Hypoxia induces differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts

Megan Short,1 Raphel A. Nemenoff,2 W. Michael Zawada,3 Kurt R. Stenmark,1 and Mita Das1

1Department of Pediatrics, Developmental Lung Biology Research Laboratories, 2Renal Division, and 3Department of Medicine, Division of Clinical Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Submitted 29 April 2003 ; accepted in final form 13 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of the {alpha}-smooth muscle actin ({alpha}-SMA) gene during the conversion of fibroblasts into myofibroblasts is an essential feature of various fibrotic conditions. Microvascular compromise and thus local environmental hypoxia are important components of the fibrotic response. The present study was thus undertaken to test the hypothesis that hypoxia can induce transdifferentiation of vascular fibroblasts into myofibroblasts and also to evaluate potential signaling mechanisms governing this process. We found that hypoxia significantly upregulates {alpha}-SMA protein levels in bovine pulmonary artery adventitial fibroblasts. Increased {alpha}-SMA expression is controlled at the transcriptional level because the {alpha}-SMA gene promoter activity, assayed via a luciferase reporter, was markedly increased in transfected fibroblasts exposed to hypoxia. Hypoxic induction of the {alpha}-SMA gene was mimicked by overexpression of constitutively active G{alpha}i2 ({alpha}i2Q205L) but not G{alpha}16 ({alpha}-16Q212L). Blockade of hypoxia-induced {alpha}-SMA expression with pertussis toxin, a G{alpha}i antagonist, confirmed a role for G{alpha}i in the hypoxia-induced transdifferentiation process. c-Jun NH2-terminal kinase (JNK) inhibitor II and SB202190, but not U0126, also attenuated {alpha}-SMA expression in hypoxic fibroblasts, suggesting the importance of JNK in the differentiation process. Hypoxia-induced increase in bromodeoxyuridine incorporation, which occurred concomitantly with hypoxia-induced differentiation, was blocked by U0126, suggesting that DNA synthesis and {alpha}-SMA expression take place through simultaneously activated parallel signaling pathways. Neutralizing antibody against transforming growth factor-{beta}1 blocked only 30% of the hypoxia-induced {alpha}-SMA promoter activity. Taken together, our results suggest that hypoxia induces differentiation of vascular fibroblasts into myofibroblasts by upregulating the expression of {alpha}-SMA, and this increase in {alpha}-SMA level occurs through G{alpha}i- and JNK-dependent signaling pathways.

{alpha}-smooth muscle actin promoter activity; G{alpha}i; Gq; extracellular regulated kinase; c-Jun NH2-terminal kinase; bromodeoxyuridine incorporation; transforming growth factor-{beta}1


ACTIVATION OF FIBROBLASTS with various stimuli, e.g., transforming growth factor (TGF)-{beta}, thrombin, IL-4, and IL-13, induces the expression of {alpha}-smooth muscle actin ({alpha}-SMA), the development of a cytoplasmically enriched microfilamentous system, and other features shared by smooth muscle cells (3, 17, 19, 20). These hybrid nonmuscle-smooth muscle cells are known as myofibroblasts and are identified as key participants in the tissue remodeling that occurs during wound healing and various fibrotic disorders (9, 33, 43, 48). Differentiation of fibroblasts into myofibroblasts has also been speculated to be an essential step in the pathophysiology of a variety of vascular diseases (36). For instance, in experimental models of balloon catheter-induced injury and hypoxia-induced pulmonary vascular remodeling, adventitial fibroblasts have been shown to convert into myofibroblasts in the vascular wall (27, 36, 38, 39). These myofibroblasts are believed to contribute to many of the functional abnormalities reported in remodeled vessels (36). The factors that regulate the expression and fate of myofibroblasts are unknown, but alterations in the expression of a variety of growth factors and cytokines, e.g., TGF-{beta} and thrombin, during the inflammatory and postinflammatory states are likely contributors (9, 42). Little attention, however, has been paid to environmental factors such as hypoxia, which might induce fibroblast differentiation even though hypoxia has been shown to activate signaling pathways associated with cellular differentiation processes (47). Regional hypoxia is a critical component of various fibrotic diseases and thus might play a role in the fibroblast transdifferentiation process (15, 25, 26, 36). The signaling pathways operating to induce {alpha}-SMA expression during transdifferentiation of fibroblasts into myofibroblasts under hypoxic conditions have not yet been established.

One potential candidate for mediating a hypoxia-induced phenotypic switch of fibroblasts into myofibroblasts is the family of trimeric G proteins. G proteins are signal transducers that couple a large number of membrane-bound receptors to a variety of intracellular effector systems. Muscle-specific gene expression in vascular smooth muscle cells is regulated through the Gq protein family (30). In embryonic heart, the transdifferentiation of epithelial cells into a mesenchymal phenotype, which is characterized by the expression of {alpha}-SMA, is dependent on G proteins (4). Activation of G proteins has been speculated to be a critical early event in hypoxia-induced responses in many cell types (10, 14, 23, 31). Recently, we have demonstrated that pertussis toxin-sensitive G proteins play an important role in the hypoxia-induced proliferative responses of vascular fibroblasts (5). However, whether G proteins play a critical role in hypoxia-induced transdifferentiation of vascular adventitial fibroblasts into myofibroblasts is unknown.

