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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 603-610, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4851

Carbon Monoxide Inhibits Human Airway Smooth Muscle Cell Proliferation via Mitogen-Activated Protein Kinase Pathway

Ruiping Song, Raja S. Mahidhara, Fang Liu, Wen Ning, Leo E. Otterbein and Augustine M. K. Choi

Division of Pulmonary, Allergy and Critical Care Medicine, and Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Address correspondence to: Augustine M. K. Choi, M.D., Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, MUH628, 3459 5th Avenue, Pittsburgh, PA 15213. E-mail: choiam{at}msx.upmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The gaseous molecule carbon monoxide (CO) is elevated in the breath of individuals with asthma. The physiologic function of CO in asthma is poorly understood. Here we demonstrate that CO (250 ppm) markedly inhibits human airway smooth muscle cell (HASMC) proliferation, arresting cells at the G0/G1 phase. This CO-induced cell growth arrest of HASMC was associated with upregulation of p21 and downregulation of cyclin D1 expression. It is generally believed that the signaling pathway by which CO affects biologic processes is primarily mediated via the guanylyl cyclase/3',5'-Guanylate cyclic monophosphate (cGMP) pathway. To examine whether guanylyl cyclase/cGMP was involved in CO-induced growth arrest of HASMC, Rp-8-Br-cGMP, a selective inhibitor of cGMP-dependent protein kinase and ODQ, a selective inhibitor of soluble guanylate cyclase, were administered to HASMC in the presence of CO. Interestingly, CO-induced cell growth arrest was not reversed by these inhibitors. We next examined whether the extracellular signal-regulated kinase (ERK) 1/ERK2 mitogen-activated protein kinase (MAPK) signaling pathway may regulate the antiproliferative effect of CO. We first showed time-dependent activation of the various MAPKs in HASMC in response to serum, including phosphorylated ERK1/ERK2, p38, and JNK and then demonstrated that CO exerted negligible effect on activated p38 and JNK; however, ERK activation was significantly attenuated in the presence of CO. These data suggest that CO can inhibit HASMC proliferation via the ERK1/ERK2 MAPK pathway, independent of a guanylyl cyclase/cGMP independent pathway. CO may act as an important mediator of remodeling of human airways in asthma via its ability to regulate cell growth of airway smooth muscle cells.

Abbreviations: carbon monoxide, CO • 3',5'-guanylate cyclic monophosphate, cGMP • extracellular signal-regulated kinase, ERK • fetal bovine serum, FBS • human airway smooth muscle cell, HASMC • heme oxygenases, HO • mitogen-activated protein kinase, MAPK • phosphate-buffered saline, PBS • vascular smooth-muscle cells, VSMC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Airway wall remodeling contributes significantly to airway hyperresponsiveness that characterizes asthma (1). The increase in airway smooth muscle volume is believed to be important in airway remodeling, and can also perpetuate airway inflammation in asthma (1). Plasma and inflammatory cell–derived mediators are also known to induce airway smooth muscle proliferation (1, 2), resulting in further increase in airway smooth muscle cell volume. Thus, an improved understanding in the mechanism(s) that regulate airway smooth muscle proliferation will help us better elucidate the biology and physiology of airway remodeling in asthma. To this end, human airway smooth muscle cells (HAMSC) have been used by many investigators as a suitable cell culture model to investigate the regulation of airway smooth muscle proliferation and the signal transduction pathways that mediate cell proliferation.

Heme oxygenases (HOs) are the rate-limiting enzymes in heme degradation, catalyzing the cleavage of the heme ring to form ferrous iron, carbon monoxide (CO) and biliverdin (3, 4). A catalytic byproduct of HO enzyme activity that is receiving increasing attention is CO. CO is a gaseous molecule with known toxicity and lethality to living organisms exposed to industrial doses. However, against this known paradigm of CO toxicity, there has been renewed interest in recent years in CO behaving as a signaling regulatory molecule in cellular and biologic processes (5–10). Mammalian cells have the ability to generate endogenous CO primarily through the catalysis of heme by HO, with a much smaller amount produced following peroxidation of lipids (3). Accumulating data by our laboratory and others suggest that CO exerts key physiologic function in neuronal and cardiovascular biology, and most recently involved in providing cytoprotection via its anti-apoptotic and anti-inflammatory effects (7–13).

