Atrial Natriuretic Peptide Inhibits Transforming Growth Factor β–Induced Smad Signaling and Myofibroblast Transformation in Mouse Cardiac Fibroblasts
This study tested the hypothesis that activation of atrial natriuretic peptide (ANP)/cGMP/protein kinase G signaling inhibits transforming growth factor (TGF)-β1–induced extracellular matrix expression in cardiac fibroblasts and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Left ventricular hypertrophy and fibrosis, collagen deposition, and myofibroblast transformation of cardiac fibroblasts in response to pressure overload by transverse aortic constriction were exaggerated in ANP-null mice compared with wild-type controls. ANP and cGMP inhibited TGF-β1–induced myofibroblast transformation, proliferation, collagen synthesis, and plasminogen activator inhibitor-1 expression in cardiac fibroblasts isolated from wild-type mice. Following pretreatment with cGMP, TGF-β1 induced phosphorylation of Smad3, but the resultant pSmad3 could not be translocated to the nucleus. pSmad3 that had been phosphorylated with recombinant protein kinase G-1α was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry. The analysis revealed phosphorylation of Ser309 and Thr388 residues, sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-β receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3. These results suggest that phosphorylation of Smad3 by protein kinase G is a potential molecular mechanism by which activation of ANP/cGMP/protein kinase G signaling disrupts TGF-β1–induced nuclear translocation of pSmad3 and downstream events, including myofibroblast transformation, proliferation, and expression of extracellular matrix molecules in cardiac fibroblasts. We postulate that this process contributes to the antifibrogenic effects of the natriuretic peptide in heart.
- atrial natriuretic factor
- transforming growth factor
- cardiac fibroblast
- cardiac fibrosis and remodeling
- signal transduction
Our previous studies have shown that endogenous atrial natriuretic peptide (ANP) and transforming growth factor (TGF)-β play important counterregulatory roles in pressure overload–induced cardiac hypertrophy, remodeling, and fibrosis.1–4 ANP and TGF-β expression are upregulated in heart with pressure-overload stress.1 The functional significance of this stress-induced increase in ANP is supported by observations of exaggerated left ventricular hypertrophy, fibrosis, and remodeling in ANP-deficient (Nppa−/−) mice compared with wild-type controls in response to pressure overload.1,2,4 Our recent studies in a novel mouse model that expresses an inducible dominant negative mutation of the TGF-β receptor type II gene (DnTGFβRII), and thus cannot activate the TGF-β/Smad signaling cascade, demonstrated that disruption of TGF-β signaling greatly attenuated the pressure overload–induced MF transformation and interstitial fibrosis in heart, supporting a critical role for TGF-β signaling in the pathogenesis of pressure overload–induced cardiac hypertrophy and remodeling.3
ANP increases intracellular cGMP levels and activates cGMP-dependent protein kinase (PK)G, with resultant growth inhibiting and antigrowth/-proliferative effects in a variety of cell types, including cardiac fibroblasts (CFs),5 whereas activated TGF-β stimulates cellular differentiation, transformation, proliferation, migration, and extracellular matrix (ECM) expression.6–8 TGF-β signals through membrane-bound heteromeric type I (TGFβRI) and type II (TGFβRII) receptor kinases that transduce intracellular signals via phosphorylation and nuclear translocation of receptor-activated Smad2 and Smad3 proteins, which modulate the transcription of many genes.9 Phosphorylation of the C-terminal Ser423/425 of Smad3 by the TGF-β receptor kinase is critical for its nuclear translocation and downstream signaling. The molecular mechanisms of the counterregulatory effects of ANP/cGMP/PKG signaling on activated TGF-β–induced Smad signaling in cardiac cells have not been studied.
