flow from the basal to the apical portion of the lung (3). Chronic hypoxia experienced by persons suffering from advanced respiratory diseases or living at high altitude can lead to vascular remodeling of pulmonary arteries and, eventually, to pulmonary hypertension (4, 5). The effects of exposure to low O2 are also evident in the upper airway. In vivo, chronic hypoxia results in hyperplasia of bronchial smooth muscle (SM) (6) and attenuation of contractile responses in rat isolated trachea (7). In vitro, O2 levels are suggested to modulate airway SM (ASM) cell growth, with moderate hypoxia inducing and severe hypoxia inhibiting the proliferation of ASM cells (8).

At the cellular level, hypoxia modulates the expression of genes that mediate physiologic and cellular adaptive responses, including oxygen transport and iron metabolism, angiogenesis, SM tone, ventilation, and cell proliferation (reviewed in Refs. 9 and 10). The key mediator of the hypoxic response is the hypoxia-inducible factor (HIF)-1, which consists of two subunits: HIF-1{alpha} and HIF-1ß. HIF-1ß, also known as the aryl hydrocarbon receptor nuclear translocator (ARNT) is constitutively expressed. In contrast, HIF-1{alpha} expression is regulated. At normoxia and according to current models, modification by HIF-1{alpha} prolyl hydroxylases causes binding of HIF-1{alpha} to the tumor suppressor von Hippel-Lindau (VHL) protein and subsequent degradation by the proteasome. In the absence of oxygen, prolyl-hydroxylation is blocked and HIF-1{alpha} is stabilized. Induction of HIF-1{alpha} is also achieved by cobalt, desferrioxamine, and a number of factors, including insulin, thrombin, and angiotensin II, depending on the cell type or tissue (11–16). However, the mechanism of HIF-1{alpha} induction in some of these cases is not well understood. In particular, activation of HIF-1{alpha} by growth factors appears to involve both transcription- and translation-dependent mechanisms.

As far as the respiratory tract is concerned, HIF-1{alpha} is induced by hypoxia in many cultured pulmonary cell types (3), but appears to be expressed also under nonhypoxic conditions in pulmonary arterial SM cells (3, 17). However, the response of SM cells from the upper airway to hypoxia or cobalt has not been investigated thus far. We therefore tested whether SM cells derived from rabbit trachea express HIF-1{alpha} when exposed to cobalt and studied the mechanism of the regulation of HIF-1{alpha} protein expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Trachea Contraction Studies and Statistical Analysis
Adult male rabbits were killed by intravenous administration of sodium thiopentone (Abbott, Italy), exothoracic tracheal tissue was removed and placed in Krebs solution (110.9 mM NaCl, 5.9 mM KCl, 1.1 mM MgCl2, 2 mM CaCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, 9.6 mM glucose, pH 7.4 at 37°C), and gassed with 95% O2 and 5% CO2. Tracheal rings 2 mm in width were dissected and strips were cut longitudinally through the cartilage, opposite the SM layer. Strips were epithelium-denuded and placed luminal-side up in an organ bath, with one end fixed and the other attached to the transducer tip, stretched manually to 1 g resting tension, and were allowed to equilibrate for at least 60 min. Strips were continuously perfused with oxygenated Krebs solution at 37°C and cobalt chloride (CoCl2; 100 µM) was added where indicated.

Contraction experiments with tracheal strips under control conditions or treated with CoCl2 were performed in parallel as previously described (18). KCl or acetylcholine (Ach)-induced contractions were obtained every 30 min in both preparations. Changes in tension were recorded on a Grass FT03C force displacement transducer and displayed via a Grass 7400 Physiological Recorder (Astro-med, West Warwick, RI).

Values are expressed as a percentage of reference contraction induced by 80 mM KCl or 10–5 M Ach. All data are presented as means ± SE. Statistical differences between all the responses were assessed using paired and unpaired t tests; P values < 0.05 were considered to be significant.

