heme oxygenase inhibits human airway smooth muscle proliferation

1

Heme oxygenase inhibits human airway smooth muscle

proliferation via a bilirubin-dependent modulation

of ERK1/2 phosphorylation

by

Camille Taillé, Abdelhamid Almolki, Moussa Benhamed,

Christine Zedda, Jérôme Mégret,

Patrick Berger*, Guy Lesèche**, Elie Fadel ***, Tokio Yamaguchi ****,

Roger Marthan*, Michel Aubier and Jorge Boczkowski.

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 408,

Faculté de Médecine Xavier Bichat, Paris ; * INSERM, E9937, Université Victor Ségalen, Bordeaux 2;

** Service de Chirurgie Vasculaire et Thoracique, Hôpital Beaujon, Clichy ; *** Service de Chirurgie Thoracique, Centre Chirurgical Marie Lannelongue,

le Plessis Robinson , France ; **** Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

Correspondance to : Jorge Boczkowski, MD, PhD. INSERM U408 Faculté de Médecine 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 : [email protected]

Running title : Bilirubin and Human Airway Smooth Muscle proliferation.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 10, 2003 as Manuscript M300364200 by guest on February 24, 2018

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Summary

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 (FCS) or 50 ng/ml

PDGF-AB induced cell proliferation, extra and intracellular ROS production and ERK 1/2

phosphorylation. Pharmacological HO-1 induction (by hemin 10 µM or cobalt-

protoporphyrin, CoPP, 20 µM), and HO inhibition (by tin-protoporphyrin, SnPP-IX, 25 µM or

by an antisense oligonucleotide), respectively reduced and enhanced significantly both cell

proliferation and ROS production. Nor the carbon monoxide (CO) scavenger myoglobin (5-

20µM) neither the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one

(ODQ) could reverse ASM proliferation induced by SnPP, making unlikely a role of the CO-

cGMP pathway in HO-modulated proliferation. By contrast, bilirubin (1µM) and the

antioxidant N-acetyl-cysteine (1mM), significantly reduced mitogen-induced cell

proliferation, ROS production and ERK1/2 phosphorylation. Furthermore, both bilirubin and

N-acetyl-cysteine, and the ERK1/2 inhibitor PD 98059 significantly reversed the effects of

HO inhibition on ASM proliferation. These results could be relevant to ASM alterations

observed in asthma since 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.

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Introduction

An increase in ASM1 mass is one of the features that characterise airway remodelling 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 or 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]. As ASM can be

continuously exposed to large amounts of exogenous or endogenous ROS produced by

inhaled agents, inflammatory or ASM cells themselves [5], redox signalling 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 [6,7,8]. But, if the

oxidant signalling involved in ASM proliferation is well characterised, 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].

1 Abbreviations used in the text are : ASM : airway smooth muscle; CO : carbon monoxide; CoPP : Cobalt-protoporphyrin; H2DCFH-DA : 2’-7’dichlorodihydrofluorescein diacetate; DMEM : Dulbecco’s modified Eagle’s medium; ERK 1/2 : extracellular signal-regulated kinases 1/2; FCS: fetal calf serum; HO : heme oxygenase; ODN : PDGF : platelet derived growth factor; PD 98059 : 2’-Amino-3’-methoxyflavone; PI : propidium iodide; ROS : reactive oxygen species; SM1 : smooth muscle myosin heavy chain isoform 1; SnPP IX : tin-protoporphyrin IX; SOD : superoxide dismutase.


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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

signalling in ASM. Furthermore, HO has shown an antiproliferative effect in vascular smooth

muscle in vitro and in vivo [17,18]. However, no data is 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 2 mitogens, PDGF or FCS

[6], and the mechanism(s) involved in this phenomenon, with special attention to the role of

ROS signalling and ERK1/2 activation. We also investigated which of 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 remodelling secondary to multiple

ovalbumin challenge in immunized guinea pigs.