Several groups have demonstrated that HO-1 expression is induced in preclinical models of asthma and in human asthma (14–16). Others have shown that CO levels in the breath of patients with asthma are elevated (3, 17). The physiologic function of HO-1 or CO in the pathogenesis of asthma, however, is unknown. To better understand the functional role of CO in human asthma, we have initiated studies to examine this important question. Our laboratory has recently demonstrated that CO can attenuate aeroallergen-induced airway inflammation in mice (13), implicating a potential important functional role of CO in human asthma. In the present study we tested the hypothesis that CO exerts antiproliferative effects on HASMC and that CO mediates this effect via the mitogen-activated protein kinase (MAPK) signaling pathway. We show that CO can inhibit HASMC proliferation via the extracellular signal-regulated kinase (ERK) 1/ERK2 MAPK pathway, independent of the guanylyl cyclase/3',5'-Guanylate cyclic monophosphate (cGMP) pathway. CO may act as an important mediator of remodeling of human airways in asthma via its ability to regulate proliferation of airway smooth muscle cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Treatment and Reagents
Primary HASMC (Clonetics, Walkersville, MD) were cultured in Smooth Muscle Cell Basal Medium (SmBM; Clonetics) containing 5% fetal bovine serum and insulin, hFGF, GA-1000, hEGF (Clonetics). The guanylate cyclase inhibitor IH (1, 2, 4) oxadiazolo (4,3-a) quinoxalin-1 (ODQ; Calbiochem-Novabiochem, San Diego, CA) (10–100 mM) was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO). The cGMP analog 8-bromo-cGMP sodium salt (8-Br-cGMP; Sigma-Aldrich) (10–100 mM) was dissolved in dH2O. SB203580 (20 µM), a selective inhibitor of p38 MAPK, and PD98059 and UO126, selective inhibitors of MEK1 and MEK1/2 (Calbiochem, Darmstadt, Germany), were dissolved in DMSO (5–20 mM). All reagents were added to the culture medium 1 h before the treatments.

CO Exposure
For cell culture experiments CO at a concentration of 1% (10,000 ppm) in compressed air was mixed with compressed air containing 5% CO2 in a stainless steel mixing cylinder before being delivered into the exposure chamber. Flow into the chamber was at a rate of 2 L/min. The chamber was humidified and maintained at 37°C. A CO analyzer (Interscan Corporation, Chatsworth, CA) was used to measure CO levels in the chamber and there were no fluctuations in the CO concentrations after the chamber had equilibrated.

cGMP Immunoassays
Cellular levels of cGMP were quantified using a commercially available immunoassay (Biomol, Plymouth Meeting, PA). HASMC were incubated in the presence or absence of CO (250 ppm) and cell lysates were analyzed for cGMP content, as suggested by the vendor.

Cell Counts and [3H]Thymidine Incorporation
Proliferation assays were performed as described previously (4). Briefly, cells were seeded at 5 x 103 cells/well and cultured overnight in Smooth Muscle Cell Basal Media with insulin, hFGF, GA-1000, hEGF, and 5% fetal bovine serum (FBS). Cells were serum-starved for 24 h (0% serum) before induction of cell proliferation (10% FBS; Hyclone, Logan, UT). Cells were counted daily using a Neubauer hemocytometer. Viability was assessed with trypan blue exclusion methods. For [3H]thymidine incorporation studies cells were serum-starved overnight and then stimulated with 20% serum containing 5 µCi/ml [3H]thymidine (New England Nuclear, Boston, MA) and measured by scintillation spectroscopy. Data are presented as the mean counts/min/well. Experiments were done in quadruplicate.

Cell Extracts and Western Blot Analysis
Cellular protein extracts were electrophoresed under denaturing conditions (10–12.5% polyacrylamide gels) and transferred onto nitrocellulose membranes (BioRad, Hercules, CA). Total and activated/phosphorylated forms of MAPK were detected using rabbit polyclonal antibodies directed against the total and phosphorylated forms of these MAPK, according to the manufacturer's suggestions (New England Biolabs Inc., Beverly, MA). Phosphorylated p38, JNK, and ERK were normalized to the total amount of ERK, detected in the same membrane. p21Cipl was detected using a rabbit anti-human polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Cyclin D1 was detected using a rabbit anti-human polyclonal antibody (Santa Cruz Biotechnology). ß-Actin was detected using anti-human ß-actin monoclonal antibody (Sigma Chemical Co., St. Louis, MO). Primary antibodies were detected using horseradish peroxidase conjugated donkey anti-rabbit IgG secondary antibodies (Pierce, Rockford, IL). Peroxidase was visualized using the Enhanced ChemiLuminescence assay (Amersham Life Science Inc., Arlington Heights, IL) according to manufacturer's instructions and stored in the form of photoradiograph (Biomax MS; Eastrnan Kodak, Rochester, NY). Digital images were obtained using an image scanner equipped with FotoLook and Photoshop software. When indicated, membranes were stripped (62.5 mM Tris.HCl, pH 6.8, 2% SDS, and 100 mM P-mercaptoethanol, 30 min, 50°C)

Cell Cycle Analysis by Flow Cytometry
After serum starvation of 48 h, HASMC are stimulated with 5% FBS for 24 and 48 h. Cells were harvested by trypsin digestion (0.025% Trypsin/0.01% EDTA; BioWhittaker Inc., Walkersville, MD), washed with PBS, and fixed at -20°C overnight with 70% ethanol. The next day cells were washed with PBS and then incubated at 37°C for 30 min with RNase (100 µg/ml; Sigma). After centrifugation, cells are resuspended in 2.0 ml of propidium iodide (100 µg/ml; Sigma) in PBS for at least 1 h. Fluorescent labeling was evaluated using a FACsort equipped with Cell Quest Software (Becton Dickinson, Palo Alto, CA). Cells (2 x 104) were collected at a sample flow rate of 10 µl/min. Experiments were performed in triplicate.

