Fibroblast growth factor mediates hypoxia-induced endothelin-A ...

J Appl Physiol 95: 643–651, 2003; 10.1152/japplphysiol.00652.2002.

Fibroblast growth factor mediates hypoxia-induced endothelin-A receptor expression in lung artery smooth muscle cells Peng Li,1 Suzanne Oparil,1 Ju-Zhong Sun,1 John A. Thompson,2 and Yiu-Fai Chen1 1

Department of Medicine, Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, and 2Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294 Submitted 17 July 2002; accepted in final form 7 April 2003

Li, Peng, Suzanne Oparil, Ju-Zhong Sun, John A. Thompson, and Yiu-Fai Chen. Fibroblast growth factor mediates hypoxia-induced endothelin-A receptor expression in lung artery smooth muscle cells. J Appl Physiol 95: 643–651, 2003; 10.1152/japplphysiol.00652.2002.—We have previously demonstrated that endothelin (ET)-1 and its subtype A receptor (ET-AR) expression are increased in lung under hypoxic conditions and that activation of ET-AR by ET-1 is a major mediator of hypoxia-induced pulmonary hypertension in the rat. The present study tested the hypothesis that the hypoxia-responsive tyrosine kinase receptoractivating growth factors fibroblast growth factor (FGF)-1, FGF-2, and platelet-derived growth factor (PDGF)-BB stimulate expression of the ET-AR in pulmonary arterial smooth muscle cells (PASMCs). Quiescent rat PASMCs were incubated under hypoxia (1% O2), or with FGF-1, FGF-2, PDGFBB, vascular endothelial growth factor, ET-1, angiotensin II, or atrial natriuretic peptide under normoxic conditions for 24 h. FGF-1 and -2 and PDGF-BB, but not hypoxia, vascular endothelial growth factor, ET-1, angiotensin II, or atrial natriuretic peptide, significantly increased ET-AR mRNA levels. FGF-1-induced ET-AR expression was inhibited by FGF-receptor inhibitor PD-166866, MEK inhibitor U-0126, transcription inhibitor actinomycin D, and translation inhibitor cycloheximide. In contrast, the stimulatory effect of FGF-1 on ET-AR mRNA expression was not altered by PI3 kinase, PKA, PKC, or adenylate cyclase inhibitors. PASMC ET-AR gene transcription, assessed by nuclear-runoff analysis, was increased by FGF-1. These results provide novel finding that ET-AR in PASMCs in vitro is unresponsive to hypoxia per se but is robustly simulated by tyrosine kinase receptor-associated growth factors (FGF-1, FGF-2, PDGFBB) that themselves are stimulated by hypoxia in lung. This observation suggests a novel signaling mechanism that may be responsible for overexpression of ET-AR in lung, and may contribute to the hypoxia-induced pulmonary vasoconstriction, hypertension, and vascular remodeling in hypoxiaadapted animal.

OUR LABORATORY’S PREVIOUS STUDIES

(ET)-1 and its subtype A receptor (ET-AR) in lung and main pulmonary artery but not in organs supplied by the systemic vascular bed. The finding of concomitant increases in ET-AR expression in lung suggests the possibility that ET-1 synthesized by pulmonary vascular endothelial cells under hypoxic conditions acts locally on ET-AR on smooth muscle cells (SMCs) in the pulmonary arterial tree to produce vasoconstriction, vascular remodeling, and hypoxic pulmonary hypertension (8, 27). We and others have also demonstrated that increased ET-1 expression in pulmonary vascular endothelial cells appears to be transcriptionally mediated by hypoxia (15, 17, 19, 21, 40). In contrast, it is unclear whether the responses of ET-AR gene expression in lung to hypoxia are mediated directly by hypoxia or indirectly by alterations in tissue levels of hypoxiasensitive mediators or by hemodynamic alterations in the pulmonary circulation. The present study tested the hypothesis that hypoxia-induced growth factors, rather than hypoxia per se, mediate increased expression of ET-AR in pulmonary arterial SMCs (PASMCs) in vitro. Because most of the ET-AR in lung is located in PASMCs (12, 20, 22, 28, 33, 38), this cell type provides an ideal in vitro system to dissociate the effects of hypoxia per se from those of various hypoxia-induced vasoactive and/or growth factors, as well as hemodynamic, neural, and hormonal influences, on gene expression in lung. The present study focused on the effects of fibroblast growth factor (FGF)-1, FGF-2, and platelet-derived growth factor (PDGF)-BB on ET-AR gene expression in PASMCs. Our data demonstrated that the hypoxia responsive tyrosine kinase receptor-associated growth factors FGF-1, FGF-2, and PDGF-BB, but not the G protein-coupled receptor-associated growth factors angiotensin (ANG) II or ET-1, or hypoxia per se, upregulate ET-AR mRNA expression in PASMC via a mitogenactivated protein (MAP) kinase-mediated pathway.

Address for reprint requests and other correspondence: Y.-F. Chen, 1008 Zeigler Research Bldg., Univ. of Alabama at Birmingham, UAB Station, Birmingham, AL 35294 (E-mail: [email protected]).