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Experimental procedures

Reagents

[methyl-3H]- thymidine was purchased from NEN (Perkin-Elmer, Courtaboeuf, France) and

PDGF-AB was from R&D System (Abingdon, United-Kingdom). CoPP, hemin and SnPP IX

were from Porphyrin Products (London, England). Sense and antisense oligonucleotides were

supplied by Invitrogen (Cergy Pontoise, France), and transfected with the Superfect

Transfection Reagent (Qiagen SA, Courtaboeuf, France). H2DCFH-DA and PI were from

Molecular Probes (Eugene, OR). The selective MEK inhibitor PD 98059 was purchased from

Calbiochem (Merk 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 SM1 antibody was from Seikagaku America (Palmouth, MA) and anti α-actin antibody

was from Sigma (Saint Quentin Fallavier, France). 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 Life Technologies SARL (Cergy Pontoise, France). Tissue

culture plastic ware was supplied by Costar Corp. (Cambridge, MA). Reagents for Western

blot were from BioRad Laboratories (Richmond, CA). Reagents for immunohistochemistry

were from Dako (Carpinteria, CA). Other reagents were from Sigma Chemical Co. (Saint

Quentin Fallavier, France).

Human bronchial smooth muscle isolation and cell culture

Primary cultures of human bronchial smooth muscle were established as described [19,20].

Briefly, human bronchi (5 to 15 mm internal diameter) 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 mm square pieces.


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These muscular pieces, termed explants, were incubated in DMEM supplemented with 10%

heat inactivated FCS, antibiotics (streptomycin 10 mg/ml, penicillin G 10 000 UI/ml) and

amphotericin B (25µg/ml) in a humidified atmosphere of 5% CO2-95% air at 37°C, as

described [19]. On reaching confluence, cells were passed by lifting the cells with 0.05%

trypsin - 0.5mM EDTA. Cultures from passages 3 to 5 were used for experiments. At

confluence, cells exhibited the typical “hill and valley” aspect [20]. Cell characterization was

assessed by immunostaining using a monoclonal antibody against α-smooth muscle actin and

against the specific smooth muscle myosin heavy chain isoform 1 (SM1) [19, 22]. Greater

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 : cells were seeded at an initial density

104/cm2 , grown to 70% - 80% confluence and serum deprived (1% FCS) for 24 hours. Then,

they were stimulated with 2 different mitogens : either 10% FCS or 50 ng/ml PDGF-AB.

Control non-stimulated 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. Experiments were carried out in at least 3 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, 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 β-actin was evaluated using a monoclonal anti β-

actin antibody. Optical densities were measured with a Perfect Image 2.01 image analysis


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system (Iconix, Courtaboeuf, France). Results were expressed as the ratio of the expression of

HO-1 to that of β-actin.

For immunohistochemistry, 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 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 equivalent protein concentration and by

omitting the primary antibody.

[methyl-3H]-thymidine incorporation in ASM cells

Cells were cultured in 24-well plates and stimulated with mitogens for 24 h. For the final 18

hour incubation with the mitogen, 4µCi/ml of [methyl-3H]-thymidine was added to measure

DNA synthesis by scintillation counting. Results of individual treatments were obtained on

quadruplicate and were expressed as count per minute. Then the percent 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


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treated cells from the same 24 well plate. In experiments with the MEK inhibitor PD 98056,

the control condition was FCS+DMSO, explaining the lower proliferative response. However,

we checked in preliminary experiments, that DMSO 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 3 different methods : cell count and trypan

blue exclusion, LDH release in the medium, and incorporation of the fluorescent dye

propidium iodide (PI). For PI incorporation, cells seeded in 96-well plates were incubated

with 5 µM PI for 30 minutes and then washed in PBS before reading at 480-520 nm with a

multiwell fluorescence plate reader (Fluorostar BMG, Netherlands). Results are expressed in

fluorescent arbitrary units.

Measurement of ROS production by ASM cells

Intracellular ROS production : H2DCFH-DA oxidation.