Statistical Analysis
Data are expressed as the mean ± SE. Differences in measured variables between experimental and control group were assessed using Student's t tests. Statistical calculations were performed on a Macintosh personal computer using the Statview II Statistical Package (Abacus Concepts, Berkeley, CA). Statistical difference was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CO Suppresses HASMC Proliferation
We conducted cell growth curve experiments to determine the effect of CO on HASMC proliferation. Figure 1A demonstrates the cell growth curve of HASMC over 6 d in the presence or absence of CO. Low concentration of CO (250 ppm) suppressed HASMC proliferation (Figure 1A). We then performed [3H]thymidine incorporation studies to further assess effects of CO on cell proliferation. We demonstrate that CO (250 ppm) markedly suppressed HASMC proliferation by 24 h (Figure 1B). We also demonstrate that CO can inhibit HASMC proliferation at concentrations ranging from 50–500 ppm. (Figure 1C). Inhibition of HASMC proliferation was not associated with cell death, as assessed by trypan blue and propidium iodide exclusion analyses (data not shown).



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  		Figure 1. CO inhibits HASMC proliferation. (A) Growth curve by cell counts. Growth of HASMC was analyzed by cell counts in the presence or absence of CO (250 ppm) for 6 d, as described in MATERIALS AND METHODS. Data represent the mean value ± SE of samples from three independent experiments (*P < 0.05 versus CO exposure). Circles, control; squares, CO. (B) [3H]Thymidine incorporation. Proliferation of HASMC was analyzed by [3H]thymidine incorporation assay (CPM, counts per minute) in the absence or presence of CO (250 ppm) for 24 h, as described in MATERIALS AND METHODS. Data represent the mean value ± SE of samples from three independent experiments (*P < 0.05 versus room air control). (C) Concentration response of CO on inhibition of HASMC proliferation. Proliferation of HASMC was analyzed by [3H]thymidine incorporation assay (CPM) in the absence or presence of different concentrations of CO, ranging from 10–250 ppm. Data represent the mean value ± SE of samples from three independent experiments (*P < 0.05 versus room air control).

 
CO Affects HASMC Cycle Progression at the G0/G1 Phase of the Cell Cycle
After synchronization of HASMC by serum starvation for 48 h, cells were stimulated to proliferate with 10% FBS. HASMC were stained with propidium iodide and cell cycle was analyzed at 24 and 48 h after stimulation by flow cytometry. In the absence of CO, there was an increase in the percentage of cells in S and G2/M phase. This increase was blocked by exposing cells to 250 ppm CO (Figure 2A). Quantitation of cells in each phase of the cell cycle showed that CO decreased the percentage of cells in S and G2/M phase by 71% (24% in control versus 7% in CO) at 24 h and 52% (25% in control versus 12% in CO) at 48 h. (Figure 2B). These data suggest that the effect of CO is associated with arrest of HASMC cycle progression at the G0/G1 phase of the cell cycle.



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  		Figure 2. Cell cycle analysis of CO-induced growth arrest. (A) CO-induced growth arrest phase at G0/G1. HASMC were starved for 48 h and stimulated with 10% serum in the presence and absence of CO (250 ppm). Cell cycle distribution of HASMC at 24 and 48 h after serum stimulation was analyzed by flow cytometry, as described in MATERIALS AND METHODS. G0/G1 represents resting/first growth phase, S represents synthesis phase, and G2/M represents second growth/mitosis phase. Data are representative of three independent experiments.(B) Quantitative analysis of cells at S-G2/M phase. Percentage of cells in S-G2/M phase obtained by flow cytometry analysis. Data represent the mean ± SE (n = 3). (*P < 0.05 versus room air control). Black, control; white, CO.

 
The Antiproliferative Effect of CO Is Associated with Increased Expression of the Cell Cycle Inhibitor p21CIP1 and Decreased Expression of Cyclin D1
To assess the role of CO on cell cycle protein expression, we serum starved HASMC before stimulation with serum in the presence and absence of CO. Exposure of HASMC to CO increased expression of p21Cip1, a potent inhibitor against cell cycle progression (Figure 3A). Interestingly, we also observed that cyclin D1, a key regulator of G1 progression, showed a time-dependent attenuation in HASMC exposed to 250 ppm of CO (Figure 3B).



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  		Figure 3. (A) p21Cip1 level increased in presence of 250ppm CO. HASMC were starved for 24 h, and stimulated with 5% serum in the presence and absence of CO (250 ppm). p21Cip1 protein expression at time 0, 15, 30 and 60 min was detected by Western blot, as described in MATERIALS AND METHODS. The same membranes were probed with an antibody against ß-actin to assure equal loading of the gel. Results shown are representative of 3 independent experiments. (B) Cyclin D1 decreased in the presence of 250 ppm CO. HASMC were exposed to CO (250 ppm), and cyclin D1 protein expression at time 0, 1, 8, 24 and 48 h was detected by Western blot, as described in MATERIALS AND METHODS. The same membranes were probed with an antibody against ß-actin to assure equal loading of the gel. Data are representative of three independent experiments.

