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Heterotrimeric G Proteins and the Platelet-Derived Growth Factor Receptor- Contribute to Hypoxic Proliferation of Smooth Muscle Cells

Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana; and Northern Ontario School of Medicine, Sudbury, Ontario, Canada

Correspondence and requests for reprints should be addressed to Carita Lannér, Northern Ontario Medical School, Division of Medical Sciences, Willet Green Miller Center, 8th floor, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6 Canada. E-mail: carita.lanner{at}normed.ca

	Abstract

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Hypoxic proliferation of pulmonary arterial smooth muscle cells (PASMC) is mitogen dependent, but the signaling pathways mediating hypoxia-induced cell growth are not well understood. We investigated hypoxic proliferation in primary cultures from porcine pulmonary artery smooth muscle. The cells were grown in medium with or without platelet-derived growth factor (PDGF)-B, a potent smooth muscle cell mitogen. Hypoxia induced upregulation of PDGF receptor- expression, the primary receptor for PDGF-B. However, PDGF-B–mediated hypoxic enhancement of proliferation was abolished by pertussis toxin, indicating (1) involvement of heterotrimeric G_i proteins and (2) minimal effect of increased PDGF receptor expression in hypoxic enhancement of proliferation. We treated PASMC with labeled, nonhydrolyzable analogs of GTP to determine directly if GTP binding proteins were activated by hypoxia in PASMC. We show that hypoxia stimulates GTP incorporation in PASMC both in the presence and absence of PDGF-B. Serum-starved PASMC are able to increase their incorporation of GTP after only 10 min of hypoxia, and this response is not pertussis toxin sensitive. In serum-starved PASMC, we show that hypoxia stimulates incorporation of GTP into a 44-kD protein. The results show that heterotrimeric G proteins are involved in hypoxia-induced signaling in pulmonary vascular smooth muscle cells.

Key Words: hypoxia • vascular remodelling • GTP binding protein • smooth muscle growth

	Introduction

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Hypoxia contributes to the regulation of many physiologic processes such as glycolysis, angiogenesis, and erythropoiesis. In the circulatory system, hypoxia exerts the opposite effect on systemic and pulmonary circulation. Hypoxia induces vasodilatation in the systemic vessels. In sharp contrast, hypoxia is one of the most important stimuli for pulmonary vasoconstriction, and regional hypoxia serves as an important regulatory mechanism in redirecting blood flow to better-oxygenated lung regions. However, chronic hypoxia, which is often associated with chronic obstructive pulmonary disorders, causes pulmonary hypertension in humans. The hypoxia-induced pulmonary hypertension is characterized by a marked increase in pulmonary vascular resistance, with significant vessel wall remodeling and a marked proliferation of pulmonary artery smooth muscle cells (PASMC) (1). Hypoxia has been shown to enhance smooth muscle cell proliferation in various systems and is thought to be a primary signaling mechanism in smooth muscle proliferation, which appears to be a key component of the remodeling seen in pulmonary hypertension (2, 3).

In many cell types, hypoxia induces gene expression by stabilizing a hypoxia-inducible transcription factor, HIF-1, thereby coordinating a tissue-specific response to hypoxia (4, 5). While vascular smooth muscle cells induce HIF-1 in response to hypoxia, smooth muscle growth cannot be induced without the presence of exogenous growth factors; indicating that HIF-1 transcription does not lead to autocrine growth factor production (6–8). However, when a mitogenic stimulus is present, hypoxia is able to stimulate the proliferative response to the mitogen in vascular smooth muscle cells (9). While an autocrine production of growth factors may not occur, an increase in receptor levels for mitogens may contribute to the hypoxic enhancement of proliferation. Regulation of mitogen receptor levels is one feature of the intrinsic signaling mechanisms functioning in the hypoxia response. In this study, we have investigated the effect of hypoxia on receptor levels for a smooth muscle mitogen, platelet-derived growth factor (PDGF), and show that hypoxia significantly increases receptor levels on PASMC.

Although the hypoxic enhancement of proliferation is a well-documented phenomenon, the mechanism of hypoxic enhancement is not clear (6, 9, 12). An increasing body of evidence implicates the GTP-binding heterotrimeric G proteins, in both cell proliferation and aspects of hypoxic-induced proliferation such as activation of mitogen-activated protein kinases (12, 13). Previous work in our laboratory and by others have shown that hypoxia is able to activate the small GTP-binding protein Rho and its downstream effector, Rho-kinase, in PASMC (14, 15). PASMC possess an intrinsic ability to contract in response to acute hypoxia, a response shown to be mediated by Rho and Rho kinase (10, 11). Several studies of Rho activity have implicated the heterotrimeric G-protein subunits, G₁₂ and G₁₃, in the activation of Rho (16–19). Although hypoxia is known to activate Rho and it is known that Rho activation is typically mediated via the heterotrimeric G_12/13 protein family, it is not yet known whether hypoxia is able to activate G_12/13 proteins. Therefore, we have investigated the role of heterotrimeric G proteins in the response of PASMC to hypoxia. In particular, we show that heterotrimeric G proteins of the G_i/o family play an important role in hypoxic proliferation, based on the observation that the hypoxic proliferative response is ablated with pertussis toxin (an inhibitor of G proteins of the G_i/o family). In addition, we observed activation of a 44 kD GTP-binding protein in direct response to acute hypoxia in quiescent porcine PASMC.