The present study tested the hypotheses that TGF-β1 accelerates myofibroblast (MF) transformation and ECM production in CFs and that activation of the ANP signaling cascade inhibits these processes by interrupting TGF-β1 signaling. Specifically, we tested whether ANP/cGMP/PKG signaling interrupts specific downstream events in the TGF-β1 signaling pathway, including phosphorylation and nuclear translocation of Smad2 and Smad3. We demonstrated that, following pretreatment with cGMP, TGF-β1 induced phosphorylation of Smad2 and Smad3, but the resultant phosphorylated (p)Smads could not be translocated to the nucleus. We then tested the hypothesis that PKG has the potential to phosphorylate Smad3 at sites different from those required for its TGF-β1–induced nuclear translocation, thus disrupting its entry into the nucleus and downstream signaling. Two novel sites of Smad3 phosphorylation were identified (Ser309 and Thr388) by use of high-resolution mass spectrometry (MS). These differ from the C-terminal Ser423/425 residues that are substrates for phosphorylation by the TGF-β receptor kinase, suggesting a novel mechanism by which the ANP/cGMP/PKG signaling pathway can inhibit the profibrotic effects of TGF-β.
Materials and Methods
Male ANP null (Nppa−/−) mice1 and wild-type controls (Nppa+/+) of the C57BL/6 strain were studied. Mice were fed a standard diet (Harlan-Teklad) and were housed in rooms maintained at constant humidity (60±5%), temperature (24±1°C), and light cycle (6:00 am to 6:00 pm). All protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for the Care and Use of Laboratory Animals published by the NIH (Department of Health, Education, and Welfare Publication No. 96-01, revised in 2002).
Nppa−/−, 9 to 10 weeks of age, and age-matched wild-type Nppa+/+ mice underwent transverse aortic constriction (TAC) or sham surgery under ketamine/xylazine (8 mg/1.2 mg per 100 g IP) anesthesia.1,2,4 Using this methodology, we have previously demonstrated reproducible pressure gradients across the TAC of 50 to 65 mm Hg.1
Effects of TAC on Collagen Deposition and MF Transformation in Hearts of Nppa+/+ and Nppa−/− Mice
One week after TAC, mice were killed with an overdose of pentobarbital and by cervical dislocation. Heart were dissected and weighed. The left ventricle (LV) was fixed with 4% paraformaldehyde, paraffin embedded, and sectioned for morphological and immunohistochemical examination of collagen deposition and MF transformation. Collagen was assessed using Picrosirius red staining, and MFs were identified by α-smooth muscle actin (α-SMA) (using clone1A4 anti–α-SMA antibody, Dako) immunochemical staining.2,4,10 Adjacent cross-sections (5 μm) from LVs were examined to assess colocalization of collagen and α-SMA in or near the same cell types. In a subgroup of wild-type mice, TGF-β1 (total and active) protein levels in LVs were measured using an ELISA kit (R&D Systems).
CFs were isolated from hearts of adult male C57BL/6 mice weighing 18 to 25 g. Hearts were excised, rinsed in cold Hank’s balanced salt solution, minced, and digested with collagenase type 4 (100 U/mL) and trypsin (0.6 mg/mL) at 37°C for 30 minutes. The first digestion was discarded. The collagenase medium from the second digestion containing the CFs was centrifuged for 10 minutes at 180g and resuspended in DMEM with 15% FBS. The digestion was repeated until the digestion fluid became clear (5 to 6 times). Cells were plated in laminin-coated 60-mm dishes (Becton Dickinson) and allowed to attach for 45 minutes before the first media change, which removed weakly adherent cells, including myocytes and endothelial cells. Passage 1 CFs were used for the experiments.
Effects of cGMP on TGF-β1–Induced MF Transformation in Cultured Mouse CFs
CFs isolated from wild-type mice were grown in 15% FBS-DMEM to 85% confluence on 18-mm2 glass cover slides and then made quiescent by culturing them in 0.1% FBS medium for 48 hours. Quiescent CFs were pretreated with cGMP (8-bromo-cGMP) (a cGMP analog, 10−3 mol/L) (Sigma) or vehicle for 30 minutes and then exposed to TGF-β1 (1 ng/mL) (Sigma) or vehicle for an additional 24 hours. Cells were then fixed with 4% paraformaldehyde and immunostained for α-SMA as a marker of MF transformation and counterstained with hematoxylin for visualization of nuclei. Quantitative analysis for α-SMA–positive cell/total cell ratios was performed by light microscopy with a Qimaging QiCam color camera (Qimaging) interfaced with a computer system running MetaMorph software (Universal Imaging Corp).