 
The Antiproliferative Effect of CO Is Independent of the Activation of Guanylate Cyclase or the Generation of cGMP
It is well established that CO mediates a variety of cell functions, including cell proliferation in vascular smooth muscle cells via the guanylyl/cGMP pathway (3, 9, 10, 18, 19). To investigate whether CO mediates its antiproliferative effects via this pathway, we first exposed HASMC to CO and observed that CO increased the levels of intracellular cGMP in a time-dependent manner (Figure 4A). 1H(1,2,4) Oxadiazolo (4,3-a) Quinoxalin-1 (ODQ), a selective inhibitor of soluble guanylate cyclase, blocked the production of cGMP in the presence of CO (Figure 4B). Importantly, the ability of CO to suppress HASMC proliferation was not reversed by ODQ (Figure 4C) or under inhibition of guanylate cyclase activity by Rp-8-Br-cGMP, a selective inhibitor of cGMP-dependent protein kinase (Figure 4C). 8-Br-cGMP, a cell-permeable cGMP analog that activates cGMP-dependent protein kinase, also did not suppress the proliferation of HASMC (Figure 4D). Taken together, these observations suggest that the antiproliferative effect of CO is independent of the activation of guanylate cyclase or the generation of cGMP.



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  		Figure 4. The antiproliferative effect of CO is independent of the activation of guanylate cyclase or the generation of cGMP. (A) Increased cGMP in HASMC exposed to CO. HASMC were exposed to CO (250 ppm) for 8, 16, 24 h and cell extracts were analyzed for cGMP content, as described in MATERIALS AND METHODS. Data represent the mean value ± SE of samples from three independent experiments (*P < 0.05 versus room air control). Black, control; white, CO. (B) ODQ blocked cGMP in the presence of CO. ODQ (50 µM), a selective inhibitor of soluble guanylate, was administered to HASMC 1 h before exposure to CO (250 ppm). Cell extracts were analyzed for cGMP content 8 h after CO exposure, as described in MATERIALS AND METHODS. Data represent the mean value ± SE of samples from three independent experiments (*P < 0.05 versus room air control). (C) Rp-8-Br-CGMP and ODQ do not reverse the CO-induced growth arrest. HASMC were exposed to CO (250 ppm) in the presence or absence of ODQ (50 µM), a guanylate cyclase inhibitor, and Rp-8-Br-CGMP (30 µM), a selective inhibitor of cGMP-dependent protein kinase. Growth of HASMC was analyzed by 3H-thymidine incorporation assay, as described in MATERIALS AND METHODS. Data represent the mean value ± SE of samples from three independent experiments (*P < 0.05 versus room air control). Black, control; white, CO. (D) 8-Br-cGMP does not suppress the proliferation of HASMC. HASMC were exposed to CO (250 ppm) in the presence or absence of 8-Br-cGMP, a cell-permeable cGMP analog that activates cGMP-dependent protein kinase. Growth of HASMC was analyzed by 3H-tymidine corporation assay, as described in MATERIALS AND METHODS. Data represent the mean value ± SE of samples from three independent experiments (*P < 0.05 versus room air control).

 
CO Exerts its Antiproliferative Effect via the Inhibition of the Activation of ERK MAPK Pathway
Based on our observations that CO mediates its antiproliferative effects via a cGMP-independent pathway, we then sought out an alternative signaling pathway. We examined whether the MAPK pathway may be involved in the antiproliferative effects of CO. We first show time-dependent activation of the various MAPKs in HASMC in response to serum, including phosphorylated ERK1/ERK2, p38, and JNK (Figure 5). We then examined the activation of phosphorylated p38, JNK, and ERK in the absence or presence of CO. CO exerted a negligible effect on activated p38 and JNK; however, ERK activation was significantly attenuated in the presence of CO (250 ppm) (Figure 6). We also examined the effects of CO at different time points and different concentrations. We demonstrate that at both 5 and 15 min after serum stimulation, ERK activation was attenuated in the presence of CO (250 ppm) (Figure 7A). ERK activation was also attenuated by CO at a lower concentration (50 ppm) (Figure 7B). We also examined the effect of CO on non-starved cells, subconfluent HASMC growing in normal medium with 5% FBS. When cells were exposed to 250 ppm of CO for 30 min, 1 h, and 2 h, we observed that the basal level of phosphorylated ERK that is required for HASMC proliferation was markedly decreased (Figure 7C). These data indicate that the inhibition of ERK MAPK activation is critical in the suppression of HASMC proliferation by CO. Importantly, PD98059 (20 µM) and UO126 (20 µM), selective inhibitors of MEK1 and MEK1/2 (upstream mediators of ERK1/ERK2), decrease the proliferation of HASMC in response to serum, indicating that ERK1/2 MAPK activation is required for HASMC proliferation (Figure 8).



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  		Figure 5. Activation of MAPK by serum stimulation. HASMC were starved for 24 h, and stimulated with 10% serum. Phosphorylation of ERK1/ERK2, JNK and p38 were measured by Western blot as described in MATERIALS AND METHODS. Total ERK (ERK1/ERK2), JNK and p38 were used as normalization controls. Data shown is representative blot from three to four independent experiments.