ASM Cell Isolation and Culture
Tracheas were placed into sterile, ice-cold, low-Ca2+ Krebs solution (139 mM NaCl, 5.4 mM KCl, 1.47 mM MgSO4, 11 mM glucose, 1.47 mM KH2PO4, 2.8 mM Na2HPO4, 1.4 mM NaHCO3, 0.2 mM CaCl2). Tracheal muscle was epithelium-denuded, dissected with scissors from cartilage, and washed in low-Ca2+ Krebs solution. Myocytes were isolated enzymatically. Briefly, tracheal SM was digested in 2 ml low-Ca2+ Krebs solution (0.25% bovine serum albumin [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, centrifuged (1,000 x g for 10 min), and subsequently incubated in low-Ca2+ Krebs solution (0.25% BSA, 1 mg/ml collagenase I, and 20 U/ml elastase IV) for 60 min. Dispersed myocytes were washed twice in Dulbecco's modified Eagle's medium/Ham's F12 medium, containing L-glutamine, 10% fetal bovine serum, 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 on passages 3–6.

The SM origin of the cells was confirmed by immunofluorescence with the monoclonal antibody A104 (Sigma, St. Louis, MO) against SM{alpha}-actin (19).

Treatment of ASM Cells
Cells were grown to 50–80% confluence and stimulated with 100 µM CoCl2 for the indicated times. Pretreatment of cells with 0.5 µg/ml actinomycin D, 100–500 nM wortmannin, 50 µM LY294002, and 10 µM MG132 was performed 15 min before CoCl2 addition, and with 5 mM N-acetyl-L-cysteine (NAC) 2 h before CoCl2 addition.

Cycloheximide (10 µg/ml) was added after 4 h of CoCl2 or MG132 induction and cells were harvested at the indicated times.

For hypoxia, cells were exposed for 4 h to 1% O2–5% CO2–balance N2 in a tightly sealed modular incubation chamber (Billups-Rothenberg, Inc., Del Mar, CA).

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 phenylmethylsulfonyl fluoride, and were incubated on ice for 15 min. The cell lysate was obtained by centrifugation at 10,000 x g for 20 min at 4°C. 40 µg of protein were resolved by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by Western blot with an anti–HIF-1{alpha} mouse monoclonal antibody (1:500; BD Transduction Laboratories, San Diego, CA) or an anti–SM{alpha}-actin mouse monoclonal antibody (1:400; Sigma). Membranes were then incubated with horseradish peroxidase conjugated anti-mouse IgG (Amersham) followed by ECL (Amersham). A quantitation of HIF-1{alpha} and actin bands was performed using the Histogram function of the Adobe Photoshop program.

Plasmid Construction and Transfection
Plasmid pEGFP–HIF-1{alpha} was constructed by inserting the full length HIF-1{alpha} cDNA into the BamHI position of the multicloning site of the pEGFP-C1 plasmid (Clontech). ASM cells were cultured on coverslips and transfected with 1.5 µg of pEGFP-C1 or 1.5 µg of pEGFP–HIF-1{alpha} and 15 µl of DOTAP liposomal transfection reagent (Roche) according to the manufacturer's instructions. Expression of GFP–HIF-1{alpha} and GFP was assayed 24 h after transfection. The fluorescent signal was analyzed with an Optiphot-2 microscope and UFX-DX camera system (Nikon).

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 anti-HIF-1{alpha} mouse monoclonal antibody (1:200) or anti-SM {alpha}-actin mouse monoclonal antibody (1:400), washed and incubated further with FITC-coupled goat anti-mouse IgG (Amersham). The fluorescent signal was analyzed with an Optiphot-2 microscope and UFX-DX camera system (Nikon).

Reagents
Unless otherwise stated, chemicals and other reagents were purchased from Sigma-Aldrich. Dulbecco's modified Eagle's medium/Ham's F12 medium with L-glutamine, fetal bovine serum, penicillin/streptomycin, and trypsin were from Gibco BRL.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nuclear HIF-1{alpha} is Induced by Cobalt in ASM Cells
Primary cultures were established from ASM cells of rabbit trachea. Immunocytochemical localization confirmed the SM origin of cells in the primary culture, because > 95% expressed the SM contractile protein SM{alpha}-actin (Figure 1A).