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.

tyrosine kinase receptor; pulmonary arterial smooth muscle cells; signal transduction pathway; gene expression

(20, 22) have shown that exposure of rats or mice to normobaric hypoxia is associated with increased expression of endothelin

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GROWTH FACTORS UPREGULATE PULMONARY ET-A RECEPTOR

MATERIALS AND METHODS

Animals and cell culture. All animals used in this study were cared for and handled according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH) [Department of Health, Education, and Welfare Publication No. (NIH) 96-01, revised 1996]. PASMCs were initially cultured in DMEM (GIBCO BRL, Rockville, MD) with 10% heat-inactivated low endotoxin fetal bovine serum (FBS) (GIBCO BRL), 2 mM glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin at 37°C in a humidified atmosphere containing 95% air-5% CO2 and were passaged by harvesting with trypsin-EDTA and seeding in 6-cm cell culture dishes. Passaged cells were grown in DMEM with 10% FBS. PASMCs were used for experiments between passages 4 and 8. To confirm the characteristics of SMCs in culture, immunohistochemical staining of ␣-actin was performed by using specific ␣-actin antibody and peroxidase-labeled anti-IgM or anti-IgG antibodies. Before each study, PASMCs were grown to 100% confluence and then made quiescent by placing them in medium containing 0.1% FBS for 24 h. To examine the effects of hypoxia on ET-AR mRNA expression, PASMCs were transferred into an air-tight hypoxic chamber (model 2710 cell culture incubator, Queue Systems) containing 1% O2-5% CO2-94% N2 as previously described (7, 37). For treatment with FGF-1 or FGF-2, heparin was added to the medium at a final concentration of 5 U/ml. Heparin protects FGF-1 and FGF-2 against enzymatic digestion and promotes FGF-receptor binding (42). For the study of signal transduction pathways, PASMCs were pretreated with selective inhibitors for 45 min before addition of growth factors to the medium and incubation for an additional 24 h. RNA extraction and Northern blot analysis. PASMCs were lysed, and total RNA was extracted by the TRIZOL total RNA isolation reagent (GIBCO BRL, Life Technologies). Northern analysis was performed by using a 32P-labeled selective cDNA probe for ET-AR, which had been generated in our laboratory by reverse transcription followed by the DNA PCR using lung RNA as the template, as previously described (20, 22). In studies carried out under normoxic conditions, RNA loading was quantitated by stripping 32P-labeled ET-AR cDNA off the membrane and rehybridizing with the control probe, a 32P-labeled GAPDH probe (36, 37). Because GAPDH is a hypoxia-responsive gene (13), a 32P-labeled 18S rRNA oligonucleotide (5⬘-ACGGTATCTGATCGTCTTCGAACC-3⬘) was used as the control probe to normalize data from studies performed under hypoxic conditions. Autoradiographic signals were scanned with an optical densitometer (model GS670 Imaging Densitometer, Bio-Rad, Hercules, CA). To estimate steady-state ET-AR mRNA levels, ET-AR mRNA/18S rRNA ratios were determined by dividing the absorbance corresponding to the ET-AR cDNA probe hybridization by the absorbance corresponding to the 18S rRNA probe hybridization. The results were expressed as the ratios of ET-AR mRNA to 18S rRNA. Quantitation of FGF-1 (Western analysis) and FGF-2 (ELISA) protein levels. FGF-1 in lung was measured by a Western blot procedure developed in our laboratory. Briefly, 1.5 g of frozen lung tissue was homogenized in 6 ml of 9 M urea. The homogenate was diluted to 6 M urea with TrisEDTA buffer (TE; 20 mM Tris, 1 mM EDTA, pH 8.0) and mixed with 1-ml heparin-sepharose beads by rotating the mixture at room temperature for 1 h. The beads were pelleted at low-speed centrifugation and washed with TE (3 times) until the supernatant appeared clear. The beads were loaded onto a column and washed with TE (3 volumes) and 6 M urea J Appl Physiol • VOL

