Tyrosine kinase receptor activation inhibits NPR-C in lung arterial smooth muscle cells

Ju-Zhong Sun1, Suzanne Oparil1, Pamela Lucchesi2, John A. Thompson3, and Yiu-Fai Chen1

1 Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, Department of Medicine, 2 Department of Physiology and Biophysics, and 3 Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294


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We have previously demonstrated that expression of the atrial natriuretic peptide (ANP) clearance receptor (NPR-C) is reduced selectively in the lung of rats and mice exposed to hypoxia but not in pulmonary arterial smooth muscle cells (PASMCs) cultured under hypoxic conditions. The current study tested the hypothesis that hypoxia-responsive growth factors, fibroblast growth factors (FGF-1 and FGF-2) and platelet-derived growth factor-BB (PDGF-BB), that activate tyrosine kinase receptors can reduce expression of NPR-C in PASMCs independent of environmental oxygen tension. Growth-arrested rat PASMCs were incubated under hypoxic conditions (1% O2) for 24 h; with FGF-1, FGF-2, or PDGF-BB (0.1-20 ng/ml for 1-24 h); or with ANG II (1-100 nM), endothelin-1 (ET-1, 0.1 µM), ANP (0.1 µM), sodium nitroprusside (SNP, 0.1 µM), or 8-bromo-cGMP (0.1 mM) for 24 h under normoxic conditions. Steady-state NPR-C mRNA levels were assessed by Northern blot analysis. FGF-1, FGF-2, and PDGF-BB induced dose- and time-dependent reduction of NPR-C mRNA expression within 1 h at a threshold concentration of 1 ng/ml; hypoxia, ANG II, ET-1, ANP, SNP, or cGMP did not decrease NPR-C mRNA levels in PASMCs under the above conditions. Downregulation of NPR-C expression by FGF-1, FGF-2, and PDGF-BB was inhibited by the selective FGF-1 receptor tyrosine kinase inhibitor PD-166866 and mitogen-activated protein/extracellular signal-regulated kinase inhibitors U-0126 and PD-98059. These results indicate that activation of tyrosine kinase receptors by hypoxia-responsive growth factors, but neither hypoxia per se nor activation of G protein-coupled receptors, inhibits NPR-C gene expression in PASMCs. These results suggest that FGF-1, FGF-2, and PDGF-BB play a role in the signal transduction pathway linking hypoxia to altered NPR-C expression in lung.

fibroblast growth factor; platelet-derived growth factor-BB; hypoxia; pulmonary; signal transduction


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INTRODUCTION
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SYNTHESIS AND RELEASE OF atrial natriuretic peptide (ANP) in cardiac atria and ventricles are increased under stressful conditions such as pulmonary hypertension, congestive heart failure, and hypoxia (11, 25). ANP binds to receptors in the pulmonary circulation (12, 19), relaxing preconstricted pulmonary arteries (31) and reducing pulmonary artery pressure (12, 16-19). Natriuretic peptide receptors of the A and B subtype (NPR-A and NPR-B, respectively) are membrane-bound guanylate cyclases that mediate most of the biological actions of ANP, including pulmonary arterial relaxation, through regulation of intracellular cGMP levels (12, 19). The NPR-C subtype, which lacks the intracellular guanylate cyclase domain and is uncoupled from cGMP (3), mediates the cellular internalization and subsequent lysosomal degradation of ANP and is thus thought to function as a clearance receptor (37). NPR-C plays a central role in modulating the biological actions of ANP by regulating its plasma and local tissue concentrations. NPR-C accounts for the overwhelming majority of ANP-binding sites in most rat tissues, including lung, kidney, and brain, as assessed by radioligand binding techniques and mRNA quantitation (19, 21). Thus NPR-C is thus termed the high-capacity natriuretic peptide clearance receptor (19, 37).