	MATERIALS AND METHODS

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Smooth muscle growth medium with growth factors (SmGM2), smooth muscle base medium without growth factors (SmBM), trypsin, and trypsin neutralizing solution were purchased from Clonetics-Biowhittaker-Cambrex (Walkersville, MD). FBS was from Atlanta Biologicals (Norcross, GA). Penicillin-streptomycin-amphotericin solution for tissue culture was from GIBCO-Invitrogen (Grand Island, NY). Dulbecco's modified minimal essential medium (DMEM), insulin, PBS, and general laboratory chemicals were from Sigma (St. Louis, MO). Antibodies against heterotrimeric G protein subunits (G_i-3, G₁₂, G₁₃ G_q/11, G_s, G_o) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Reagents for polyacrylamide gel electrophoresis were purchased from BioRad (Hercules, CA). Antibodies for Western blotting of the PDGF receptor (PDGFR-) were rabbit polyclonal IgG anti-PDGF receptor Type B from Upstate Biotechnology (Lake Placid, NY). The secondary antibody for Western blotting was anti-rabbit IgG horseradish peroxidase conjugate from Santa Cruz Biotechnology. Antibodies for flow cytometry staining of PDGFR- were mouse monoclonal IgG anti-PDGF Receptor B (Ab-2) and monoclonal rabbit anti-mouse IgG fluorescein conjugate from Calbiochem (San Diego, CA). Isotype control antibodies were phycoerythrin-conjugated mouse IgG from BD Biosciences (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-smooth muscle -actin (clone 14A) was from Sigma. Radioactively labeled GTP--³⁵S (1,250 Ci/mmol) was purchased from Perkin Elmer Life Sciences (Boston, MA). The radioactively labeled photo affinity analog, azidoanilide GTP--³²P (10–15 Ci/mmol), was from Affinity Labeling Technologies, Inc. (Lexington, KY). The tritium-labeled compound ³H-thymidine (2.0 Ci/mmol) was from ICN Biomedicals, Inc. (Irvine, CA).

Isolation and Culture of Porcine PASMC
Smooth muscle cells were isolated from the main branches of pulmonary arteries of 6-mo-old pigs. Porcine pulmonary artery sections were kindly provided by Dr. Gordon McLennan and Dr. Bradley Allen (Indiana University School of Medicine). Explants were cut from strips taken from the media of vessel walls. The explants were cultured on 10-cm tissue culture dishes in DMEM supplemented with 10% FBS, 1% PAS (10,000 U/ml penicillin G sodium, 10 mg/ml streptomycin sulfate, 25 µg/ml amphotericin B), and 10 nM insulin for 10–14 d before being transferred to tissue culture flasks (T75) for expansion of the culture. Media were changed every 48 h. Samples of isolates were stained for expression of smooth muscle -actin with FITC-conjugated anti–smooth muscle -actin, and all isolates exhibited robust expression of smooth muscle -actin as determined by fluorescence microscopy. The purity of the smooth muscle cells was > 90%, based on expression of smooth muscle -actin. After expansion of the cultures, cells were frozen and kept in liquid nitrogen. Upon retrieval from the liquid nitrogen, cells were adapted to the optimal culture medium, SmGM2, from Clonetics: 5% FBS in SmBM base medium, 0.5 ng/ml hEGF, 5 ng/ml insulin, 2 ng/ml hFGF-B, 50 µg/ml Gentamicin, 50 ng/ml Amphotericin-B. The SmGM2 medium is an optimized medium for the culture of smooth muscle cells, and our cells exhibited more robust responses to treatment after recovery in SmGM2 medium. After recovery from storage in liquid nitrogen, all experiments were conducted with cells in the SmBM medium, as described for each experiment. Cells in passages 3 to 4 were used in the experiments.

