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J Appl Physiol 102: 390-398, 2007. First published October 12, 2006; doi:10.1152/japplphysiol.00468.2006
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ANP signaling inhibits TGF-beta-induced Smad2 and Smad3 nuclear translocation and extracellular matrix expression in rat pulmonary arterial smooth muscle cells

Peng Li,1 Suzanne Oparil,1 Lea Novak,2 Xu Cao,2 Weibin Shi,2 Jason Lucas,1 and Yiu-Fai Chen1

1Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, Department of Medicine; and 2Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 24 April 2006 ; accepted in final form 24 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atrial natriuretic peptide (ANP) and transforming growth factor (TGF)-beta play important counterregulatory roles in pulmonary vascular adaptation to chronic hypoxia. To define the molecular mechanism of this important interaction, we tested whether ANP-cGMP-protein kinase G (PKG) signaling inhibits TGF-beta1-induced extracellular matrix (ECM) expression and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Rat pulmonary arterial smooth muscle cells (PASMCs) were treated with ANP (1 µM) or cGMP (1 mM) with or without pretreatment with PKG inhibitors KT-5823 (1 µM) or Rp-8-bromo-cGMP (Rp-8-Br-cGMP 50 µM), then exposed to TGF-beta1 (1 ng/ml) for 5–360 min (for pSmad nuclear translocation and protein analysis) or 24 h (for ECM mRNA expression). Nuclear translocation of pSmad2 and pSmad3 was assessed by fluorescent confocal microscopy. ANP and cGMP inhibited TGF-beta1-induced pSmad2 and pSmad3 nuclear translocation and expression of periostin, osteopontin, and plasminogen activator inhibitor-1 mRNA and protein, but not TGF-beta1-induced phosphorylation of Smad2 and Smad3. KT-5823 and Rp-8-Br-cGMP blocked ANP/cGMP-induced activation of PKG and inhibition of TGF-beta1-stimulated nuclear translocation of pSmad2 and pSmad3 in PASMCs. These results reveal for the first time a precise site at which ANP-cGMP-PKG signaling exerts its antifibrogenic effect on the profibrogenic TGF-beta1 signaling pathway: by blocking TGF-beta1-induced pSmad2 and pSmad3 nuclear translocation and ECM expression in PASMCs. Blocking nuclear translocation and subsequent binding of pSmad2 and pSmad3 to TGF-beta-Smad response elements in ECM genes may be responsible for the inhibitory effects of ANP on TGF-beta-induced expression of ECM molecules.

lung; vascular hypertrophy and remodeling; atrial natriuretic factor; transforming growth factor; signal transduction

HYPOXIA-INDUCED PULMONARY hypertension and vascular remodeling result from an imbalance in the normal relationships between vasoconstrictor and vasodilator and between mitogenic and growth-inhibiting pathways in the pulmonary vasculature. Our previous studies in rodent models have provided convincing evidence that endogenous atrial natriuretic peptide (ANP) and transforming growth factor (TGF)-beta play important counterregulatory roles in pulmonary vascular adaptation to chronic hypoxia (3, 29). ANP and TGF-beta expression are upregulated in heart and lung, respectively, of hypoxia-adapted animals (1, 2, 26, 29). The functional significance of the hypoxia-induced increase in ANP is supported by observations of worsening pulmonary hypertension and vascular remodeling in ANP-deficient (Nppa –/–) mice compared with wild-type controls in response to hypoxic stress (29). Our recent studies in a novel mouse model that expresses an inducible dominant negative mutation of the TGF-beta receptor type II gene (DnTGFbetaRII), and thus cannot activate the TGF-beta-Smad signaling cascade, demonstrate that disruption of TGF-beta signaling greatly attenuates hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, pulmonary arterial remodeling and muscularization, and expression of extracellular matrix (ECM) in lung, supporting a critical role for TGF-beta signaling in the pathogenesis of chronic hypoxia-induced pulmonary hypertension and vascular remodeling (3).

ANP, via activation of guanylate cyclase-coupled membrane receptors, increases intracellular cGMP levels and activates cGMP-dependent protein kinase (PKG), with resultant growth-inhibiting and antiproliferative effects in a variety of cell types, including pulmonary arterial smooth muscle cells (PASMCs) (2, 10, 22). In contrast, activated TGF-beta participates in pulmonary morphogenesis and in the pathogenesis of pulmonary fibrosis and vascular remodeling by stimulating PASMC proliferation and ECM expression (1, 23, 33). TGF-beta signals through membrane-bound heteromeric type I (TGFbetaRI) and type II (TGFbetaRII) receptor kinases that transduce intracellular signals via phosphorylation and nuclear translocation of receptor-activated Smad2 and Smad3 proteins, which modulate the transcription of a large number of genes (20, 28). The molecular mechanisms of the counterregulatory effects of ANP-cGMP-PKG signaling on activated TGF-beta-induced Smad signaling in PAMSCs have not been studied.