Cells were cultured in 96-well plates as described previously. H2DCFH-DA (10µM final in

DMSO) was added one hour before stimulation. Immediately after addition of the mitogen,

fluorescence was measured every 5 minutes during a 45 minute period with a multiwell

fluorescence plate reader at 480-555 nm. Intracellular ROS (especially H2O2 or hydroxyl

radical) oxidize DCFH, yielding the fluorescent product DCF [24]. Results are expressed as

the ratio between fluorescence measured at 45 minutes 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, cells were

cultured in 6-well plates. Before the addition of the mitogen, the medium was replaced with


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HBSS without phenol red and incubated in 1 ml of the same buffer with and without 300

U/ml SOD. Subsequently, ferricytochrome c was added at a final concentration of 80µM to

the reaction buffer solution, followed by addition of the mitogens. After 1 h, the buffer was

removed and absorbance at 550 nm was measured immediately. Superoxide anion production

was calculated from the differences in the absorbances between samples with and without

SOD, using an extinction coefficient of 21.1 mM-1 cm–1 for reduced ferricytochrome c.

Measurement of ERK1/2 phosphorylation in ASM cells

Cells were cultured in 75 cm2 plates. In a first series of experiments, cells were stimulated for

different times with 50 ng/ml PDGF-AB or serum. Then the medium was removed, cells were

washed twice with cold PBS and removed in lysis buffer containing phosphatase inhibitors

(natrium fluoride 2.5 mM, β-glycerophosphate 1 mM, orthovanadate 1mM and para-

nitrophenol-phosphate 1mM). In a second series of experiments, cells were pre-treated with

different pharmacological agents before a 10 minutes-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 �-actin expression.

Immunohistochemichal 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 2 bronchi utilised 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 oligonucleotides (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’-


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CGCCTTCATGGTGCC-3’, whereas the sense sequence was 5’-GGCACCATGAAGGCG-

3’. ODNs were phosphorothioated on the first 3 bases on the 3’ end. Cells were transfected

using the Superfect Transfection Reagent (Qiagen SA, Courtaboeuf, France) following

manufactor’s instructions. Briefly, cells were seeded in 24-well plates at a density of 20 000

cells /well 24 hours before transfection. The proportions used were 1µg DNA/ 5 µl

transfection reagent/ well. Cells were incubated for 3 hours with the ODNs; then the medium

was replaced with fresh medium containing 10% serum. Experiments for proliferation and

western blot were performed 48 hours after transfection.

Induction of airway remodelling in ovalbumin -sensitized and aerosol- challenged guinea pig

Pathogen-free male Hartley guinea pigs (250-300 g body wt; Charles River, France) 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% wt/vol NaCl (saline) containing 100 mg ovalbumin (OVA), injected subcutaneously on

the neck, and another 0.5 ml intraperitoneally (i.p.) 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 minute

exposure to aerosolized OVA (0.1% OVA in 10 ml 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 OVA 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 2 groups. One group received the

inhibitor of HO activity SnPP IX, given i.p. at a dose of 50 µmol/kg on days 8, 10, 13 and 15,

6 hours before the challenge (OO-SnPP animals); a second group received received the HO-1


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inductor, hemin, given i.p. 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).

Animals were sacrificed 24 hours after the last challenge. They were anesthetized with

sodium pentobarbital (Nesdonal®, Specia-Rhone-Poulenc, Romainville, France), (50 mg/kg

of body weight i.p.), then the lung was inflated through a tracheal canula at 25 cm H2O with

10% formol and fixed in paraffine.

Evaluation of ASM area and HO-1 and bilirubin immunohistochemistry

Hematoxylin and eosin-stained tissues sections were examined microscopically and smooth

muscle area was measured with a microscope (Leitz, Germany) related to a camera (Olympus,

France) and a computerized image analysis system (AnalySIS, Soft Imaging System Münster,

Germany) as described by Palmans and coworkers [30]. 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 5 animals in each group and 3 to 4 different bronchi for each

animal.