Porcine PASMC were cultured under normoxia (21% O₂, 5% CO₂, balance nitrogen) at 37°C. Hypoxic culture or exposure to hypoxia was performed in an InVivo₂ Hypoxia Workstation (Russkin Technologies Limited, Leeds, UK) at 3% O₂ (PO₂ = 23 ± 2 mm Hg), 5% CO₂, balance nitrogen at 37°C. A 3% O₂ concentration, corresponding to a PO₂ of 23 ± 2 mm Hg, was used to stimulate an adaptive response to moderate hypoxia. The hypoxia workstation permits chamber access through ports fitted with gas-impermeable sleeves and airlocks, so that hypoxic conditions can be maintained during experiments.

Proliferation Assay of PASMC under Hypoxia
To determine proliferation rates for hypoxia versus normoxia, cells were seeded at a density of 5 x 10⁴ cells per well (5.2 x 10³ cells/cm²) in a 6-well tissue culture plate. Each culture condition was plated in triplicate. In our preliminary experiments, cells were cultured in the SmBM base medium from Clonetics with 5% FBS, with or without PDGF-B (Sigma) at 10 ng/ml. In later experiments, proliferation was assayed in low serum (0.1% FBS), with or without PDGF-B, to minimize the nonspecific mitogenic effects of serum. Pertussis toxin (Calbiochem) was used at 100 ng/ml to inhibit heterotrimeric G proteins of the G_i/o subtype.

Following seeding of the cells, plates were cultured under either normoxia or hypoxia for 48, 72, or 96 h. At each assay time point, a set of three wells was harvested and the cell number determined in each well by Coulter counting (Beckman Coulter, Fullerton, CA). Medium was changed every 48 h. Cells exposed to hypoxia did not alter the pH of the medium, nor were significant numbers of dead cells observed over the time course of the proliferation assay. Average cell numbers were plotted for each day of culture under either normoxia or hypoxia.

DNA Synthesis Assay
PASMC were seeded at a density of 5 x 10⁴ cells per well in a 24-well plate. A row of six wells was used per treatment, four wells for ³H-thymidine labeling and two wells for counting to control for plating numbers. After 24 h, medium was removed and replaced with medium containing 0.1% FBS. Cells were serum-starved for 72 h to induce complete growth arrest. Fresh medium containing either 0.1% FBS or 0.1% FBS with PDGF (10 ng/ml) and 0.5 µCi ³H-thymidine (2.0 Ci/mmol; ICN Biomedicals) was added to each of the assay wells. After a 24-h incubation in either normoxia or hypoxia, medium was removed. Cells were washed with PBS, and acid-insoluble material was fixed with fresh 0.2% perchloric acid for 10 min. The acid-precipitated material was solubilized in 200 µl 0.01 M NaOH-0.1% SDS per well. The radioactivity of the total contents of each well was determined by liquid scintillation counting. After averaging the counts across the four replicates, the values were normalized to the cell number per row.

Flow Cytometry Analysis of PDGF Receptor Expression by PASMC
Porcine PASMC were plated in triplicate in 6-well tissue culture plates at a density of 3 x 10⁵ cells per well and cultured overnight (18 h) in SmGM2. After the overnight culture, medium was removed and replaced with 0.1% FBS–SmBM for 24 h to induce quiescence while cells were cultured under normoxia. The following day, medium was replaced with fresh 0.1% FBS–SmBM and cells were cultured under normoxia or hypoxia for 24 h. Cells were harvested by trypsinization, washed, and stained with mouse monoclonal IgG anti-PDGF Receptor B (Ab-2), as the primary antibody. After washing, the cells were stained with anti-mouse monoclonal IgG fluorescein conjugate as the secondary antibody. Isotype staining was done using phycoerythrin-conjugated mouse IgG. The amount of fluorescent label present on normoxic and hypoxic cells was determined by flow cytometry using a Facscalibur fluorescence-activated cell sorter from Becton Dickinson (San Diego, CA). Mean fluorescence values were taken for samples.

Labeling of GTP-Binding Proteins
Two nonhydrolyzable GTP analogs, GTP--³⁵S and azidoanilide GTP--³²P (AAGTP--³²P), were used in the experiments. Upon stimulation of a cell, the GTP--³⁵S compound will accumulate after associating with a GTP-binding protein, due to the inability of GTPases to cleave the terminal sulfate group. This allows us to measure the total change in GTP binding induced by a stimulus, e.g., hypoxia. The AAGTP--³²P will also accumulate in a stimulated cell due to the location of an azidoanilide group on the terminal phosphate, which prevents cleavage by GTPases. In addition, the AAGTP--³²P is a photo affinity compound, which can be covalently cross-linked to the GTP-binding protein active site by irradiation with shortwave ultraviolet (254 nm) light. In experiments using the photo affinity analog, AAGTP--³²P, cells were irradiated with (254 nm) ultraviolet light, on ice, for 2 min at a distance of 1 cm to covalently link the AAGTP to the GTP-binding protein.