The mitogen-activated protein (MAP) kinases, downstream effectors of G proteins, have been shown to regulate {alpha}-SMA expression in different cell types (12, 19, 20, 30, 45). Three major subfamilies of mammalian MAP kinases have been molecularly characterized to date: extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase (8). Regulation of the {alpha}-SMA promoter in vascular smooth muscle cells is dependent on both JNK and p38 MAP kinase (30). In contrast, a recent report (20) demonstrated that IL-4 and IL-13 induce {alpha}-SMA expression through JNK but not p38 or ERK pathways in human lung fibroblasts. Therefore, the role of the MAP kinase signaling pathways in regulating {alpha}-SMA gene expression can be cell, stimulus, and species specific. Hypoxia has been shown to stimulate the activation of all three MAP kinases in a distinct manner in different cell types (8). In vascular adventitial fibroblasts, hypoxia activates ERK and JNK through G{alpha}i/o-dependent pathways and also p38 MAP kinase via, as yet, unidentified signaling events (5). However, the role of MAP kinases in hypoxia-induced differentiation of vascular adventitial fibroblasts into myofibroblasts is not known.

The goal of the present study was to determine whether hypoxia could induce differentiation of vascular adventitial fibroblasts (as assessed by increase in {alpha}-SMA expression) and, if so, to examine the possible signaling mechanisms responsible for this process. Our approach was first to establish that exposure of cultured fibroblasts derived from pulmonary artery adventitia to hypoxia resulted in increased {alpha}-SMA expression. Different molecular strategies and pharmacological inhibitors were then used to identify the pathways involved in hypoxia-induced differentiation of fibroblasts. We show that hypoxia regulates activation of the {alpha}-SMA gene at the transcriptional level in vascular fibroblasts. We also demonstrate that, in response to chronic hypoxia, G{alpha}i and JNK are at least two of the signaling mediators involved in the conversion of vascular adventitial fibroblasts into myofibroblasts.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Monoclonal antibody against {alpha}-SMA and Eagle's MEM were purchased from Sigma Chemical (St. Louis, MO). Goat antimouse IgG, conjugated with Cy3, was obtained from Jackson Immuno Research Laboratories (West Grove, PA). Biotinylated anti-mouse IgG, anti-mouse IgG linked to Alexa 488, streptavidin Alexa 594 conjugate, and propidium iodide were purchased from Molecular Probes (Eugene, OR). Mounting media with and without 4',6-diamino-2-phenylindole dihydrochloride (DAPI) were obtained from Vector Laboratories (Burlingame, CA). Fetal bovine serum (FBS) was from Gemini Bio-Products (Woodland, CA). Different inhibitors were purchased from the following companies: JNK inhibitor II and SB-202190 from Calbiochem (La Jolla, CA), PD-98059 from Cell Signaling (Beverly, MA), U0126 from Alexis Biochemicals (San Diego, CA), and pertussis toxin from List Biologicals Laboratories (Campbell, CA).

Isolation and growth of pulmonary artery adventitial fibroblasts from neonatal bovine calves. Adventitia from the main pulmonary artery was harvested from 15-day-old neonatal calves. Tissue was collected, carefully dissected free of blood vessels and fat under a dissecting microscope, and then cut into small pieces. Fibroblasts from the tissue were isolated and characterized according to previously described methods (7). Cells were maintained in MEM, pH 7.4, supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and incubated in a humidified atmosphere with 5% CO2 at 37°C. Medium was changed twice per week, and cells were harvested with trypsin (0.2 g/l) containing EDTA (0.5 g/l). Passages ranging from 3 to 11 were used for all experiments. The growth characteristics and the appearance of the cells, examined under light microscopy, did not change for the passages studied (up to passage 11).

Immunofluorescent staining for {alpha}-SMA in isolated pulmonary artery adventitial fibroblasts. Cells were plated in the medium containing 10% FBS at a density of ~10,000 cells/well in eight-well glass chamber slides (Life Sciences, Denver, CO). After overnight attachment was allowed, fibroblasts were growth arrested with medium containing 0.1% FBS for 72 h. Quiescent cells were treated with either 21 or 1% O2 for 24 or 72 h. For hypoxic experiments, cells were placed in sealed humidified gas chambers as previously described (6). The chambers were repurged with fresh gas every 24 h. After treatment, cells were fixed in methanol, blocked with 100% FBS, and incubated with an anti-{alpha}-SMA antibody (1:200). Immunofluorescent staining of {alpha}-SMA was completed by incubating the cells with a rabbit anti-mouse IgG-conjugated with Cy3 (1:300). Cellular nuclei were stained with Hoechst dye (1:1,000). Slides were mounted with H1000 mounting medium. All steps were done at room temperature. Zeiss epifluorescence microscope was used to visualize the stained cells, and photographs were taken by using a Zeiss digital camera and Axiovision 2.05 software (Munchen-Hallbergmoos, Germany).

Double immunofluorescent staining for bromodeoxyuridine and {alpha}-SMA in vascular fibroblasts. Cells were plated in eight-well glass chamber slides and growth-arrested according to the aforementioned method. Bromodeoxyuridine (BrdU; 10 µM) was added to the wells, and cells were exposed to normoxia (21% O2) and hypoxia (1% O2) for 24 h. At the end of the treatment, cells were fixed with 4% paraformaldyhyde, blocked with goat serum, treated with 2 N HCl and then incubated with anti-BrdU antibody (Becton Dickinson, San Jose, CA). BrdU staining was completed with Alexa 488-conjugated goat anti-mouse IgG. Then immunoflourescent staining of {alpha}-SMA was performed according to the aforementioned method. Hoechst dye was used to stain the nuclei, and the slides were mounted with mounting media.

To evaluate the role of ERK1/2 in hypoxia-induced proliferation and differentiation responses of vascular fibroblasts, quiescent cells were preincubated with U0126 (10 µM)for1hat37°C. Then the cells were exposed to normoxia or hypoxia in the presence or absence of BrdU for 24 h. Cells were fixed, double-stained for BrdU and {alpha}-SMA, and visualized by using the Zeiss fluorescence microscope as described above. BrdU-positive cells were counted to determine the proliferative index of the fibroblasts.