HO-1 and bilirubin immunohistochemistry was performed as previously described [23].

Concentration of the anti-bilirubin antibody was 1µg/ml.

Statistical analysis

Values are given as mean ± S.E.M. Data were analysed by one way ANOVA; differences

between means were analysed with the Fisher's protected least-significant difference multiple

comparison test. Significance for all statistics was accepted at p<0.05.


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Results

HO-1 is expressed in human ASM

HO-1 was expressed in non stimulated ASM cells in culture (Figure 1, panel A). Incubation

with the mitogens FCS (10%) or PDGF (50 ng/ml) for 24 hours significantly and quite

similarly increased HO-1 expression and HO activity (p<0.05 as compared with 1% FCS;

Figure 1, panels A and B). CoPP (20 µM) could further potentiate HO activity induced by the

mitogens. As attempted, SnPP IX (25 µM) inhibited significantly HO activity (Figure 1,

panel B).

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

(Figure1, panel C). No expression was observed with a control isotype antibody. HO-1 was

also expressed in ASM in human bronchi (Figure 1, panel D).

The HO pathway and bilirubin modulate human ASM cells proliferation

Mean thymidine incorporation in non-stimulated 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 (Figure 2, panel 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 (Figure 2, panels

A and B, p<0.05 respectively). Hemin (10µM), another HO inductor, has a similar effect on

ASM proliferation (data not shown). On the opposite, the HO inhibitor SnPP IX induced a

523% and 690% increase in proliferation induced by FCS and by PDGF (Figure 2, panels 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


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with the HO-1 antisense effectively blocked HO-1 expression (Figure 3, panel A) and, like

pharmacological blockade, enhanced PDGF-induced proliferation (Figure 3, panel B).

No toxicity was observed with metalloporphyrins at the doses we used. Neither blue trypan

exclusion test nor LDH measurement or propidium iodide incorporation was modified (LDH

content between 6 ± 2 and 8 ± 4 UI/ml; propidium iodide incoporation between 32566 ± 1250

and 34559 ± 1452 arbitrary fluorescence units, NS, 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 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one

ODQ (10 µM, given 1 hour before mitogenic stimulation) nor the CO scavenger myoglobin

(5-20 µM for 24 hours) could reverse inhibition of proliferation induced by SnPP IX, making

unlikely a role of the CO-cGMP pathway in HO-modulated proliferation (Figure 4, panels A

and B respectively).

By contrast, bilirubin (1 µM, 1 hour before stimulation) was able to significantly reduce FCS-

and PDGF-induced proliferation (Figure 2, panel C and D, p<0.05 vers 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 (Figure 2, panels C and D). Considering the antioxidant properties of bilirubin, we

investigated if 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 hour

before stimulation) mimicked the effect of CoPP and bilirubin on proliferation and reversed

the effect observed after treatment with SnPP IX (Figure 2, panels C and D).

The HO pathway modulates both intra and extracellular ROS production

Considering the inhibitory effect of bilirubin and NAC on cellular proliferation, we


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investigated the role of the HO pathway in intra and extracellular ROS production by

measurement of the oxidation of H2DCFH-DA and the reduction of cytochrome c

respectively.

Both FCS and PDGF increased significantly intra and extracellular ROS production. Indeed,

FCS and PDGF increased H2-DCFH-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 1). 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 to 1% FCS respectively, Table

1). The HO inhibitor SnPP IX increased ROS production by 53 and 58% for H2-DCFH-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 to 1% FCS respectively, Table

1). 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 1).

The HO pathway modulates ERK 1/2 phosphorylation by a redox mechanism.

Considering that ROS modulate ERK 1/2 activation, and that these kinases are involved in

ASM cell proliferation, we investigated the role of the HO pathway in the modulation of ERK

1/2 phosphorylation.