Effects of ANP on cGMP Levels and Effects of ANP/cGMP on Collagen Synthesis, Cell Proliferation, and Plasminogen Activator Inhibitor-1 Expression in Mouse CFs
Quiescent mouse CFs were treated with ANP (1 μmol/L) (Sigma) for 30 minutes and then harvested for cGMP measurement using a standard radioimmunoassay kit (Amersham). CFs isolated from adult male Sprague–Dawley rats were isolated and treated similarly and used as controls.
Separate groups of quiescent CFs were pretreated with ANP (1 μmol/L) or cGMP (1 mmol/L) for 30 minutes before exposure to TGF-β1 (5 ng/mL) for an additional 24 hours, and de novo collagen synthesis in CFs was evaluated by measuring [3H]-proline incorporation into cells using the method of Maki et al.11
To test the hypothesis that cGMP inhibits TGF-β1–stimulated cell proliferation, quiescent CFs were pretreated with cGMP (1 mmol/L) for 30 minutes before exposure to TGF-β1 (5 ng/mL). Cell proliferation was measured using a CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega) at 30, 90, and 240 minutes after TGF-β1 treatment.
The effects of cGMP on TGF-β1–stimulated plasminogen activator inhibitor (PAI)-1 mRNA expression (a biomarker of TGF-β action in cells) was tested in quiescent CFs treated with TGF-β1 (5 ng/mL) for 24 hours with or without pretreatment with cGMP (1 mmol/L for 30 minutes) and/or the PKG inhibitor KT5823 (1 μmol/L, 15 minutes before cGMP) (Calbiochem). Northern blot analysis for PAI-1 and GAPDH (internal control) mRNA levels was performed.1,12
Effects of ANP/cGMP on TGF-β1–Induced Nuclear Translocation of pSmad3 in Mouse CFs
To test the hypothesis that inhibition of TGF-β1–stimulated MF transformation and 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-β1–stimulated nuclear translocation of pSmad2 and pSmad3 in CFs. Quiescent mouse CFs were pretreated with ANP (1 μmol/L), cGMP (1 mmol/L), or vehicle for 30 minutes and then exposed to TGF-β1 (1 ng/mL) for an additional 30 minutes. Subgroups of CFs were pretreated with the PKG inhibitor KT5823 (1 μmol/L) for 15 minutes before ANP or cGMP. CFs were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS. The fixed CFs were stained with selective anti–pSmad2, anti-pSmad3 or anti-Smad2/3 primary antibodies (1:400×, Cell Signaling Technology) overnight at 4°C and then with a Texas-conjugated donkey anti-rabbit IgG secondary antibody (1:500×, Jackson ImmunoResearch Laboratory) for 1 hour at room temperature to assess nuclear translocation of pSmad2 and pSmad3 using confocal fluorescence microscopy with a computerized Zeiss Axioskop system.
Effects of TGF-β1 and cGMP on Phosphorylation of Smad Proteins
Quiescent CFs were pretreated with cGMP (1 mmol/L) or vehicle for 30 minutes before addition of TGF-β1 (1 ng/mL) to the medium and incubated for an additional 30 minutes and then harvested for assessment of pSmad2 and pSmad3 using Western blot analysis.12
We then tested the hypothesis that cGMP/PKG activation can phosphorylate Smad3 protein at sites other than the C-terminal SSXS motif, in which the 2 end serines (Ser423/425) are phosphorylated by the TGF-β type I receptor kinase.13 A plasmid with a glutathione S-transferase (GST)-Smad3 fusion gene was transfected into Escherichia coli and expression of fusion proteins was induced by isopropyl-B-d-thiogalactoside. Proteins were harvested with glutathione beads and cleaved with thrombin to yield purified Smad3 as described previously.14 Purified Smad3 (10 μg) was first phosphorylated with a recombinant PKG-1α (1000 U for 20 minutes, Calbiochem) using γ-32P-ATP or nonradioactive ATP as substrate and then digested with carboxypeptidase Y (a C-terminal peptidase, 1 ng/μL, Sigma) for 1, 30, and 60 minutes. The reaction mixture was subjected to 10% SDS-PAGE. The gel with 32P-Smad3 was used for radioautography to identify the phosphorylated peptides, and the gel with nonradioactive pSmad3 was used for Western blot analysis to identify the Smad3 peptides. The Western blot was probed with either a selective anti–N-terminal Smad3 antibody or an anti–C-terminal Smad3-pSer423/S425 antibody.