(3 volumes). FGF-1 bound to heparin was then eluted with 300 ␮l of 10% SDS in water followed by 3 ml of TE. The eluate was precipitated with four volumes of acetone at ⫺20°C overnight and then centrifuged at 3,000 g for 30 min. The acetone was decanted, and the pellet was resuspended in SDS-PAGE loading buffer and then subjected to electrophoresis on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked for 2 h in 1% casein (Hammarsten-prepared), 0.05% Tween 20, and 0.05% azide in PBS. Western blot analysis was performed by using monoclonal anti-FGF-1 primary antibodies (Santa Cruz) and a horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad). Immunoreactive bands were visualized by using enhanced chemiluminescence reagent (Amersham). Autoradiograms exposed in the linear range of film density were scanned by using a densitometer (model GS-670 imaging densitometer, Bio-Rad). FGF-2 in lung was measured by ELISA with FGF-2 immunoassay kit (R&D Systems, Minneapolis, MN). Briefly, 0.5 g of lung tissue was homogenized in 3 ml of lysis buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 ␮g/ml leupeptin, 10 ␮g aprotinin, 50 mM Tris 䡠 HCl, pH 7.5). Homogenate was centrifuged at 3,000 g at 4°C for 30 min. The supernatant was used for FGF-2 ELISA. The sensitivity of the FGF-2 ELISA was 10 pg/assay tube. Western blot analysis for mitogen-activated protein kinase kinase, ERK1/2, and Akt. To evaluate the effects of FGF-1 and ANG II on activities of the MAP or ERK-mitogen-activated protein kinase kinase (MEK)-ERK1/2 pathway, and the phosphatidylinosital 3 kinase (PI3K)-protein kinase B (Akt) pathway, as well as selectivities of the MEK1/2 inhibitor U-0126 and the PI3K inhibitor LY-294002, Western blot analysis for MEK1/2, phosphorylated p44, and p42 MAP kinases (ERK1 and ERK2, respectively), and total and phosphorylated Akt was performed by using the Phospho-ERK1/2 pathway sample kit and PhosphoPlus Akt antibody kit (Cell Signaling Technology, Beverly, MD), respectively, as previously described (37). PASMCs were made quiescent by placing them in medium containing 0.1% FBS for 24 h. For the study of signal transduction pathways, PASMCs were pretreated with the selective MEK1/2 inhibitor U-0126 (5 ␮M) or the selective PI3K inhibitor LY-294002 (5 ␮M) for 45 min before addition of FGF-1 (10 ng/ml) or ANG II (100 nM) to the medium and incubation for an additional 5, 10, or 15 min. After treatment with FGF-1 or ANG II, cells were lysed by the addition of 0.5 ml of ice-cold lysis buffer containing (in mM) 50 NaCl, 50 NaF, 50 sodium pyrophosphate, 5 EDTA, 5 EGTA, 2 Na3VO4, 0.5 phenylmethylsulfonyl fluoride, and 10 HEPES at pH 7.4 along with 0.1% Triton X-100 and 10 ␮g/ml leupeptin, followed by immediate freezing on ethanol or dry ice. The cell lysates were thawed on ice, scraped, sonicated, and centrifuged at 14,000 g at 4°C for 30 min. Cell lysates (25 ␮g of protein) were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked for 2 h in 1% casein (Hammarsten-prepared), 0.05% Tween 20, and 0.05% azide in PBS. Western blot analysis was performed by using anti-phospho-MEK1/2, anti-phospho-ERK1 and anti-phospho-ERK2, and anti-Akt and anti-phospho-Akt-specific primary antibodies, and a horseradish peroxidase-conjugated goat anti-rabbit IgG. Immunoreactive bands were visualized by using enhanced chemiluminescence reagent (Amersham). Autoradiograms exposed in the linear range of film density were scanned by using a densitometer (model GS-670 imaging densitometer, Bio-Rad).

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Nuclear runoff assay. To evaluate the effect of FGF-1 on ET-AR gene transcriptional rate in PASMCs, nuclear runoff assays were performed with nuclei isolated from FGF-1 (24 h) or vehicle-treated PASMCs, as described previously (23). Linearized ET-AR and ␣-tubulin cDNAs were anchored on 0.45-␮m nitrocellulose membranes. The HindIII digested ␭DNA was used as a negative control. The harvested PASMCs were lysed in NP-40 lysis buffer [10 mM Tris 䡠 Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% (vol/vol) NP-40] by vortexing and centrifuging at 500 g at 4°C for 5 min. Nuclear pellets were resuspended in 200 ␮l of glycerol storage buffer [50 mM Tris 䡠 Cl, pH 8.3, 40% (vol/vol) glycerol, 5 mM MgCl2, 0.1 mM EDTA]. Equal amounts of nuclei (equivalent to 300 ␮g of DNA) were incubated with nucleotides and [␣-32P]UTP at 30°C for 30 min. Total RNA was isolated by using TRIZOL reagent and dissolved in N-tris(hydroxymethy)methyl-2-aminoethanesulfonic acid (TES) solution [10 mM TES, pH 7.4, 10 mM EDTA, 0.2% (wt/vol) SDS], cleaned of free [32P]UTP, and then hybridized to linearized ET-AR cDNA immobilized on nitrocellulose filter. Hybridization was carried out with equal amounts of 32P-labeled RNA from nuclei of FGF-1- or vehicletreated PASMCs at 65°C for 36 h. Filter was then washed and exposed to a phosphor imaging screen for 3 days and read by a Cyclone Storage Phosphor System (Packard BioScience, Meriden, CT). ET-AR mRNA levels were standardized to ␣-tubulin mRNA to correct for variations in loading. Reagents. Human recombinant FGF-1, FGF-2, PDGF-BB, vascular endothelial growth factor, and ANG II were purchased from Sigma Chemical (St. Louis, MO). Rat ET-1 and atrial natriuretic peptide (ANP) were purchased from Peninsula Laboratories (Belmont, CA). MAP kinase/MEK inhibitor U-0126, the PI3K inhibitor LY-294002, the PKA inhibitor H-89, the PKC inhibitor calphostin C, and the adenylate cyclase inhibitor SQ-22536 were purchased from Calbiochem-Novabiochem (San Diego, CA). The FGF-1 receptor tyrosine kinase inhibitor PD-166866 was provided by ParkeDavis Pharmaceutical Research, Division of Pfizer (30). Statistical analysis. Results were expressed as means ⫾ SE. Statistical analyses were carried out by using the SigmaStat package (Jandel Scientific Software, San Rafael, CA) on a personal computer. Statistical comparisons of mRNA levels were performed with unpaired t-test or one-way ANOVA. If ANOVA results were significant, a post hoc comparison among groups was performed with the NewmanKeuls test. Differences were reported as significant if the P value was of ⬍0.05. RESULTS