ASM cells exhibited a basal level of phosphorylated ERK 1/2 (Figure 5, panel A). PDGF

induced a time-dependent increase in ERK 1/2 phosphorylation in HASM cells, that peaked

after 10 minutes of stimulation (Figure 5, panel A). Pre-incubation with CoPP decreased

PDGF-induced ERK 1/2 phosphorylation at 10 minutes, while SnPP increased it (Figure 4,

panel B). 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 ERK 1/2 activation. Similar results were observed with serum-stimulated cells (data


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not shown).

The MEK inhibitor PD98059 inhibited cell proliferation (Figure 5, panels D and E) and

blocked both FCS- and PDGF-induced ERK 1/2 phosphorylation at 10 minutes (Figure 5,

panel C), confirming involvement of the ERK 1/2 pathway in ASM cells growth. Moreover,

PD98059 inhibited the increase in cell proliferation induced by SnPP IX (Figure 5, panel E)

and reversed SnPP-enhanced ERK 1/2 phosphorylation, thus confirming that the effect of HO

on cell proliferation involves the MAP kinase 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 to ON

animals, along with expression in epithelium and inflammatory cells (Figure 6, panel A).

Bilirubin, one of the end step product of heme catabolism, was found in a similar manner by

immunohistochemistry in bronchial smooth muscle, confirming the in vivo activity of

muscular HO (Figure 6, panel A).

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 vs 119.32 ± 18.45 µm2, p<0.05 OO vs ON, Figure 6, panel B).

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 ± 11.12µm2 for hemin treated- and

158.02 ± 18.23µm2 in SnPP-treated OO animals, p<0.05 vs OO animals, Figure 6, panel B).

In ON animals, the modulation of bronchial smooth mucle area induced by hemin and SnPP

was slight but non significant (data not shown).


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Discussion

The main results of this study indicate that HO acts in an autocrine negative feedback manner

to limit ROS-dependent phosphorylation of the ERK 1/2 MAP kinase 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 signalling, 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

remodelling 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], then confirmed in vivo in animal models of

vascular remodeling after hypoxia or balloon-induced wall injury [32,33]. Indeed, to our

knowledge, all 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-dependant, since 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, while 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 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


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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 2 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 supports this interpretation. To finish

with, ASM transfection with an HO-1 antisense oligonucleotide induced a similar effect than

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, since 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, that is considered to be comparable to 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-


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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 since

application of different concentrations (5 to 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, neural and hepatoma cells

[36]. 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 and associates [38] shows that biliverdin and bilirubin can induce apoptosis in rat vascular

smooth muscle cells, as done by HO induction, while CO and iron failed to induce such

phenomenon. In this study, bilirubin required concentrations as high as 500 µM to induce

apoptosis, while 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 suggests 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, stressing therefore 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


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ROS scavenging properties [39], but also to the modulation of ROS production. Since 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 if bilirubin

modulates NAD(P)H oxidase in ASM and the mechanism(s) involved in this effect.

Involvement of the ERK 1/2 MAP kinase 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 [6,7,40,42-44]. 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 ERK 1/2

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-dependant

signaling pathway, 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 investigations.


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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

remodelling, 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 remodelling and hyperreactivity.


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Acknowledgements

We are in debt to Drs Roberta Foresti and Roberto Motterlini (Harrow, Middlesex, United

Kingdom.) and Pr Bruno Crestani (Paris, France) for their helpful and encouraging comments.


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Table 1. Cellular ROS production.

H2DCFHDA oxidation (T45/T0 ratio)

SOD-inhibitable

extracellular production of ROS

(nmol of superoxide anion/ 106 cells/hour)