In the present study, we tested the hypothesis that ANP signaling inhibits TGF-beta1-stimulated ECM expression in isolated PASMCs and probed the specific site(s) at which molecular merging of these pro- and antifibrogenic signaling cascades occurs. Specifically, we tested whether ANP-cGMP-PKG signaling interrupts downstream events in the TGF-beta1 signaling pathway by inhibiting TGF-beta1-induced phosphorylation of Smad2 and Smad3 proteins and/or preventing nuclear translocation of phosphorylated Smad2 and Smad3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Cell Culture

Young adult (8 wk old) male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were used. All experimental protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHHS Publication No. 96–01, 1996).

PASMCs were isolated from distal segments of pulmonary arteries (2nd–3rd branches, 0.1–0.2 mm external diameter) using the explant method described previously (14, 15, 30). To confirm the characteristics of smooth muscle cells (SMCs) in culture, immunohistochemical staining of {alpha}-SMC actin was performed using selective {alpha}-SMC actin antibody and secondary horseradish peroxidase (HRP)-labeled anti-IgG antibodies. All cultures were examined by phase-contrast microscopy before and after each experimental period to assess cell viability. PASMCs were used for experiments at passage 3 or 4.

Before each study, PASMCs were grown in 10% FBS-DMEM to 95% confluence and then made quiescent by placing them in medium containing 0.1% FBS for 24 h. For Northern, real-time quantitative RT-PCR, or Western blot analyses, PASMCs were cultured in 60-mm cell culture dishes. For quantitative analysis of nuclear translocation of phosphorylated Smad2 (pSmad2) and Smad3 (pSmad3), PASMCs were cultured on 18 mm x 18 mm glass cover slides.

Experimental Protocols

Experiment 1: effects of ANP signaling on TGF-beta1-stimulated ECM expression.   To test the hypothesis that ANP and cGMP inhibit TGF-beta1-stimulated expression of mRNA for the ECM proteins periostin (PN), osteopontin (OPN), and plasminogen activator inhibitor 1 (PAI-1, a biomarker of TGF-beta1 action in cells), quiescent PASMCs were treated with TGF-beta1 (0.1–5 ng/ml) (Sigma) for 24 h with or without pretreatment with ANP (1 µM) (Sigma), cGMP (8-Br-cGMP, 1 mM) (Sigma), and/or PKG inhibitors KT-5823 (1 µM) (CalBiochem) or Rp-8-Br-cGMP (50 µM) (Alexis) for 30 min. PASMCs were then harvested for Northern blot, real-time quantitative RT-PCR, or Western blot analyses.

Experiment 2: effects of TGF-beta1 and cGMP on phosphorylation of Smad3 and Smad2 proteins in PASMCs.   To test the hypothesis that cGMP inhibits TGF-beta1-induced phosphorylation of Smad2 and Smad3 in PASMCs, quiescent PASMCs were pretreated with cGMP (0.01–1 mM) or vehicle for 30 min before addition of TGF-beta1 (1 ng/ml) to the medium and incubation for an additional 30 min. After treatment with TGF-beta1 and/or cGMP, cells were harvested for assessment of pSmad2, pSmad3, and Smad4 (internal control that cannot be phosphorylated) levels using Western blot analysis.

Experiment 3: effects of ANP/cGMP on TGF-beta1-induced nuclear translocation of pSmad2 and pSmad3 in rat PASMCs.   To test the hypothesis that inhibition of TGF-beta1-stimulated ECM expression by ANP-cGMP-PKG signaling is dependent on events downstream from phosphorylation of Smad2 and Smad3, we examined the effects of ANP and cGMP on TGF-beta1-stimulated nuclear translocation of pSmad2 and pSmad3 in PASMCs. Quiescent rat PASMCs were pretreated with ANP (1 µM), cGMP (1 mM), or vehicle for 30 min and then exposed to TGF-beta1 (1 ng/ml) for an additional 5 min to 6 h. PASMCs were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS. The fixed PASMCs were stained with selective anti-pSmad2, anti-pSmad3, and anti-Smad4 primary antibodies and a Texas Red-labeled donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Lab) to assess nuclear translocation of pSmad2 and pSmad3 by using confocal fluorescence microscopy with a computerized Zeiss-Axioskop system.