NPR-C in the pulmonary vascular bed plays an important role in ANP clearance, accounting for removal of ~50% of injected ANP in a single pass through the lung (23). Furthermore, the ANP system modulates the development of hypoxic pulmonary hypertension (16, 17). Steady-state NPR-C mRNA levels are selectively downregulated in the lung of hypoxia-adapted rats and mice in association with decreased 125I-labeled ANP binding to pulmonary tissue and increased circulating levels of ANP (21, 35). In contrast, mRNA levels of NPR-A and NPR-B are unchanged or increased during hypoxic exposure. Nuclear run-off analysis showed greatly decreased transcription of the NPR-C gene in the lung of hypoxia-exposed rats (21). Downregulation of NPR-C expression in the setting of hypoxia may contribute to an increase in plasma ANP and enhance the vasodilator effects of ANP in lung, thus modulating hypoxic pulmonary vasoconstriction/hypertension.

The present study explored the mechanism(s) of the downregulation of NPR-C gene expression in lung of hypoxia-adapted animals. We consider the alternative hypotheses that either hypoxia per se, hemodynamic alterations in the hypoxia-adapted pulmonary circulation, and/or the indirect effects of hypoxia-induced mediators such as ANP, nitric oxide (NO), ANG II, endothelin-1 (ET-1), fibroblast growth factors-1 and -2 (FGF-1 and FGF-2, respectively), and platelet-derived growth factor-BB (PDGF-BB) are responsible for reduced expression of NPR-C in the hypoxia-adapted lung (1, 5, 8, 26). Because most of the NPR-C in lung is located in pulmonary vascular smooth muscle cells (VSMCs; see Ref. 36), in vitro cultured pulmonary arterial smooth muscle cells (PASMCs) provide an ideal system to dissociate the effects of hypoxia from those of various hypoxia-induced vasoactive and/or growth factors on gene expression in lung. Accordingly, we used PASMCs cultured in vitro to test the hypothesis that hypoxia-induced growth factors, but not hypoxia per se, can suppress the NPR-C gene expression. This in vitro model system allowed us to study the response of NPR-C gene expression to hypoxia or growth factors in the absence of hemodynamic, neural, and hormonal influences. Our data demonstrated that hypoxia-responsive tyrosine kinase receptor-associated growth factors (FGF-1, FGF-2, and PDGF-BB), but not G protein-coupled receptor-associated growth factors (ANG II and ET-1) or hypoxia per se, can downregulate NPR-C mRNA expression in PASMC via a mitogen-activated protein (MAP) kinase-mediated pathway.


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Animal 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) 85-23, revised 1985]. Vascular PASMCs of male Sprague-Dawley rats were isolated by the modification of the explant technique of Ross (32). Isolated PASMCs were initially cultured in DMEM (GIBCO BRL, Rockville, MD) with 10% heat-inactivated low-endotoxin FBS (GIBCO BRL), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/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 10-cm 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 smooth muscle cells (SMCs) in culture, immunohistochemical staining of alpha -actin was performed using specific alpha -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 NPR-C mRNA expression, PASMCs were transferred to an airtight hypoxic chamber (model 2710, Cell Culture Incubator; Queue Systems) containing 1% O2-5% CO2-94% N2. For treatment with FGF-1 or FGF-2, heparin was added to the medium at a final concentration of 5 U/ml. Heparin provides protection of FGF-1 and FGF-2 against enzymatic digestion and promotes FGF receptor binding (39). For the study of signal transduction pathways, PASMCs were pretreated with inhibitors for 45 min before addition of growth factors to the medium and incubation for an additional 6 h.

RNA extraction and Northern blot analysis. PASMCs were lysed, and total RNA was extracted by the guanidine thiocyanate method. Northern analysis was performed using a 32P-labeled selective cDNA probe for NPR-C that had been generated in our laboratory by RT followed by DNA PCR using lung RNA as the template, as previously described (35). Hybridization with the NPR-C cDNA revealed three major bands of 8, 5, and 3 kb, identical to the Northern analyses previously reported for bovine and rat NPR-C (29, 34). In studies carried out under normoxic conditions, RNA loading was quantitated by stripping 32P-labeled NPR-C cDNA off the membrane and rehybridizing with the control probe, a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (35). Autoradiographic signals were scanned with an optical densitometer (model GS-670 Imaging Densitometer; Bio-Rad, Hercules, CA). To estimate steady-state NPR-C mRNA levels, NPR-C-to-GAPDH mRNA ratios were determined by dividing the absorbance corresponding to the NPR-C cDNA probe hybridization by the absorbance corresponding to the GAPDH cDNA probe hybridization. The results were expressed as the ratios of NPR-C mRNA to GAPDH mRNA. Because GAPDH is a hypoxia-responsive gene (14), a 32P-labeled 18S rRNA oligonucleotide (5'-ACGGTATCTGATCGTCTTCGAACC-3') was used as the control probe to normalize data from studies performed under hypoxic conditions.

