Heme Oxygenase Inhibits Human Airway Smooth Muscle Proliferation via a Bilirubin-dependent Modulation of ERK1/2 Phosphorylation*

Camille Taillé {ddagger}, Abdelhamid Almolki {ddagger}, Moussa Benhamed {ddagger}, Christine Zedda {ddagger}, Jérôme Mégret {ddagger}, Patrick Berger §, Guy Lesèche ¶, Elie Fadel ||, Tokio Yamaguchi **, Roger Marthan §, Michel Aubier {ddagger} and Jorge Boczkowski {ddagger} {ddagger}{ddagger}

From the {ddagger}INSERM, Unité 408, Faculté de Médecine Xavier Bichat, 75018 Paris, §INSERM, E9937, Université Victor Ségalen, 33076 Bordeaux 2, the Service de Chirurgie Vasculaire et Thoracique, Hôpital Beaujon, 92118 Clichy, and the ||Service de Chirurgie Thoracique, Centre Chirurgical Marie Lannelongue, 92350 le Plessis Robinson, France, and the **Medical Research Institute, Tokyo Medical and Dental University, 113-8549 Tokyo, Japan

Received for publication, January 13, 2003 , and in revised form, March 24, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 µM hemin or by 20 µM cobalt-protoporphyrin) and HO inhibition (by 25 µM tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5–20 µM) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 µM) and the antioxidant N-acetyl-cysteine (1 mM) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
An increase in ASM1 mass is one of the features that characterize airway remodeling in asthmatic patients (1). Moreover, bronchial smooth muscle cells from asthmatic patients have shown abnormal cellular proliferation in vitro (2). Therefore, knowing the factors and the mechanisms that modulate ASM proliferation can have important pathophysiological implications. In the last years, various types of mitogens have been shown to induce human ASM proliferation, including growth factors, contractile agonists, and inflammatory mediators, such as ROS (3). Indeed, evidence is growing that in physiological conditions, ROS induce signal transduction leading to gene transcription and cell growth (4). Because ASM can be continuously exposed to large amounts of exogenous or endogenous ROS produced by inhaled agents, inflammatory or ASM cells themselves (5), redox signaling might be of particular importance in ASM proliferation. A mitogen-induced ROS production leading to cell proliferation via activation of ERK1/2 has been described in ASM (68). But, if the oxidant signaling involved in ASM proliferation is well characterized, little is known about involvement of antioxidant systems in the control of muscle proliferation.

Heme oxygenase, the enzyme responsible for heme degradation, is a powerful cytoprotective antioxidant system (9, 10). Heme degradation produces CO and biliverdin, reduced into bilirubin by the biliverdin reductase. In the airways, HO is expressed in the epithelium, the smooth muscle, macrophages, parasympathic ganglia, and endothelium (11, 12) and is involved in the protection against oxidative-mediated airway inflammation and hyperreactivity (13, 14). Bilirubin is one of the most powerful antioxidant system in the organism (15), mainly known for its cytoprotective effect in oxidative stress models (16). Moreover, we have recently shown that bilirubin could also modulate ROS production in guinea pig tracheal smooth muscle in physiological conditions (14). In this model, bilirubin also modulated oxidant signaled phosphorylation of myosin light chain. This suggests that HO, by the way of its antioxidant properties, could also play a role in the modulation of redox signaling in ASM. Furthermore, HO has shown an antiproliferative effect in vascular smooth muscle in vitro and in vivo (17, 18). However, no data are available in the current literature investigating the effects of HO on ASM proliferation and the mechanism(s) involved in this phenomenon.

Therefore, the aim of this study was to investigate whether the HO pathway could modulate proliferation of human bronchial smooth muscle cells exposed to two mitogens, PDGF or FCS (6), and the mechanism(s) involved in this phenomenon, with special attention to the role of ROS signaling and ERK1/2 activation. We also investigated which of the HO end products, CO and bilirubin, was responsible for the effect of HO. Finally, we evaluated the functional role of the HO pathway on ASM in vivo in a model of airway remodeling secondary to multiple ovalbumin challenge in immunized guinea pigs.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
[Methyl-3H]thymidine was purchased from PerkinElmer Life Sciences, and PDGF-AB was from R & D System (Abingdon, UK). CoPP, hemin, and SnPP IX were from Porphyrin Products (London, UK). Sense and antisense oligonucleotides were supplied by Invitrogen and transfected with the Superfect® transfection reagent (Qiagen). H2DCFH-DA and propidium iodide were from Molecular Probes (Eugene, OR). The selective MEK inhibitor PD98059 was purchased from Calbiochem (Merck Eurolab SA, Fontenay-sous-Bois, France). Anti-phosphorylated p42/44 antibody was purchased from New England Biolabs (Ozyme, Saint-Quentin-en-Yvelines, France), anti-HO-1 antibodies were from StressGen (Tebu, Le-Perray-en-Yvelines, France), anti-smooth muscle myosin heavy chain isoform 1 antibody was from Seikagaku America (Palmouth, MA), and anti-{alpha}-actin antibody was from Sigma. Except for the anti-phosphorylated p42/44 antibody, which was polyclonal, all of the antibodies used were monoclonal. Culture media, supplements, and FCS were from Invitrogen. Tissue culture plasticware was supplied by Costar Corp. (Cambridge, MA). Reagents for Western blot were from Bio-Rad. The reagents for immunohistochemistry were from Dako (Carpinteria, CA). The other reagents were from Sigma.