To determine if proliferating PASMC exhibited an increased incorporation of GTP, PASMC were cultured under hypoxia or normoxia with PDGF-B (10 ng/ml) for 24 h before treatment with GTP--³⁵S. After introduction of the GTP analog into the cells, cells were washed, and medium containing PDGF-B was replaced. Hypoxic cells were treated inside the InVivo hypoxia chamber and normoxic cells were treated in a tissue culture cabinet, then replaced inside a cell culture incubator. PASMC were left under hypoxia or normoxia for an additional 1 h. For acute hypoxic exposure in the absence of growth factors, PASMC were made quiescent in 0.1% FBS under normoxia, in SmBM for 24 h before treatment with GTP analog. After treatment with GTP analog, a plate of cells was introduced into the InVivo hypoxia chamber and exposed to hypoxia for 10–30 min or put into a cell culture incubator for exposure to normoxia for 10–30 min in serum-free medium. Lysophosphatidic acid (LPA) was used as a positive control for G protein activation, since LPA is known to simultaneously stimulate the three heterotrimeric G proteins G_i/o, G_q, and G_12/13 (20, 21). PASMC were stimulated with 10 µM LPA under normoxia after permeabilization and introduction of radiolabeled GTP analog into the cells.

The GTP analogs were introduced into the PASMC cells using a permeabilization method developed by Schelling and coworkers (22). Schelling and colleagues measured GTP activation of phospholipase C in smooth muscle cells by using the saponin -escin to generate pores in cell membranes and to introduce the GTP analog GTP--S into the cells. Briefly, cells are permeabilized for 3–5 min at 37°C in a buffer (100 mM KCl, 3 mM NaCl, 25 mM HEPES pH 7.2, 5 mM MgCl₂, 1 mM EGTA, 75 nM CaCl₂, 1 mM ATP) containing 30 µg/ml -escin and 60 µCi per 1 x 10⁶ cells of GTP--³⁵S or 10 µCi per 1 x 10⁶ cells of AAGTP--³²P analog. Permeabilization was performed in the hypoxia chamber using hypoxia-equilibrated reagents, when cells had been cultured for 24 h under hypoxia. The permeabilization buffer was removed, cells were washed in buffer without -escin or GTP analog, and fresh medium (either 0.1% FBS–PDGF-B or serum-free SmBM) was put on the cells. The cells were exposed to either normoxia or hypoxia for different lengths of time. At the end of the incubation period, medium was removed, the plates were placed on ice to stop the reaction, and cells were lysed in RIPA buffer applied directly to the plates. PASMC treated with AAGTP--³²P were irradiated with ultraviolet light before lysis.

Preparation of Cell Lysates
The cells were scraped loose from the plates, on ice, in a RIPA lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 5 µg/ml aprotinin, 2 µg/ml leupeptin) and transferred to a 1.5-ml microfuge tube. The cell suspension was sonicated for three 5 s bursts at a setting 3 on a Sonic Dismembrator 550 from Fisher Scientific (Suwanee, GA) and incubated on ice for 30 min. Protein concentration of cells lysates was determined using the Bicinchoninic acid assay. For the GTP-binding experiments, the amount of radioactivity incorporated was determined by liquid scintillation counting of an aliquot of each cell lysate.

Western Blot Analysis and Autoradiography
Equal amounts of total protein (20 µg/lane) were loaded for cell lysates and resolved by 10% polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto nitrocellulose membranes (BioRad). Membranes were exposed to Biomax MS film (Eastman Kodak Co., Rochester, NY) for 18–48 h to detect binding of radioactively labeled AAGTP--³²P to protein components of the cell lysates. After the film exposure, membranes were blotted with rabbit polyclonal IgG antibodies against heterotrimeric G proteins (Santa Cruz Biotechnology) followed by anti-rabbit IgG antibody conjugated to horseradish peroxidase. Proteins bound to antibodies were detected using enhanced chemoluminescence (ECL) reagents from Amersham. Chemoluminescent exposure times were 30 s to 1 min.

Positive signals on the western blots and autoradiograms were quantified by densitometry scanning (GS-670; BioRad).

Statistical Treatment of Data
Student's t test was used to compare results from control and experimental groups. All experiments were repeated at least three times. Results were expressed as mean values ± SE. A value of P < 0.05 was considered statistically significant.