Experiment 4: effects of ANP and cGMP on PKG activity in rat PASMCs.   To test the hypothesis that ANP and/or cGMP activates PKG in rat PASMCs, quiescent PASMCs were pretreated with PKG inhibitors KT-5823 (1 µM) or Rp-8-Br-cGMP (50 µM), or vehicle for 15 min before adding ANP (1 µM) or cGMP (1 mM). Fifteen minutes after beginning treatment with ANP or cGMP, cells were exposed to TGF-beta1 (1 ng/ml) for an additional 15 min. Cells were then harvested for PKG activity measurement.

Experiment 5: ANP/cGMP signaling inhibits TGF-beta1-induced nuclear translocation of pSmad2 and pSmad3 in rat PASMCs by activating PKG in rat PASMCs.   To test the hypothesis that ANP/cGMP signaling inhibits TGF-beta1-induced nuclear translocation of pSmad3 via activation of PKG, quiescent PASMCs cultured on slides were pretreated with PKG inhibitors KT-5823 (1 µM) or Rp-8-Br-cGMP (50 µM), or vehicle for 15 min before adding ANP (1 µM) or cGMP (1 mM). Cells were incubated with TGF-beta1 for an additional 60 min, then fixed and stained with selective anti-pSmad3, as in experiment 3. Nuclear translocation of pSmad3 was assessed using confocal phase-contrast fluorescence microscopy, as in experiment 3.

RNA Isolation for Northern blot

PASMC were homogenized, and total RNA was extracted using the TRIZOL total RNA isolation reagent (Invitrogen). Northern analysis was performed by using 32P-labeled selective cDNA probes for PN, OPN, PAI-1, and GAPDH that had been generated in our laboratory by reverse transcription (RT) followed by the DNA PCR by using lung RNA as the template, as previously described (14, 15). A 32P-labeled 18S rRNA-oligonucleotide (5'-ACGGTATCTGATCGTCTTCGAACC-3') was used as the control probe to normalize data. Autoradiographic signals were scanned with an optical densitometer (Bio-Rad, Model GS-670 Imaging Densitometer). To estimate steady-state specific mRNA levels, PN and OPN mRNA/18S rRNA and PAI-1 mRNA/GAPDH mRNA ratios were determined by dividing the absorbance corresponding to the specific cDNA probe hybridization by the absorbance corresponding to the 18S rRNA or GAPDH cDNA probe hybridization.

Western Blot Analysis

Standard Western blot analysis for phosphor-Smad2, phosphor-Smad3, Smad4, PN, vasodilator-stimulated phosphoprotein (VASP, a substrate of PKG), phosphor-VASP (pVASP [Ser239], a selective product of PKG), PN, OPN, and beta-actin was performed by using anti-pSmad2, anti-pSmad3, anti-Smad4, anti-Smad2/3, anti-VASP, anti-pVASP (Cell Signaling), anti-PN (Abcam), and anti-OPN (a gift from Dr. P.-L. Chang, Ref. 9) specific primary antibodies and a HRP-conjugated goat anti-rabbit IgG. Immune complexes were detected by using a Phototop-HRP Western Detection Kit (Cell Signaling). Autoradiograms exposed in the linear range of film density were scanned by using a densitometer (Bio-Rad Model GS-670 Imaging Densitometer) as described previously (14, 15).

PKG Activity Assay

PASMC lysates were assayed for PKG activity by measuring 1) the incorporation of 32P from [{gamma}-32P]ATP into a specific PKG substrate, Glasstide (CalBiochem) using a modified method of Lincoln et al. (17); and 2) the phosphorylation at Ser239 of VASP (Cell Signaling) by using a modified method of Lawrence and Pryzwansky (12) and Li et al. (16). To measure the PKG-stimulated incorporaton of 32P to Glasstide, PASMCs were lysed in CelLytic-M Lysis Reagent (Sigma) plus 10 mM dithiothreitol (DTT), 1 mM isobutylmethylxanthine, and 1x Halt Protease inhibitor Cocktail (Pierce). Lysates were sonicated and centrifuged at 14,000 rpm for 15 min at 4°C, and supernatants were assayed for PKG activity without adding exogenous cGMP. PKG activity was normalized to the protein concentration of the supernatant, measured by Bradford’s procedure, using BSA as a standard. To measure the PKG-stimulated Ser239 phosphorylation of VASP, PASMCs were lysed in 1x SDS buffer (2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue in 62.5 mM Tris·HCl, pH 6.8). Lysates were sonicated and subjected to Western blot analysis for quantitation of VASP and p-Ser239-VASP (Cell Signaling).