Human Bronchial Smooth Muscle Isolation and Cell Culture
Primary cultures of human bronchial smooth muscle were established as described (19, 20). Briefly, human bronchi (internal diameters, 5–15 mm) were obtained from lobes resected during thoracotomy for lung cancer in 11 different patients and dissected from the surrounding parenchyma. The epithelium was removed, and bands of airway smooth muscle were isolated by dissection under binocular microscope and cut into 1-mm2 pieces. These muscular pieces, termed explants, were incubated in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FCS, antibiotics (10 mg/ml streptomycin, 10,000 UI/ml penicillin G), and amphotericin B (25 µg/ml) in a humidified atmosphere of 5% CO2, 95% air at 37 °C as described (19). On reaching confluence, the cells were passed by lifting the cells with 0.05% trypsin, 0.5 mM EDTA. The cultures from passages 3–5 were used for the experiments. At confluence, cells the exhibited the typical "hill and valley" aspect (20). Cell characterization was assessed by immunostaining using a monoclonal antibody against {alpha}-smooth muscle actin and against the specific smooth muscle myosin heavy chain isoform 1 (19, 22). More than 95% of cells from each patient used in the different experiments displayed positive immunohistochemical staining for both antibodies.

The general experimental protocol was as follows. The cells were seeded at an initial density of 104/cm2, grown to 70–80% confluence, and serum-deprived (1% FCS) for 24 h. Then they were stimulated with one of two different mitogens, 10% FCS and 50 ng/ml PDGF-AB. Control unstimulated cells were grown on 1% FCS. In some experiments, the cells were incubated with different pharmacological agents and their respective vehicles for the indicated time before the addition of the mitogen. The experiments were carried out in at least three different cell lines, each one derived from a different individual.

HO Expression and Activity in ASM Cells
HO-1 protein expression was measured by Western blot and immunohistochemistry. For Western blot experiments, the cells were cultured in 75-cm2 plates. Western blot was performed as described previously (21, 22). The concentration of the anti-HO-1 antibody was 1/1000. Detection was performed by a chemiluminescence substrate. Using the same blots, the expression of the housekeeping protein {beta}-actin was evaluated using a monoclonal anti-{beta}-actin antibody. Optical densities were measured with a Perfect Image 2.01 image analysis system (Iconix, Courtaboeuf, France). The results were expressed as the ratio of the expression of HO-1 to that of {beta}-actin.

For immunohistochemistry, the cells were cultured in a Lab-Tek chamber slide (Nunc, Naperville, IL). Immunohistochemistry was performed as described before (24), with a 1/1000 dilution of the anti-HO-1 antibody. The specificity of the immunostaining was evaluated by replacement of the primary antibody by a control isotype antibody at an equivalent protein concentration and by omitting the primary antibody.

HO activity was assessed by bilirubin production as already described (23) in cells grown on 75-cm2 plates. In addition, HO activity was evaluated in situ by immunohistochemistry using the anti-bilirubin IX monoclonal antibody, 24G7 (25). This monoclonal antibody specifically recognizes the conjugated and unconjugated forms of bilirubin IX but not other isomers of bilirubin (26). Bilirubin IX is produced by the reduction of biliverdin IX, a product of the HO-1 reaction. Thus, immunohistochemistry with the use of 24G7 allowed us to assess the HO-1-specific heme degradation in situ in fixed cells. The antibody was used at 10 µg/ml concentration. The specificity of the immunostaining was evaluated by replacement of the primary antibody by a control isotype antibody at an equivalent protein concentration and by omitting the primary antibody.

[Methyl-3H]thymidine Incorporation in ASM Cells
The cells were cultured in 24-well plates and stimulated with mitogens for 24 h. For the final 18 h of incubation with the mitogen, 4 µCi/ml of [methyl-3H]thymidine was added to measure DNA synthesis by scintillation counting. The results of individual treatments were obtained in quadruplicate and were expressed as the number of counts/min. Then the percentage of change from the response of 1% FCS for each individual result was calculated. To minimize the influence of variability between tissue donor, the value was calculated from the response of 1% FCS-treated cells from the same 24-well plate. In experiments with the MEK inhibitor PD98056, the control condition was FCS + Me2SO, explaining the lower proliferative response. However, we checked in preliminary experiments that Me2SO does not change the way of proliferative response to mitogens but just decrease by about one-third the absolute value of thymidine incorporation.