Effects of hypoxia and FBS on ET-AR expression. Exposure of growth-arrested PASMCs to 1% oxygen (PO2 ⫽ 20–30 Torr in media) for 24 h did not alter ET-AR mRNA expression but was associated with increased GAPDH mRNA levels (Fig. 1). The ET-AR receptor cDNA probe detected two bands of ⬃5.2 and 4.2 kb. The sizes of these bands were identical to those of authentic ET-AR mRNA previously described (16, 20, 24). The densities of these two bands were identically regulated by hypoxia and FGF-1 (see Figs. 1, 4, 5, and 8), suggesting that the two gene transcripts are regulated similarly. Whether the different sizes of the ET-AR transcripts represent alternative splicing or specific degradation of ET-AR mRNA is currently not understood. No significant cell death [as assessed by trypan blue (0.4%) exclusion] in PASMCs exposed to hypoxia or normoxia was observed. In contrast, addiJ Appl Physiol • VOL

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Fig. 1. Effects of 24-h exposure to hypoxia (1% O2) on steady-state endothelin-A receptor (ET-AR) mRNA expression in pulmonary artery smooth muscle cells (PASMCs). Controls were PASMCs cultured in 0.1% FBS medium for 24 h and exposed to room air for an additional 24 h. Numbers in parentheses are the numbers of plates contributing data to each group. Northern blot analysis was carried out with 15 ␮g of total RNA extracted from each plate. mRNA from each plate was quantitated individually. A representative Northern blot is shown at top. The Northern blot membrane was probed with ET-AR and GAPDH cDNA and 18S rRNA oligonucleotide sequentially. ET-AR and GAPDH mRNA data were normalized to the 18S rRNA to allow for variations in RNA loading. Results are means ⫾ SE. *P ⬍ 0.05 vs. respective air control group by unpaired t-test.

tion of 10% FBS to growth-arrested PASMCs resulted in a significant increase (⬃80%) in steady-state ET-AR mRNA levels within 24 h (Fig. 2), suggesting that growth factors and cytokines in FBS might contribute to the upregulation of ET-AR mRNA expression in PASMCs. Effects of growth factors on ET-AR expression. To determine whether growth factors that activate membrane tyrosine kinase receptors contribute to the upregulation of ET-AR gene expression, PASMCs were treated with FGF-1, FGF-2, PDGF-BB, or vascular endothelial growth factor (Fig. 2). Quantitative Northern blot analysis demonstrated that ET-AR mRNA expression increased in PASMCs treated with FGF-1 (10 ng/ml ⫽ 0.67 nM), FGF-2 (10 ng/ml ⫽ 0.60 nM), or PDGF-BB (20 ng/ml ⫽ 0.8 nM) for 24 h (Fig. 2). Vascular endothelial growth factor at a concentration of 30 ng/ml (0.72 nM for 24 h) did not alter ET-AR mRNA expression in PASMCs (Fig. 2). To determine whether growth factors that activate membrane G protein-coupled receptors alter ET-AR gene expression, PASMCs were treated with ET-1 or ANG II under conditions similar to those described for the tyrosine kinase receptor-activating factors above. ET-1 (10⫺7 M or 0.25 ␮g/ml for 24 h) or ANG II (10⫺7 M or 0.1 ␮g/ml for 24 h) did not affect ET-AR mRNA levels in PASMCs (Fig. 2).

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Fig. 2. Effects of 24-h exposure to hypoxia (1% O2), FBS (10%), human recombinant fibroblast growth factor (FGF)-1 (10 ng/ml), human recombinant FGF-2 (10 ng/ml), platelet-derived growth factor (PDGF)-BB (20 ng/ml), vascular endothelial growth factor (VEGF; 30 ng/ml), atrial natriuretic peptide (ANP; 10⫺6 M), endothelin (ET)-1 (10⫺7 M), or ANG II (10⫺7 M) on steadystate ET-AR mRNA expression in PASMCs. Controls were PASMCs cultured in 0.1% FBS medium for 24 h and exposed to room air for an additional 24 h. Northern blot analysis was carried out with 15 ␮g of total RNA extracted from each plate, which was quantitated individually. mRNA loading was normalized to the 18S rRNA. Numbers in parentheses are the numbers of plates contributing data to each group. Results are means ⫾ SE. *P ⬍ 0.05 vs. control group by 1-way ANOVA.

To determine whether cGMP-associated antiproliferative factors alter ET-AR gene expression, PASMCs were treated with ANP under conditions similar to those described for the tyrosine kinase receptor-activating factors above. ANP (10⫺6 M or 3 ␮g/ml for 24 h) did not affect the ET-AR mRNA levels in PASMCs (Fig. 2). Effects of hypoxic exposure on FGF-1 and FGF-2 protein levels in rat lung. To determine whether hypoxic exposure increases FGF-1 and FGF-2 production in vivo, the effects of hypoxia (10% O2, 1 atm for 2 wk) on lung FGF-1 and FGF-2 protein levels were measured by using Western blot analysis and ELISA, respectively. Two-week hypoxic exposure significantly increased FGF-1 and FGF-2 protein levels in rat lung (Fig. 3). Heart and kidney FGF-2 levels (by ELISA) were not significantly changed in response to hypoxic exposure (data not shown). These data indicate that FGF-1 and FGF-2 are hypoxia-responsive growth fac-