Identification of the PKG Phosphorylated Sites on Smad3 Protein by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Two micrograms of PKG-1α-phosphorylated Smad3 (n=6) and unphosphorylated (reaction without PKG in the buffer, as a negative control, n=2) were dissolved in 100 μL of 50 mmol/L ammonium bicarbonate solution. pSmad3 was first reduced in 10 mmol/L dithiothreitol (by adding 5 μL of 200 mmol/L dithiothreitol to the reaction mixture) for 30 minutes, alkylated in 50 mmol/L iodoacetamide (by adding 5 μL of 1 mol/L iodoacetamide to the reaction mixture) for 30 minutes, and then digested with 0.2 μg (by adding 5 μL of 40 μg/mL enzyme to the reaction mixture) of trypsin (Promega) or GluC (Roche) for 24 hours at room temperature. The trypsin- or GluC-digested pSmad3 peptides were then analyzed by use of reversed-phase C18 liquid chromatography (RP-C18 LC)-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis and ion trap tandem MS (MS/MS), as described previously.15,16
Results are expressed as means±SE. Statistical analyses were performed using the SigmaStat package (Jandel Scientific Software, San Rafael, Calif) on a personal computer. The primary statistical test was ANOVA. If ANOVA results were significant, a post hoc comparison among groups was performed with the Newman–Keuls test. Differences in mean values were reported as significant if the probability value was <0.05.
MF Transformation and Collagen Deposition Are Increased in Hearts of Nppa−/− Mice Compared With Nppa+/+ Mice Subjected to TAC
In response to TAC, Nppa−/− mice developed significant cardiac hypertrophy (left ventricular weight [adjusted by body weight]: 209±6 mg in TAC-Nppa−/− mice [n=10] versus 124±3 mg in Sham-Nppa−/− mice [n=8]), with greatly increased numbers of MFs (α-SMA antibody–stained cells that are not vascular smooth muscle cells or cardiomyocytes [CMs]) and more robust collagen protein expression (Picrosirius stain) compared with wild-type (Nppa+/+) mice (left ventricular weight [adjusted by body weight]: 113±3 mg in TAC-Nppa+/+ mice [n=9] versus 99±2 mg in Sham-Nppa+/+ mice [n=9]) (Figure 1). MFs were colocalized with collagen in the hypertrophic LVs in both genotypes (Figure 1A versus 1B and 1C versus 1D). No MFs (data not shown) or significant collagen deposition was observed in sham-operated Nppa−/− or Nppa+/+ mice (Figure 1E). Higher active TGF-β1 protein levels were observed in LVs of wild-type TAC mice than in sham-operated controls (Figure 1F).
Expression of α-SMA was also induced in some CMs in LVs of TAC mice (data not shown). The α-SMA in CMs was not colocalized with collagen, suggesting that these cells are likely not synthesizing ECM.
These results suggest that ANP is a negative modulator of MF transformation in response to pressure-overload stress and that the exaggerated cardiac hypertrophy/remodeling observed in Nppa−/− mice in response to TAC is related to increased MF transformation and ECM deposition.
cGMP Inhibits TGF-β1–Induced MF Transformation of Mouse CFs In Vitro
MFs in vehicle-treated cultures (0.1% FBS medium) were sparse and characterized by disorganized intracellular α-SMA (percentage of α-SMA–positive cells, 27±5%; n=4 plates/group) (Figure 2A and 2E), whereas most (95±3%; n=4) cells in TGF-β1 treated cultures were α-SMA–positive and contained well-organized α-SMA filaments, indicating nearly complete transformation of CFs to MFs (Figure 2B). cGMP completely inhibited TGF-β1–induced MF transformation (percentage of α-SMA–positive cells, 21±5%; n=4) but did not change basal (% α-SMA positive cells=23±5%, n=4) MF numbers (Figure 2C through 2E). Together with our in vivo data, these results support the hypothesis that ANP (through cGMP) inhibits TGF-β–induced MF transformation and ECM deposition.