Evaluation of {alpha}-SMA expression in vascular fibroblasts by using immunoblotting. Pulmonary artery adventitial fibroblasts were plated at a density of 50,000/35-mm Petri dish in medium containing 10% FBS and then growth arrested for 72 h. Quiescent cells were exposed to hypoxia (1% O2) for 24 or 72 h. After treatment, cells were harvested with lysis buffer (Cell Signaling), freeze-thawed to disrupt the membrane, and centrifuged at 10,000 g. Lysates were collected and protein concentrations were estimated by using the Bradford method (Bio-Rad, Hercules, CA). Cellular proteins were separated by electrophoresis, transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotechnologies, Piscataway, NJ), and probed with an antibody against {alpha}-SMA (1:1,000). Antibody against vimentin (1:1,000) was used as an internal loading standard. Immunoreactivity of the proteins was detected by using chemiluminescent reagents (Amersham Pharmacia Biotech). Images of Western blots were scanned and visualized by using a vista scan software package. Densitometric quantitation of the bands was performed by using National Institutes of Health Image 1.58 program. The area under the curve of each scanned band was determined. This value represents the band intensity in arbitrary units. The value at 0 h time was considered as 100%, and fold increases in response to hypoxic exposure were calculated with respect to 0 h.

The levels of {alpha}-SMA protein were also evaluated by Western blot analysis in cells treated with pertussis toxin (100 ng/ml) and JNK inhibitor II (10 µM). Fibroblasts were plated and growth arrested as previously described. Quiescent cells were preincubated with pertussis toxin and JNK inhibitor II for 1 h and exposed to normoxia or hypoxia for 48 h. The samples were analyzed for {alpha}-SMA expression according to the aforementioned protocol.

Measurement of {alpha}-SMA promoter activity in pulmonary artery adventitial fibroblasts. Cells were plated in medium containing 10% FBS at a density of 50,000/well in six-well plates. The next day, fibroblasts were growth arrested with medium containing 0.1% FBS for 72 h. Transient transfections of the cells were carried out with Optimem medium (Invitrogen, Carlsbad, CA) by using 2 µg/ml of each: {alpha}-SMA-luciferase, CMV-{beta}-galactosidase (to monitor transfection efficiency), and when necessary either a dominant negative ERK1 or constitutively active constructs of Gq ({alpha}-16Q212L) or Gi ({alpha}i2-Q205L) in the presence of lipofectin for 5–6 h at 37°C. A region coding for 713 bases of 5' sequence of the rat {alpha}-SMA promoter was isolated by polymerase chain reaction and ligated into a promoterless luciferase vector (41). We used this rat {alpha}-SMA promoter to transfect bovine cells because a high degree of evolutionary conservation is exhibited in the {alpha}-SMA transcription regulatory region since a 5'-specific probe containing 206 base pairs of the mouse {alpha}-SMA regulatory region cross hybridizes with genomic DNA from human, bovine, porcine, and chicken tissues (28). Constitutively active Gq ({alpha}-16Q212L) was designed by mutation of glutamine 212 to leucine in the conserved guanosine triphosphate-guanosine diphosphate binding domain of {alpha}16, similar to {alpha}q, which inhibits its intrinsic GTPase activity (35). Also, GTPase-deficient mutant of G{alpha}i2 ({alpha}i2Q205L) was prepared by mutating glutamine 205 to leucine (24). The cells were allowed to recover from transfection overnight and then were either treated with inhibitors followed by hypoxic exposure or, if no chemical treatment was necessary, were immediately exposed to hypoxia for 48 h. All samples were run with the appropriate vector controls or chemical agent controls.

Transiently transfected cells were also preincubated with TGF-{beta}1 neutralizing antibody (5 µg/ml) for 1 h at 37°C and exposed to normoxia (21% O2) or hypoxia (1% O2). Next day, another dose of TGF-{beta}1-neutralizing antibody (5 µg/ml) was re-added to the cells being exposed to normoxia or hypoxia. After a total of 48 h of hypoxic exposure, cells were harvested for luciferase and {beta}-gal assays.

Cells from each experiment were lysed in a reporter lysis buffer (Promega, Madison, WI). The luciferase assay was performed by using a Promega kit according to the manufacturer's recommendations. {beta}-Galactosidase assay was run according to the previously described method (21). {alpha}-SMA promoter activity of fibroblasts was expressed as luciferase units per {beta}-galactosidase units.

Evaluation of activation of ERK in the presence of U0126 in hypoxic fibroblasts. Quiescent fibroblasts were preincubated with U0126 (antagonist of mitogen-activated protein kinase kinase 1/2, upstream kinases of ERK1/2) at a concentration of 10 µM for an hour and then were exposed to hypoxia for 10 min. Control cells were treated with dimethyl sulfoxide, the vehicle of U0126, for 1 h and also subjected to 10 min of hypoxic exposure. Cells were harvested with cell lysis buffer, and an equal amount of protein from each condition was immunoblotted with antibody against phospho-ERK1/2 (1:1,000, Cell Signaling Technology). Alkaline phosphatase-linked anti-rabbit IgG (1:2,000) and lumi-phos (Pierce, Rockford, IL) were used for the detection of the immunoreactivity of the proteins.

Analysis of hypoxia-induced activation of c-Jun and ATF2 in pulmonary artery adventitial fibroblasts. Cells were plated at a density of 500,000/100-mm petri dish and were growth arrested in medium containing 0.1% FBS for 72 h. Quiescent cells were preincubated with SB-202190 (10 µM) and JNK inhibitor II (10 µM)for 1 h at 37°C and then exposed to hypoxia for 30 min. Cell lysates were prepared and separated by Western blot analysis. Membranes were immunolabeled against phospho-c-Jun and phospho-activating transcription factor (ATF2) (1:1,000; Cell Signaling Technology). Immunoreactivity was detected by using alkaline phosphatase-linked anti-rabbit IgG (1:2,000) and lumi-phos.