Western blot analysis. Western blot analysis for the p42 and p44 MAP kinases [extracellular signal-regulated kinase (ERK) 2 and ERK1, respectively] was performed as previously described (22). After treatment with FGF-1 for 6 h, 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-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, and 0.05% azide in PBS. Western blot analysis was performed using anti-ERK1- and anti-ERK2-specific primary antibodies (Santa Cruz) and a horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad). Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Amersham). Autoradiograms exposed in the linear range of film density were scanned using a densitometer (model GS-670 Imaging Densitometer; Bio-Rad). Because of insufficient resolution of the ERK1 bands, only ERK2 phosphorylation was quantified. Percent ERK2 activation was defined as the autoradiographic density (measured in arbitrary units) of phosphorylated MAP kinase (p42) divided by the total autoradiographic density of both the unphosphorylated and phosphorylated MAP kinases (p42/42) × 100%.

Reagents. Human recombinant FGF-1, FGF-2, PDGF-BB, and ANG II were purchased from Sigma (St. Louis, MO). Rat ANP (28 amino acids) was purchased from Peninsula Laboratories (Belmont, CA). Human ET-1, MAP kinase/ERK kinase (MEK) inhibitors PD-98059 and U-0126, the adenylate cyclase inhibitor SQ-22536, the protein kinase C (PKC) inhibitor calphostin C, the protein kinase A (PKA) inhibitor H-89, the phosphatidylinositol 3-kinase (PI 3-kinase) kinase inhibitor LY-294002, and 8-bromo-cGMP 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 (28).

Statistical analysis. Results were expressed as means ± SE. Statistical analyses were carried out using the CRUNCH statistical package (CRUNCH Software, Oakland, CA) on an IBM 486 compatible computer. Statistical comparisons of mRNA levels were performed with the 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 <0.05.


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Effects of hypoxia and FBS on NPR-C expression. Exposure of growth-arrested PASMCs to 1% oxygen (PO2 = 20-30 mmHg in medium) for 24 h did not alter NPR-C mRNA expression (Fig. 1A). 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 reduction (~40%) in steady-state NPR-C mRNA levels within 24 h (Fig. 1B), suggesting that growth factors/cytokines in FBS might contribute to the downregulation of NPR-C mRNA expression in PASMCs.


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Fig. 1.   Effects of 24-h exposure to hypoxia (1% O2; A) and 10% FBS (B) on steady-state natriuretic peptide receptor (NPR)-C mRNA expression in pulmonary artery smooth muscle cells (PASMCs). Controls were PASMCs cultured in 0.1% FBS medium (SFM) for 24 h and exposed to room air or vehicle for an additional 24 h. Nos. in parentheses are the no. of plates contributing data to each group. Northern blot analysis was carried out with 15 µg of total RNA extracted from each plate. mRNA from each plate was quantitated individually. mRNA data were normalized by 18S rRNA (A) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (B) levels to allow for variations in RNA loading. Results are means ± SE. *P < 0.05 vs. respective control groups by unpaired t-test. Note that 18S RNA was used for normalization in the hypoxia study because GAPDH is a hypoxia-responsive gene; GAPDH was used in all studies carried out under normoxic conditions.

Effects of tyrosine kinase receptor activation on NPR-C expression. To determine whether growth factors that activate membrane tyrosine kinase receptors contribute to the downregulation of NPR-C gene expression, PASMCs were treated with FGF-1, FGF-2, and PDGF-BB. Quantitative Northern blot analysis demonstrated that NPR-C mRNA expression was decreased in a dose-dependent fashion in PASMCs treated with FGF-1 for 24 h (Fig. 2A). The threshold concentration was 1 ng/ml (~67 pM). The densities of the 8-, 5-, and 3-kb bands previously shown to hybridize with NPR-C cDNA were uniformly reduced by treatment with FGF-1, suggesting that levels of the various gene transcripts are regulated similarly. Whether the different sizes of the NPR-C transcripts represent alternative splicing or specific degradation of NPR-C mRNA is currently not understood. Similarly, steady-state NPR-C mRNA levels were reduced in a dose-dependent fashion (dose range 0.1-20 ng/ml) by incubation with FGF-2 for 24 h (Fig. 2B). The threshold dose of FGF-2 was 1 ng/ml (~60 pM); the maximal dose was 10 ng/ml. Treatment with PDGF-BB for 24 h (dose range 0.1-20 ng/ml) resulted in a similar dose-dependent reduction in steady-state NPR-C mRNA levels (Fig. 2C).


