doi: 10.1161/CIRCRESAHA.107.170217
© 2008 American Heart Association, Inc.
Editorials |
Cyclic GMP/Protein Kinase G Phosphorylation of Smad3 Blocks Transforming Growth Factor-β–Induced Nuclear Smad Translocation
A Key Antifibrogenic Mechanism of Atrial Natriuretic Peptide
From the Department of Pharmacology, University of Nevada School of Medicine, Reno.
Correspondence to Dayue Duan, MD, PhD, FAHA, Laboratory of Functional Genomics and Proteomics, Center of Biomedical Research Excellence, Department of Pharmacology, University of Nevada School of Medicine, Manville Medical Building Room #9/MS 318, Reno, NV 89557-0270. E-mail dduan{at}medicine.nevada.edu
See related article, pages 185–192
Key Words: cardiac remodeling • signal transduction • atrial natriuretic factor • transforming growth factor • protein kinase G • Smad phosphorylation • phosphoproteome
In response to hemodynamic overload, the heart undergoes a complex adaptive remodeling process that involves cardiac myocyte hypertrophy, transformation of fibroblast into myofibroblast, high-level expression of extracellular matrix (ECM) proteins, interstitial fibrosis, and cell death.1 Differentiation of cardiac fibroblasts into myofibroblasts is critical to the production and deposition of collagens and plays a decisive role in myocardial fibrosis and morphological alterations during the progression of adaptive myocardial hypertrophy to decompensation and heart failure.1,2
Among the plethora of identified fibrogenic factors, transforming growth factor (TGF)-β3 plays a fundamental role in hypertrophic and fibrotic remodeling of the heart, where it regulates cardiomyocyte growth, fibroblast activation, and ECM deposition.4,5 The expression of ventricular TGF-β mRNA and protein is increased in numerous models of pathological cardiac hypertrophy and in cardiac cells in response to putative hypertrophic stimuli.6,7 In vitro, TGF-β activates myofibroblast transformation and increases ECM production.7 Blockade of TGF-β signaling is predicted to blunt fibrosis.5 Recent studies from Chen et al8 convincingly demonstrated that disruption of TGF-β signaling by inducible dominant-negative mutation of the TGF-β receptor type II (TβRII) gene significantly reduced the pressure overload–induced myofibroblast transformation and interstitial fibrosis in mouse heart. Thus, there is considerable interest in understanding how signaling by TGF-β receptors is transduced and how the inevitable damage produced could be mitigated.
Of the 3 isoforms of TGF-β expressed in mammals,9,10 TGF-β1 is expressed in the adult heart, where it is secreted by cardiomyocytes and myofibroblasts and retained in significant amounts in ECM as a latent cytokine. Recent advances have led to clear description of downstream of TGF-β signal transduction pathways initiated by binding of TGF-β to membrane-bound heteromeric receptor kinases (TβRI and TβRII) that transduce intracellular signals via both Smad and non-Smad pathways (Figure).9,10 The primary TGF-β signal transduction pathway is the highly conserved Smad pathway.9 Activated TβRII and TβRI receptors phosphorylate receptor-regulated Smads (R-Smads, such as Smad2/3), which form homomeric complexes and heteromeric complexes with co-Smad (Smad4).9,10 These active Smad complexes translocate into the nucleus, where they accumulate and bind to target genes to directly regulate their transcription. A third class of Smad proteins is capable of inhibiting TGF-β signaling (I-Smads, such as Smad6/7). Posttranslational modifications of TGF-β/Smads pathway components, including receptor–receptor interactions, receptor-activated phosphorylation/dephosphorylation, Smad–Smad interactions, Smad shuttling and nuclear transportation, and accumulation of Smad complexes that bind to specific promoter elements to alter gene transcription, have been found to be very important for both positive and negative feedback in TGF-β signaling pathways.9,10
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Nuclear translocation of active Smad complex is fundamental for TGF-β signaling. In the absence of TGF-β ligands, monomeric R-Smads are predominantly cytoplasmic,10 whereas Smad4 is equally distributed between the nucleus and cytoplasm.11 Continuous shuttling of Smads between the nucleus and cytoplasm maintains a "basal shuttling equilibrium." Receptor kinase activation causes cytoplasmic R-Smads phosphorylation and Smad complex formation. Activated Smad complexes enter nucleus and associate with other transcription factors to positively or negatively regulate transcriptions of target genes. Constitutive Smad dephosphorylation by C-terminal phosphatases in the nucleus antagonizes Smad complex formation and releases monomeric Smads, which reenter the basal Smad shuttling equilibrium and, hence, mostly exit into the cytoplasm (Figure). This cycle continues and dynamically maintains a pool of cytoplasmic Smads, which keeps the cell responsive to changing signal intensities. Therefore, prevention of de novo formation of active Smad complexes will disrupt nuclear translocation and thus terminate TGF-β signaling.9,12