Data analysis. All data are expressed as arithmetic means ± SE; n equals the number of replicate wells per test condition in representative experiments. One- and two-way analyses of variance followed by the Student-Newman-Keuls multiple-comparisons tests within and between groups of data points were utilized. Data are considered significantly different if P <= 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia induces a redistribution of and an increase in {alpha}-SMA protein in cultured adventitial fibroblasts. We performed indirect immunofluorescent staining for {alpha}-SMA to determine whether hypoxia increases {alpha}-SMA protein levels in isolated vascular adventitial fibroblasts. In quiescent control cells, {alpha}-SMA expression was detectable in a disorganized cytoplasmic manner (Fig. 1A). Hypoxic exposure increased the intensity of fluorescent staining for {alpha}-SMA in cells cultured for both 24 and 72 h (Fig. 1A). {alpha}-SMA was redistributed along newly apparent cytoskeletal stress fibers within 24 h of hypoxic exposure. Cells exposed to hypoxia for 72 h exhibited a highly elongated morphology compared with the fibroblasts maintained under normoxic conditions. At this time, {alpha}-SMA was organized along very distinctly defined stress fibers. The nuclear membrane of the hypoxic cells (both at 24 and 72 h of hypoxia) stained much brighter for {alpha}-SMA than that of the corresponding normoxic cells.



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Fig. 1. {alpha}-Smooth muscle actin ({alpha}-SMA) level is increased in vascular adventitial fibroblasts in response to hypoxia. A: hypoxia induces redistribution of {alpha}-SMA along stress fibers in fibroblasts. Cells were plated at the density of 10 x 103 per well per 0.4 ml of 10% FBS/MEM in 8-well glass chamber slides (Lab-Tek), which were allowed to attach overnight and then were growth arrested with 0.1% FBS/MEM for 72 h. Quiescent cells were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 and 72 h and fixed with cold methanol. Indirect immunofluorescent staining was performed with an anti-{alpha}-SMA antibody. Nuclei were stained with Hoechst dye. Magnification: x40. B: {alpha}-SMA protein level is upregulated in quiescent pulmonary artery adventitial fibroblasts exposed to hypoxia. Quiescent fibroblasts were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24–72 h and harvested with lysis buffer. Western blot analysis of the cell lysates was performed by using an anti-{alpha}-SMA antibody. An immunoblot representative of 3 independent experiments is shown. C: quantitative data of {alpha}-SMA level in normoxic and hypoxic fibroblasts. *P < 0.05 compared with normoxic values.

 

The effects of hypoxia on {alpha}-SMA protein levels in vascular fibroblasts were also evaluated by immunoblotting. {alpha}-SMA was detectable in cultured fibroblasts under quiescent normoxic conditions, as has been reported previously (9). Hypoxic exposure upregulated {alpha}-SMA protein levels in a time-dependent manner (Fig. 1B). Figure 1C shows quantitatively that chronic hypoxic exposure (72 h) significantly upregulates (3-fold) the level of {alpha}-SMA protein in fibroblasts. Taken together, these results suggest that hypoxia induces the phenotypic conversion of fibroblasts into myofibroblasts in vitro by redistributing {alpha}-SMA into directionally defined stress fibers and concurrently raising the amount of detectable {alpha}-SMA.

Hypoxia increases {alpha}-SMA promoter activity in cultured adventitial fibroblasts. To examine whether the hypoxia-induced upregulation of {alpha}-SMA is controlled transcriptionally in vascular fibroblasts, {alpha}-SMA promoter activity was evaluated under hypoxic conditions. Quiescent bovine pulmonary artery adventitial fibroblasts were transiently transfected with a rat {alpha}-SMA promoter construct and then exposed to normoxia or hypoxia for 48 h. In cells cultured under the normoxic condition, low levels of promoter function, as evaluated by luciferase activity, were detectable and were consistent with the low levels of {alpha}-SMA protein observed in quiescent fibroblasts shown in Fig. 1. The promoter activity was increased by 26-fold in fibroblasts exposed to hypoxia (Fig. 2). These data suggest that hypoxia-induced upregulation of {alpha}-SMA expression in vascular fibroblasts is controlled at the transcriptional level.



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Fig. 2. Activity of rat {alpha}-SMA promoter is increased in bovine pulmonary artery adventitial fibroblasts exposed to hypoxia. Fibroblasts were plated at the density of 50 x 103 cells per well per 3.0 ml of 10% FBS/MEM in 6-well plates. The next day, cells were growth arrested with 0.1% FBS/MEM for 72 h. Quiescent fibroblasts were transiently transfected with 2 µg of rat {alpha}-SMA promoter tagged with luciferase, 2 µg of pCMV {beta}gal by using lipofectin, and exposed to normoxia (21% O2) or hypoxia (1% O2) for 48 h. At the end of the treatment, cells were harvested in a reporter lysis buffer, and luciferase and {beta}gal activities were assayed. n = 6 replicate wells. *P < 0.01 compared with normoxic values. Results were reproduced with at least 2 independent cell populations.

 

Hypoxia-induced increase in {alpha}-SMA promoter activity in vascular fibroblasts is dependent on G{alpha}i but not on Gq proteins. To examine the early signaling events mediating hypoxia-induced expression of {alpha}-SMA in adventitial fibroblasts, we used a variety of genetic and pharmacological strategies targeting the {alpha}-subunit of heterotrimeric G proteins. Quiescent fibroblasts were co-transfected with constitutively active constructs encoding for G{alpha}i2 ({alpha}-16Q212L) or G{alpha}16 ({alpha}i2-Q205L) proteins and {alpha}-SMA-luciferase promoter as a transcriptional reporter. Expression of constitutively active G{alpha}i protein in vascular fibroblasts mimicked the effects of hypoxia on the induction of the {alpha}-SMA gene by increasing the promoter activity by 10-fold (Fig. 3A). In contrast, constitutively active G{alpha}16 protein did not affect the level of {alpha}-SMA promoter activity in the vascular fibroblasts (Fig. 3B).