Cellular Toxicity and Viability
Cellular toxicity and viability were assessed by three different methods: cell count and trypan blue exclusion, lactate dehydrogenase release in the medium, and incorporation of the fluorescent dye propidium iodide. For propidium iodide incorporation, the cells seeded in 96-well plates were incubated with 5 µM propidium iodide for 30 min and then washed in phosphate-buffered saline before reading at 480–520 nm with a multiwell fluorescence plate reader (Fluorostar BMG). The results are expressed in fluorescent arbitrary units.

Measurement of ROS Production by ASM Cells
Intracellular ROS Production: H2DCFH-DA Oxidation—The cells were cultured in 96-well plates as described previously. H2DCFH-DA (final concentration in Me2SO, 10 µM) was added 1 h before stimulation. Immediately after the addition of the mitogen, fluorescence was measured every 5 min during a 45-min period with a multiwell fluorescence plate reader at 480–555 nm. Intracellular ROS (especially H2O2 or hydroxyl radical) oxidized dichlorodihydrofluorescein, yielding the fluorescent product dichlorodifluorescein (24). The results are expressed as the ratio between fluorescence measured at 45 min and that measured at the first point (27).

Extracellular Superoxide Anion Release: Cytochrome c Reduction— Ferricytochrome c reduction was measured as previously described (24). Briefly, the cells were cultured in 6-well plates. Before the addition of the mitogen, the medium was replaced with Hanks' balanced salt solution without phenol red and incubated in 1 ml of the same buffer with and without 300 units/ml superoxide dismutase. Subsequently, ferricytochrome c was added at a final concentration of 80 µM to the reaction buffer solution, followed by the addition of the mitogens. After 1 h, the buffer was removed, and the absorbance at 550 nm was measured immediately. Superoxide anion production was calculated from the differences in the absorbances between samples with and without superoxide dismutase, using an extinction coefficient of 21.1 mM-1 cm-1 for reduced ferricytochrome c.

Measurement of ERK1/2 Phosphorylation in ASM Cells
The cells were cultured in 75-cm2 plates. In a first series of experiments, the cells were stimulated for different times with 50 ng/ml PDGF-AB or serum. Then the medium was removed, and the cells were washed twice with cold phosphate-buffered saline and removed in lysis buffer containing phosphatase inhibitors (2.5 mM natrium fluoride, 1 mM {beta}-glycerophosphate, 1 mM orthovanadate, and 1 mM para-nitrophenol-phosphate). In a second series of experiments, the cells were pretreated with different pharmacological agents before a 10-min stimulation with PDGF or serum.

Western blot was performed as already described (14, 24). The phosphorylated forms of the enzyme were detected with a polyclonal antibody used at 1/1000 dilution. Optical density of the band was measured as described (see below) and compared with {beta}-actin expression.

Immunohistochemical Detection of HO-1 in Human Bronchial Smooth Muscle
Detection of HO-1 protein in airway smooth muscle was examined in human bronchi. Segments of two bronchi utilized for ASM cells isolation and culture were fixed in formol 10% and embedded in paraffin. Immunohistochemistry was performed as previously published (23, 24).

HO-1 Sense and Antisense Oligonucleotide (ODN) Treatment
The sense/antisense ODNs for HO-1 were directed against the flanking translation initiation codon in the human HO-1 cDNA (28). The antisense sequence was 5'-CGCCTTCATGGTGCC-3', whereas the sense sequence was 5'-GGCACCATGAAGGCG-3'. ODNs were phosphorothioated on the first three bases on the 3' end. The cells were transfected using the Superfect® transfection reagent (Qiagen) following the manufacturer's instructions. Briefly, the cells were seeded in 24-well plates at a density of 20,000 cells/well 24 h before transfection. The proportions used were 1 µg of DNA/5 µl of transfection reagent/well. The cells were incubated for 3 h with the ODNs; then the medium was replaced with fresh medium containing 10% serum. The experiments for proliferation and Western blot were performed 48 h after transfection.

Induction of Airway Remodeling in Ovalbumin-sensitized and Aerosol-challenged Guinea Pig
Pathogen-free male Hartley guinea pigs (250–300 g of body wt; Charles River) were housed in individual cages in climate-controlled animal quarters and were given water and food ad libitum. As previously described (29), the animals were immunized with 0.5 ml of 0.9% (w/v) NaCl (saline) containing 100 mg of ovalbumin, injected subcutaneously on the neck, and with another 0.5 ml intraperitoneally on day 1. On days 8, 9, 10, 13, 14, and 15, the animals (called OO animals) were challenged in a 5-liter plastic chamber by a 10-min exposure to aerosolized ovalbumin (0.1% ovalbumin in 10 ml of saline) using a Devilbiss nebulizer (Sunrise, Devilbiss Medical, Nantes, France). The time of exposure was determined by the appearance of respiratory distress signs (polypnea, bronchospasm, contraction of accessory respiratory muscles, and cyanose). Another group of animals were immunized to ovalbumin as described above and exposed to aerosolized saline (ON animals). This model is characterized by bronchial hyperreactivity to histamine (measured by pulmonary inflation pressure) and an increased number of polynuclear eosinophils in bronchoalveolar lavage in OO animals (29). Both OO and ON animals were randomly divided into two groups. One group received the inhibitor of HO activity SnPP IX, given intraperitoneally 6 h before the challenge at a dose of 50 µmol/kg on days 8, 10, 13, and 15 (OO-SnPP animals); a second group received the HO-1 inductor, hemin, given intraperitoneally at a dose of 50 mg/kg on days 7, 10, and 13 (OO-Hemin animals). In preliminary experiments we verified that SnPP IX and hemin's vehicle did not modify the parameters measured in the study. We also verified that hemin and SnPP IX significantly increased and decreased HO activity, respectively, by measurement of bilirubin production in lung microsomes, as previously described (23) (data not shown).