Statistical Analysis

Results were expressed as means ± SE. Statistical analyses were carried out using the SigmaStat package (Jandel Scientific Software, San Rafael, CA) on a PC. Statistical comparisons of mRNA levels were performed with the one-way ANOVA or unpaired t-test. 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.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ANP and cGMP Inhibit TGF-beta1-Stimulated PN, OPN, and PAI-1 Expression in PASMCs

Northern blot analysis demonstrated that PN and OPN mRNA expression increased in a dose-dependent fashion in PASMCs treated with TGF-beta1 for 24 h (Fig. 1, A and B). The threshold concentration was between 0.1 and 1 ng/ml, and the maximum effect was observed at doses of 5 ng/ml for PN (a 3.3-fold increase) and OPN (a 3.7-fold increase), respectively. Pretreatment with ANP (1 µM) or cGMP (1 mM) decreased baseline levels of PN and OPN and significantly attenuated TGF-beta1-induced expression of PN and OPN mRNAs (Fig. 1, C and D) and proteins (Fig. 1F).


Figure 1
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  		Fig. 1. A and B: dose-dependent stimulatory effects of transforming growth factor-beta1 (TGF-beta1) on steady-state periostin (PN) and osteopontin (OPN) mRNA expression in rat pulmonary arterial smooth muscle cells (PASMCs). Quiescent PASMCs cultured in 0.1% FBS medium for 24 h were exposed to graded doses of TGF-beta1 or vehicle for an additional 24 h before being harvested. C–E: inhibitory effects of ANP or cGMP on TGF-beta1-stimulated PN, OPN, and plasminogen activator inhibitor (PAI)-1 mRNA expression in rat PASMCs. Quiescent PASMCs were pretreated with ANP (1 µM) or cGMP [8-bromo-cGMP (8-Br-cGMP), a cGMP analog, 1 mM] for 30 min before TGF-beta1 (1 ng/ml) for an additional 24 h before being harvested. In E, subgroups of PASMCs were pretreated with protein kinase G (PKG) inhibitors KT-5823 (1 µM) or Rp-8-Br-cGMP (50 µM) for 15 min before ANP or cGMP. Nos. in parentheses are the nos. 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. The Northern blot membrane was probed with PN and OPN cDNAs and 18S rRNA oligonucleotide or with PAI-1 and GAPDH cDNAs sequentially. PN and OPN mRNA data were normalized to the 18S rRNA, and PAI-1 mRNA to GAPDH mRNA to allow for variation in RNA loading. F: cell lysates (25 µg) were size fractionated by SDS-PAGE, and Western blot analysis was performed with selective anti-PN, -OPN, and -beta-actin antibodies. Results demonstrating that pretreatment with cGMP (1 mM for 30 min) blocked TGF-beta1 (1 ng/ml for 24 h)-stimulated PN and OPN protein expression in rat PASMCs. beta-Actin protein levels were measured to show protein loading in each lane. Results are means ± SE. *P < 0.05 vs. respective vehicle groups; # P < 0.05 vs. respective TGF-beta1-alone groups by 1-way ANOVA.

 
Similarly, pretreatment with cGMP (1 mM for 30 min) blocked TGF-beta1 (1 ng/ml for an additional 24 h)-stimulated PAI-1 mRNA expression, assessed by real-time quantitative RT-PCR (Fig. 1E). Pretreatment with PKG inhibitors KT-5823 (1 µM) or Rp-8-Br-cGMP (50 µM) blocked the inhibitory effects of cGMP on TGF-beta1-stimulated PAI-1 expression in PASMCs, suggesting that the action of cGMP was through the activation of PKG. These data support the hypothesis that ANP-cGMP-PKG signaling has antifibrogenic effects that antagonize TGF-beta-induced stimulation of ECM expression in PASMCs.

cGMP Does Not Inhibit TGF-beta1-Induced Phosphorylation of Smad3 and Smad2 in PASMCs

Western blot analysis demonstrated that TGF-beta1 treatment significantly increased pSmad3 and pSmad2 levels in PASMCs and that pretreatment with cGMP did not inhibit TGF-beta1-induced phosphorylation of Smad3 or Smad2 in these cells. Smad4 levels were not altered by either TGF-beta1 or cGMP treatment (Fig. 2A). Neither cGMP nor TGF-beta1 treatment altered total Smad2/3 levels in these cells (Fig. 2B). These results indicate that activation of ANP-cGMP signaling does not block TGF-beta1-induced phosphorylation of Smad2 and Smad3 and thus that disruption of Smad2 and Smad3 phosphorylation does not account for the inhibitory effects of ANP and cGMP on TGF-beta1-induced ECM expression.