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Fig. 3. Constitutively active G{alpha}i, but not Gq protein, mimics hypoxia-induced activation of the {alpha}-SMA gene in vascular fibroblasts. A: {alpha}-SMA promoter activity is increased in fibroblasts in the presence of {alpha}i2Q205L, a constitutively active G{alpha}i. Quiescent cells were cotransfected with plasmids (2 µg each) containing {alpha}-SMA promoter, pCMV{beta}-gal, and {alpha}i2Q205L with lipofectin for 6 h. After 48 h, transfected cells were processed for luciferase and {beta}-gal assays. n = 6 replicate wells. *P < 0.05 compared with control value. B: {alpha}16Q212L (constitutively active Gq protein) does not affect the promoter activity of the {alpha}-SMA gene in fibroblasts. n = 6 replicate wells. Similar results were obtained from 3 independent experiments using fibroblasts from 2 different animals.

 

To further evaluate the role of G{alpha}i protein in the hypoxia-induced fibroblast transdifferentiation process, transiently transfected cells were treated with pertussis toxin, an antagonist of G{alpha}i. Fibroblasts were then exposed to normoxia or hypoxia for 48 h. The hypoxia-induced increase in {alpha}-SMA promoter activity was attenuated in the pertussis toxin-treated cells (Fig. 4A). To confirm that pertussis toxin-sensitive G proteins are critical upstream mediators of hypoxia-induced {alpha}-SMA expression, we evaluated {alpha}-SMA protein expression in the presence and absence of the toxin. The increase in {alpha}-SMA protein levels induced by hypoxia was inhibited by pertussis toxin (Fig. 4B). These data suggest that hypoxia-induced activation of the {alpha}-SMA gene in pulmonary artery adventitial fibroblasts is mediated through activation of G{alpha}i protein.



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Fig. 4. Hypoxia-induced activation of {alpha}-SMA gene in fibroblasts is abolished by pertussis toxin, an antagonist of G{alpha}i. A: pertussis toxin blocks the hypoxia-induced increase in {alpha}-SMA promoter activity. Growth-arrested fibroblasts were transiently transfected with {alpha}-SMA promoter tagged with luciferase and pCMV{beta}-gal. The next day, cells were treated with pertussis toxin (100 ng/ml) for 1 h at 37°C, exposed to normoxia or hypoxia for 48 h, and then harvested for luciferase and {beta}-gal assays. n = 6 replicate wells. *P < 0.001 compared with normoxic control values. **P < 0.001 compared with hypoxic control values. B: increased level of {alpha}-SMA protein in hypoxic fibroblasts is also attenuated by pertussis toxin. Quiescent fibroblasts were exposed to normoxic or hypoxic conditions in the presence or absence of pertussis toxin (100 ng/ml) for 48 h. {alpha}-SMA protein levels in cell extracts were determined by Western blot analysis. Representative data from 1 of the 3 independent experiments are shown.

 

Hypoxia-induced {alpha}-SMA gene upregulation requires JNK. Previously, our laboratory demonstrated that hypoxia stimulates the activation of ERK, JNK, and p38 MAP kinase in pulmonary artery adventitial fibroblasts (5). To determine the role of these MAP kinases in hypoxia-induced increases in {alpha}-SMA expression in fibroblasts, genetic and pharmacological inhibitor strategies were used. U0126, an inhibitor of mitogen-activated protein kinase kinase 1/2, blocked the hypoxia-induced activation of ERK1/2 (Fig. 5A) but had no effect on the enhanced activity of {alpha}-SMA promoter in the hypoxic fibroblasts (Fig. 5B). Similar results were obtained when the cells were treated either with PD-98059, another antagonist of the ERK pathway, or cotransfected with a dominant negative construct of ERK1 (data not shown).



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Fig. 5. Inhibitor of mitogen-activated protein kinase kinase (MEK) 1 and MEK2, U0126, has no effect on the activation of the {alpha}-SMA gene in hypoxic fibroblasts. A: U0126 blocks the activation of ERK1/2 in hypoxic fibroblasts. Quiescent fibroblasts were preincubated with U0126 (10 µM) for 1 h at 37°C, exposed to hypoxia for 10 min, and then harvested for Western blot analysis. Cell lysates were immunoblotted for phospho-ERK1/2. Representative immunoblot of 3 independent experiments is shown. B: hypoxia-induced increase in {alpha}-SMA promoter activity is not inhibited by U0126. Fibroblasts were plated and transiently transfected with {alpha}-SMA promoter and pCMV{beta}-gal according to the above-mentioned method. Transfected cells were then treated with U0126 (10 µM) for 1 h at 37°C and exposed to normoxia or hypoxia for 48 h. Luciferase and {beta}-gal activities were assayed in the cell lysates. n = 6 replicate wells. *P < 0.001 compared with normoxic values. Similar results were obtained in 2 other experiments.

 

JNK inhibitor II is a new highly selective inhibitor of JNK signaling (2). The specificity of the blockade of hypoxia-induced JNK activation by this inhibitor was evaluated by examining the attenuation of increased phosphorylation of its substrate c-Jun in response to hypoxia. The JNK inhibitor II effectively blocked the hypoxia-induced c-Jun phosphorylation (data not shown) and also inhibited the increase in {alpha}-SMA promoter activity in hypoxic fibroblasts (Fig. 6A). To confirm the role of JNK in the hypoxia-induced activation of the {alpha}-SMA gene, the protein levels of {alpha}-SMA in the presence and absence of JNK inhibitor II were evaluated. Hypoxia-induced upregulation of {alpha}-SMA protein was blocked by JNK inhibitor II (Fig. 6B).