Fig. 3. Effects of hypoxic exposure (10% O2, 1 atm for 2 wk) on FGF-1 and FGF-2 protein levels in lung of rats adapted to hypoxia. A: Western analysis for FGF-1 was carried out with 1.5 g of pooled lung tissue from air control and hypoxic rats (n ⫽ 3 rats for each lane). Std, standard human recombinant FGF-1. B: ELISA for FGF-2 was carried out with 2 mg of total protein extracted from each lung. Data were normalized to allow for variations in total protein content in each sample and expressed as FGF-2-to-protein ratios. FGF-2 content in each rat was quantitated individually. Numbers in parentheses are the numbers of rats contributing data to each group. Results are means ⫾ SE. *P ⬍ 0.05 vs. air control group by unpaired Student’s t-test. J Appl Physiol • VOL

tors whose expression is increased selectively in lung under hypoxic conditions. This is the first time that FGF-1 protein has been measured in lung of hypoxiaadapted animals with the use of Western blot analysis. Because the effects of FGF-1 and FGF-2 are mediated through the same membrane tyrosine kinase receptor and the FGF-2 molecule lacks a secretory signal peptide sequence and is presumably not secreted, we tested FGF-1 only in the following experiments. Dose- and time-dependent effects of FGF-1 on ET-AR expression. ET-AR mRNA expression increased in a dose-dependent fashion (dose range of 0.1–20 ng/ml) in PASMCs treated with FGF-1 for 24 h (Fig. 4A). The threshold concentration was 1 ng/ml (⬃67 pM), and largest effect was observed (a 2.7-fold increase) at the maximal dose of 20 ng/ml. The time course for enhancement of ET-AR gene expression with FGF-1 was examined at a concentration of 10 ng/ml (Fig. 4B). Significant increases in ET-AR mRNA levels in PASMCs were observed after 12 h of FGF-1 treatment. ET-AR mRNA levels increased progressively over the 24 h period of FGF-1 exposure. Effects of FGF-1 receptor inhibition on FGF-1-induced upregulation of ET-AR expression. PASMCs were pretreated with PD-166866 (5 ␮M), a novel potent and selective inhibitor of FGF-1-receptor tyrosine kinase (30), for 45 min before FGF-1 (10 ng/ml) was added to the medium for an additional 24 h of incubation. PD-166866 completely blocked the FGF-1 induced upregulation of ET-AR mRNA expression (Fig. 5A). These data provide evidence that the upregulation of ET-AR gene expression by FGF-1 in PASMCs is mediated through the activation of FGF-1 receptor. Recovery of ET-AR expression after removal of FGF-1 stimulation. Quiescent PASMCs were pretreated with FGF-1 (10 ng/ml) for 24 h to upregulate ET-AR mRNA expression. The FGF-1-containing medium was replaced with fresh 0.1% FBS medium, and cells were

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phostin C (1 ␮M), and the adenylate cyclase inhibitor SQ-22536 (100 ␮M) did not alter the stimulatory effect of FGF-1 on ET-AR mRNA expression. Western blot analysis confirmed that FGF-1 or ANG II significantly increased activation of MAP kinase and PI3K within 5 min in PASMCs (Fig. 7). The FGF-1 or ANG II-stimulated phosphorylation of ERK1/2 was blocked by pretreatment with the selective MEK inhibitor U-0126 (Fig. 7A), and the FGF-1 or ANG II-stimulated phos-

Fig. 4. Dose- (A) and time-dependent (B) effects of FGF-1 on regulation of steady-state ET-AR mRNA expression in PASMCs. Growtharrested PASMCs were exposed to different concentrations of FGF-1 for 24 h (A) and to 10 ng/ml FGF-1 for 1, 6, 12 and 24 h before being harvested (B). Controls were PASMCs cultured in 0.1% FBS medium for 24 h and exposed to room air for an additional 24 h. A representative Northern blot is shown in A and B at top. The Northern analysis was carried out with 15 ␮g of total RNA extracted from each plate, which was quantitated individally. mRNA loading was normalized to the 18S rRNA. Numbers in parentheses are the numbers of plates contributing data to each group. Results are means ⫾ SE. *P ⬍ 0.05 vs. control group by 1-way ANOVA.

incubated for an additional 1, 3, 6, and 12 h before being harvested for analysis. ET-AR mRNA levels in PASMCs completely recovered within 6 h after FGF-1 was removed from the culture medium (Fig. 5B). The rapid recovery of ET-AR mRNA levels suggests that FGF-1 could be a physiological regulator of ET-AR gene expression in PASMCs. Effects of signal transduction pathway inhibitors on FGF-1-induced upregulation of ET-AR expression. PASMCs were pretreated with inhibitors for 45 min before FGF-1 (10 ng/ml) was added to the medium for an additional 24 h of incubation. U-0126 (5 ␮M), a MEK inhibitor, completely blocked the FGF-1-induced upregulation of ET-AR mRNA expression (Fig. 6). In contrast, the PI3K inhibitor LY-294002 (5 ␮M), the PKA inhibitor H-89 (1 ␮M), the PKC inhibitor calJ Appl Physiol • VOL

Fig. 5. A: effects of PD-166866, a selective inhibitor of the FGF-1receptor tyrosine kinase, on upregulation of ET-AR expression in PASMCs by FGF-1. Control was growth-arrested PASMCs grown in 0.1% FBS medium. Groups 2 and 3 were PASMCs treated with FGF-1 (10 ng/ml) or PD-166866 (5 ␮M), respectively, for 24 h. Group 4 was PASMCs pretreated with PD-166866 (5 ␮M) for 45 min before FGF-1 (10 ng/ml) was added to the medium, and an additional 24 h incubation was carried out before harvesting for analysis. B: recovery of ET-AR mRNA levels in PASMCs after FGF-1 (10 ng/ml for 24 h) was removed from the culture medium (groups 3–6). Control was growth-arrested PASMCs cultured in 0.1% FBS; FGF-1 group was PASMCs treated with FGF-1 (10 ng/ml) for 24 h. A representative Northern blot is shown in A and B at top. The Northern analysis was carried out with 15 ␮g of total RNA extracted from each plate, which was quantitated individally. mRNA loading was normalized to the 18S rRNA. Numbers in parentheses are the numbers of plates contributing data to each group. Results are means ⫾ SE. *P ⬍ 0.05 vs. control group; #P ⬍ 0.05 vs. FGF-1 group by 1-way ANOVA.