Figure 2
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  		Fig. 2. Representative Western blot analysis demonstrating that pretreatment with cGMP does not block TGF-beta1-induced phosphorylation of Smad2 or Smad3 (A) and that neither cGMP nor TGFbeta1 treatment altered total Smad2/3 levels in rat PASMCs (B). Quiescent PASMCs were stimulated with TGF-beta1 (1 ng/ml) for 30 min with or without pretreatment with cGMP (8-Br-cGMP, 0.01–1 mM for 30 min). Smad4 levels were not altered by TGF-beta1 or cGMP treatment. Cell lysates (25 µg) were size fractionated by SDS-PAGE, and Western blot analysis was performed with selective anti-phospho-Smad2 (pSmad2), anti-phospho-Smad3 (pSmad3), anti-Smad4, or anti-Smad2/3 antibodies. Anti-Smad2/3 antibody cannot distinguish Smad2 and Smad3. Vehicle control (Veh): PASMCs cultured in 0.1% FBS medium.

 
ANP and cGMP Inhibit TGF-beta1-Induced Nuclear Translocation of pSmad2 and pSmad3 in PASMCs

In vehicle-treated cells, immunostaining of pSmad2 and pSmad3 was weak and distributed evenly in cytoplasm and nucleus, suggesting that pSmad2 and pSmad3 levels were low and without significant nuclear translocation (Figs. 3 and 4). TGF-beta1 treatment significantly stimulated nuclear translocation of pSmad2 and pSmad3, indicated by strong pSmad2 and pSmad3 staining in the nucleus. Pretreatment with ANP or cGMP significantly attenuated nuclear translocation of pSmad2 and pSmad3.


Figure 3
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  		Fig. 3. Representative fluorescent micrographs show that ANP and cGMP inhibit TGF-beta1-stimulated nuclear translocation of pSmad2 (A) and pSmad3 (B) in rat PASMCs. PASMCs were cultured on slides. After starvation in 0.1% FBS medium for 24 h, PASMCs were pretreated with ANP (1 µM), cGMP (8-Br-cGMP, 1 mM), or vehicle for 30 min and then exposed to TGF-beta1 (1 ng/ml) for an additional 1 h. Fixed cells were stained with selective anti-pSmad2 or anti-pSmad3 antibodies and a Texas Red-labeled secondary antibody.

 

Figure 4
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  		Fig. 4. Confocal fluorescent micrographs show that cGMP inhibits TGF-beta1-stimulated nuclear translocation of pSmad2 (A) and pSmad3 (B) in rat PASMCs. PASMCs were cultured on slides. Quiescent PASMCs were pretreated with cGMP (8-Br-cGMP, 1 mM) or vehicle for 30 min and then exposed to TGF-beta1 (1 ng/ml) for an additional 1 h. Fixed cells were stained with selective anti-pSmad2 or anti-pSmad3 antibodies and a Texas Red-labeled secondary antibody. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).

 
The number of cells with pSmad3 nuclear translocation and intensity of the pSmad3 signal in TGF-beta1 (1 ng/ml for 30 min)-treated PASMCs were significantly greater than that of pSmad2 (Fig. 5, A and B). Pretreatment with ANP (1 µM for 30 min) or cGMP (1 mM for 30 min) blocked TGF-beta1-induced nuclear accumulation of pSmad2 and pSmad3 (Fig. 5, A and B), with a substantial level of pSmad2 and Smad3 staining remaining in the cytoplasm in most cells (Figs. 3 and 4).