ANP Increases cGMP Expression and ANP and/or cGMP Inhibits TGF-β–Stimulated Collagen Synthesis, Cell Proliferation, and PAI-1 mRNA Expression in CFs In Vitro
Exposure to ANP (10−7 mol/L) for 30 minutes increased cGMP levels in both mouse and rat CFs (Figure 3A). TGF-β increased collagen synthesis (3[H]-proline incorporation into CFs) (Figure 3B) and cell proliferation (total dehydrogenase activity) (Figure 3C), and pretreatment with ANP or cGMP decreased basal collagen synthesis and inhibited TGF-β–induced collagen synthesis and proliferation of mouse CFs.
Pretreatment with cGMP decreased baseline levels of PAI-1 and significantly attenuated TGF-β1–stimulated PAI-1 mRNA expression (Figure 3D). Pretreatment with the PKG inhibitor KT5823 blocked the inhibitory effects of cGMP on TGF-β1–stimulated PAI-1 mRNA expression in CFs, suggesting that cGMP was acting through activation of PKG. These data support the hypothesis that ANP/cGMP/PKG signaling has antifibrogenic effects that antagonize TGF-β–induced stimulation of ECM expression in CFs.
ANP and cGMP Inhibit TGF-β–Induced Nuclear Translocation of pSmad3 in Mouse CFs
In vehicle-treated cells, immunostaining of pSmad3 (Figure 4A and 4E) and pSmad2 (data not shown) was weak and distributed evenly in cytoplasm and nucleus, suggesting that pSmad3 and pSmad2 levels were low and without significant nuclear translocation. TGF-β1 (1 ng/mL for 30 minutes) treatment significantly stimulated nuclear translocation of pSmad3, indicated by strong pSmad3 staining in the nucleus (Figure 4B and 4E). We did not observe significant pSmad2 nuclear translocation after 30 minutes of TGF-β1 treatment in mouse CFs (data not shown). Nearly 100% of TGF-β1–treated CFs had accumulated pSmad3 in their nuclei, and >30% of CFs had double nuclei, indicating cell proliferation. Pretreatment with ANP (1 μmol/L for 30 minutes) or cGMP (1 mmol/L for 30 minutes) inhibited TGF-β1–induced pSmad3 translocation, with substantial levels of Smad3 staining remaining in the cytoplasm in most cells (Figure 4C through 4E).
To test whether activation of PKG mediates the inhibitory effects of ANP and cGMP on TGF-β1–induced nuclear translocation of pSmad3, pSmad3 nuclear translocation was measured in the presence of the PKG inhibitor KT5823. Pretreatment with KT5823 blocked the inhibitory effects of ANP and cGMP on TGF-β1–stimulated pSmad3 nuclear translocation (Figure 4F).
cGMP Does Not Inhibit TGF-β1–Induced pSmad3 and pSmad2 Phosphorylation
Western blot analysis demonstrated that TGF-β1 treatment significantly increased pSmad3 and pSmad2 levels in CFs and that pretreatment with cGMP did not inhibit this process (Figure 5A). Neither cGMP nor TGF-β altered total Smad2/3 levels in these cells. Thus, disruption of Smad3 and Smad2 phosphorylation does not account for the inhibitory effects of ANP and cGMP on TGF-β1–induced ECM expression in CFs.
PKG Phosphorylates Smad3 Protein at Sites Other Than the C-Terminal Residues
As an initial test of the hypothesis that PKG can phosphorylate Smad3 protein at novel sites other than the C-terminal Ser423/425 residues that are phosphorylated by the TGF-β type I receptor kinase, PKG-phosphorylated Smad3 was digested with carboxypeptidase Y and size fractionated by SDS-PAGE. Autoradiographic analysis and Western blots of pSmad3 fragments probed with either a selective anti–N-terminal Smad3 antibody or an anti–C-terminal Smad3-pSer423/S425 antibody indicated that PKG did phosphorylate Smad3 when the C-terminal residues were removed by a C-terminal peptidase (Figure 5B), supporting the hypothesis that PKG can phosphorylate Smad3 at novel sites other than its C-terminal residues.