	RESULTS

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Hypoxic Enhancement of Mitogen-Mediated Proliferation
Porcine PASMC were cultured in SmBM, a base medium, to which growth factors can be added. Proliferation studies were performed with PASMC cultured in SmBM containing 5% FBS in the absence or presence of PDGF-B (10 ng/ml). Hypoxia significantly enhanced the proliferation of PASMC when the cells were cultured in medium containing PDGF-B. As shown in Figure 1, culture of PASMC in absence of PDGF-B did not lead to significantly increased cell proliferation under hypoxia, whereas hypoxia-enhanced proliferation of PASMC occurred in medium containing serum and PDGF-B. In 5% FBS medium there was a 6-fold increase in cell number by Day 4, for both the normoxic and hypoxic cultures (n = 29.5 ± 2.0, H = 32.2 ± 1.6, P = 6.00 x 10^–2), demonstrating that 5% FBS serum is a mitogenic stimulus under both conditions. However, in 5% FBS-PDGF medium there was a 13-fold increase in cell number by Day 4 under normoxia and a 20-fold increase in cell number by Day 4 under hypoxia (n = 71.3 ± 5.8, H = 104.3 ± 11.0, P = 3.87 x 10^–3). The results indicate that while PDGF together with serum is a more potent mitogenic stimulus than serum alone under both normoxia and hypoxia, proliferation is significantly enhanced in the presence of PDGF-B only under hypoxia.

View larger version (17K): [in this window] [in a new window]   Figure 1. Hypoxic enhancement of proliferation in the presence of PDGF-B. PASMC were cultured for up to 4 d under normoxia and hypoxia. Cells were harvested at time points as indicated and counted using a Coulter counter. Proliferation is presented as increase in cell number over 4 d. (A) Proliferation in SmBM medium supplemented with 5% FBS. (B) Proliferation in SmBM supplemented with 5% FBS and PDGF-B (10 ng/ml) (n = 4; * P 0.05 hypoxia versus normoxia).

View larger version (17K): [in this window] [in a new window]   Figure 2. Proliferation in PDGF-B and/or 0.1% FBS under normoxia and hypoxia. PASMC were cultured in 0.1% FBS–SmBM or 0.1% FBS–SmBM with PDGF-B (10 ng/ml) for up to 4 d. Cells were harvested at time points as indicated and counted using a Coulter counter. Proliferation is presented as increase in cell number over 4 d (n = 4; * P 0.05 hypoxia versus normoxia).

View larger version (9K): [in this window] [in a new window]   Figure 3. Hypoxic enhancement of DNA synthesis. PASMC were serum starved for 72 h. Tritiated thymidine was added to either 0.1%FBS–SmBM or 0.1% FBS–SmBM with PDGF-B (10 ng/ml), and cells were cultured in the tritium medium for 24 h. Acid-insoluble material was precipitated with 0.2% perchloric acid and proteins solubilized in 0.01 M NaOH–0.1% SDS. Incorporation of tritium into acid-insoluble material was determined by liquid scintillation counting. Data are presented as a ratio to normoxia of the dpm/1 x 104 cells (n = 3; * P 0.05 hypoxia versus normoxia).

The total amount of PDGFR- protein in cell lysates exposed to hypoxia for up to 4 d in the presence and absence of PDGF-B (10 ng/ml) was determined by Western blotting. The data showed a tendency to increase at each time point (24, 48, 72, and 96 h) in both the presence and absence of PDGF-B. However, the trend was not statistically significant (data not shown).

A more sensitive method of measuring cell surface expression only was used to investigate the effect of hypoxia on PDGF receptors. This method eliminated measurement of the total amount of receptor protein, which would include internalized receptors and those targeted for degradation. The expression of PDGFR- on the cell surface was investigated using flow cytometry of intact cells, stained with an anti–PDGFR- antibody and a fluorescein-conjugated secondary antibody. The anti–PDGFR- antibody was raised against an extracellular epitope of the PDGFR-. Serum-starved PASMC were exposed to normoxia or hypoxia for 24 h before staining with the anti–PDGFR-B antibody. Exposure to hypoxia for 24 h significantly increased the level of expression of the PDGFR- on the surface of PASMC by 28% (Figure 4) as detected by flow cytometry. The mean fluorescence of PDGF receptors on normoxic cells was 69.87 ± 7.2, while hypoxic cells displayed a mean fluorescence of 89.64 ± 12.9 (P = 3.8 x 10^–2, N versus H). The specificity of the anti–PDGFR- antibody staining of the receptor was confirmed by staining PASMC with a phycoerythrin-conjugated IgG of the same isotype as the anti–PDGFR- antibody (Figure 4). Isotype staining was typically one tenth the level of staining achieved by the anti–PDGFR- antibody.