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  		Figure 1. Hypoxic stimuli induce HIF-1{alpha} accumulation in ASM cells. (A) Immunofluorescence of ASM cells with an anti-human SM{alpha}-actin mouse monoclonal antibody A104. More than 95% of the cells stained positively for the SM-specific protein. The position of the cell nucleus is visualized by 4,6-Diamidino-2-phenylindole staining. (B) ASM cells express HIF-1{alpha}, when treated with 100 µM CoCl2 (lane 2) or exposed to 1% O2 (lane 4) for 4 h. (C) Kinetics of HIF-1{alpha} accumulation in ASM cells. ASM cells were treated with 100 µM CoCl2 for the indicated times, up to 24 h. Total cell extracts were analyzed by SDS-PAGE and Western blot with anti–HIF-1{alpha} antibody and anti–SM{alpha}-actin antibody as control for equal loading.

 
We then investigated if HIF-1{alpha} can be induced in primary ASM cells. Under control conditions, expression of HIF-1{alpha} protein was very low. However, when cells were exposed for 4 h to CoCl2, HIF-1{alpha} protein levels increased dramatically (Figure 1B).

A similar increase was also observed when cells were exposed for 4 h to hypoxia (Figure 1B) or treated with the iron chelator desferrioxamine (data not shown). To further assess the pattern of HIF-1{alpha} protein induction by treatment with CoCl2, we performed a kinetic analysis. As seen in Figure 1C, induction of HIF-1{alpha} was rapid, being visible at only 30 min after the addition of CoCl2. Maximal expression was observed after 4 h, and HIF-1{alpha} protein levels remained elevated for up to 24 h after addition of CoCl2 to the culture medium.

In order for HIF-1 to be active, HIF-1{alpha} has to enter the nucleus, where it can dimerize with aryl hydrocarbon receptor nuclear translocator and activate transcription of hypoxia-regulated genes (20). We therefore tested if induction of HIF-1{alpha} by CoCl2 is followed by accumulation of the protein inside the nucleus. Figure 2A shows the intracellular localization of HIF-1{alpha} in ASM cells by indirect immunofluorescence before and after treatment with CoCl2. Control cells showed very weak HIF-1{alpha} immunoreactivity, in accordance with the low HIF-1{alpha} signal observed under control conditions by Western blot. In contrast, a strong nuclear signal is visible after cells are incubated with CoCl2 for 4 h. Because it was difficult to assess the intracellular localization of the endogenous HIF-1{alpha} under nonhypoxic conditions, ASM cells were also transfected with plasmid pEGFP–HIF-1{alpha} overexpressing the chimeric GFP (enhanced version of the green fluorescent protein)–HIF-1{alpha} protein. In the absence of CoCl2, GFP–HIF-1{alpha} localizes mainly in the nucleus and, to a lesser extent, also in the cytoplasm (Figure 2B). GFP expressed in control cells, on the other hand, shows even distribution between the two compartments. Upon treatment of cells with CoCl2, GFP–HIF-1{alpha} becomes exclusively nuclear, whereas, in the control cells, the distribution of GFP remains unchanged. This indicates that, although nuclear import of GFP–HIF-1{alpha} occurs under normal conditions, treatment of ASM cells with CoCl2 increases the efficiency of this transport process and results in a strictly nuclear localization of GFP–HIF-1{alpha}, as observed also in the case of the endogenous protein (see above).



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  		Figure 2. Intracellular localization of HIF-1{alpha} in ASM cells. (A) ASM cells were maintained under control conditions, or in the presence of CoCl2 (100 µM) for 4 h and analyzed by immunofluorescence using an anti–HIF-1{alpha} mouse monoclonal antibody. (B) ASM cells were transfected with pEGFP-C1 or pEGFP–HIF-1{alpha}. Twenty-four hour posttransfection cells were treated (or not) with CoCl2 (100 µM) for 4 h. GFP–HIF-1{alpha} and GFP localization was analyzed by direct fluorescence microscopy.