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Fig. 6. Effects of signaling pathway inhibitors on the upregulation of ET-AR expression in PASMCs by FGF-1. Control was growth-arrested PASMCs cultured in 0.1% FBS medium. Experimental groups were PASMCs pretreated with mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) inhibitors U-0126 (5 ␮M), phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY-294002 (5 ␮M), PKA inhibitor H-89 (1 ␮M), PKC inhibitor calphostin C (1 ␮M), or adenylate cyclase inhibitor SQ-22536 (100 ␮M) for 45 min before FGF-1 (10 ng/ml) was added to the medium, and an additional 24 h incubation was carried out before harvesting for analysis. The Northern analysis was carried out with 15 ␮g of total RNA extracted from each plate, which was quantitated individally. mRNA loading was normalized to the 18S rRNA. Numbers in parentheses are the numbers of plates contributing data to each group. Results are mean ⫾ SE. *P ⬍ 0.05 vs. control group; #P ⬍ 0.05 vs. FGF-1 group by 1-way ANOVA.

Fig. 7. A: effects of the selective MEK1/2 inhibitor U-0126 on FGF-1 or ANG II-stimulated MAP and PI3-kinases in PASMCs. C, control: PASMCs cultured in 0.1% FBS medium. Growth-arrested PASMCs were stimulated with FGF-1 (10 ng/ml) or ANG II (100 nM) for 5, 10, or 15 min without (⫺) or with (⫹; 10 min only) pretreatment with U-0126 (5 ␮M, 45 min). Cell lysates (25 ␮g) were size fractionated by SDS-PAGE, and Western blot analysis was performed with antiphospho-MEK1/2, anti-phospho-ERK1/2, or anti-phospho-Akt specific antibodies. U-0126 selectively blocked the FGF-1- or ANG IIinduced phosphorylation of ERK1/2 but not the phosphorylation of MEK1/2 or Akt. B: effects of the selective PI3-kinase inhibitor LY-294002 on FGF-1- or ANG II-stimulated MAP and PI3-kinases in PASMCs. C, Control: PASMCs cultured in 0.1% FBS medium. The growth-arrested PASMCs were stimulated with FGF-1 (10 ng/ml) or ANG II (100 nM) for 5, 10, or 15 min without (⫺) or with (⫹; 10 min only) pretreatment with LY-294002 (5 ␮M, 45 min). Cell lysates (25 ␮g) were size fractionated by SDS-PAGE, and Western blot analysis was performed with anti-phospho-Akt-, anti-Akt-, or anti-phosphoERK1/2-specific antibodies. LY-294002 selectively blocked the FGF-1 or ANG II-induced phosphorylation of Akt but not the phosphorylation of ERK1/2.

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phorylation of Akt was blocked by pretreatment with the selective PI3K inhibitor (Fig. 7B). U-0126 did not block FGF-1 or ANG II-stimulated phosphorylation of MEK1/2 or Akt, and LY-294002 did not block FGF-1 or ANG II-stimulated phosphorylation of ERK1/2, indicating the selectivity of these signaling pathway inhibitors. Together, these results indicate that the stimulatory effects of FGF-1 on ET-AR mRNA gene expression are mainly mediated through the activation of MAP kinase. Effects of transcriptional and translational inhibitors on FGF-1-induced upregulation of ET-AR expression. PASMCs were pretreated with actinomycin D (5 ␮M), a transcriptional inhibitor, or cycloheximide (30 ␮g/ml), a protein synthesis inhibitor, for 45 min before FGF-1 (10 ng/ml) was added to the medium for an additional 24 h of incubation. Actinomycin D (data not shown) and cycloheximide (Fig. 8A) alone decreased ET-AR mRNA expression below baseline and completely blocked the FGF-1-induced upregulation of ET-AR mRNA expression. These results indicate that both transcription and translation are essential to maintain basal and FGF-1 stimulated ET-AR gene expression. Transcriptional rate of the ET-AR gene was significantly increased in FGF-1-treated (10 ng/ml for 24 h) PASMCs compared with control PASMCs incubated in medium with 0.1% FBS (Fig. 8B). ET-AR gene transcription was increased ⬃3.8-fold relative to an ␣-tubulin control in nuclei derived from FGF-1-treated PASMCs. This relative increase in transcription paralleled the increase in steady-state ET-AR mRNA levels in PASMCs treated with FGF-1 for 24 h. DISCUSSION

FGF-1, FGF-2, and PDGF-BB are hypoxia-responsive growth factors whose biological actions are mediated mainly through the tyrosine kinase receptor Ras-Raf-MEK-MAP kinase-transcriptional regulator pathway. The present study is the first to show that expression of FGF-1 and FGF-2 increases in lung of rats adapted to hypoxia and that FGF-1, FGF-2, and