The animals were sacrificed 24 h after the last challenge. They were anesthetized with sodium pentobarbital (Nesdonal®, Specia-Rhone-Poulenc, Romainville, France) (50 mg/kg of body weight intraperitoneal), and then the lung was inflated through a tracheal canula at 25 cm H2O with 10% formol and fixed in paraffin.

Evaluation of ASM Area and HO-1 and Bilirubin Immunohistochemistry
Hematoxylin- and eosin-stained tissues sections were examined microscopically, and the smooth muscle area was measured with a microscope (Leitz, Germany) related to a camera (Olympus) and a computerized image analysis system (AnalySIS Soft Imaging System, Münster, Germany) as described by Palmans et al. (30). The muscular area was determined by delimitating the outer side of the basal membrane and the outer side of the muscular area. We analyzed bronchi with similar diameter and with a ratio of minimal to maximal diameter of more than 0.6. We analyzed five animals in each group and three or four different bronchi for each animal.

HO-1 and bilirubin immunohistochemistry was performed as previously described (23). The concentration of the anti-bilirubin antibody was 1 µg/ml.

Statistical Analysis
The values are given as the means ± S.E. The data were analyzed by one-way analysis of variance; the differences between the means were analyzed with the Fisher's protected least significant difference multiple comparison test. The significance for all statistics was accepted at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
HO-1 Is Expressed in Human ASM—HO-1 was expressed in unstimulated ASM cells in culture (Fig. 1A). Incubation with the mitogens FCS (10%) or PDGF (50 ng/ml) for 24 h significantly and quite similarly increased HO-1 expression and HO activity (p < 0.05 as compared with 1% FCS; Fig. 1, A and B). CoPP (20 µM) could further potentiate HO activity induced by the mitogens. As attempted, SnPP IX (25 µM) significantly inhibited HO activity (Fig. 1B).



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FIG. 1.
HO-1 expression in human airway smooth muscle cells. A, Western blot analysis of HO-1 expression. Whole cell proteins were extracted from human ASM cells after a 24-h serum deprivation followed by a stimulation with 10% FCS, 50 ng/ml PDGF, or 20 µM CoPP for 24 h. The histogram represents the ratio between HO-1 optical density and that of {beta}-actin for the above typical experiment. B, HO activity was assessed by measuring bilirubin production by human ASM cells with a spectrophotometer according to the method described under "Experimental Procedures." n = 4–6 for each condition. The bars are the means ± S.E. *, p < 0.05 versus 1% FCS; #, p < 0.05 versus mitogen alone. C, immunohistochemical analysis of HO-1 and bilirubin expression in ASM cells. The cells were cultured in a chamber slide, fixed with acetone, and stained by immunoreaction with monoclonal antibodies. No staining was observed with isotype antibodies. D, immunohistochemical analysis of HO-1 expression in airway smooth muscle in human bronchi. Staining was observed in airway smooth muscle (asterisks) and epithelium (arrow). No staining was observed with the isotype antibody.

 

HO-1 expression and activity in human ASM cells in culture was further confirmed with immunohistochemical analysis using anti-HO-1 and anti-bilirubin antibodies, respectively (Fig. 1C). No expression was observed with a control isotype antibody. HO-1 was also expressed in ASM in human bronchi (Fig. 1D).

The HO Pathway and Bilirubin Modulate Human ASM Cell Proliferation—Mean thymidine incorporation in unstimulated cells was 5448 ± 1228 cpm. ASM cell proliferation was significantly enhanced by 10% FCS and PDGF, being 360 and 372% higher, respectively, than observed with 1% FCS (Fig. 2, A and B, p < 0.05, respectively). HO induction by CoPP significantly decreased the proliferative response to FCS and PDGF. Indeed, in CoPP-treated cells, proliferation induced by 10% FCS and PDGF was only 135 and 155% higher, respectively, than observed with 1% FCS (Fig. 2, A and B, p < 0.05, respectively). Hemin (10 µM), another HO inductor, has a similar effect on ASM proliferation (data not shown). On the other hand, the HO inhibitor SnPP IX induced a 523 and 690% increase in proliferation induced by FCS and by PDGF (Fig. 2, A and B, p < 0.05, respectively). For both mitogenic factors, SnPP IX reversed the antiproliferative effect of CoPP, thus confirming a specific effect of HO activity. Transfection with the HO-1 antisense effectively blocked HO-1 expression (Fig. 3A) and, like pharmacological blockade, enhanced PDGF-induced proliferation (Fig. 3B).