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Fig. 2.   Dose-dependent effects of human recombinant fibroblast growth factor (FGF)-1 (A), human recombinant FGF-2 (B), and platelet-derived growth factor (PDGF)-BB (C) on regulation of the steady-state NPR-C mRNA expression in PASMCs. The growth-arrested PASMCs were exposed to different concentrations of FGF-1, FGF-2, and PDGF-BB for 24 h. Controls were PASMCs cultured in 0.1% FBS medium without FGF-1, FGF-2, or PDGF-BB treatment. A representative Northern blot is shown in A-C, top. Three major NPR-C mRNA bands (~8, 5, and 3 kb) were detected on the Northern blots. Northern blot analysis was carried out with 15 µg of total RNA extracted from each plate that was quantitated individually. The mRNA loading was normalized by GAPDH mRNA. Nos. in parentheses are the no. of plates contributing data to each group. Results are means ± SE. *P < 0.05 vs. control group by 1-way ANOVA.

The temporal course for suppression of NPR-C gene expression with FGF-1 was examined at a concentration of 5 ng/ml (Fig. 3A) and with PDGF-BB at a concentration of 10 ng/ml (Fig. 3B). Reductions in NPR-C mRNA levels in PASMCs were observed as early as 1 h after FGF-1 and PDGF-BB exposure. With FGF-1 treatment, NPR-C mRNA levels decreased progressively over the 24-h period of exposure; with PDGF-BB treatment, NPR-C mRNA reached trough levels at 6 h and then slowly increased over the remaining 18 h of exposure.


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Fig. 3.   Time-dependent effects of FGF-1 (5 ng/ml; A) and PDGF-BB (10 ng/ml; B) on the regulation of steady-state NPR-C mRNA expression in PASMCs. Controls were growth-arrested PASMCs cultured in 0.1% FBS medium for 24 h without FGF-1 or PDGF-BB treatment. Experimental groups were PASMCs exposed to FGF-1 or PDGF-BB for 1, 3, 6, or 24 h before being harvested. A representative Northern blot is shown in A and B, top. Northern blot analysis was carried out with 15 µg of total RNA extracted from each plate, which was quantitated individually. The mRNA loading was normalized by GAPDH mRNA. Nos. in parentheses are the no. of plates contributing data to each group. Results are means ± SE. *P < 0.05 vs. control group by 1-way ANOVA.

Effects of G protein-linked receptor activation on NPR-C expression. To determine whether growth factors that activate membrane G protein-coupled receptors alter NPR-C gene expression, PASMCs were treated with ANG II and ET-1 under conditions similar to those described for tyrosine kinase receptor-activating factors above. At a physiological concentration (1 nM or 1.05 ng/ml for 24 h), ANG II did not affect the NPR-C mRNA levels (120 ± 15% of the baseline levels, n = 3, data not shown); at a high concentration (0.1 µM or 0.1 µg/ml for 24 h), ANG II significantly increased NPR-C mRNA levels (135 ± 9% of the baseline levels, Fig. 4); and at a pharmacological concentration (1 µM or 1.05 µg/ml for 24 h), ANG II significantly decreased (42 ± 2% of the baseline levels, n = 6, P < 0.05, data not shown) NPR-C mRNA levels in PASMCs. ET-1 at a high concentration (0.1 µM or 0.25 µg/ml for 24 h) did not affect NPR-C mRNA levels in PASMCs (Fig. 4).