 


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  		Figure 6. Selective inhibition of ERK by CO. HASMC were starved for 24 h, and stimulated with 10% serum in the presence and absence of CO (250 ppm). Phosphorylation of ERK1/ERK2, JNK and p38 were measured at 15 min by Western blot as described in MATERIALS AND METHODS. Total ERK (ERK1/ERK2), JNK and p38 were used as normalization controls. Data shown is a representative blot from three to four independent experiments.

 


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  		Figure 7. Time and concentration response of CO on ERK inhibition. (A) HASMC were starved for 24 h, and stimulated with 10% serum in the presence and absence of CO (250 ppm). Phosphorylation of ERK1/ERK2 was measured at 5 and 15 min after serum stimulation by Western blot as described in MATERIALS AND METHODS. Total ERK (ERK1/ERK2) was used as normalization controls. Data shown is representative blot from three to four independent experiments. (B) HASMC were starved for 24 h, and stimulated with 10% serum in the presence and absence of 50 ppm CO. Phosphorylation of ERK1/ERK2 was measured at 15 min by Western blot as described in MATERIALS AND METHODS. Total ERK (ERK1/ERK2) was used as normalization controls. Data shown is a representative blot from three to four independent experiments. (C) Subconfluent HASMC were exposed to CO (250 ppm), and phosphorylation of ERK1/ERK2 was measured at 0, 30 min, 1 h and 2 h by Western blot as described in MATERIALS AND METHODS. Membranes were subsequently probed with an antibody against total ERK to assure equal loading. Data shown is a representative blot from three to four independent experiments.

 


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  		Figure 8. Effects of inhibition of MEK on the proliferation of HASMC. SB203580 (20 µM), PD98059 (20 µM) and UO126 (20 µM) were administered to starved HASMC 1 h before serum stimulation. HASMC proliferation was analyzed by 3H-thymidine incorporation assay at 24 h, as described in MATERIALS AND METHODS. Results shown are the mean ± SE (n = 3). (*P < 0.05 versus control group)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HOs are the rate-limiting enzymes in heme degradation, catalyzing the cleavage of the heme ring to form ferrous iron, CO, and biliverdin (3–5). Expression of the inducible form of this enzyme, HO-1, has been shown to protect endothelial cells from undergoing apoptosis, inhibit monocyte-macrophage chemotaxis, drive monocyte-macrophage activation into an anti-inflammatory and anti-thrombotic phenotype, induce the expression of vasodilator molecules in SMC, and suppress platelet aggregation (20–23). These anti-inflammatory and anti-apoptotic effects of HO-1 can be reproduced by CO, one of the catalytic products of HO-1 on heme. For example, exogenous CO can prevent the deleterious effects associated with inflammatory reactions such as those that occur during the rejection of transplanted organs, hyperoxic lung injury, or ischemia reperfusion injury (11, 24–26), in a manner that mimics that of HO-1.

In view of these cytoprotective effects of HO-1 and CO in a variety of models of oxidative stress and tissue injury, it is interesting to note that recent data demonstrate increased HO-1 expression in preclinical models of asthma and in human asthma (14, 15), and increased CO in the breath of individuals with asthma (3, 17). Although the physiologic function of HO-1 or CO is poorly understood in either preclinical models of asthma or human asthma, we have recently reported that CO can markedly attenuate aeroallergen-induced airway inflammation in mice (13). CO markedly attenuated aeroallergen-induced BAL eosinophils, and selective proinflammatory mediators such as IL-5, PGE2, and LTB4 (13). In this report, we demonstrate another possible role of CO in human asthma; that is, CO could play an important role in the remodeling process in human asthma by its ability to modulate cell proliferation of airway smooth muscle cells. An important question regarding exogenous administration of CO, which challenges the scientific community, currently lies in the physiologic concentration of CO to be used in vivo. This question will require rigorous preclinical and clinical studies in the future; however, accumulating data document the relative safety of CO concentration similar or equivalent to ours (11–13, 24–26). The concentration of CO (50–500 ppm) used in our studies are much lower than the levels used in humans (3,000 ppm) during measurement of DLCO, a standard pulmonary function test, albeit our studies involved continuous CO exposure. Previous studies have demonstrated that animals exposed to CO (200–500 ppm) for 3–6 mo did not exhibit any significant alterations in physiologic or biochemical parameters (38, 39). Interestingly, exogenous administration of the gaseous molecule NO in human subjects involve concentrations in the range of 1–5 ppm, which represent ~ 1,000 fold higher concentration than the level of NO detected in the breath of human subjects (ppb range) (40–42). In contrast, the concentration used for our CO studies (e.g., 250 ppm) is ~ 50 fold higher than the level of CO detected in the breath of human subjects (40, 43).