 
Induction of HIF-1{alpha} by CoCl2 in ASM Cells Requires Active Protein Synthesis
The induction of HIF-1{alpha} by CoCl2 in ASM cells may be due to stimulation of transcription, upregulation of mRNA translation, or a posttranslational event, such as inhibition of degradation. It is generally assumed that induction of HIF-1{alpha} by hypoxia is caused by protein stabilization through inhibition of proteolysis (9, 10). However, many HIF-1{alpha}–inducing factors do not act through protein stabilization, but affect instead HIF-1{alpha} transcription and/or translation (11–16, 21). To investigate the possibility that the increase of HIF-1{alpha} in ASM cells by CoCl2 is due to enhanced transcription of the HIF-1{alpha} gene, we used actinomycin D. Cells were pretreated for 15 min with actinomycin D, followed by 4 h CoCl2 treatment. Figure 3A shows that actinomycin D had no significant effect on the induction of HIF-1{alpha}, demonstrating that active transcription is not required for the CoCl2-induced accumulation of HIF-1{alpha}. To test if ongoing protein synthesis is important for the induction of HIF-1{alpha} by cobalt in ASM cells, we checked the effect of the translation inhibitor cycloheximide. ASM cells were incubated with CoCl2 for 4 h, cycloheximide was added, and incubation was continued for another 15–60 min. A decrease of HIF-1{alpha} levels was already evident 15 min after cycloheximide addition, and prolonged (60 min) incubation resulted in a significant reduction of HIF-1{alpha} (Figure 3B, lanes 2–5). Interestingly, when CoCl2 is washed away from the cells before cycloheximide addition, HIF-1{alpha} protein decreases with very similar kinetics (Figure 3B, lanes 6–8), exhibiting a half-life not significantly different from that in the presence of cobalt. Therefore, the ability of CoCl2 to increase HIF-1{alpha} levels does not appear to be due to an increase in protein stability, but rather to an increase of mRNA translation.



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  		Figure 3. Effect of actinomycin D and cycloheximide on the induction of HIF-1{alpha} in ASM cells. (A) Inhibition of transcription does not affect HIF-1{alpha} induction in ASM cells. Cells were pretreated for 15 min with actinomycin D (0.5 µg/ml) and stimulated with CoCl2 (100 µM) for 4 h. (B) The half-life of HIF-1{alpha} does not change in the presence of cobalt. ASM cells were stimulated with CoCl2 (100 µM) for 4 h (lane 2). Cycloheximide (10 µg/ml) was added and incubation continued for the indicated time in the presence (+, lanes 3–5) or absence (–, lanes 6–8) of CoCl2. (C) MG132 stabilizes HIF-1{alpha}. ASM cells were treated with MG132 (10 µM) for 4 h (lane 2). Cycloheximide (10 µg/ml) was added and incubation continued for the indicated time in the presence (+, lanes 3–5) or absence (–, lanes 6–8) of MG132. Total cell extracts were analyzed by SDS-PAGE and Western blot with anti–HIF-1{alpha} and anti–SM{alpha}-actin antibodies as controls for equal loading. Values represent the HIF-1{alpha} to actin ratio, expressed as the percentage of the point before the addition of cycloheximide.

 
To test this idea further, we used the proteosomal inhibitor MG132, which was shown to stabilize HIF-1{alpha} by inhibiting its degradation in other cell systems (12). Incubation of ASM cells with MG132 for 4 h caused a strong increase in the amount of HIF-1{alpha} (Figure 3C, compare lanes 1 and 2), comparable to the one induced by cobalt. After addition of cycloheximide, the level of HIF-1{alpha} remained essentially unchanged for up to 60 min (Figure 3C, lanes 3–5), demonstrating that MG132 increases the half-life of HIF-1{alpha}. When MG132 is washed off before the addition of cycloheximide, HIF-1{alpha} levels are reduced with a similar rate as in the previous experiment with CoCl2 (Figure 3C lanes 6–8, compare with Figure 3B, lanes 6–8). This distinction between the effects of CoCl2 and MG132 on the stability of HIF-1{alpha} in the presence of cycloheximide is suggestive of a role for CoCl2 in affecting HIF-1{alpha} protein synthesis in ASM cells.