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FIG. 2.
Role of the HO pathway on human ASM cells proliferation. The cells were stimulated for 24 h with 10% FCS (A and C) or PDGF (B and D). [3H]Thymidine incorporation (4 µCi/ml) was measured by scintillation counting. Each condition was done in quadruplicate. Three different muscular explants were tested for each experiment. The values are the means ± S.E. *, p < 0.05 versus 1% FCS; #, p < 0.05 versus mitogen alone; &, p < 0.05 versus mitogen + SnPP.

 


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FIG. 3.
Effect of cell transfection with HO sense and antisense ODNs. A, effect of HO-1 ODNs on PDGF-induced HO-1 expression. The experimental procedure is the same described for Fig. 1. B, thymidine incorporation in ODN-transfected cells. The cells were stimulated for 24 h with PDGF. [3H]Thymidine incorporation (4 µCi/ml) was measured by scintillation counting. Each condition was done in quadruplicate. The values are the means ± S.E. *, p < 0.05 versus 1% FCS; #, p < 0.05 versus mitogen alone.

 

No toxicity was observed with metalloporphyrins at the doses we used. Neither blue trypan exclusion test nor lactate dehydrogenase measurement or propidium iodide incorporation was modified (lactate dehydrogenase content between 6 ± 2 and 8 ± 4 UI/ml; propidium iodide incorporation between 32566 ± 1250 and 34559 ± 1452 arbitrary fluorescence units; non-significant; data not shown).

Having demonstrated that HO modulated ASM proliferation, we wondered which of the HO end products, CO and bilirubin, was responsible for its antiproliferative effect. Concerning CO, neither the guanylate cyclase inhibitor ODQ (10 µM, given 1 h before mitogenic stimulation) nor the CO scavenger myoglobin (5–20 µM for 24 h) could reverse inhibition of proliferation induced by SnPP IX, making unlikely a role of the CO-cGMP pathway in HO-modulated proliferation (Fig. 4, A and B, respectively).



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FIG. 4.
Role of the guanylyl cyclase-cGMP pathway. A, thymidine incorporation by human ASM cells stimulated for 24 h with serum or PDGF plus Me2SO (that is used as the solvent for ODQ). The cells were treated with the HO inductor CoPP (20 µM) and by the guanylate cyclase inhibitor ODQ (10 µM). B, role of myoglobin, a scavenger for CO, on cell proliferation. For both panels, p < 0.05 in all conditions. n = 12 for each condition. The bars are the means ± S.E. *, p < 0.05 versus 1% FCS; #, p < 0.05 versus mitogen.

 

By contrast, bilirubin (1 µM, 1 h before stimulation) was able to significantly reduce FCS- and PDGF-induced proliferation (Fig. 2, C and D, p < 0.05 versus 1% FCS). Furthermore, bilirubin significantly reversed the mitogenic effect of SnPP IX; proliferation in SnPP + bilirubin cultured cells was similar to that observed in cells cultured with the mitogen alone (Fig. 2, C and D). Considering the antioxidant properties of bilirubin, we investigated whether the antioxidant N-acetylcysteine (NAC) could mimic the effect of CoPP and bilirubin. These experiments showed that pretreatment of cells with NAC (1 mM, 1 h before stimulation) mimicked the effect of CoPP and bilirubin on proliferation and reversed the effect observed after treatment with SnPP IX (Fig. 2, C and D).

The HO Pathway Modulates Both Intracellular and Extracellular ROS Production—Considering the inhibitory effect of bilirubin and NAC on cellular proliferation, we investigated the role of the HO pathway in intracellular and extracellular ROS production by measurement of the oxidation of H2DCFH-DA and the reduction of cytochrome c, respectively.

Both FCS and PDGF significantly increased intracellular and extracellular ROS production. Indeed, FCS and PDGF increased H2DCFH-DA oxidation by 41 and 38% and cytochrome c reduction by 108 and 152%, respectively, compared with 1% FCS (p < 0.05 in each case; Table I). Treatment with the HO inductor CoPP prior to cell stimulation decreased mitogen-induced H2DCFH-DA oxidation by 29 and 25%, respectively, and cytochrome c reduction by 68 and 48% (p < 0.05 for 10% FCS and PDGF as compared with 1% FCS, respectively; Table I). The HO inhibitor SnPP IX increased ROS production by 53 and 58% for H2DCFH-DA oxidation and by 51 and 44.6% for cytochrome c reduction compared with stimulation with mitogens alone (p < 0.05 for 10% FCS and PDGF as compared with 1% FCS respectively; Table I). Bilirubin (1 µM) and N-acetylcysteine (1 mM) mimicked the effect observed with CoPP and reversed the pro-oxidative effect of SnPP (p < 0.05 respectively; Table I).