In our present study of HASMC, we observed that continuous exposure to low concentration of CO (250 ppm) directly suppresses HASMC proliferation as assessed by cell growth curves and thymidine incorporation assays. CO-induced cell growth arrest was associated with upregulation of p21Cip1 and downregulation of cyclin D1. Most of the biologic effects attributed to CO have been linked to its ability to modulate the activity of guanylate cyclase and to increase the levels of cellular cGMP (3, 18, 19). Interestingly though, we show here that CO-induced antiproliferative effects in HASMC is not via the guanylate cyclase/cGMP pathway, but rather through the ERK1/ERK2 MAPK pathway. The MAPK pathway is commonly activated by growth factors and has been shown to play a crucial role in cell proliferation (27–29). MAPK, and ERK1/ERK2 in particular, are believed to play an obligatory role in the signaling cascade that enables cells to pass through the restriction point of the cell cycle, although the relative contributions of the other MAPKs remain unclear. Several studies support an important regulatory role for the ras/raf/MEK1/ERK pathway as an essential upstream signaling cascade, ultimately leading to cell cycle progression to S-phase and increase in cyclin D1 levels and persistent ERK activation is required for DNA synthesis (27).

Our current observation that CO mediates antiproliferative effects via the ERK MAPK pathway is interesting in that our laboratory has recently demonstrated the critical role of the MKK3/p38 MAPK pathway in mediating the anti-inflammatory and anti-apoptotic effects of CO (11, 12). For example, in endothelial cells the anti-apoptotic effect of CO is strictly dependent on the activation of p38 MAPK, independent of the guanylate cyclase/cGMP system (12). However, in fibroblasts the same anti-apoptotic effect is dependent on the activation of guanylate cyclase (30). Thus, it appears that CO signals anti-inflammatory and anti-apoptotic effects via the p38 MAPK pathway or GC pathway, whereas CO-induced antiproliferative effect is via the ERK MAPK pathway.

HO-1 has recently been shown to modulate cell proliferation in vascular smooth muscle cells (31). Infection of vascular smooth muscle cells (VSMC) with AdHO-1 inhibited serum-stimulated VSMC proliferation in a dose-dependent manner (31). Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, and the effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1 (31). To elucidate the mechanisms by which HO mediates these antiproliferative effects, our laboratory has examined the effect of CO on VSMC growth and found it to have a similar effect as HO-1, as growth arrest was induced and mediated via a cGMP and p38 MAPK pathway (unpublished data). Interestingly, we show that the antiproliferative effects of CO in HASMC is mediated by the ERK MAPK in contrast to the critical role of the p38 MAPK pathway in vascular smooth muscle cell proliferation. The critical role of the ERK MAPK pathway in mediating CO-induced cell growth arrest in HASMC supports the current paradigm that indeed the ERK pathway represents one major pathway which regulates HASMC proliferation (32–34).

Cyclin-dependent kinases (cdks) inhibitors are implicated in the regulation of cyclin-cdk activity such as p21Cip1, p16Ink4, and p27Kip1 (35). p21Cip1 is upregulated in arteries after vascular injury and overexpression of p21Cip1 in VSMCs results in G1 arrest and inhibition of cell growth (31). In this study, we observed that exposure of HASMC to CO also induced upregulation of p21Cip1 protein expression strongly by 1 h (Figure 3A), indicating that the antiproliferative effect of CO is dependent on the expression of the cell cycle inhibitor p21Cip1. p21Cip1 has an ability to inhibit cyclin D1 nuclear export and correlates with its ability to bind to Thr-286 phosphorylated cyclin D1 (35) and thereby prevent cyclin D1/CRM1 association. In this study, subconfluent HASMC growing in normal medium with 5% FBS were exposed to 250 ppm of CO for up to 48 h, we observed a time-dependent cyclin D1 reduction (Figure 3B). The critical role of p21Cip1 and cyclin D1 have been observed by others in HASMC in the regulation of cell proliferation (36, 37)

In summary, our data suggest that CO induces significant antiproliferative effects in HASMC, primarily via the ERK MAPK pathway, which currently is believed to be a major signaling pathway by which HASMC mediates cellular proliferation. The elucidation of the signaling pathways involved in the antiproliferative effects of CO is important not only for our basic understanding of the mechanism of action of CO, but also may be considered as novel targets for new therapeutic approaches to overcome airway remodeling, such as it occurs in asthma.


   Acknowledgments
 
The work by A.M.K.C. was supported by the NIH Grants HL55330, NIH HL60234, NIH AI-42365, and AHA EIA. L.E.O. was supported by AHA GIA.

