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1Department 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
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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tyrosine kinase receptor; pulmonary arterial smooth muscle cells; signal transduction pathway; gene expression
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 hypoxia-sensitive 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 mitogen-activated protein (MAP) kinase-mediated pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-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 GS-670 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 Tris-EDTA 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 (3 volumes). FGF-1 bound to heparin was then eluted with 300 µl 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 µg/ml leupeptin, 10 µg aprotinin, 50 mM Tris · 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 µM) or the selective PI3K inhibitor LY-294002 (5 µM) 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 µg/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 µg 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).
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-µm 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
· 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 µl of glycerol storage buffer [50 mM Tris
· Cl, pH 8.3, 40% (vol/vol) glycerol, 5 mM MgCl2, 0.1 mM
EDTA]. Equal amounts of nuclei (equivalent to 300 µg 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 vehicle-treated 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
Bio-Science, 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 Parke-Davis 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 Newman-Keuls test. Differences were reported as significant if the P value was of <0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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, addition 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.
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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 µg/ml for 24 h) or ANG II (10-7 M or 0.1 µg/ml for 24 h) did not affect ET-AR mRNA levels in PASMCs (Fig. 2).
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 µg/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 factors 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 hypoxia-adapted 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.
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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 µM), 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 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 µM), 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 µM), the PKA inhibitor H-89 (1 µM), the PKC inhibitor calphostin C (1 µM), and the adenylate cyclase inhibitor SQ-22536 (100 µM) 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 phosphorylation 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.
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Effects of transcriptional and translational inhibitors on FGF-1-induced upregulation of ET-AR expression. PASMCs were pretreated with actinomycin D (5 µM), a transcriptional inhibitor, or cycloheximide (30 µg/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.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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 medium-sized 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 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 µM), the PKC inhibitor calphostin C (1 µM), and the adenylate cyclase inhibitor SQ-22536 (100 µM) 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 cycloheximide were utilized. Actinomycin D or cycloheximide administered alone decreased ET-AR mRNA expression below baseline and completely blocked FGF-1-induced upregulation of ET-AR mRNA expression. These results suggest that FGF-1 stimulates ET-AR expression at the transcriptional level and that FGF-1-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.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-stimulated
upregulation of platelet-derived growth factor -receptor on rat
pulmonary myofibroblasts. Am J Respir Cell Mol Biol
20: 433-440,
1999.This article has been cited by other articles:
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P. Li, S. Oparil, W. Feng, and Y.-F. Chen Hypoxia-responsive growth factors upregulate periostin and osteopontin expression via distinct signaling pathways in rat pulmonary arterial smooth muscle cells J Appl Physiol, October 1, 2004; 97(4): 1550 - 1558. [Abstract] [Full Text] [PDF]
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