View larger version (9K): [in this window] [in a new window]   Figure 4. Hypoxic increase in expression of PDGFR- on cell surface. PASMC were serum starved for 24 h in 0.1% FBS–SmBM, fresh medium was put on the cells, and PASMC were exposed to hypoxia or normoxia for 24 h. After harvesting, the cells were stained with anti–PDGFR- mouse monoclonal IgG, washed and stained with fluorescein-conjugated anti-mouse monoclonal IgG. Isotype staining was done using phycoerythrin-conjugated mouse IgG. The amount of fluorescent label present on the cells was determined by FACS analysis. Data are presented as average mean fluorescence (n = 3; * P 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 5. Effect of pertussis toxin on hypoxic enhancement of proliferation. PASMC were cultured in SmBM 0.1% FBS–PDGF-B (10 ng/ml) for up to 4 d. Pertussis toxin (PTX) was added at a concentration of 100 ng/ml. Cells were harvested at time points as indicated and counted using a Coulter counter. Proliferation is presented as increase in cell number over 4 d (n = 4; P 0.05 indicated as follows: *Day 2, H versus H + PTX; **Day 3, N versus N + PTX; Day 4, N versus H).

View larger version (20K): [in this window] [in a new window]   Figure 6. Binding of GTP by PASMC under hypoxia. Data is presented as ratio to normoxia of dpm/ug total protein. LPA (10 µM) was used as a positive control for stimulation of binding of GTP by heterotrimeric G-proteins. (A) PASMC were cultured in 0.1% FBS–SmBM with PDGF (10 ng/ml) for 24 h. Pertussis toxin was added at 100 ng/ml for 24 h. After treatment with the GTP analog, GTP--35S, fresh 0.1%FBS–PDGF medium was added and cells were maintained in normoxia or hypoxia for 1 h (n = 3; * P 0.05 treatment versus normoxia). (B) PASMC were cultured in 0.1% FBS–SmBM for 24 h. Pertussis toxin was added at 100 ng/ml for 24 h. After treatment with GTP analog, serum-free medium was added and cells were kept in normoxia or hypoxia for 10 min (n = 4; * P 0.05 treatment versus normoxia).

To determine if PASMC possessed an intrinsic ability to activate GTP-binding proteins under hypoxia, quiescent PASMC were exposed to acute hypoxia for 10 min in the absence of mitogens. With this treatment protocol, cells were not conditioned to hypoxia or affected by mitogens before uptake of GTP analog and exposure to hypoxia or normoxia. Serum-starved PASMC demonstrated an increased level of GTP incorporation upon exposure to hypoxia compared with normoxia (Figure 6B). The amount of GTP incorporated increased on average by 30% (H/N = 1.30 ± 0.03, P = 3.01 x 10^–5 for H versus N). When serum-starved PASMC were incubated with pertussis toxin 24 h before acute hypoxia exposure, the hypoxic increase in GTP incorporation persisted (H + PTX/N = 1.25 ± 0.04, P = 3.30 x 10^–3, H + PTX versus N) (Figure 6B). Normoxic cells showed a slight decrease in GTP incorporation, but the change was not significant (N+PTX/N = 0.86 ± 0.07, P = 8.81 x 10^–2, N + PTX versus N). In the positive control cells, treated with LPA, GTP incorporation increased by 26% (Figure 6B).

These results provide strong evidence that hypoxia can activate the GTP-binding proteins in PASMC, both during acute exposure to hypoxia and during chronic hypoxia-enhanced proliferation. The GTP-binding protein involved in the proliferative response, but not the acute response, is most likely a heterotrimeric G protein of the G_i/o family based on inhibition by pertussis toxin.

Acute Hypoxia Stimulates a Heterotrimeric G Protein
The GTP-binding activity induced by acute hypoxia was investigated in quiescent PASMC following exposure to hypoxia in the presence of a cross-linkable GTP analog, AAGTP--³²P. In our first experiments, antibodies against the heterotrimeric G proteins G_i-1, G_i-3, G₁₂, G₁₃, G_q/11, G_s were used to immunoprecipitate the G proteins from normoxic and hypoxic cell lysates. The antibodies were not able to immunoprecipitate consistently detectable amounts of radioactively labeled G proteins compared with the total amount of G protein immunoprecipitated from the lysates, but they could be used successfully for detection of total G protein on Western blots. Therefore, aliquots of lysates from cells treated with labeled GTP analogs and exposed to hypoxia or normoxia were resolved by SDS-PAGE on 10% acrylamide gels and transferred to nitrocellulose membranes. The membranes were exposed to X-ray film for up to 48 h. The resulting autoradiograms indicated that a significant amount of the labeled GTP analog had been incorporated into a protein of 44 kD (Figure 7A). The radioactive signal was stronger from the hypoxic lysates. Blotting the radioactively labeled membrane with the heterotrimeric G protein antibodies produced bands in the same region of the membrane as the radioactive band, with the antibodies for the G₁₂ and G₁₃ proteins superimposing upon the 44-kD radioactively labeled band. This indicated that the radioactively labeled band was most likely to be a heterotrimeric G protein of the G_12/13 subtype. Therefore, as an example, a blot of the membrane using the antibody against the heterotrimeric G protein G₁₂, which represents G proteins with a size of 44 kD, is shown to indicate that in serum-starved PASMC, a heterotrimeric G protein, likely of the G_12/13 subtype, is activated by hypoxia (Figure 7B).