Figure 5
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  		Fig. 5. A and B: inhibitory effects of ANP (1 µM for 30 min) and cGMP (8-Br-cGMP, 1 mM for 30 min) on TGF-beta1 (1 ng/ml for an additional 30 min)-stimulated nuclear translocation of pSmad2 and pSamd3 in rat PASMCs. Vehicle was PASMCs cultured in 0.1% FBS medium for 24 h. Fixed cells were incubated with selective anti-pSmad2 or anti-pSmad3 antibody and a Texas Red-labeled secondary antibody. Results are means ± SE. A total of >500 cells in 10 plates was counted. *P < 0.05 vs. respective vehicle groups; # P < 0.05 vs. respective TGF-beta1-alone group by 1-way ANOVA. C and D: time course of the inhibitory effects of cGMP (8-Br-cGMP, 1 mM for 30 min) on TGF-beta1 (1 ng/ml for additional 5 min to 6 h)-stimulated nuclear translocation of pSmad2 and pSmad3 in rat PASMCs. Results are means ± SE. A total of >300 cells in 3 independent experiments was counted.

 
When PASMCs were exposed to TGF-beta1 (1 ng/ml) for 5 to 360 min, the nuclear accumulation of pSmad2 and pSmad3 began at 15 min, peaked at 30 min (pSmad2) and 60 min (pSmad3), and decreased but did not reach pretreatment levels by 3 and 6 h of TGF-beta1 treatment (Fig. 5, C and D). The maximal percentages of PASMCs with pSmad2 and pSmad3 nuclear translocation were 23% and 94%, respectively. Pretreatment with cGMP (1 mM for 30 min) decreased both the peak and duration of TGF-beta1-induced pSmad2 and pSmad3 nuclear translocation (Fig. 5, C and D).

Inhibition of PKG Attenuates the Inhibitory Effects of ANP and cGMP on TGF-beta1-Induced Nuclear Translocation of pSmad3 in PASMCs

Both ANP (1 µM) and cGMP (1 mM) increased PKG activity in PASMCs within 15 min of treatment. Pretreatment with PKG inhibitors KT-5823 or Rp-8-Br-cGMP blocked the ANP and cGMP-induced increases in PKG activity, and TGF-beta1 did not alter cellular PKG activity in these cells (Fig. 6, A and B).


Figure 6
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  		Fig. 6. Effects of ANP, cGMP, TGF-beta1, and/or PKG inhibitors on PKG activity in rat PASMCs. PASMCs were pretreated with PKG inhibitors KT-5823 (1 µM) or Rp-8-Br-cGMP (50 µM) for 15 min before ANP (1 µM) or cGMP (8-Br-cGMP, 1 mM), with or without TGF-beta1 (1 ng/ml) treatment for an additional 15 min before being harvested for analysis. Vehicle control (lane 1) was growth-arrested PASMCs grown in 0.1% FBS medium. A: cell lysates were assayed for PKG activity by measuring the incorporation of 32P from [{gamma}-32P]ATP into a selective PKG substrate, Glasstide. B: cell lysates were assayed for PKG activity by measuring the phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Ser239 is the major PKG phosphorylation site on VASP. Results are means ± SE; n = no. of plates. *P < 0.05 vs. vehicle control group (lane 1); # P < 0.05 vs. respective cGMP or ANP groups by 1-way ANOVA.

 
To test whether activation of PKG mediates the inhibitory effects of ANP and cGMP on TGF-beta1-induced nuclear translocation of pSmads, pSmad3 nuclear translocation was measured in the presence of PKG inhibitors KT-5823 or Rp-8-Br-cGMP. Pretreatment with KT-5823 or Rp-8-Br-cGMP blocked the inhibitory effects of ANP and cGMP on TGF-beta1-stimulated pSmad3 nuclear translocation (Fig. 7).


Figure 7
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  		Fig. 7. PKG inhibitors KT-5823 and Rp-8-Br-cGMP block the inhibitory effects of ANP and cGMP on TGF-beta1 (1 ng/ml)-stimulated nuclear translocation of pSmad3 in rat PASMCs. Vehicle control (lane 1) was growth-arrested PASMCs grown on slides in 0.1% FBS medium for 24 h. The order of the treatment was: KT-5823 (1 µM), Rp-8-Br-cGMP (50 µM) or vehicle for 15 min; ANP (1 µM), cGMP (8-Br-cGMP, 1 mM) or vehicle for 15 min; TGF-beta1 (1 ng/ml) or vehicle for an additional 1 h. Cells were fixed and stained with selective anti-pSmad3 antibody and a Texas Red-labeled secondary antibody. Results are means ± SE. A total of >300 cells in 3 independent experiments was counted. *P < 0.05 vs. vehicle control group (lane 1); # P < 0.05 vs. respective groups with TGF-beta1 by 1-way ANOVA.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrate for the first time that ANP has an antifibrogenic effect on PASMCs treated with TGF-beta1. ANP and cGMP suppressed TGF-beta-stimulated ECM gene expression by interfering with Smad signaling through a PKG-dependent mechanism. The most striking finding of this study is that ANP and cGMP inhibit TGF-beta1-induced nuclear translocation of pSmad2 and pSmad3, but not the TGF-beta1-induced phosphorylation of Smad2 and Smad3 in PASMCs, thus defining a novel molecular mechanism by which ANP signaling intercepts the TGF-beta signaling pathway and blocks TGF-beta-induced ECM expression.