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Fig. 6. c-Jun NH2-terminal kinase (JNK)-specific inhibitor, JNK inhibitor II, blocks the upregulation of {alpha}-SMA expression in hypoxic fibroblasts. A: hypoxia-induced increase in {alpha}-SMA promoter activity is attenuated by JNK inhibitor II. Quiescent fibroblasts were transfected with plasmids containing {alpha}-SMA promoter and {beta}-gal, treated with JNK inhibitor II (10 µM) and then exposed to normoxia or hypoxia for 48 h. Luciferase and {beta}-gal assays were performed in the cell lysates. n = 6 replicate wells. *P < 0.001 compared with vehicle-treated normoxic values. **P < 0.001 compared with vehicle-treated hypoxic values. B: JNK inhibitor II also blocks the increase in {alpha}-SMA protein level in hypoxic fibroblasts. Lysates were prepared from the cells exposed to normoxia or hypoxia in the absence or presence of JNK inhibitor II (10 µM). {alpha}-SMA expression was evaluated by Western blot analysis. Representative data from 1 of the 3 independent experiments are shown.

 

To examine the potential role of yet another MAP kinase, p38 MAP kinase in the hypoxia-induced activation of the {alpha}-SMA gene, we utilized a p38 MAP kinase inhibitor, SB-202190. The inhibitor (10 µM) significantly attenuated the {alpha}-SMA promoter activity observed under hypoxic conditions (Fig. 7A). At this concentration, SB-202190 blocked hypoxia-induced activation (ascertained by phospho-immunoblots) of both c-Jun, a substrate of the JNK pathway, and ATF2, a downstream target of p38 and JNK MAP kinases (Fig. 7, B and C). Others have also shown that SB-202190 and a related imidazole inhibitor of p38, SB-203580, when applied in micromolar concentrations, in addition to blocking the activity of p38 MAP kinase also reduces the activity of JNK (Ref. 46 and our unpublished data). The attenuation of hypoxia-induced phosphorylation of c-Jun and ATF2 was specific, because the compound at this concentration (10 µM) did not affect the hypoxia-induced phosphorylation of c-Myc, a substrate of ERK pathway (data not shown). Taken together, the experiments utilizing JNK inhibitor II and SB-202190 allow us to conclude that hypoxia-induced activation of JNK plays an important role in the conversion of fibroblasts into cells with a myofibroblast phenotype.



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Fig. 7. SB-202190 inhibits the hypoxia-induced activation of the {alpha}-SMA gene in fibroblasts. A: enhanced {alpha}-SMA promoter activity in hypoxic fibroblasts is diminished in the presence of 10 µM SB-202190. Transiently transfected fibroblasts were treated with SB-202190 and then exposed to normoxia or hypoxia for 48 h. At the end of the treatment, cells were harvested and lysates were used for luciferase and {beta}-gal assays. n = 6 replicate wells. *P < 0.01 compared with vehicle-treated normoxic values. **P < 0.01 compared with vehicle-treated hypoxic values. B: SB-202190 blocks hypoxia-induced activation of both JNK and p38 mitogen-activated protein (MAP) kinase pathways. Quiescent fibroblasts were treated with SB-202190 (10 µM) and then exposed to hypoxia for 30 min. Cellular lysates were prepared and immunoblotted for phospho-c-Jun, downstream target of JNK pathway and phospho-ATF2, substrate of p38 MAP kinase, and JNK. Similar results were obtained in 2 independent experiments.

 

In vascular adventitial fibroblasts, hypoxia induces increase in BrdU incorporation and {alpha}-SMA expression simultaneously, but independently. To examine whether hypoxia-induced differentiation of vascular fibroblasts occurs concomitantly with and is dependent on proliferation, BrdU incorporation and {alpha}-SMA expression were assessed by double immunofluorescent staining in normoxic and hypoxia-exposed fibroblasts. Consistent with our previous reports (5), we found that hypoxic exposure significantly increased BrdU incorporation in adventitial fibroblasts (Fig. 8A). BrdU-positive fibroblasts exhibited high levels of {alpha}-SMA expressed along stress fibers (Fig. 8B). To determine whether increase in BrdU incorporation was necessary for the hypoxia-induced increase in {alpha}-SMA expression, cells were treated with U0126, antagonist of ERK pathway previously shown to block hypoxia-induced fibroblast proliferation (5). We found that U0126 blocked the hypoxia-induced increase in BrdU-labeling index (normoxic control: 10%; hypoxia: 24%; hypoxia + U0126: 6%) but had no effect on the {alpha}-SMA expression (Fig. 8, A and B). These data suggest that hypoxia initiates both growth and differentiation promoting signals simultaneously in vascular fibroblasts, but the responses are mediated through different parallel pathways.



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Fig. 8. Hypoxia induces increase in DNA synthesis and {alpha}-SMA expression in vascular adventitial fibroblasts simultaneously but through different signaling mechanisms. Top: bromodeoxyuridine (BrdU) incorporation and {alpha}-SMA expression are increased in hypoxic cells. BrdU (10 µM) was added to the quiescent fibroblasts, which then were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 h. At the end of the treatment, cells were fixed with 4% paraformaldehyde and stained for BrdU (green color) and {alpha}-SMA (red color). Bottom: U0126, antagonist of ERK pathway, blocked only the BrdU incorporation but not the {alpha}-SMA expression. Fibroblasts were preincubated with U0126 (10 µM) for 1 h at 37°C and then exposed to normoxia and hypoxia in the presence of BrdU for 24 h. Cells were fixed and stained for BrdU (green color) and {alpha}-SMA (red color). n = 4 Replicate wells. Representative data from 1 of the 3 independent experiments are shown.