Received in original form February 26, 2002

Received in final form June 7, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ammit, A. J., and R. A. Panettieri, Jr. 2001. The circle of life: cell cycle regulation in airway smooth muscle. J. Appl. Physiol. 3:1431–1437.
  2. Zelazny, D. S., E. T. Souhrada, and J. F. Souhrada. 1993. Interleukin-1ß stimulates the proliferation of cultured airway smooth muscle cells via platelet-derived growth factor. Am. J. Respir. Cell Mol. Biol. 9:645–651.[Medline]
  3. Otterbein, L. E., and A. M. K. Choi. 2000. Heme oxygenase: colors of defense against cellular stress. Am. J. Respir. Cell Mol. Biol. 279:L1029–L1037.
  4. Tenhunen, P., H. S. Marver, and R. Schmidt. 1970. The enzymatic catabolism of hemoglobin: stimulation of microsomal heme oxygenase by hemin. J. Lab. Clin. Med. 75:410–412.[Medline]
  5. Maines, M. D. 1997. The heme oxygenase system: a regulator of second messenger gases. Annu. Rev. Pharmacol. Toxicol. 37:517–554.[Medline]
  6. Soares, M. P., Y. Lin, J. Anrather, E. Csizmadia, K. Takigami, K. Sato, S. T. Grey, R. B. Colvin, A. M. K. Choi, K. D. Poss, and F. H. Bach. 1998. Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat. Med. 4:1073–1077.[Medline]
  7. Verma, A., D. J. Hirsch, C. E. Blatt, G. V. Ronnett, and S. H. Syneder. 1993. Carbon Monoxide: a putative neural messenger. Science 259:381–384.[Abstract/Free Full Text]
  8. Prabhakar, N. R., J. L. Dinerman, F. H. Agani, and S. H. Snyder. 1995. Carbon monoxide: a role in carotid body chemoreception. Proc. Natl. Acad. Sci. USA 92:1994–1997.[Abstract/Free Full Text]
  9. Ingi, T., J. Cheng, and G. V. Ronnett. 1996. Carbon monoxide an endogenous modulator of the nitric oxide-cyclic GMP signaling system. Neuron 4:835–842.
  10. Morita, T., M. A. Perrella, M. Lee, and S. Kourembanas. 1995. Smooth muscle cell-derived carbon monoxide. J. Clin. Invest. 96:2676–2682.[Medline]
  11. Otterbein, L. E., F. H. Bach, J. Alam, M. Soares, H. Tao Lu, M. Wysk, R. J. Davis, R. Flavell, and A. M. K. Choi. 2000. Carbon monoxide has anti-inflammatory effects involving the Mitogen activated protein kinase pathway. Nat. Med. 6:422–428.[Medline]
  12. Brouard, S., L. E. Otterbein, J. Anrather, E. Tobiasch, F. H. Bach, A. M. K. Choi, and M. P. Soares. 2000. Carbon monoxide generated by heme oxygenase-1 suppresses endothelial cell apoptosis. J. Exp. Med. 192:1015–1026.[Abstract/Free Full Text]
  13. Chapman, J. T., L. E. Otterbein, J. A. Elias, and A. M. K. Choi. 2001. Exogenous carbon monoxide attenuates aeroallergen- induced inflammation in mice. Am. J. Physiol. 281:L209–L216.
  14. Kitada, O., T. Kodama, K. Kuribayashi, D. Ihaku, M. Fujita, T. Matsuyama, and M. Sugita. 2001. Heme oxygenase-1 (HO-1) protein induction in a mouse model of asthma. Clin. Exp. Allergy 9:1470–1477.
  15. Donnelly, L. E., and P. J. Barnes. 2001. Expression of heme oxygenase in human airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 3:295–303.
  16. Lim, S., D. Groneberg, A. Fischer, T. Oates, G. Caramori, W. Mattos, I. Adcock, P. J. Barnes, and K. F. Chung. 2000. Expression of heme oxygenase isoenzymes 1 and 2 in normal and asthmatic airways: effect of inhaled corticosteroids. Am. J. Respir. Crit. Care Med. 5:1912–1918.
  17. Horvath, I., L. E. Donnelly, A. Kiss, P. Paredi, S. A. Kharitonov, and P. J. Barnes. 1998. Raised levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax 8:668–672.
  18. Morita, T., and S. Kourembanas. 1995. Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. J. Clin. Invest. 6:2676–2682.
  19. Morita, T., S. A. Mitsialis, H. Koike, Y. Liu, and S. Kourembanas. 1997. Carbon monoxide controls the proliferation of hypoxic vascular smooth muscle cells. J. Biol. Chem. 52:32804–32809.
  20. Hancock, W. W., R. Buelow, M. H. Sayegh, and L. A. Turka. 1998. Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes. Nat. Med. 4:1392–1396.[Medline]
  21. Lee, P. J., J. Alam, G. W. Wiegand, and A. M. K. Choi. 1996. Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia. Proc. Natl. Acad. Sci. USA 93:10393–10398.[Abstract/Free Full Text]
  22. Otterbein, L. E., J. K. Kolls, L. L. Mantell, J. L. Cook, J. Alam, and A. M. K. Choi. 1999. Exogenous administration of of heme oxygenas-1 by gene transfer provides protection against hyperoxia-induced lung injury. J. Chin. Invest 103:1047–1054.