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Fig. 8. A: effects of cycloheximide (CHX), a protein synthesis inhibitor, on FGF-1-induced increases in ET-AR mRNA expression in PASMCs. Groups 2 and 3 were growth-arrested PASMCs treated with FGF-1 (10 ng/ml) or CHX (30 ␮g/ml), respectively, for 24 h. Group 4 was PASMCs pretreated with CHX (30 ␮g/ml) for 45 min before FGF-1 (10 ng/ml) was added to the medium, and an additional 24-h incubation was carried out before harvesting for analysis. mRNA loading was normalized to the 18S rRNA. Numbers in parentheses are the numbers of plates contributing data to each group. B: transcriptional rate of ET-AR and ␣-tubulin gene measured by nuclear runoff assay using nuclei isolated from PASMCs treated with FGF-1 (10 ng/ml) or vehicle for 24 h. Results are means ⫾ SE. *P ⬍ 0.05 vs. respective control groups; #P ⬍ 0.05 vs. respective FGF-1 group by 1-way ANOVA.

PDGF-BB stimulate the expression of ET-AR mRNA in PASMCs. FGF-1 upregulated the expression of ET-AR mRNA in PASMCs in a dose- and time-dependent manner. The rapid increases in steady-state ET-AR mRNA levels observed in our study in response to low concentrations of FGF-1 in vitro suggest that this growth factor could be a physiological regulator of ET-AR gene expression in PASMCs. Studies using inhibitors of the FGF-1 tyrosine kinase receptor (FGFR-1) and signal transduction pathways indicate that the stimulatory effects of FGF on ET-AR mRNA expression are mediated through FGFR-1 receptors and activation of MAP kinase. In contrast, hypoxia per se, the G protein-coupled receptor growth factors ET-1 and ANG II, and the cGMP-associated antiproliferative factor ANP did not stimulate ET-AR mRNA expression in PASMCs. Upregulation of ET-AR mRNA expression by FGF-1 was totally blocked by the transcription inhibitor actinomycin D and the protein synJ Appl Physiol • VOL

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thesis inhibitor cycloheximide, indicating that FGF-1 stimulates ET-AR gene expression through a transcriptional mechanism in PASMCs. These results suggest that FGF-1 may play an important role in the signal transduction pathway linking hypoxia to altered ET-AR expression in lung. Because ET-AR is the predominant receptor subtype that mediates the contractile effects of ET-1 in lung, our present finding that FGF-1 induces increased expression of ET-AR in PASMCs is consistent with a role for FGF-1 signaling via a FGFR-1-dependent mechanism in the pathogenesis of hypoxia-induced pulmonary vasoconstriction and hypertension. Hypoxia mediates acute pulmonary vasoconstriction, chronic pulmonary hypertension, and pulmonary vascular remodeling (8, 27). We have previously reported that ET-1 released from pulmonary vascular endothelium and ET-AR in lung are important mediators of these processes (8, 27). We have demonstrated selective increases in ET-1 gene expression in lung of rats exposed to short-term and chronic normobaric hypoxia; that receptors for ET in lung, both ET-AR and ET-BR, are upregulated under hypoxic conditions in the presence of increased concentrations of their agonist, ET-1 (20, 22); and that hypoxic pulmonary vasoconstriction and vascular remodeling can be prevented and reversed by administration of selective ET-AR, but not selective ET-BR, antagonists (7, 10, 39). In the present study, we used PASMCs as a model system to study the regulation of ET-AR gene expression during hypoxia. Our results indicate that hypoxia per se does not alter ET-AR mRNA levels in PASMCs in vitro, suggesting that the response of ET-AR gene expression in lung of hypoxia-exposed subjects in vivo might not be mediated directly by hypoxia but by alterations in tissue levels of hypoxia-sensitive growth factors, such as FGFs and PDGFs. The FGFs (at least isoforms FGF-1, -2, and -7) and PDGFs (PDGF-AB and -BB) are expressed in lung and play important roles in diverse aspects of pulmonary development and growth, including lung epithelial cell, vascular SMC, and myofibroblast proliferation, differentiation, and angiogenesis, as well as adaptation to hypoxic environments (1, 25, 32). Using RT-PCR technology, we recently demonstrated that cultured PASMCs express FGFR1-␤, but not FGFR1–1␣, FGFR2-␣, or FGFR2-␤ receptors (data not shown). PDGF receptors (both ␣ and ␤ subtypes) are also expressed in cultured lung myofibroblasts, and smooth muscle and endothelial cells derived from mediumsized and small arteries of rat lung (4, 6). Binding of FGFs or PDGFs to their receptors results in autophosphorylation of the tyrosine residues in the FGF (e.g., FGFR1-␤) or PDGF (e.g., PDGF-␣ and -␤) receptor kinase domain, respectively. This triggers a cascade of phosphorylation events involving sequential activation of Ras-Raf-MEK-ERK-Elk (34), resulting in the regulation of cell proliferation, differentiation, and migration (5, 9, 18, 32). It has been hypothesized that these growth factors may contribute to hyperproliferation of