RP-C18-LC FT-ICR-MS/MS analysis of trypsin- or Gluc-digested pSmad3 that was phosphorylated by PKG confirmed that Ser309 and Thr388 on pSmad3 was phosphorylated by PKG (Figure 6). Figure 6B shows the FT-ICR MS spectrum of a doubly charged ion species at m/z 822.3542, corresponding to the mass of the pSmad3 tryptic peptide L296-S309 plus the addition of a phosphate group (theoretical, 1643.7063 m/z; 3.18 ppm mass error). Figure 6C shows the linear quadrupole ion trap (LTQ) MS/MS product ion spectrum of the same ion after fragmentation with the dominant fragment corresponding to the neutral loss of 80 Da, confirming the phosphorylated pSmad3 peptide. Phosphorylation of Thr388 was confirmed in similar fashion (data not shown).
This study yields novel insights into precise sites of “molecular merging” of pro- and antifibrogenic pathways in heart and characterizes CFs as the target cell in which pro- and antifibrogenic signaling cascades converge and regulate responses of the heart to hemodynamic stress. We demonstrate crosstalk between ANP and TGF-β signaling pathways in CFs in vitro such that ANP and cGMP, through a PKG-dependent mechanism, block induction of ECM and PAI-1 expression by TGF-β1. Our observation that cGMP inhibits TGF-β1–induced nuclear translocation of pSmad2 and pSmad3 in CFs defines for the first time a precise molecular mechanism by which ANP/cGMP/PKG signaling interferes with downstream signaling from TGF-β and thus protects against cardiac remodeling/fibrosis and failure in response to hemodynamic stress. These findings suggest the intriguing hypothesis that ANP signaling results in phosphorylation of Smad proteins on sites other than their C-terminal residues, thus blocking their nuclear translocation and binding to TGF-β–Smad responsive elements in the promoter regions of ECM genes.
TGF-β1, the major isoform in heart,17 is produced in CFs and CMs under stressful conditions and stimulates CF transformation (to MFs) and proliferation, as well as ECM production in response to hypertrophic stimuli.17,18 Thus, it plays a major role in cardiac remodeling under stress conditions.
Activated TGF-β ligands bind to a heteromeric complex of type II (TGFβRII) and type I (TGFβRI) receptors that transduce intracellular signals via phosphorylation of TGFβRI-associated Smad2 and Smad3.14,18 Phosphorylation of TGFβRI-associated Smad2 and Smad3 and nuclear translocation of pSmad2 and pSmad3 are critical steps in TGF-β signaling.14,18 Smad2 (467 aa) and Smad3 (425 aa) contain predominantly serine, with some threonine residues, in the C-terminal, linker, and MH1 regions that are accessible for phosphorylation. After ligand binding, phosphorylation by TGFβR1 kinase of the 2 most C-terminal serine residues drives the activation of Smad2 and Smad3 is required for nuclear translocation and subsequent binding of pSmad2 and pSmad3 to nuclear transcriptional factors and DNA that regulate the transcriptional expression of downstream genes.14,18
The interplay between Smads and other signaling pathways in the cytoplasm and nucleus is a critical mechanism by which the activities and expression of Smad proteins are modulated and is responsible for the diverse effects of the TGF-β family of proteins.14 Mice overexpressing TGF-β have cardiac hypertrophy and interstitial fibrosis,8 and studies in rat and human CFs have shown that B-type natriuretic peptide (BNP) and NO attenuate expression of TGF-β through activation of cGMP. However, the signaling cascades involved remain to be elucidated.8,19
Both inhibition and overphosphorylation of Smad2 or Smad3 have been reported to disrupt their heterodimerization with Smad4 and nuclear translocation, resulting in repression of transcriptional activation of TGF-β responsive promoters.20,21 Thus, phosphorylation not only activates Smad proteins but also modulates their activity. This provides a potential mechanism for integration of the Smad pathway with ANP/cGMP/PKG signaling pathway that modulates TGF-β signal transduction. The current study demonstrates that the PKG acts as a stronger protein kinase than TGFβRI and phosphorylates additional serine or threonine residues on Smad3 (and/or Smad2), thus disrupting their nuclear translocation, resulting in repression of transcriptional activation of TGF-β response promoters, eg, on collagen and PAI-1 genes. This represents a novel molecular merging mechanism by which ANP/cGMP/PKG signaling intercepts the TGF-β signaling cascade and may contribute to the antifibrogenic effects of ANP in the stressed heart.