  23. Abraham, N. G., Y. Lavrovsky, M. L. Schwartzman, R. A. Stoltz, R. D. Levere, M. E. Gerritsen, S. Shibarhara, and A. Kappas. 1995. Transfection of the human heme oxygenase gene into rabbit coronay microvessel endothelial cells: protective effects against heme and hemoglobin toxicity. Proc. Natl. Acad. Sci. USA 92:6798–6802.[Abstract/Free Full Text]
  24. Otterbein, L. E., L.L. Mantell, and A. M. K. Choi. 1999. Carbon monoxide provides protection against hyperoxic lung injury. Am. J. Physiol. 276: L688–L694.[Medline]
  25. Sato, K., J. Balla, L. Otterbein, R. N. Smith, S. Brouard, Y. Lin, E. Csizmadia, J. Sevigny, S. C. Robson, G. Vercellotti, A. M. Choi, F. H. Bach, and M. P. Soares. 2001. Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J. Immunol. 166:4185–4194.[Abstract/Free Full Text]
  26. Fujita, T., T. Koichi, A. Karimova, S. F. Yan, Y. Naka, S. F. Yet, and D. J. Pinsky. 2001. Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat. Med. 7:598–604.[Medline]
  27. Robinson, M. J., and M. H. Cobb. 1997. Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9:180–186.[Medline]
  28. Schaeffer, H. J., and M. J. Weber. 1999. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol. Cell. Biol. 19:2435–2443.[Free Full Text]
  29. Dhanasekaran, N., and E. P. Reddy. 1998. Signaling by dual specificity kinases. Oncogene 17:1447–1455.[Medline]
  30. Petrache, I., L. E. Otterbein, J. Alam, G. W. Wiegand, and A. M. K. Choi. 2000. Heme Oxygenase-1 inhibits TNF-{alpha}-induced apoptosis in cultured fibroblasts. Am. J. Physiol. 278:L312–L319.
  31. Duckers, H. J., M. Boehm, A. L. True, S. F. Yet, H. San, J. L. Park, R. Webb, M. E. Lee, G. J. Nabel, and E. G. Nabel. 2001. Heme oxygenase-1 protects against vascular constriction and proliferation. Nat. Med. 7:693–698.[Medline]
  32. Lee, J. H., P. R. Johnson, M. Roth, N. H. Hunt, and J. L. Black. 2001. ERK activation and mitogenesis in human airway smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 5:L1019–L1029.
  33. Orsini, M. J., V. P. Krymskaya, A. J. Eszterhas, J. L. Benovic, and R. A. Panettieri, Jr. and R. B. Penn. 1999. MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation. Am. J. Physiol. Lung Cell. Mol. Physiol. 277:L479–L488.[Abstract/Free Full Text]
  34. Karpova, A. K., M. K. Abe, J. Li, P. T. Liu, J. M. Rhee, W. L. Kuo, and M. B. Hershenson. 1997. MEKI is required for PDGF-induced ERK activation and DNA synthesis in tracheal monocytes. Am. J. Physiol. Lung Cell. Mol. Physiol. 272:L558–L565.[Abstract/Free Full Text]
  35. Sherr, C. J. 1994. G1 phase progression: cycling on cue. Cell 79:551–555.[Medline]
  36. Ravenhall, C., E. Guida, T. Harris, V. Koutsoubos, and A. Stewart. 2000. The importance of ERK activity in the regulation of cyclin D1 levels and DNA synthesis in human cultured airway smooth muscle. Br. J. Pharmacol. 131:17–28.[Medline]
  37. Page, K. J., and M. B. Hershenson. 1999. Platelet-derived growth factor stimulation of mitogen-activated protein kinases and cyclin D1 promoter activity in cultured airway smooth-muscle cells: role of Ras. Am. J. Respir. Crit. Care Med. 20:1294–1302.
  38. Jones, R. A., J. A. Strickland, J. A. Stunkard, and J. Siegel. 1971. Effects on experimental animals of long-term inhalation exposeure to carbon monoxide. Toxicol. Appl. Pharmacol. 19:46–53.[Medline]
  39. Stupfel, M., and G. Bouley. 1970. Physiological and biochemical effects on rats and mice exposed to small concentrations of carbon monoxide for long periods. Ann. N.Y. Acad. Sci. 174:342–368.[Medline]
  40. Biernacki, W. A., S. A. Kharitonov, and P. J. Barnes. 2001. Exhaled carbon monoxide in patients with lower respiratory tract infection. Respir. Med. 95:1003–1005.[Medline]
  41. Taylor, D. A., J. L. McGrath, L. M. Orr, P. J. Barnes, and B. J. O'Connor. 1998. Effect of endogenous nitric oxide inhibition on airway responsiveness to histamine and adenosine-5'-monophosphate in asthma. Thorax 53:483–489.[Abstract/Free Full Text]
  42. Katayama, Y., T. W. Higenbottam, G. Cremona, S. Akamine, E. A. Demoncheaux, A. P. Smith, and T. E. Siddons. 1998. Mnimizing the inhaled dose of NO with breath-by-breath delivery of spikes of concentrated gas. Circulation 98:2429–2432.[Abstract/Free Full Text]
  43. Zayasu, K., K. Sekizawa, S. Okinaga, M. Yamaya, T. Ohrui, and H. Sasaki. 1997. Increased carbon monoxide in exhaled air of asthmatic patients. Am. J. Respir. Crit. Care Med. 156:1140–1143.[Abstract/Free Full Text]



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