TGF-beta is a key mediator of pulmonary morphogenesis and of the pathogenesis of pulmonary fibrosis and vascular remodeling (1, 7, 23, 33). Increases in the local abundance of TGF-beta1 promote vascular wall remodeling, arterial lesion growth, and vascular cell differentiation (25). Small amounts of TGF-beta are present in a latent, inactive form in the normal adult lung, and expression of TGF-beta is increased in pathological conditions, including cystic fibrosis, asthma, and hypoxia-induced pulmonary hypertension and vascular remodeling (1, 6, 26). TGF-beta1 is involved in fibrotic tissue remodeling and is overexpressed in areas of active fibrosis in lung (4), as well as in several animal models of pulmonary hypertension (5, 24).

Activated TGF-beta ligands bind to a heteromeric complex of type II (TGFbetaRII) and type I (TGFbetaRI) receptors that transduce intracellular signals via activation of Smad2 and Smad3 (21, 28). Phosphorylation of TGF receptor-associated Smad2 and Smad3 and nuclear translocation of pSmad2 and pSmad3 are critical steps in TGF-beta signaling (28, 32) (Fig. 8). Smad2 (467 amino acids) and Smad3 (425 amino acids) contain predominantly serine, with some threonine residues, in the COOH-terminal, linker, and MH1 regions that are accessible for phosphorylation. On ligand binding, phosphorylation by TGFbetaR1 kinase of the two most COOH-terminal serine residues drives the activation of Smad2, and Smad3 is required for nuclear translocation and subsequent binding of pSmads to nuclear transcriptional factors and DNA that regulate the transcriptional expression of downstream genes (28, 32).


Figure 8
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  		Fig. 8. Schematic illustration of TGF-beta1 signal transduction and postulated mechanisms of inhibition by ANP-cGMP-PKG signaling, i.e., overphosphorylation of Smad2 and Smad3, resulting in inhibition of nuclear translocation and subsequent interaction with transcriptional regulatory factors and binding to DNA to regulate expression of downstream genes. TGFRI and TGFRII, type I and type II TGF-beta receptors; NPRA, natriuretic peptide receptor type A; TF, transcriptional factors.

 
Input from other receptor systems, e.g., natriuretic peptide receptor type A (NPRA)-cGMP-PKG, influences the Smad signaling network, and the cross-talk created by the interplay between Smads and other signaling pathways is largely responsible for the diverse and context-specific effects of the TGF-beta family of proteins (11, 19, 28). Both inhibition of phosphorylation and overphosphorylation of Smad2 and Smad3, disruption of their heterodimerization with Smad4 and nuclear translocation, as well as inhibition of their binding to transcription factors and TGF-beta-responsive promoters on DNA have been reported to suppress TGF-beta-stimulated gene expression (11, 19, 27, 28, 34). Thus phosphorylation not only activates Smad proteins but also modulates their activity. This provides a novel mechanism for integration of the Smad pathway with ANP-NPRA-cGMP-PKG signaling that could modulate TGF-beta signal transduction (Fig. 8). The finding that ANP and cGMP inhibit TGF-induced nuclear translocation of pSmad2 and pSmad3, but not the TGF-beta-stimulated phosphorylation of Smad2 and Smad3, in PASMCs suggests a novel molecular mechanism by which ANP-cGMP-PKG signaling intercepts the TGF-beta signaling cascade. The results suggest that the PKG may act as a stronger protein kinase than TGFbetaRI and phosphorylates additional serine residues (i.e., in the linker or MH1 regions) on Smad2 and/or Smad3, thus disrupting their nuclear translocation, resulting in repression of transcriptional activation of TGF-beta response promoters, e.g., on PN, OPN, and PAI-1 genes.