View larger version (38K): [in this window] [in a new window]   Figure 7. Hypoxic stimulation of binding of GTP analog to heterotrimeric G protein. PASMC were maintained in 0.1% FBS–SmBM medium for 24 h, washed, and permeabilized together with the labeled, nonhydrolyzable photo affinity GTP analog, azidoanilide GTP--32P (AAGTP). The buffer containing the GTP was removed, cells were washed, and serum-free SmBM medium was added to the cells. PASMC were exposed to normoxia or hypoxia for 10 or 30 min. LPA (10 µM) was used as a positive control for stimulation of binding of GTP by heterotrimeric G proteins. AAGTP bound to GTP-binding proteins was cross-linked by shortwave ultraviolet irradiation. Lysates were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes and exposed to X-ray film. After film exposure, membranes were blotted with antibodies to heterotrimeric G proteins. Densitometry was performed and values were calculated as ratios to normoxia (n = 3; *P 0.05 hypoxia versus normoxia). (A) Autoradiogram of AAGTP-labeled proteins. (B) Western blot of membrane with anti-G12 rabbit polyclonal antibodies. (C) Ratios of densitometry values for autoradiograms showing incorporation of AAGTP under acute hypoxia.

	DISCUSSION

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The present investigation shows that hypoxia-enhanced proliferation of porcine PASMC involves signaling through the receptor for PDGF-B and activation of heterotrimeric G proteins by hypoxia.

In our experimental model, porcine PASMC required a mitogen to exhibit enhanced proliferation to hypoxia. A strong proliferative response to hypoxia was observed with the growth factor, PDGF-B (Figure 1). This growth factor is especially significant in the context of hypoxia, since it is known that endothelial cells produce PDGF-B in response to hypoxia (23), but smooth muscle cells do not (24). Two separate approaches were used to determine the effect of hypoxia on receptor levels. Western blotting of total cell protein with anti–PDGFR- antibody did not show a significant upregulation of receptor amount under hypoxia, although a trend for increased receptor amounts was present. However, examining the total amount of protein, which includes receptors that have been internalized and are either targeted for degradation or recycled to the cell surface, and would not reflect functional receptor levels on the cell available for active signaling. Therefore, the level of expression of PDGFR- on the cell surface under hypoxia was examined using flow cytometry of intact PASMC stained with anti–PDGFR- antibody. Hypoxia caused an upregulation of the PDGFR- levels expressed on the cell surface, even in the absence of PDGF-B, indicating that hypoxia alone is sufficient to upregulate receptor expression (Figure 4). These findings are in contrast to the report by Humar and coworkers (9), in which rat aortic smooth muscle cells (ASMC) do not upregulate PDGFR- in response to hypoxia. The increase in expression of PDGFR- on the cell surface of porcine PASMC reported by us could be a result of the different techniques used to assay receptor expression. Humar and colleagues used Northern and Western blotting techniques to assay receptor expression in rat ASMC. We also did not observe a significant upregulation of the PDGFR- when we applied a Western blotting assay to porcine PASMC. An alternative explanation for the difference between porcine PASMC and rat ASMC expression of PDGFR- under hypoxia is that there may be a true difference between the two cell types. This would support a cell type–specific response to hypoxia.

Although receptor levels likely play a role in the hypoxic enhancement of mitogen-mediated proliferation, pertussis toxin treatment of PASMC in PDGF was able to abate the hypoxic enhancement of proliferation to PDGF-B (Figure 5). This indicates that heterotrimeric G proteins of the G_i/o family play a crucial role in the signaling of hypoxia in PDGF-B–mediated proliferation.