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TABLE I
Cellular ROS production

 

The HO Pathway Modulates ERK1/2 Phosphorylation by a Redox Mechanism—Considering that ROS modulate ERK1/2 activation and that these kinases are involved in ASM cell proliferation, we investigated the role of the HO pathway in the modulation of ERK1/2 phosphorylation. ASM cells exhibited a basal level of phosphorylated ERK1/2 (Fig. 5A). PDGF induced a time-dependent increase in ERK1/2 phosphorylation in HASM cells that peaked after 10 min of stimulation (Fig. 5A). Preincubation with CoPP decreased PDGF-induced ERK1/2 phosphorylation at 10 min, whereas SnPP increased it (Fig. 4B). Bilirubin (1 µM) strongly decreased SnPP-enhanced phosphorylation. A similar effect was observed with N-acetylcysteine (1 mM), confirming the role of ROS in SnPP-induced ERK1/2 activation. Similar results were observed with serum-stimulated cells (data not shown).



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FIG. 5.
Role of HO on phosphorylation of the ERK1/2 MAPK. A, time course of phosphorylation of ERK1/2 MAPK induced by 50 ng/ml PDGF, assessed by Western blot. B, typical Western blot analysis of ERK1/2 phosphorylation after a 10-min stimulation with 50 ng/ml PDGF-AB. The cells were pretreated with the different reagents for one h before stimulation. The results were similar with cells from three different muscular explants. The graph represents the optical density of the bands of one typical Western blot, compared with the expression of {beta}-actin. C, effects of the MEK inhibitor PD98059 on ERK phosphorylation induced by a 10-min stimulation with serum and PDGF. The histogram represents the ratio between phosphorylated ERK1/2 optical density and that of {beta}-actin for a typical experiment. D and E, assessment of the role of PD98059 on cell proliferation by incorporation of [3H]thymidine after a 24-h stimulation with 10% serum (D) and PDGF (E). The results are expressed as the means ± S.E. of three different experiments with cells from three different explants. *, p < 0.05 versus 1%FCS+ Me2SO (DMSO); #, p < 0.05 versus mitogen + Me2SO; &, p < 0.05 versus mitogen + Me2SO + SnPP.

 

The MEK inhibitor PD98059 inhibited cell proliferation (Fig. 5, D and E) and blocked both FCS- and PDGF-induced ERK1/2 phosphorylation at 10 min (Fig. 5C), confirming involvement of the ERK1/2 pathway in ASM cell growth. Moreover, PD98059 inhibited the increase in cell proliferation induced by SnPP IX (Fig. 5, D and E) and reversed SnPP-enhanced ERK1/2 phosphorylation, thus confirming that the effect of HO on cell proliferation involves the MAPK pathway.

HO Protects against the Increase in ASM Area in Ovalbumin-sensitized and -challenged Guinea Pig—HO-1 expression in bronchial smooth muscle was increased in OO as compared with ON animals, along with expression in epithelium and inflammatory cells (Fig. 6A). Bilirubin, one of the end products of heme catabolism, was found in a similar manner by immunohistochemistry in bronchial smooth muscle, confirming the in vivo activity of muscular HO (Fig. 6A).



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FIG. 6.
Role of the HO pathway on airway smooth muscle increase in allergic airway inflammation. A, HO-1 and bilirubin were expressed in ASM (asterisk) in OO guinea pigs. Expression was also observed in inflammatory cells and epithelium (arrows). No staining was observed with isotype antibodies. B, airway smooth muscle area measurement. The animals were either sensitized with ovalbumin and challenged with NaCl (ON animals) or sensitized and challenged with ovalbumin (OO animals) and chronically treated or not with the HO inductor hemin or the HO inhibitor SnPP as described under "Experimental Procedures." The results represent the means ± S.E. of three or four bronchi in five different animals in each group. *, p < 0.05 versus ON; #, p < 0.05 versus OO.

 

The diameters of bronchi examined for measurement of smooth muscle area were not statistically different, ranging from 375.78 ± 47.95 to 452.23 ± 66.72 µm within the various experimental groups. Repeated allergen challenge in immunized animals induced a significant increase in ASM area (88.40 ± 16.21 versus 119.32 ± 18.45 µm2; p < 0.05 OO versus ON; Fig. 6B). Treatment of OO animals with the HO inductor hemin inhibited the increase in bronchial smooth muscle area, and administration of the HO inhibitor SnPP produced the opposite effect. Indeed, bronchial smooth muscle area was 90.25 ± 11.12 µm2 for hemin-treated and 158.02 ± 18.23 µm2 in SnPP-treated OO animals (p < 0.05 versus OO animals; Fig. 6B). In ON animals, the modulation of bronchial smooth muscle area induced by hemin and SnPP was slight but insignificant (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The main results of this study indicate that HO acts in an autocrine negative feedback manner to limit ROS-dependent phosphorylation of the ERK1/2 MAPK and the ensuing proliferation induced by serum and PDGF in human ASM cells. These effects are secondary to a reduction in ROS production by the action of the HO end product bilirubin. These data provide the first evidence that HO takes part in the control of ASM proliferation by modulating ROS signaling via the effect of bilirubin. Furthermore, these results could be relevant to the increase in ASM mass observed in asthma. Indeed, using a model of airway remodeling secondary to multiple ovalbumin challenges in immunized guinea pigs, we found that activation of the HO pathway prevents the increase in bronchial smooth muscle area, whereas inhibition of HO has the opposite effect.