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Fig. 4.   Effects of 24-h exposure to ANG II, endothelin-1 (ET-1), atrial natriuretic peptide (ANP), sodium nitroprusside (SNP), or 8-bromo-cGMP on steady-state NPR-C mRNA expression in PASMCs. Controls were growth-arrested PASMCs cultured in 0.1% FBS medium for 24 h. Northern blot analysis was carried out with 15 µg of total RNA extracted from each plate, which was quantitated individually. The mRNA loading was normalized by GAPDH mRNA. Nos. in parentheses are the no. of plates contributing data to each group. Results are means ± SE. *P < 0.05 vs. control group by 1-way ANOVA.

Effects of cGMP-associated antiproliferative factors on NPR-C expression. ANP (0.1 µM or 0.3 µg/ml), sodium nitroprusside (SNP; an NO donor, 0.1 µM), and cGMP (the intracellular second messenger of ANP and NO, 0.1 mM) did not affect NPR-C mRNA levels in PASMCs (Fig. 4). At pharmacological concentrations, ANP (1 µM), SNP (1 µM), and cGMP (1 mM) significantly decreased NPR-C mRNA levels to 81 ± 5% (n = 9, P < 0.05), 83 ± 6% (n = 3, P < 0.05), and 54 ± 4% (n = 10, P < 0.05), respectively, of baseline levels (data not shown).

Effects of MEK inhibitors on growth factor-induced downregulation of NPR-C expression. PASMCs were pretreated with inhibitors for 45 min before FGF-1 (2 ng/ml) or PDGF (10 ng/ml) was added to the medium for an additional 6 h of incubation. Inhibitors of MEK, PD-98059 (50 µM) and U-0126 (5 µM), completely blocked the FGF-1-induced downregulation of NPR-C mRNA expression (Fig. 5). However, the PKC inhibitor calphostin C (1 µM), the PI 3-kinase inhibitor LY-294002 (20 µM), and the PKA inhibitor H-89 (1 µM) did not alter the inhibitory effect of FGF-1 on NPR-C mRNA. Because the FGF-2 molecule lacks a secretory signal peptide sequence and is presumably not secreted and the effect of FGF-2 on NPR-C expression is similar to FGF-1, we only tested FGF-1 in these experiments.


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Fig. 5.   Effects of signaling pathway inhibitors on the downregulation of NPR-C expression in PASMCs by FGF-1. Control was growth-arrested PASMCs cultured in 0.1% FBS medium. Experimental groups were PASMCs pretreated with protein kinase C (PKC) inhibitor calphostin C (1 µM), phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY-294002 (20 µM), protein kinase A (PKA) inhibitor H-89 (1 µM), or mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) inhibitors U-0126 (5 µM) and PD-98059 (50 µM) for 45 min before FGF-1 (2 ng/ml) was added to the medium, and an additional 6-h incubation was carried out before harvesting for analysis. A representative Northern blot is shown at top. Northern blot analysis was carried out with 15 µg of total RNA extracted from each plate, which was quantitated individually. The mRNA loading was normalized by GAPDH mRNA. Nos. in parentheses are the no. of plates contributing data to each group. Results are means ± SE. *P < 0.05 vs. control group by 1-way ANOVA.

PDGF-BB-induced downregulation of NPR-C expression in PASMCs was significantly attenuated by the MEK inhibitors PD-98059 (50 µM) and U-0126 (5 µM) but not by the PKC inhibitor calphostin C (1 µM), the PKA inhibitor H-89 (1 µM), or the adenylate cyclase inhibitor SQ-22536 (100 µM; Fig. 6). Western blot analysis confirmed that FGF-1 significantly increased activation of MAP kinase (phosphorylation of ERK1/2) in PASMCs without affecting total ERK1/2 protein expression. These effects were blocked by pretreatment with the MEK inhibitor U-0126 (5 µM; Fig. 7).


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Fig. 6.   Effects of signaling pathway inhibitors on the downregulation of NPR-C expression in PASMCs by PDGF-BB. Control was growth-arrested PASMCs cultured in 0.1% FBS medium. Experimental groups were PASMCs pretreated with PKC inhibitor calphostin C (1 µM), PKA inhibitor H-89 (1 µM), adenylate cyclase inhibitor SQ-22536 (100 µM), or MAP/ERK (MEK) inhibitors PD-98059 (50 µM) and U-0126 (5 µM) for 45 min before PDGF-BB (10 ng/ml) was added to the medium, and an additional 6-h incubation was carried out before harvesting for analysis. A representative Northern blot is shown at top. Northern blot analysis was carried out with 15 µg of total RNA extracted from each plate, which was quantitated individually. The mRNA loading was normalized by GAPDH mRNA. Nos. in parentheses are the no. of plates contributing data to each group. Results are means ± SE. *P < 0.05 vs. control group by 1-way ANOVA. # P < 0.05 vs. U-0126 group by 1-way ANOVA.