These data were confirmed at the single-cell level by immunofluorescence. The high levels of intranuclear HIF-1{alpha} expressed in cells that have been exposed to CoCl2 (Figure 4A) greatly diminish upon subsequent incubation with cycloheximide (Figure 4B). In contrast, the accumulation of HIF-1{alpha} caused by MG132 is not affected when protein synthesis is inhibited by cycloheximide (Figures 4A and 4B).



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  		Figure 4. Effect of cycloheximide on the intracellular accumulation of HIF-1{alpha} in ASM cells. ASM cells were treated for 4 h with: (upper panels) CoCl2 (100 µM), the cell culture medium was removed and the incubation continued for 30 min in medium containing CoCl2 (A) or CoCl2 and 10 µg/ml cycloheximide (B), as indicated, or (lower panels) MG132 (10 µM), the cell culture medium was removed and the incubation continued for 30 min in medium containing MG132 (C), or MG132 and 10 µg/ml cycloheximide (D), as indicated. Analysis was done by immunofluorescence using an anti–HIF-1{alpha} mouse monoclonal antibody.

 
Reactive Oxygen Species and the Phosphatidylinositol 3-Kinase Signaling Pathway Are Involved in the Induction of HIF-1{alpha} by Cobalt
Translation of HIF-1{alpha} mRNA has been previously suggested to be under the control of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in various cell types (12, 13, 16, 21). Moreover, reactive oxygen species (ROS) produced by cobalt treatment of Hep3B cells, are believed to act upstream of the PI3K pathway in the cascade leading to HIF-1{alpha} induction (22). To test whether the PI3K pathway is also involved in the induction of HIF-1{alpha} by CoCl2 in ASM cells, we used the PI3K inhibitors wortmannin and LY294002. Treatment of ASM cells with either LY294002 (Figure 5A) or wortmannin (Figure 5B) significantly impaired the induction of HIF-1{alpha} by CoCl2. In addition, when ROS are scavenged by the antioxidant NAC, cobalt fails to upregulate HIF-1{alpha} in ASM cells (Figure 5C), suggesting that ROS and the function of PI3K are essential for upregulating the synthesis of HIF-1{alpha} in the presence of cobalt.



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  		Figure 5. Effect of PI3K inhibitors and NAC on the induction of HIF-1{alpha} in ASM cells. (A) and (B): inhibition of PI3K prevents HIF-1{alpha} induction by cobalt in ASM cells. Cells were pretreated for 15 min with the indicated concentrations of (A) wortmannin and (B) LY294002. (C) Antioxidants abolished HIF-1{alpha} induction, when ASM cells were pretreated for 2 h with NAC. In all cases, 100 µM CoCl2 were added after pretreatment of ASM cells (+) and incubation continued for 4 h. Total cell extracts were analyzed by SDS-PAGE and Western blot with anti–HIF-1{alpha} and anti–SM{alpha}-actin antibodies as controls for equal loading.