1% FCS

1.09 ± 0.02

4.96 ± 0.82

10 % FCS 1.55 ± 0.05 * 10.01 ± 1.52 *

+ CoPP (20µM) 1.09 ± 0.09 # 3.03 ± 1.02 # + bilirubin (1µM) 1.08 ± 0.07 # 2.25 ± 0.65 # + NAC (1mM) 1.06 ± 0.02 # 4.30 ± 0.41 # + SnPP (25 µM) 2.37 ± 0.22 # 15.10 ± 1.10 # + CoPP (20µM)+ SnPP (25µM) 1.57 ± 0.42 & 9.51 ± 0.61 & + SnPP (25µM)+ Bilirubin (1µM) 1.30 ± 0.07 & 5.25 ± 1.35 & + SnPP (25µM)+ NAC (1mM) 1.01 ± 0.08 & 5.01 ± 0.55 & PDGF (50 ng/ml) 1.52 ± 0.04

* 12.10 ± 1.81

*

+ CoPP (20µM) 1.14 ± 0.02 # 6.25 ± 0.51 # + bilirubin (1µM) 1.13 ± 0.09 # 4.85 ± 0.41 # + NAC (1mM) 0.99 ± 0.19 # 5.81 ± 1.45 # + SnPP (25 µM) 2.41 ± 0.21 # 17.51 ± 0.72 # + CoPP (20µM)+ SnPP (25µM) 1.98 ± 0.15 & 9.65 ± 0.71 & + SnPP (25µM)+ Bilirubin (1µM) 1.23 ± 0.07 & 3.51 ± 0.51 & + SnPP (25µM)+ NAC (1mM)

1.17 ± 0.05 & 8.15 ± 2.61 &

Intra- and extracelular ROS production was measured by H2DCFHDA oxidation and SOD-

inhibitable reduction of cytochrome c respectivelly, as described in the Experimental

Procedures section. Results are expressed as mean ± SEM. n=6-12 for each condition. *:

P<0.05 vs 1% FCS ; # : P<0.05 vs 10% FCS or PDGF ; &: P<0.05 vs SnPP+10% FCS or

SnPP+PDGF


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Figure legends

Figure 1

HO-1 expression in human airway smooth muscle cells.

Panel A : Western blot analysis of HO-1 expression. Whole-cell proteins were extracted from

human ASM cells after a 24-hour serum deprivation followed by a stimulation with 10% FCS,

50 ng /ml PDGF or 20µM CoPP for 24 hours. The histogram represents the ratio between

HO-1 optical density and that of β-actin for the above typical experiment. Panel B : HO

activity was assessed by measuring bilirubin production by human ASM cells with a

spectrophotometer according to the method described in the “Experimental procedures”

section. n=4-6 for each condition. Bars are mean ± SE. * P<0.05 vs 1% FCS; # P<0.05 vs

mitogen alone. Panel C : immunohistochemical analysis of HO-1 and bilirubin expression in

ASM cells. Cells were cultured in a chamber slide, fixed with acetone and stained by

immunoreaction with monoclonal antibodies. No staining was observed with isotype

antibodies. Panel D : immunohistochemical analysis of HO-1 expression in airway smooth

muscle in human bronchi. Staining was observed in airway smooth muscle (*) and epithelium

(arrow). No staining was observed with the isotype antibody.

Figure 2

Role of the HO pathway on human ASM cells proliferation. Cells were stimulated for 24

hours with 10% FCS (panel A and C) or PDGF (panel B and D). 3H-thymidine incorporation

(4µCi/ml) was measured by scintillation counting. Each condition was done on quadruplicate.

Three different muscular explants were tested for each experiment. Values are mean ± SE.

*:P<0.05 vs 1% FCS, #: P<0.05 vs mitogen alone, &: P<0.05 vs mitogen+SnPP.


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Figure 3

Effect of cell transfection with HO sense and antisense oligonucleotides (ODNs).

Panel A : effect of HO-1 ODNs on PDGF-induced HO-1 expression. Experimental procedure

is the same than described in Figure 1.

Panel B : Thymidine incorporation in ODN transfected cells. Cells were stimulated for 24

hours with PDGF. 3H-thymidine incorporation (4µCi/ml) was measured by scintillation

counting. Each condition was done on quadruplicate. Values are mean ± SE. *:P<0.05 vs 1%

FCS, #: P<0.05 vs mitogen alone.