An antiproliferative effect of the HO pathway in smooth muscle was first described in rat vascular smooth muscle cells in vitro (17, 31) and then confirmed in vivo in animal models of vascular remodeling after hypoxia or balloon-induced wall injury (32, 33). Indeed, to our knowledge, all of the data concerning HO and smooth muscle proliferation were obtained in vascular smooth muscle cells either from rat or guinea pig. This antiproliferative effect is cell type-dependent, because opposite effects of HO on cell cycle have been described in endothelial and in smooth muscle cells (34); HO inhibition decreases S and G2/M phases in endothelial cells and increases it in smooth muscle cells, whereas HO induction exhibits opposite effects. To our knowledge, no information is available concerning the effect of HO on the proliferation of ASM. Our results confirm and expand the data obtained on vascular smooth muscle. Indeed, induction of HO-1 protein expression and HO activity may represent a mechanism by which mitogens such as serum or PDGF regulate ASM growth, as described in vascular smooth muscle (35). This hypothesis is supported by our data showing that serum- or PDGF-stimulated DNA synthesis was augmented in the presence of the HO inhibitor SnPP and reduced in the presence of the HO inductors hemin or CoPP, indicating a growth inhibitory effect of HO on human ASM. It must be noted, however, that definitive conclusions from drug-based experiments should not be drawn without verifying the biological activity of the drug in a particular experimental setting. In the present study, we used two different HO-1 inductors (hemin and CoPP), and we verified that both inductors and the HO inhibitor SnPP IX effectively modulated HO-1 protein expression and HO activity in ASM cells and lung microsomes. Moreover, the effect of CoPP was inhibited by the HO inhibitor SnPP IX, thus confirming the specificity of these compounds. The similar inhibitor effects of the two different HO-1 inductors on ASM proliferation further support this interpretation. To finish with, ASM transfection with an HO-1 antisense oligonucleotide induced an effect similar to that of the HO inhibitor SnPP IX on cell proliferation. Furthermore, we also found HO-1 protein expression in both human and guinea pig airway smooth muscle in situ, thus ensuing the in vivo relevance of the cellular data.

In vascular smooth muscle, the antiproliferative effect of HO has been mainly related to carbon monoxide production, because hemoglobin, a scavenger for CO, was able to reverse the effect of HO induction (17, 31). The antiproliferative effect of CO has been suggested to be secondary to soluble guanylyl cyclase activation, because inhibition of this enzyme or of its end product, cGMP, can restore DNA synthesis in vascular smooth muscle cells transfected with HO-1 cDNA (18). Exogenous administered CO has also an antiproliferative effect on vascular as well as airway smooth muscle. Indeed, exogenous CO at low doses (between 100 and 250 ppm, which is considered to be comparable with the gaseous production by the enzyme itself) arrests rat vascular smooth muscle cells at the G1/S transition of the cell cycle (17) and human ASM cells at the G0/G1 phase (36). However, in ASM, exogenous CO acts independently from a guanylyl cyclase/cGMP pathway (36). This result is in line with our data showing that the soluble guanylyl cyclase inhibitor ODQ did not impair the anti-proliferative effect of HO activation. However, in the present study, a predominant role of the endogenous CO-HO pathway in the control of ASM cell proliferation is unlikely because application of different concentrations (5–20 µM) of the CO scavenger myoglobin did not modify the antiproliferative effect of HO activation. Differences in cell type (airway versus vascular), species (human versus rat), and exogenous versus endogenous HO-produced CO may explain these discrepancies.