 
Exposure to CoCl2 Attenuates Contractile Responses of Rabbit Trachea Evoked by KCl In Vitro
Prompted by our findings on the effect of cobalt exposure on HIF-1{alpha} expression in ASM cells, we tested whether CoCl2 affected rabbit trachea SM contractile properties. To investigate the direct effect of cobalt on trachea SM, tracheal strips were epithelium-denuded to avoid effects mediated by various substances, including NO and prostaglandins, which are released from the epithelium and affect contraction (18, 23, 24). Contractions evoked by KCl decreased significantly in the presence of CoCl2 compared with control strips. The decrease of KCl-induced contractions was evoked as early as 60 min after the addition of CoCl2 to the perfusion medium, and was evident for 2.5 h (Figure 6A). The maximal difference between control and CoCl2-treated preparations was observed after 90 min of CoCl2 treatment. The difference between control and CoCl2-treated preparations diminished after approximately 4 h, as KCl-induced contractions in both preparations were successively weaker, presumably due to tissue fatigue, especially because the epithelium had been removed. To obtain a better understanding of the mechanism involved in the attenuation by CoCl2 of the trachea contractile response, we tested the effect of CoCl2 on Ach-induced contractions. In contrast to KCl-induced contractions, CoCl2 had no statistically significant effect on trachea contractions evoked by 10–5 M Ach (Figure 6B, see also DISCUSSION).



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  		Figure 6. CoCl2 affects the contraction of tracheal strips by KCl but not Ach. Epithelium-denuded tracheal strips were contracted by (A) KCl or (B) Ach in the presence (closed bars) or absence (open bars) of 100 µM CoCl2. Contractions were obtained every 30 min. Values are expressed as a percentage of reference contraction induced by 80 mM KCl or 10–5 M Ach. Data are given as means ± SE (n = 6). Statistical differences between all the responses were assessed using paired and unpaired t tests; P < 0.05 was considered to be significant (*P < 0.05; **P < 0.01; ***P < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The upper airway serves the delivery of O2 to the body, and is an important point for the regulation of the quantity of oxygen that reaches the lungs and is then delivered to the organs. To achieve this, the upper airway has to adopt a number of physiologic mechanisms in response to hypoxia. It has been recently reported that exposure to a moderate level of hypoxia acts as a mitogenic stimulus for ASM cells, whereas severe hypoxia inhibits ASM cell proliferation (8).

HIF-1, an essential mediator of homeostatic responses to hypoxia, may play an important role in these physiologic adaptations; before this study, however, the regulation of HIF-1{alpha} expression in ASM cells had not been determined.

The purpose of this study was to investigate the effect of hypoxic stimuli and, in particular, the effect of CoCl2 on the expression of HIF-1{alpha} protein in SM cells of the upper airway. Initially, we established primary cultures from rabbit trachea ASM cells (Figure 1A). Our results show that ASM cells respond to CoCl2 treatment with a significant and rapid increase of HIF-1{alpha} protein levels (Figure 1C). The fast HIF-1{alpha} induction, together with the results obtained by the treatment of ASM cells with the transcription inhibitor actinomycin D, indicate the existence of a posttranscriptional mechanism. This can be either an increase of the rate of HIF-1{alpha} mRNA translation, or an increase in HIF-1{alpha} protein stabilization in the presence of cobalt. Although the induction of HIF-1{alpha} at low oxygen concentrations is believed to occur by inactivation of the prolyl hydroxylases that targets HIF-1{alpha}, the induction mechanism of the hypoxia-mimetic agents has not been clarified. In the case of cobalt, it has been assumed that its effect on HIF-1{alpha} levels is due to depletion of Fe2+, a critical cofactor of the HIF-1{alpha} prolyl hydroxylases (2). However, a recent report suggests that cobalt may directly interact with HIF-1{alpha}, thereby inhibiting the HIF-1{alpha}–VHL protein interaction (25).

Our results suggest a third mechanism for the cobalt effect, which, at least in the case of ASM cells, involves translational upregulation of HIF-1{alpha} synthesis through a PI3K signaling pathway. This is first shown by comparing the stability of the protein in the presence of cobalt or the proteasome inhibitor MG132, which only affects the rate of protein degradation. When new protein synthesis is blocked by cycloheximide, the presence of MG132 preserves HIF-1{alpha} protein levels for at least 60 min (Figure 3C). In contrast, the presence of cobalt does not have a significant effect on the rate of HIF-1{alpha} degradation (Figure 2B). Because cobalt does not appear to increase the half-life of HIF-1{alpha} protein, our data suggest that the induction of HIF-1{alpha} by cobalt may involve stimulation of protein synthesis.