Figure 4

Role of the guanylyl cyclase-cGMP pathway.

Panel A : thymidine incorporation by human ASM cells stimulated for 24 hours with serum or

PDGF plus DMSO (that is used as the solvent for ODQ). Cells were treated with the HO

inductor CoPP (20µM) and by the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-

a)quinoxalin-1-one (ODQ, 10µM).

Panel B : role of myoglobin, a scavenger for CO, on cell proliferation. For panel A and B,

p<0.05 in all conditions. n=12 for each condition. Bars are mean ± SE. *:P<0.05 vs 1% FCS;

#: P<0.05 vs mitogen.

Figure 5

Role of HO on phosphorylation of the ERK1/2 MAPkinase.

Panel A : time course of phosphorylation of ERK1/2 MAPKinase induced by 50 ng/ml

PDGF, assessed by western blot. Panel B : typical western blot analysis of ERK1/2

phosphorylation after a 10 minute stimulation with 50 ng/ml PDGF-AB. Cells were pre

treated with the different reagents for one hour before stimulation. Results were similar with


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cells from three different muscular explants. Graph represents optical density of the bands of

one typical western blot, compared with expression of β-actin. Panel C : effects of the MEK

inhibitor PD 98059 on ERK phosphorylation induced by a 10 minutes stimulation with serum

and PDGF. The histogram represents the ratio between phosphorylated ERK1/2 optical

density and that of β-actin for a typical experiment.

Panels D and E : assessment of the role of PD 98059 on cell proliferation by incorporation of

3H-thymidine after a 24 hour stimulation with 10% serum (panel D) and PDGF (panel E).

Results are expressed as mean±SE of three different experiments with cells from three

different explants. *:P<0.05 vs 1%FCS+DMSO; #:P<0.05 vs mitogen + DMSO; &:P<0.05 vs

mitogen+DMSO+SnPP.

Figure 6

Role of the HO pathway on airway smooth muscle increase in allergic airway inflammation

Panel A : HO-1 and bilirubin were expressed in ASM (*) in OO guinea pigs. Expression was

also observed in inflammatory cells and epithelium (arrows). No staining was observed with

isotype antibodies.

Panel B : Airway smooth muscle area measurement. Animals were either sensitized with

ovalbumin and challenged with NaCl (ON animals) or sensitized and challenged with

ovalbumin (OO animals), chronically treated or not with the HO inductor hemin or the HO

inhibitor SnPP as described in “Experimental Procedures” section. Results represent the mean

± SE of 3 to 4 bronchi in 5 different animals in each group. *: P<0.05 vs ON; #: P<0.05 vs

OO.


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Figure 1

CoPP

A32k

1% FCS 10% FCS PDGF

0

0.5

1

1.5

2

2.5

HO

-1/β

act

in

(arb

itrar

y un

its)

PDGF10% FCS 1% FCS CoPP

PDGF (50 ng/ml)10% FCS

* *

#

&

#

&

0

100

200

300

400

500

600

HO

act

ivity

(p

mol

bili

rubi

n/m

g pr

otei

n/60

min

)

CoP

P (2

0µM

)

CoP

P (2

0µM

)

SnPP

(25

µM)

SnPP

(25

µM)

B by guest on February 24, 2018http://w

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Figure 1

D

C

HO-1

Bilirubin

IsotypeAntibody

HO-1

Isotype

*

* *

*


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Figure 2

1% FCS PDGF 50 ng/ml

CoP

P 20

µM

SnPP

25µ

M

CoP

P 20

µM

+ Sn

PP 2

5µM

B

*

#

#

&

3H-t

hym

idin

e in

corp

orat

ion

(per

cent

age

of c

ontr

ol)

0

200

400

600

800

1% FCS PDGF 50 ng/ml C

oPP

20µM

SnPP

25µ

M

CoP

P 20

µM

+ Sn

PP 2

5µM

3H-t

hym

idin

e in

corp

orat

ion

(per

cent

age

of c

ontr

ol)