 

Hypoxia-induced activation of {alpha}-SMA gene in vascular adventitial fibroblasts is partially inhibited by TGF-{beta}1 neutralizing antibody. To evaluate whether the increase in {alpha}-SMA promoter activity in hypoxic fibroblasts is dependent on TGF-{beta}1, a cytokine known to be capable of inducing the fibroblast differentiation process, a neutralizing antibody against TGF-{beta}1 was used. Fibroblasts were transiently transfected with the {alpha}-SMA promoter, incubated with a neutralizing antibody against TGF-{beta}1 and then exposed to hypoxia. Neutralizing antibody against TGF-{beta}1 inhibited the {alpha}-SMA promoter activity by only 30% in hypoxia-exposed fibroblasts (Fig. 9A). To confirm the effectiveness of the neutralizing antibody concentration used, transiently transfected cells were incubated with the neutralizing antibody and then treated with TGF-{beta}1. The TGF-{beta}1-induced increase in promoter activity was completely abolished by the neutralizing antibody (Fig. 9B). These results suggest that the hypoxia-induced increase in {alpha}-SMA promoter activity in vascular adventitial fibroblasts might be in part mediated through a TGF-{beta}1-independent pathway.



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Fig. 9. Hypoxia-induced activation of {alpha}-SMA gene in fibroblasts might be partly mediated through TGF-{beta}1-independent pathway. A: neutralizing antibody against TGF-{beta}1 partially inhibits {alpha}-SMA promoter activity (30%) in hypoxic cells. Quiescent fibroblasts were transfected with {alpha}-SMA promoter and pCMV{beta}gal. The transfected cells were preincubated with TGF-{beta}1 neutralizing antibody (total 10 µg/ml) for 1 h at 37°C, exposed to normoxia and hypoxia for 48 h, and harvested for luciferase and {beta}-gal assay. n = 3 replicate wells. *P < 0.001 compared with normoxic value. **P < 0.002 compared with hypoxic data. B: TGF-{beta}1-induced increase in {alpha}-SMA promoter activity is completely blocked by TGF-{beta}1 neutralizing antibody. Transfected fibroblasts were treated with the neutralizing antibody against TGF-{beta}1 (total 10 µg/ml) and then stimulated with TGF-{beta}1 (3 ng/ml) for 48 h. At the end of the treatment, cells were harvested and processed for luciferase and {beta}-gal assays. n =3 replicate wells. *P < 0.001 compared with control value. **P < 0.002 compared with TGF-{beta}1-stimulated results. Representative data of 2 independent experiments are shown.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we examined the hypothesis that hypoxia induces transdifferentiation of fibroblasts into myofibroblasts and that this response is mediated through G protein-coupled MAP kinase signaling pathways. The results demonstrate that 1) hypoxia stimulates an increase in the expression of {alpha}-SMA in vascular adventitial fibroblasts, 2) hypoxia-induced activation of the {alpha}-SMA gene occurs at the transcriptional level, 3) G{alpha}i proteins are critical upstream regulators of hypoxia-induced {alpha}-SMA expression, 4) JNK is an important signaling intermediate in the hypoxia-induced upregulation of {alpha}-SMA expression, 5) hypoxia-induced {alpha}-SMA expression is not dependent on DNA synthesis, and 6) a major portion of the hypoxic upregulation of the {alpha}-SMA gene in adventitial fibroblasts might be mediated through a TGF-{beta}1-independent pathway. These data provide strong support to the idea that hypoxia itself is capable of inducing the transdifferentiation of vascular fibroblasts into myofibroblasts.

Induction of {alpha}-SMA expression in fibroblasts has been demonstrated in response to many factors including TGF-{beta}, thrombin, IFN-{gamma}, IL-4, and IL-13 (3, 9, 19, 20). However, the role of hypoxia in {alpha}-SMA gene activation in vascular fibroblasts has never been tested even though hypoxia, as a result of microvascular compromise, may play an important role in the pathogenesis of fibrosis (15, 25, 26, 36). Numerous signaling pathways have been shown to be activated by hypoxic conditions, but direct evidence linking specific pathways to transcriptional activation of particular hypoxia-induced genes is sparse and subject to considerable cell type- and gene-dependent differences (34). Hypoxia alters gene expression via a variety of mechanisms: 1) by increasing gene transcription via binding of inducible factors to hypoxia responsive elements on 5'- or 3'-regulatory sequences, 2) by increasing mRNA stability (37), or 3) by indirectly inducing autocrine/paracrine mediators. Our results suggest that hypoxia-induced regulation of the {alpha}-SMA gene in vascular fibroblasts occurs at the transcriptional level. One potential mediator of this transcriptional regulation is TGF-{beta}, which has been shown to be induced by hypoxia and to upregulate {alpha}-SMA expression at the transcriptional level in fibroblasts (13, 17). Our results utilizing neutralizing antibody against TGF-{beta}1, however, suggest that hypoxia-induced activation of {alpha}-SMA gene is mainly independent of TGF-{beta}1. This observation is consistent with a report demonstrating that thrombin-induced expression of {alpha}-SMA in human lung fibroblasts is independent of TGF-{beta} (3). Indeed, we cannot completely rule out the role of TGF-{beta} in hypoxia-induced {alpha}-SMA expression in adventitial fibroblasts because the neutralizing antibody against TGF-{beta}1 can bind only the extracellular TGF-{beta}1 but does not affect the intracellular TGF-{beta}1. Future work should focus on more specific evaluation of the factors responsible for transcriptional upregulation of {alpha}-SMA expression in hypoxic fibroblasts.