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 SR, alteration of membrane K+ fluxes, and nuclear protein phosphorylation and translocation.22 An alternative (to overphosphorylation) explanation for the observation that ANP signaling prevents nuclear translocation of Smads is that cGMP and 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.14 The precise molecular basis for retention of pSmads in the cytosol following ANP or cGMP treatment remains to be identified and is a topic for current investigation in our laboratory.
Several recent studies have demonstrated that various cytoplasmic protein kinases and cyclic nucleotides participate in regulating responses to TGF-β.23 Extracellular signal-regulated mitogen-activated protein kinase inhibits TGF-β signaling via inhibition of nuclear accumulation of Smad2 in normal epithelial cells.24 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.21 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.25 Further, intracellular cAMP-elevating agents, such as prostaglandin E2 and the adenylate cyclase activator forskolin, inhibit TGF-β–induced Smad3/4-dependent gene expression via a cAMP-dependent, PKA-dependent mechanism in human keratinocytes.26 The current study shows that ANP and cGMP inhibit TGF-β–induced nuclear translocation of pSmad2 and pSmad3 in CFs, defining a new role for cyclic mononucleotide phosphate second messenger in regulating profibrogenic responses to TGF-β. Pretreatment with the PKG inhibitors KT5823 prevented the inhibitory effects of ANP and cGMP on TGF-β–stimulated nuclear translocation of pSmad3, supporting the hypothesis that these effects are mediated through activation of PKG.
Our previous in vivo studies have validated the importance of ANP and TGF-β and ANP as opposing influences in the pathogenesis of pressure overload–induced cardiac hypertrophy, fibrosis, and remodeling.1 We have shown that Nppa−/− mice develop cardiac enlargement and remodeling in response to pressure-overload stress, compared with Nppa+/+ mice.1,2 Similarly, other investigators have shown that Npr1−/− mice carrying gene-targeted disruption of Npr1 (encoding for the NPRA receptor) exhibit the same phenotype of cardiac hypertrophy and fibrosis as Nppa−/− mice and are associated with reduced guanylyl cyclase activity and cGMP levels and increased expression of angiotensin-converting enzyme and angiotensin II, as well as proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and TGF-β1 in the heart, suggesting that disruption of ANP/NPRA/cGMP signaling leads to increases in cardiac hypertrophic stimuli.27,28 We have shown that the excess cardiac enlargement and remodeling in Nppa−/− mice are a consequence of increased MF transformation and proliferation and deposition of ECM components, rather than excess CM hypertrophy.1 In contrast, using a novel DnTGFβRII mouse model, these processes are markedly attenuated by disruption of TGF-β signaling in heart.3–4 Taken together, these data support the hypothesis that endogenous ANP and TGF-β play important counterregulatory roles in regulating MF transformation and proliferation, ECM production, and cardiac remodeling in response to pressure-overload stress. An imbalance in the normal relationships between the antigrowth/antifibrogenic effects of ANP and mitogenic/profibrogenic effects of TGF-β results in pressure overload–induced cardiac fibrosis and remodeling.
Sources of Funding
This work was supported in part by NIH grants HL-080017, HL-044195, HL-075614, HL-07457, HL-64614, HL-075211, CA-101955, and DK60913 and American Heart Association Grant 0455197B GIA.
↵*The first two authors contributed equally to this work.
Original received June 13, 2007; revision received October 22, 2007; accepted October 31, 2007.
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