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PASMCs and muscularization of pulmonary vasculature in hypoxia-adapted animals (3, 11, 29). Hypoxia has been shown to stimulate expression of FGF-2 and PDGF-BB in lung (1, 3, 11, 29, 41). Increased FGF-2 transcription has been demonstrated in smooth muscle and adventitial tissue of rat pulmonary arteries after 14 days of hypoxic exposure (29). Furthermore, Ambalavanan et al. (1) have shown that FGF-2 in cultured neonatal porcine PASMC increases with hypoxia (1% O2). Hypoxia also increases levels of PDGF-B mRNA in lung parenchyma of rats exposed to normobaric hypoxia (3) and induces transcription of the PDGF-B gene in cultured systemic and pulmonary vascular endothelial cells (11). The present study is the first to show that FGF-1 protein levels are increased in lung under hypoxic conditions. Furthermore, our previous studies have shown that exposure of rats or mice to hypoxia is associated with selective increased expression of ET-1 and ET-AR in lung and main pulmonary artery (20, 22). The relationship between overexpression of FGF and PDGF and upregulation of ET-AR in lung under hypoxic conditions suggests that these hypoxia-responsive growth factors may potentiate ET-1 and/or ET-AR-induced pulmonary vasoconstriction, remodeling, and hypertension in animals during hypoxic exposure. Multiple intracellular signaling transduction pathways are activated by FGF (2, 35). Activation of the Ras-Raf-MEK-ERK-ELK pathway is necessary for the mitogenic activity of FGF (26), and FGFs stimulate the translocation of PKC isoforms toward perinuclear region and into the nucleus in cultured vascular SMCs (14). The adenylate cyclase-cAMP-PKA system is involved in the FGF-2-activated transcription through an FGF-inducible response element (31). Our present data demonstrated that the stimulatory effect of FGF-1 on ET-AR mRNA expression was completely blocked by the MEK inhibitor U-0126, providing evidence that the upregulation of ET-AR by FGF-1 is mediated through activation of the MAP-kinase pathway. The finding that the PKA inhibitor H-89, the PKC inhibitor calphostin C, and the PI3K inhibitor LY-294002 did not block the stimulatory effects of FGF-1 on ET-AR expression indicates that the PKA, PKC, and PI3K pathways did not mediate the enhancement of ET-AR gene expression. In addition, the adenylate cyclase inhibitor SQ-22536 did not block the FGF-1-induced enhancement of ET-AR gene expression, providing evidence that cAMP was not involved in this signal transduction pathway. We did not measure PKA, PKC, or adenylate cyclase activity in PASMCs treated with FGF-1 in the present study. However, the selectivities of the PKA inhibitor H-89 (1 ␮M), the PKC inhibitor calphostin C (1 ␮M), and the adenylate cyclase inhibitor SQ-22536 (100 ␮M) have been reported by the manufacturers and in numerous published papers (37). To test whether induction of ET-AR mRNA expression by FGF-1 is dependent on increased transcription of the ET-AR gene and/or synthesis of new protein, strategies employing the RNA polymerase inhibitor actinomycin D and the protein synthesis inhibitor cyJ Appl Physiol • VOL

cloheximide were utilized. Actinomycin D or cycloheximide administered alone decreased ET-AR mRNA expression below baseline and completely blocked FGF1-induced upregulation of ET-AR mRNA expression. These results suggest that FGF-1 stimulates ET-AR expression at the transcriptional level and that FGF1-mediated ET-AR mRNA induction requires de novo synthesis of intermediate regulatory protein(s). The molecular mechanism(s) needs further investigation. In summary, this study provides evidence that the increased expression of ET-AR mRNA observed under hypoxic conditions in lung may be mediated by the tyrosine kinase receptor-associated growth factors FGF-1, FGF-2, and PDGF-BB, whose gene expression is increased in lung of hypoxia-adapted animals. In contrast, the G protein-linked receptor-associated growth factors ET-1 and ANG II, which are also overexpressed in hypoxia-adapted lungs, do not alter ET-AR expression. We demonstrated that activation of tyrosine kinase receptors by hypoxia-responsive growth factors, but by neither hypoxia per se nor activation of G protein-coupled receptors by ET-1, increases ET-AT gene expression in PASMCs. Our results indicate that activation of FGFR-1 by FGF-1 stimulates ET-AR gene expression through a transcriptional mechanism in PASMCs and suggest that FGFs may play an important role in the signal transduction pathway linking hypoxia to altered ET-AR expression in lung. Because ET-AR is the predominant receptor subtype that mediates the contractile effects of ET in lung, our finding that FGFs induce increased expression of ET-AR in PASMCs is consistent with a role for FGF signaling via a FGFR-1-dependent mechanism in the pathogenesis of hypoxia-induced pulmonary vasoconstriction and hypertension. Our results also demonstrate that FGF-mediated upregulation of ET-AR gene expression is dependent on activation of MAP kinase in PASMCs. The mechanisms of the signal transduction and nuclear regulatory pathways involved in this tyrosine kinase receptor activation-mediated stimulation of ET-AR gene expression in PASMCs deserve further investigation. DISCLOSURES This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-44195, HL-50147, HL-45990, HL-07457, and HL-56046. REFERENCES 1. Ambalavanan N, Bulger A, and Philips JB III. Hypoxiainduced release of peptide growth factors from neonatal porcine pulmonary artery smooth muscle cells. Biol Neonate 76: 311– 319, 1999. 2. Baron W, Metz B, Bansal R, Hoekstra D, and de Vries H. PDGF and FGF-2 signaling in oligodendrocyte progenitor cells: regulation of proliferation and differentiation by multiple intracellular signaling pathways. Mol Cell Neurosci 15: 314–329, 2000. 3. Berg JT, Breen EC, Fu Z, Mathieu-Costello O, and West JB. Alveolar hypoxia increases gene expression of extracellular matrix proteins and platelet-derived growth factor-B in lung parenchyma. Am J Respir Crit Care Med 158: 1920–1928, 1998.

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