Cellular G proteins are activated by environmental stresses such as shear forces, mechanical stretch, and reactive oxygen species (31, 40). Hypoxia-induced activation of G proteins has been implicated as an early event in the modification of ion channel activity and cell depolarization (10, 29). We have reported that G{alpha}i/o proteins are necessary for hypoxia-induced proliferation of vascular fibroblasts (5). In the present study, we demonstrate that a constitutively active Gi protein, but not Gq protein, upregulates {alpha}-SMA gene expression in a manner similar to hypoxia. G proteins are also important in thrombin-induced expression of {alpha}-SMA in lung fibroblasts and activated endothelial cells in the developing heart (3, 4). Our study demonstrates for the first time the importance of G{alpha}i protein in the hypoxia-induced differentiation process of vascular fibroblasts. Activation of the Gq protein does not appear to regulate {alpha}-SMA gene in the vascular adventitial fibroblasts. This observation is in contrast with the role of Gq proteins in the regulation of {alpha}-SMA expression in rat vascular smooth muscle cells (21). Beyond species differences, it is possible that fibroblasts and smooth muscle cells use distinctly different pathways for the regulation of the {alpha}-SMA gene.

We used different antagonistic strategies to elucidate the functions of intracellular signaling intermediates ERK, JNK, and p38 MAP kinase in regulating {alpha}-SMA expression in hypoxic fibroblasts. Previously, we demonstrated that hypoxia activates ERK and JNK through G{alpha}i/o-dependent pathways and p38 MAP kinase via a G{alpha}i/o-independent mechanism in vascular fibroblasts (5). All three MAP kinases are involved in hypoxia-induced proliferative responses of fibroblasts through a complex signaling network. In the present study, we report that JNK inhibitor II and SB-202190 attenuate the hypoxia-induced increase in the {alpha}-SMA promoter activity in fibroblasts. However, SB-202190 inhibits the hypoxic activation of c-Jun (JNK substrate) as well as ATF2 (downstream target of p38 or JNK), suggesting the blockade of both JNK and p38 MAP kinase pathways by this compound. Therefore, JNK is the key intracellular signaling mediator for the {alpha}-SMA gene activation in the hypoxic fibroblasts. TGF-{beta}1-stimulated phenotypic modulation of lung fibroblasts into myofibroblasts has also been demonstrated to be mediated through JNK (19, 20).

Accumulating evidence indicates that JNK is implicated in various, often opposing cellular responses. A considerable number of studies supports the notion that JNK activation promotes cell apoptosis, whereas others report that JNK activation promotes cell proliferation and differentiation (8). Thus JNK has diverse functions, and its function strongly depends on the cell type and the context of other regulatory influences that the cell is receiving. For the vascular fibroblasts, we have reported that hypoxia-induced proliferation is dependent on JNK activation (5). Here, we demonstrate that hypoxia-induced differentiation of vascular fibroblasts is also regulated by JNK. Several transcription factors, including c-Jun, ATF2, Elk, and p53 (11, 16, 18) can be activated by JNK. Also, JNK potentiates transcriptional activity of HIF-1 through c-Jun activation in hypoxic cells (1). The functional interplay between c-Jun and HIF-1 provides a novel hypoxic regulation of VEGF, a key angiogenic factor during the development of different vascular diseases. Therefore, it will be important to understand how hypoxia-induced activation of JNK directs the same cell to perform a dual function, i.e., proliferation and differentiation.

Antagonists of the ERK pathway (PD-98059, U0126, and a dominant negative construct of ERK1) were unable to block the hypoxia-induced activation of {alpha}-SMA gene. However, inhibition of ERKs by PD-98059 results in suppression of {alpha}-SMA expression in mesangial cells (22), and ERKs appear to be essential for TGF-{beta}1-induced epithelial-mesenchymal transdifferentiation in pancreatic cancer cell lines (12), suggesting that the role for ERK in the {alpha}-SMA gene regulation is cell-type specific.

In mammalian cells, differentiation is primarily initiated by blocking cellular proliferation (44). However, numerous cytokines and growth factors can influence the proliferation level of fibroblasts as well as their differentiation to myofibroblasts by stimulating the expression of {alpha}-SMA (9). In the present study, we demonstrate that hypoxia induces an increase in BrdU incorporation as well as {alpha}-SMA expression in vascular adventitial fibroblasts. Recently, Norman et al. (32) have also reported that augmented proliferation and {alpha}-SMA expression occur concomitantly in renal fibroblasts in response to hypoxia. Importantly, we demonstrated that the hypoxia-induced increase in BrdU incorporation of vascular fibroblasts is not dependent on {alpha}-SMA expression because blockade of ERK pathway inhibits only the BrdU labeling index but not the hypoxia-induced upregulation of {alpha}-SMA level. How vascular adventitial fibroblasts divert the signal of hypoxic exposure for two different responses remains an important question to be explored in the future.

In conclusion, our study demonstrates for the first time that hypoxia upregulates {alpha}-SMA expression, a marker of myofibroblasts, in bovine pulmonary artery adventitial fibroblasts. Given recent reports that document the presence of myofibroblasts during various fibrotic and vascular diseases (3, 9, 17, 25, 26), a thorough understanding of {alpha}-SMA gene regulation in myofibroblasts offers the potential to develop novel therapeutics that target this particular cell type. Furthermore, myofibroblasts represent a significant source of other proinflammatory and angiogenic factors, as well as trophic factors (9, 14, 48). Detailed knowledge of signal transduction within these cells is thus important for a complete understanding of the pathogenesis of many diseases. Our results, which demonstrate the role of G{alpha}i and JNK in the hypoxia-induced transdifferentiation of fibroblasts into myofibroblasts, suggest that a strategy for attenuating this phenotypic modulation with specific inhibitors of either G{alpha}i protein or JNK might be beneficial for regulating various fibrotic diseases.


    ACKNOWLEDGMENTS
 
We thank Steve Hofmeister and Sandi Walchak for harvesting bovine pulmonary artery tissue, and Daniel C. Hopkins and Stephanie Fox for technical assistance.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-64917.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Das, Dept. of Pediatrics, B131, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: Mita.Das{at}uchsc.edu).

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


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