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Fig. 7.   Effects of MEK inhibitor U-0126 on FGF-1-stimulated MAP kinase in PASMCs. Control was PASMCs cultured in 0.1% FBS medium. The growth-arrested PASMCs were stimulated with FGF-1 (2 ng/ml for 6 h) with (group 4) or without (group 2) pretreatment with U-0126 (5 µM, 45 min before FGF-1). Group 3 was PASMCs treated with U-0126 (5 µM) alone. Cell lysates (25 µg) were size fractionated by SDS-PAGE, and Western blot analysis was performed with anti-ERK1/2 and anti-phospho-ERK1/2 specific antibodies. Immunoreactive bands were visualized using chemiluminescence. Representative Western blots are shown at top. Cumulative data quantifying percent activation of ERK2 Western blots were analyzed by densitometry and normalized to percent activation as described in METHODS. Nos. in parentheses are the no. of plates contributing data to each group. Results are means ± SE. *P < 0.05 vs. control group by 1-way ANOVA. PERK1 and PERK2, phosphorylated MAP kinases ERK1 and ERK2, respectively.

Effects of FGF-1 receptor inhibitor on FGF-1-induced downregulation of NPR-C expression. PASMCs were pretreated with PD-166866 (1 µM), a novel class of potent and selective inhibitor of FGF-1 receptor tyrosine kinase (28), for 45 min before FGF-1 (2 ng/ml) was added to the medium for an additional 6 h of incubation. PD-166866 completely blocked the FGF-1-induced downregulation of NPR-C mRNA expression (Fig. 8A). These data provide evidence that the downregulation of NPR-C gene expression by FGF-1 in PASMCs is mediated through the activation of FGF-1 receptor.


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Fig. 8.   A: effects of PD-166866, an inhibitor of the FGF-1 receptor tyrosine kinase, on downregulation of NPR-C expression in PASMCs by FGF-1. Control was growth-arrested PASMCs cultured in 0.1% FBS medium. Groups 2 and 3 were PASMCs treated with FGF-1 (2 ng/ml) or PD-166866 (1 µM), respectively, for 6 h. Group 4 was PASMCs pretreated with PD-166866 (1 µM) for 45 min before FGF-1 (2 ng/ml) was added to the medium, and an additional 6-h incubation was carried out before harvesting for analysis. B: recovery of NPR-C mRNA levels in PASMCs after FGF-1 (5 ng/ml for 24 h) was removed from the culture medium (groups 3-7). Control was growth-arrested PASMCs cultured in 0.1% FBS. FGF-1 group was PASMCs treated with FGF-1 (5 ng/ml) for 24 h. A representative Northern blot is shown in A and B, top. Northern blot analysis was carried out with 15 µg of total RNA extracted from each plate, which was quantitated individually. The mRNA loading was normalized by GAPDH mRNA. Nos. in parentheses are the no. of plates contributing data to each group. Results are means ± SE. *P < 0.05 vs. control group by 1-way ANOVA.