Definite confirmation of this would require direct measurement of the half-lives of both basal- and cobalt-stimulated HIF-1{alpha} using pulse labeling and pulse–chase analysis. We were, unfortunately, not able to perform such an analysis due to the unavailability of suitable antibodies that could both recognize rabbit HIF-1a and immunoprecipitate it efficiently. This leaves the possibility open that cobalt might stabilize HIF-1{alpha} by a mechanism that is distinct from that used by MG132 (i.e., inhibition of the proteasome). A hypothetical scenario could implicate activation of a labile protein that prevents proteasomal degradation of HIF-1{alpha}. In this case, inhibition of protein synthesis by cycloheximide would lead to elimination of the putative regulatory protein and, consequently, HIF-1{alpha}. Thus, the cycloheximide and MG132 experiments do not necessarily prove translational regulation of HIF-1{alpha} by cobalt.

This type of regulation, however, is further supported by the fact that the cobalt effect is sensitive to inhibitors of the PI3K signaling pathway, which is known to regulate the translation rate of particular mRNAs (26). A similar mechanism has also been proposed for the induction of HIF-1{alpha} under normoxic conditions by several biological factors, such as angiotensin II in vascular smooth muscle cells (15) or oxidized low-density lipoprotein in macrophages (27). These studies have proposed that PI3K acts upstream of a translation stimulation signal, like the phosphorylation of the ribosomal protein S6 and the regulatory eIF4E-binding protein 4E-BP1, to enhance the translation of the HIF-1{alpha} mRNA (28, 29).

Our transfection studies (Figure 2B) have also implicated a role for cobalt in enhancing the concentration of HIF-1{alpha} inside the nucleus. Nuclear protein import is a complicated multi-step process that begins with the association of the protein with an import receptor in the cytoplasm. On the other hand, nuclear proteins can also leave the nucleus and re-enter the cytoplasm escorted by nuclear export receptors. Import and export receptors are largely members of the importin ß protein family, and associate with their substrates through the recognition of specific nuclear localization or nuclear export signals, respectively, either directly or with the aid of adaptors. Another level of complexity is added by the fact that these signals can be activated or masked by posttranslational modifications (e.g., phosphorylation) and/or association with other proteins (for a review, see Refs. 30 and 31). HIF-1{alpha} has been shown to contain a nuclear localization signal at the carboxy-terminus (20, 32), but it can also exit the nucleus (33). However, the respective transport factors that mediate and probably regulate transport of HIF-1{alpha} between the nucleus and the cytoplasm remain to be discovered. It has been proposed that import of HIF-1{alpha} to the nucleus of HepG2 cells is regulated, because overexpressed GFP-tagged HIF-1{alpha} remained in the cytoplasm until it was activated by hypoxia (20). This idea was challenged by other reports that show constitutive nuclear localization of stabilized HIF-1{alpha} in renal cell carcinoma and HeLa cells (33, 34). Our data are consistent with these latter reports, but also raise the possibility that nuclear accumulation of HIF-1{alpha} can be facilitated by cobalt. Our immunofluorescence experiments show that endogenous HIF-1{alpha} localized predominantly in the nucleus both before and after induction by CoCl2 in ASM cells (Figure 2A). Chimeric overexpressed GFP–HIF-1{alpha} was also mainly localized inside the nucleus of transfected ASM cells, but a significant pool could also be detected in the cytoplasm. Treatment with cobalt reduced this cytoplasmic pool and rendered GFP–HIF-1{alpha} exclusively nuclear (Figure 2B). This makes it likely that, although not necessary, a hypoxia-mimetic agent such as cobalt may enhance the efficiency of HIF-1{alpha} nuclear accumulation by either stimulating import or impairing export. The mechanistic details of this phenomenon are hard to examine, because the basic machinery involved in the nucleocytoplamsic trafficking of HIF-1{alpha} (respective importins or exportins, see above) are, at present, unknown.