In contrast with CO, we found that bilirubin exerted a clear antiproliferative effect on human ASM cells. This result agrees with data published in osteoblasts and neural and hepatoma cells (37). The concentration of bilirubin is a very important point to consider because of the potential cellular toxicity of high concentrations of bilirubin (16). Indeed, a recent study by Liu et al. (38) shows that biliverdin and bilirubin can induce apoptosis in rat vascular smooth muscle cells, as done by HO induction, whereas CO and iron failed to induce such phenomenon. In this study, bilirubin required concentrations as high as 500 µM to induce apoptosis, whereas no effect was observed at concentrations similar to the one we used in the present study (5 µM). Although both cell type and experimental conditions were different, this can explain why we did not observe any cell toxicity and death in our model. In the present study, the simultaneous decrease in both ASM oxidants production and cell proliferation observed with 1 µM of bilirubin and the fact that this last effect was mimicked by the antioxidant NAC strongly suggest a major role of the antioxidant properties of bilirubin in the decreased proliferation. Furthermore, bilirubin was able to reverse the pro-oxidant and pro-proliferative effect of HO inhibition, therefore stressing its involvement in the effects of HO. This conclusion is further supported by the in situ immunohistochemical detection of bilirubin in ASM cells, demonstrating that this molecule was well synthesized in living cells during the different experimental conditions. The present results emphasize the concept that the antioxidant properties of the HO-bilirubin pathway are not only related to its ROS scavenging properties (39) but also to the modulation of ROS production. Because recent studies demonstrated that a NAD(P)H oxidase like system is the main source of ROS in animal and human ASM (24, 40), inhibition of NAD(P)H oxidase by the HO-bilirubin pathway could explain its inhibitory effect on ROS production. This hypothesis is supported by previous data obtained in vitro, in acellular preparations, showing that bilirubin can inhibit reconstituted NAD(P)H oxidase (41). Further studies are needed to verify whether bilirubin modulates NAD(P)H oxidase in ASM and the mechanism(s) involved in this effect.

Involvement of the ERK1/2 MAPK in PDGF- and serum-induced ASM proliferation and the role of ROS in the kinase cascade activation has been extensively described in smooth muscle cells (6, 7, 40, 4244). In our model, the critical role of ERK was confirmed by the inhibitory effect of the MEK inhibitor PD98059 on muscle proliferation. The present results show that HO is an important pathway to control activation of the ROS-sensitive ERK1/2 pathway in ASM. Indeed, ERK activation was blocked by induction of HO-1 by CoPP, by the antioxidant NAC, and by bilirubin, thus confirming the sensibility of the ERK pathway to oxygen species in human ASM cells. Moreover, the MEK inhibitor PD98059 significantly reduced the pro-proliferative effect of HO inhibition, thus confirming that the MEK-ERK1/2 pathway is a major target of the modulatory effect of HO on human ASM proliferation. However, whether or not the HO-bilirubin pathway can modulate other ROS-dependent signaling pathways, such as the JAK-STAT pathway (45, 46), remains to be investigated. Furthermore, if bilirubin antioxidant properties appear to be an important mechanism explaining the decrease in ASM proliferation, other mechanisms cannot be excluded. Indeed, bilirubin actually inhibits protein phosphorylation, probably by interacting with different domains of the kinase (47). The relative importance of these pathways in modulating ASM proliferation, as well as the role of other HO end products, such as iron and ferritin, warrants further investigation.

The antiproliferative effect of HO on ASM might be protective in vivo under conditions leading to bronchial smooth muscle proliferation. Indeed, using a model of ovalbumin-immunized and multiple aerosol-challenged guinea pigs characterized by airway inflammation (29) and an increase in ASM muscle area, we found that bronchial muscle mass was augmented in the presence of the HO inhibitor SnPP IX and reduced in the presence of the HO inductor hemin. In these animals, HO-1 protein was induced in bronchial smooth muscle, probably representing a mechanism by which HO regulates ASM growth. In this model, we cannot exclude, however, that HO may have a local anti-inflammatory effect that, in turn, could participate to the modulation of ASM growth, as observed in animals treated with anti-inflammatory drugs such as cysteinyl leukotriene or endothelin receptor antagonist (48, 49). However, immunohistochemical detection of bilirubin in bronchial muscle suggests an autocrine effect of the HO-bilirubin pathway in vivo in ovalbumin-challenged animals.

In conclusion, this study provides evidence that HO is involved in the control of ASM cells proliferation through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2. These data are relevant in terms of in vivo protection against some features of airway remodeling, such as increased ASM area. Collectively, these results and previous data showing that the HO-bilirubin pathway modulated negatively ASM contractility (14) suggest that induction of the HO pathway could be beneficial in asthma or other respiratory diseases leading to airway remodeling and hyperreactivity.


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

{ddagger}{ddagger} To whom correspondence should be addressed: INSERM U408, Faculté deMédecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France. Tel.: 33-1-44-85-62-51; Fax: 33-1-42-26-33-30; E-mail: jbb2{at}bichat.inserm.fr.

1 The abbreviations used are: ASM, airway smooth muscle; CoPP, cobalt-protoporphyrin; H2DCFH-DA, 2'-7'dichlorodihydrofluorescein diacetate; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; HO, heme oxygenase; ODN, oligonucleotide; PDGF, platelet-derived growth factor; PD98059, 2'-Amino-3'-methoxyflavone; ROS, reactive oxygen species; SnPP IX, tin-protoporphyrin IX; NAC, N-acetyl-cysteine; ODQ, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one; MAPK, mitogen-activated protein kinase; MEK, MAP kinase kinase. Back


    ACKNOWLEDGMENTS
 
We are in debt to Drs. Roberta Foresti and Roberto Motterlini (Harrow, Middlesex, UK) and Prof. Bruno Crestani (Paris, France) for helpful and encouraging comments.



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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