Recovery of NPR-C expression after removal of FGF-1 stimulation. Quiescent PASMCs were pretreated with FGF-1 (5 ng/ml) for 24 h to downregulate NPR-C mRNA expression. The FGF-1-containing medium was then replaced with fresh 0.1% FBS medium, and cells were incubated for an additional 1, 3, 6, 12, and 24 h before being harvested for analysis. NPR-C mRNA levels in PASMCs began to recover within 1 h and completely recovered within 12 h after FGF-1 was removed from the culture medium. The rapid recovery of NPR-C mRNA levels suggests that FGF-1 could be a physiological regulator of NPR-C gene expression in PASMCs.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 FGF-1, FGF-2, and PDGF-BB inhibit NPR-C gene expression in PASMCs. These results are in agreement with the report by Paul et al. (29) that serum and PDGF downregulate NPR-C mRNA expression in glomerular mesangial cells in vitro. The rapid reductions in steady-state NPR-C mRNA levels observed in our study in response to very low concentration of FGF-1, FGF-2, and PDGF-BB in vitro suggest that these growth factors could be physiological regulators of NPR-C gene expression in PASMCs. Studies using inhibitors of signal transduction pathways indicate that the inhibitory effects of FGF and PDGF on NPR-C mRNA gene expression are mediated through the activation of MAP kinase. In contrast, hypoxia per se and physiological concentrations of ANP, NO, cGMP, and G protein-coupled receptor growth factors ANG II and ET-1 did not inhibit NPR-C mRNA expression in PASMCs. These results suggest that the tyrosine kinase receptor-associated growth factors FGF-1, FGF-2, and PDGF-BB may play an important role in the signal transduction pathway linking hypoxia to altered NPR-C expression in lung, providing a cellular mechanism to account for our previous in vivo findings of decreased NPR-C gene expression in lungs and pulmonary vessels of hypoxia-adapted rats and mice.

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 cells, VSMCs, and myofibroblast proliferation, differentiation, and angiogenesis, as well as adaptation to hypoxic environments (2, 30). Immunochemical and immunohistochemical investigations showed that PDGF receptors (both alpha - and beta -subtypes) are located on cultured lung myofibroblasts and smooth muscle and endothelial cells of middle and small arterial trees in rat lung (6, 7). Using RT-PCR technology, we recently demonstrated that cultured PASMCs express FGR receptor (FGFR) 1-beta pi , but not FGFR1-alpha theta , FGFR2-alpha theta , or FGFR2-beta pi receptors (data not shown). FGFR1-beta and PDGF-alpha and -beta pi receptors stimulate cell proliferation through an MAP kinase-mediated pathway (15, 30).

Expression of a number of mammalian genes in lung, including FGF-2 (2, 26), the B-chain of PDGF, the PDGF-beta receptor (5, 10), ET-1 (1), and the ANG II type 1 (AT1) and type 2 (AT2) receptors (8), are inducible by hypoxia. It has been hypothesized that these growth factors and receptors may contribute to hyperproliferation of PASMCs and muscularization of pulmonary vasculature in hypoxia-adapted animals (1, 5, 8, 26). A relationship between overexpression of these hypoxia-responsive growth factors and downregulation of NPR-C receptors has been suggested by studies carried out in other cell types. It has been reported that NPR-C mRNA expression is decreased in mesangial cells treated with serum and PDGF (29) and in HeLa cells after PKC activation (38). These data suggest that NPR-C expression can be suppressed by growth factors and is related to the proliferative activity of SMCs.

Multiple intracellular signaling transduction pathways are activated by FGF and PDGF (4). Activation of Ras-Raf-MEK-MAP kinase is necessary for the mitogenic activity of FGF and PDGF (24). Our present data provide evidence that the downregulation of NPR-C by FGF and PDGF is mediated through activation of the tyrosine kinase receptor-MAP kinase pathway. The inhibitory effect of FGF-1 and PDGF-BB on NPR-C mRNA expression was completely blocked by two different MEK inhibitors, PD-98059 and U-0126, and the inhibitory effect of FGF-1 was blocked by the selective FGF-1 receptor inhibitor PD-166866. Furthermore, the phosphorylation of ERK MAP kinase was increased by FGF-1 and blocked by U-0126, supporting this conclusion. The finding that the PKA inhibitor H-89, the PKC inhibitor calphostin C, and the PI 3-kinase inhibitor LY-294002 did not block the inhibitory effects of FGF-1 and PDGF-BB on NPR-C expression indicates that the PKA, PKC, and PI 3-kinase pathways did not play roles in mediating the suppression of NPR-C gene expression. In addition, the adenylate cyclase inhibitor SQ-22536 did not block the FGF-1- and PDGF-BB-induced suppression of NPR-C gene expression, providing evidence that cAMP was not involved in this signal transduction pathway.