In an attempt to correlate our findings on the cellular level with the function of ASM cells within the airways, we analyzed the effect of cobalt on the contractile properties of the trachea. Our results show that direct exposure of epithelium-denuded rabbit airways to CoCl2 in vitro attenuated their contractile response to KCl, but failed to influence Ach-evoked contractions (Figure 6). KCl and Ach act through different pathways (35). High extracellular KCl concentrations cause depolarization, which leads to Ca2+ influx from the extracellular space via voltage-dependent Ca2+ channels and contraction. Ach, on the other hand, binds to its receptor and causes contraction by releasing Ca2+ from intracellular stores. The selective inhibition of tracheal ring contraction by cobalt suggests that cobalt acts by influencing the function of voltage-dependent channels. K+ channel activation and/or Ca2+ channel modulation has already been proposed to play a role in hypoxic relaxation of SM (36). Hypoxia can instantly influence the activity of O2-sensitive ion channels, which in turn change the cellular excitability, whereas prolonged cellular hypoxia is probably exerting its action through HIF-1. Along this line, the {alpha}1H gene of T-type voltage-gated Ca2+ channels was the first ion channel gene shown to be induced by an HIF-1–dependent mechanism in PC12 cells (37). Taken together, it is very likely that the cobalt-induced upregulation of HIF-1{alpha} synthesis in ASM cells is responsible for the observed changes in trachea contractility. In our experiments, attenuation of KCl contractions is initially observed 90 min after exposure to CoCl2 and is in good agreement with the kinetics of HIF-1{alpha} induction. This is a further indication that HIF-1 could be the mediator of this effect, but the target genes involved in this process remain to be identified. The cobalt- and/or hypoxia-mediated modulation of contractility may also exhibit tissue and/or species specificity. In vitro studies on the effects of hypoxia on the airway contraction report either an increase (38, 39), or a decrease in airway responsiveness (7). Likewise, cobalt and other hard metal components caused relaxation of methacholine-precontracted isolated tracheal strips from guinea pigs (40).

In conclusion, our results show that, exposure of epithelium-denuded tracheal strips to CoCl2 attenuated their contractile response to KCl by influencing the function of voltage-dependent channels, demonstrating an effect on the physiology of the ASM cells. Moreover, cobalt induces HIF-1{alpha} protein accumulation in ASM cells, probably via a translation-dependent mechanism. Furthermore, we show in this article that the induction of HIF-1{alpha} by CoCl2 is dependent on an active PI3K pathway, which modulates the activity of proteins involved in translation efficiency (Figure 7). Cobalt may activate the PI3K pathway by stimulating ROS production (22, 41). Indeed, both these and more recently published articles (27, 42) have shown that scavenging of ROS by pretreatment of cells with NAC can inhibit induction of HIF-1{alpha}. In a presumably parallel pathway, and depending on the cellular environment, CoCl2 could also prevent the interaction between HIF-1{alpha} and VHL by inhibiting the HIF-1{alpha} prolyl hydroxylases.



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  		Figure 7. Schematic model of the possible mechanism of HIF-1{alpha} induction by CoCl2 in ASM cells. Cobalt activates the PI3K pathway by stimulating ROS production. PI3K most probably acts upstream of a translation stimulation signal, to enhance the translation of cellular mRNAs, including the mRNA of HIF-1{alpha}. In a presumably parallel pathway (dotted lines), CoCl2 could also prevent the interaction between HIF-1{alpha} and VHL by inhibiting the HIF-1{alpha} prolyl hydroxylases.

 

   Acknowledgments
 
The authors thank I. Makadassis for technical assistance, and Prof. A. Germenis and Prof. N. Vamvakopoulos for the use of their fluorescent microscopes. The HIF-1{alpha} cDNA was kindly provided by Dr. P. J. Ratcliffe. This work was supported by a grant from the Greek Secretariat for Research and Technology (PENED 2001, No 01E{Delta}121 to S.B.) with the participation of the company BACACOS, A.E., and a grant from the Research Committee of the University of Thessaly (grant 2770 to E.P.).

Received in original form November 25, 2003

Received in final form July 8, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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