We recognize that overphosphorylation of Smads is not the only cellular function of PKG activation. PKG has diverse intracellular actions, including integrin signal transduction, modulation of Ca2+ release and uptake into sacroplasmic reticulum, alteration of membrane K+ fluxes, and nuclear protein phosphorylation and translocation (17). An alternative (to overphosphorylation) explanation for the observation that ANP signaling prevents nuclear translocation of Smads is that cGMP-PKG may alter the affinity of Smads for cytoplasmic anchoring molecules or nuclear export proteins. Subcellular localization of Smads has been shown to be controlled by interaction with these cytoplasmic and nuclear retention factors (28). The precise molecular basis for retention of pSmads in the cytosol following ANP or cGMP treatment remains to be identified.

Several recent studies have demonstrated that various cytoplasmic protein kinases and cyclic nucleotides participate in regulating responses to TGF-beta (18, 19). In normal epithelial cells, Erk MAP kinase inhibits TGF-beta signaling via inhibition of nuclear accumulation of Smad2 (11), but in malignant epithelial cells, Erk does not alter the function of Smad2, -3, or -4 at the level of nuclear translocation, DNA binding, or transcriptional activation (13). Protein kinase C (PKC) directly phosphorylates Smad3 and abrogates the ability of Smad3 to bind directly to DNA, leading to impairment of transcriptional responses dependent on the direct binding of Smad3 to DNA (34). Specifically, PKC has been shown to block proapoptotic action of TGF-beta in Mv1Lu mink lung epithelial cells. Activation of Ca2+-calmodulin-dependent protein kinase II prevents Smad2/4 heterodimerization and nuclear translocation and concomitant transcriptional responses in HEK-293 human kidney fibroblasts (31). Furthermore, intracellular cAMP-elevating agents such as prostaglandin E2, the adenylate cyclase activator forskolin, and the phosphodiesterase inhibitor isobutylmethylxanthine inhibit TGF-beta-induced Smad3/4-dependent gene expression via a cAMP-dependent protein kinase A (PKA)-dependent mechanism in human keratinocytes (27). Interestingly, activation of cAMP-PKA does not inhibit nuclear translocation and DNA binding of Smad3/4 complexes but abolishes interactions of Smad3/4 with transcription activators in a PKA-dependent manner in the nucleus.

The present study shows that ANP and cGMP inhibit TGF-beta-induced nuclear translocation of pSmad2 and pSmad3 in PASMCs, defining a new role for cyclic mononucleotide phosphate second messenger in regulating profibrogenic responses to TGF-beta. Pretreatment with the PKG inhibitors KT-5823 or Rp-8-Br-cGMP prevented the inhibitory effects of ANP and cGMP on TGF-beta-stimulated nuclear translocation of pSmad2 and pSmad3, supporting the hypothesis that these effects are mediated through activation of PKG.

TGF-beta mediates fibrotic tissue remodeling by increasing the production and decreasing the degradation of ECM (1, 5, 33). The present study is the first to demonstrate increased expression of PN, a novel ECM molecule originally described in bone (8), in PASMCs is part of a generalized ECM response to TGF-beta. This finding, coupled with our previous observation of increased PN expression in lung of mice adapted to hypoxia (3), suggests an involvement of this novel ECM molecule in hypoxia-induced pulmonary vascular remodeling. Our finding that ANP and cGMP inhibit TGF-beta1-induced expression of the ECM molecules PN and OPN, as well as PAI-1, a TGF-beta-Smad target gene in PASMCs, indicates that there is a functionally significant interaction between ANP and TGF-beta signaling that may play an important role in modulating hypoxia-induced pulmonary vascular remodeling.

Our previous in vivo studies have validated the importance of TGF-beta and ANP as opposing influences in the pathogenesis of chronic hypoxia-induced pulmonary hypertension (3, 29). Using a novel DnTGFbetaRII mouse model, we have demonstrated that hypoxia-induced pulmonary hypertension and vascular and parenchymal remodeling and right ventricular hypertrophy are markedly attenuated by disruption of TGF-beta signaling in lung (3). In contrast, disruption of ANP expression in ANP null mice exacerbates these hypoxia-induced processes (29). Taken together, these data support the hypothesis that endogenous TGF-beta and ANP play important counterregulatory (yin-yang) roles in regulating pulmonary artery pressure, ECM production, and pulmonary vascular remodeling in response to hypoxic stress. An imbalance in the normal relationships between the mitogenic/profibrogenic of TGF-beta and antigrowth/antifibrogenic effects of ANP results in chronic hypoxia-induced pulmonary hypertension and vascular and parenchymal remodeling.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y.-F. Chen, 1008 Zeigler Research Bldg., Dept. of Medicine, Univ. of Alabama at Birmingham, 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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