Downregulation of NPR-C during hypoxic exposure has been attributed to elevated ANP levels in the circulation, which stimulate increases in cGMP production through the activation of NPR-A or NPR-B and particulate guanylate cyclase (19). Our present findings that neither ANP, SNP, nor cGMP in high concentrations reduced NPR-C gene expression in cultured PASMCs do not support that concept. Furthermore, we have recently demonstrated that NPR-C mRNA expression is reduced in tissues of ANP knockout mice exposed to hypoxia in the absence of cardiac ANP mRNA expression and circulating ANP, indicating that hypoxia-induced downregulation of NPR-C expression is independent of ANP (35). For these reasons, and because physiological levels of ANP and NO are within the nanomolar range and those of cGMP are within the micromolar range, it is unlikely that ANP, NO, or cGMP is a physiological regulator of NPR-C gene expression in vivo.

Previous studies carried out in our laboratory and others have shown that expression of ET-1 is increased at the transcriptional level by exposure to hypoxia (10, 20). The increased pulmonary production of ET-1 contributes to the development of hypoxia-induced pulmonary vasoconstriction, vascular hypertrophy, and hypertension (1, 9). The biological actions of ET-1 and ANG II are mediated through a G protein-phospholipase C-inositol trisphosphate-Ca2+-PKC signal transduction pathway in VSMCs. This signaling pathway is distinct from the tyrosine kinase receptor-MAP kinase pathway stimulated by FGF-1 and -2 and PDGF. In the current study, ET-1 did not produce changes in NPR-C mRNA levels in PASMCs in vitro, a result that is in agreement with a previous report of Schiffrin et al. (33) that ET-1 (0.1 µM) induced no changes in the density of ANP-binding sites or the relative proportion of ANP receptor subtypes in cultured rat aortic SMCs.

Early studies related ANG II and its receptors to hypoxia and to regulation of NPR-C expression. Expression of ANG II AT1 and AT2 receptors in lung can be induced by hypoxia (8). Infusion of ANG II in vivo into rats produced a decrease in the density of NPR-A and NPR-C (13). However, studies of Schiffrin et al. (33) and Palaparti and Anand-Srivastava (27) demonstrated that treatment of rat aortic SMCs with ANG II or the AT1 receptor antagonist losartan did not alter the expression of NPR-C binding sites. Our current study demonstrated ANG II-induced inhibition of NPR-C mRNA expression in PASMCs but only at pharmacological concentrations (1 µM) of ANG II. The discrepancies among the results of these studies may be attributed to differences in protocols, origins of cultured rat VSMCs (aorta vs. pulmonary arteries), duration of treatment, and other unknown factors. Furthermore, our findings that the PKC inhibitor calphostin C did not block the FGF-1- and PDGF-BB-induced downregulation of NPR-C indicate that the PKC-mediated signaling pathway, which is known to transduce signals from the AT1 receptors, may not be involved in the regulation of NPR-C gene expression in PASMCs.

In summary, this study provides evidence that the reduced expression of NPR-C mRNA observed under hypoxic conditions in lung may be mediated by the tyrosine kinase receptor-associated growth factors FGF and PDGF, the gene expression of which is increased in lung of hypoxia-adapted animals. In contrast, the G protein-linked receptor-associated growth factors ANG II and ET-1, which are also overexpressed in hypoxia-adapted lungs, do not alter NPR-C expression. This tyrosine kinase receptor activation-mediated downregulation of NPR-C in PASMCs likely represents an adaptation aimed at reducing ANP clearance from the circulation, thus enhancing the biological effects of ANP in response to stressful conditions, such as hypoxia-induced pulmonary vasoconstriction and hypertension. Our results also demonstrate that the FGF- and PDGF-mediated downregulation of NPR-C gene expression is dependent on the activation of MAP kinase in PASMCs. We acknowledge that the levels of NPR-C mRNA expression measured in these studies cannot be equated with functional NPR-C protein levels or natriuretic peptide binding in PASMCs. The effects of tyrosine kinase receptor activation-mediated reduction of NPR-C gene expression on NPR-C protein expression and biological activity in vitro and in vivo, as well as on the biological activity of the natriuretic peptides and pulmonary hemodynamics in vivo, deserve further investigation.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-44195, HL-50147, HL-45990, HL-07457, HL-56046, and HL-45990.


    FOOTNOTES

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: YFChen{at}UAB.EDU).

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

Received 25 September 2000; accepted in final form 23 February 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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