0

200

400

600

300

500

100

*

#

#

&

A


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Figure 2

NA

C 1

mM

*# #

#

& &

0

100

200

300

400

500

SnPP 25µM 1 % FCS

10% FCS B

iliru

bin

1µM

NA

C 1

mM

Bili

rubi

n 1µ

M

C

1 % FCS

PDGF (50 ng/ml)

Bili

rubi

n 1µ

M

NA

C 1

mM

SnPP 25µM

Bili

rubi

n 1µ

M

NA

C 1

mM

D

*

# #

#

&&

3H-t

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idin

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corp

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ion

(per

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3H-t

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corp

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(per

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0

100

200

300

400

500

600


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Figure 3

HO-1

β-Actin

Positive control (spleen)

Non transfected

cells

Transfection reagent alone

HO-1 HO-1 antisense

A

0

100

200

300

400

500

600

700

1%FCS PDGF (50 ng/ml)

HO

-1 a

ntis

ense

HO

-1 se

nse

Tra

nsfe

ctio

n re

agen

t al

one

*#

3H-t

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corp

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(per

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age

of c

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B


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Figure 4

10% FCS PDGF (50 ng/ml)

CoP

P (2

0µM

)

OD

Q (1

0µM

)

C

oPP

(20µ

M)

+ O

DQ

(10µ

M)

CoP

P (2

0µM

)

OD

Q (1

0µM

)

C

oPP

(20µ

M)

+ O

DQ

(10µ

M)

A

1% FCS

*

*

# #

0

100

200

300

400

500

3H-t

hym

idin

e in

corp

orat

ion

(per

cent

age

of c

ontr

ol)

0

100

200

300

400

500

B

CoPP (20µM) Myoglobin

5 10 20µM

CoPP (20µM) Myoglobin

5 10 20µM

10% FCS PDGF (50 ng/ml) 1% FCS

**

# ## #

## #

#

3H-t

hym

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corp

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(per

cent

age

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Figure 5

PDGF 50 ng/ml

0 5 10 15 30 60 90 min

Aphospho ERK1/2

0

0.5

1

1.5

2 2.5

3

phos

pho

ER

K 1

/2 /β

act

in

Bilir

ubin

SnPP

+B

iliru

bin

PDGF

CoP

P

NA

C

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PDGF 1% FBS

10% FBS

SnPP

CoP

P

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C

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ubin

SnPP

+Bili

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n

PDGF

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0.5

1

1.5

2

Cphospho ERK1/2

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PDGF

PD98

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SnPP

+

PD98

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SnPP

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PDGF 10% FCS

PD98

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SnPP

+

PD98

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SnPP

PD98

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SnPP

+

PD98

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SnPP


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36

Figure 5

E

D

0

100

200

300

400

500

3H-t

hym

idin

e in

corp

orat

ion

(per

cent

age

of c

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PD 98 059

PDGF (50 ng/ml) + DMSO

SnPP (25µM)

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PD 98 059

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1%FCS +DMSO

# # #

#

&

& &

*

# #

#

#&

&

&

*

0

100

200

300

400

500

600

PD 98 059

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SnPP (25µM)

10 30 50µM

PD 98 059

10 30 50 µM

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3H-t

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idin

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37

Figure 6

A

B

Bilirubin

HO-1

OO ON Isotype

0

50

100

150

200

Mus

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(µm

2 )

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Michel Aubier and Jorge BoczkowskiMegret, Patrick Berger, Guy Leseche, Elie Fadel, Tokio Yamaguchi, Roger Marthan, Camille Taille, Abdelhamid Almolki, Moussa Benhamed, Christine Zedda, Jerome

bilirubin-dependent modulationof ERK1/2 phosphorylationHeme oxygenase inhibits human airway smooth muscle proliferation via a

published online April 10, 2003J. Biol. Chem.

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heme oxygenase inhibits human airway smooth muscle proliferation

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