The ability of proliferating PASMC to increase activation of GTP-binding proteins under hypoxia was observed using nonhydrolysable GTP analogs. In PASMC cultured in PDGF-B, GTP incorporation was sensitive to pertussis toxin, confirming a role for heterotrimeric G proteins of the G_i/o family in signaling of hypoxic proliferation (Figure 6A). Other studies of hypoxic stimulation have also reported a role for heterotrimeric G proteins in the hypoxia proliferation signaling pathway. Activation of ERK and JNK kinases by hypoxia in subsets of bovine neonatal adventitial fibroblasts was significantly attenuated and hypoxia-stimulated DNA synthesis in the same cells was ablated by pertussis toxin, indicating a role for the heterotrimeric subunit, G_i/o, in the proliferative response of selected adventitial fibroblasts to hypoxia (25). Contribution of G_i/o to the hypoxic proliferation of adventitial fibroblasts was further confirmed in a study indicating that extracellular ATP acts as a mitogen together with G_i/o under hypoxia (26). Hypoxic transdifferentiation of vascular fibroblasts into myofibroblasts was also found to involve activation of a heterotrimeric G protein, namely G_i2 (27). These studies support our observation that increased incorporation of GTP in proliferating PASMC under hypoxia involves activation of heterotrimeric G protein G_i/o subunits, based on sensitivity to pertussis toxin.

Serum-starved PASMC were also found to increase incorporation of GTP analogs under brief episodes of hypoxia in the absence of PDGF (Figure 6B). The response of serum-starved PASMC is different from the proliferative response in the presence of PDGF, and more representative of the cellular response during the hypoxic contraction of pulmonary smooth muscle. This response was not sensitive to pertussis toxin, indicating that a heterotrimeric G protein other than G_i/o is involved in that response. Using a photo affinity nonhydrolyzable GTP analog, the GTP-binding protein responding to acute hypoxia in serum-starved PASMC could be cross-linked to the radioactively labeled photo affinity analog and resolved by SDS-PAGE. This analysis showed that a protein in the 44-kD size range is able to increase binding of GTP upon exposure to hypoxia (Figure 7A). Western blotting further indicated that the protein is of a similar size as heterotrimeric G proteins of the G_12/13 family (Figure 7B). Support for this concept comes from studies of signaling in the hypoxic contractile response, which occurs within 10 min of exposure to hypoxia, can persist up to 1 h, and is not growth factor dependent (14, 15, 28). These studies show that the hypoxic contraction of PASMC is mediated via the small GTPase, Rho A, and its downstream effector Rho kinase. It is also known that Rho A is primarily activated by heterotrimeric G_12/13 subunits (16). Therefore, incorporation of GTP into quiescent PASMC after a brief exposure to hypoxia could involve G_12/13 subunits, which would not be affected by pertussis toxin. The hypoxic activation of the G protein is not affected by pertussis toxin, indicating that G_i proteins are not responsible for the acute response to hypoxia (Figure 6B). Based on the size of the GTP-binding protein, the lack of inhibition by pertussis toxin, and the evidence for activation of Rho A by G_12/13, it is likely that the heterotrimeric G protein mediating the acute response to hypoxia would be a G_12/13 protein.

Activation of heterotrimeric G proteins by hypoxia implies the existence of a G protein–activating mechanism that responds to hypoxia. Typically, heterotrimeric G proteins are activated by G protein–coupled receptors (GPCR) (29–31). Although no hypoxia-responsive receptor has been identified to date, the accumulating evidence of G protein involvement in the response to hypoxia lends support to the concept. The attractiveness of this idea is further supported by studies of cross-talk between GPCR and growth factor receptors, whereby stimulation of a GPCR activates associated heterotrimeric G proteins which, in turn, stimulate specific growth factor receptors resulting in a synergistic signaling response (32–34). Cross-talk between activated G proteins and growth factor receptors is a potential basis for the hypoxic enhancement of proliferation in the presence of growth factors like PDGF-B.

In summary, the present study demonstrates two signaling pathways that are involved in mediating hypoxic enhancement of PASMC proliferation. The first is activation of heterotrimeric G proteins in hypoxia-stimulated PASMC, and the second is upregulation of the PDGFR- by hypoxia alone. In the hypoxic enhancement of PDGF-B–mediated proliferation, heterotrimeric G_i/o subunits play a crucial role, while acute hypoxic exposure of quiescent PASMC indicates activation of heterotrimeric G proteins of a different type, possibly the G_12/13 family. While both signaling pathways contribute to the hypoxic response of PASMC, the immediate activation of heterotrimeric G proteins by hypoxia and the necessary involvement of G_i/o subunits in hypoxia-enhanced proliferation imply a pivotal role for this type of signaling in the hypoxic response.

	Acknowledgments

 
The authors are indebted to Dr. Gordon McLennan and Dr. Bradley Allen for tissue samples of porcine arteries. Dr. Jeffery Elmendorff is thanked for providing critical comment and discussion. The authors thank Mr. Kevin Harvey of the Methodist Research Institute, Methodist Hospital, Indianapolis, IN for his valuable assistance with the flow cytometry measurements.

	Footnotes

 
Supported in part by the Showalter Foundation.

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 5, 2005

Received in final form July 5, 2005

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