To date, little is known about pathways that may inhibit TGF-β–mediated fibrosis. Early in the 1990s, atrial natriuretic peptide (ANP), an endogenous hormone that is released by the heart in response to myocardial stretch and pressure overload, was found to inhibit collagen synthesis in cardiac fibroblasts via activation of membrane guanylyl cyclase receptors, and increase in intracellular cGMP levels.13 Recent evidence shows that endogenous ANP and TGF-β expression are both upregulated in heart with pressure overload,14 and ANP plays an important counterregulatory role against TGF-β–induced cardiac hypertrophy, remodeling, and fibrosis.8,14,15
In this issue of Circulation Research, Li et al16 studied the molecular mechanisms of the counterregulatory effects of ANP/cGMP/protein kinase G (PKG) signaling on activated TGF-β–induced Smad signaling in cardiac cells. In ANP-null (Nppa–/–) mice, transverse aortic constriction for 1 week leads to a near 10-fold increase in the deposition of interstitial collagen and increased numbers of myofibroblasts, as measured by 
-smooth muscle actin staining, as well as a concomitant increase in active TGF-β expression. In cardiac fibroblasts cultured from wild-type mice, treatment of cells for 24 hours with TGF-β caused a large increase in expression of -smooth muscle actin that was completely prevented by pretreatment of cells with the cell-permeant cGMP analog 8-bromo cGMP as a surrogate for the effect of ANP. Because both ANP and 8-bromo cGMP treatment reduced the production of collagen in these cells in the presence of TGF-β, the authors are able to propose that ANP/cGMP/PKG signaling has antifibrogenic effects that will lead to inhibition of the fibrotic damage conferred by TGF-β and, by extension, pressure-overload hypertrophy. This assertion is further supported by the effects of the cGMP/PKG to prevent the expression of mRNA for the plasminogen activator inhibitor PAI-1 as a measure of the genomic effects of TGF-β stimulation in cardiac fibroblasts. These results establish the crosstalk between TGF-β–induced profibrogenic and ANP-mediated antifibrogenic signaling pathways.
To gain further insights into the molecular mechanism by which ANP/cGMP/PKG signaling interferes with downstream signaling from TGF-β, the authors used anti-Smad3 immunostaining of cardiac fibroblasts to investigate the involvement of Smad pathway. They found that ANP and 8-bromo cGMP substantially inhibited TGF-β1–induced pSmad3 nuclear translocation, and this inhibitory effect of ANP and cGMP was effectively prevented by the PKG inhibitor KT5823.
With clear evidence in hand that disruption of the known TGF-β–induced phosphorylation of Smad3 required for nuclear localization (Ser423, Ser425) by interaction with Smad4 does not account for the inhibitory effects of ANP and cGMP on TGF-β1–induced Smad nuclear translocation, the authors elegantly probed the obvious mechanistic possibility that PKG phosphorylation of Smad3 at one or more sites distinct from that of activated TGF-β receptor may block their nuclear localization. Using MS/MS detection, the authors demonstrate two unique sites on Smad3 are phosphorylated by PKG (Ser309, Thr388). The identification of new phosphorylation sites of Smad3 and the characterization of the functional role of ANP/cGMP/PKG mediated phosphorylation of Smad3 at these sites in the inhibition of nuclear translocation of the Smad3 and cardiac fibroblast transformation and collagen synthesis have significantly advanced our understanding of a heretofore unrecognized role for ANP in regulating TGF-β Smad signaling.
Thus, the demonstration that over-phosphorylation of Smad3 by PKG as a key mechanism for the antifibrogenic effect of ANP against TGF-β–induced cardiac fibrotic damage in the heart during pressure overload offers a significant advancement in our understanding of myocardial hypertrophy and cardiac remodeling. Future studies of the phosphoproteome involved in the phosphorylation/dephosphorylation of the integrated ANP/cGMP/PKG and TGF-β and other signaling pathway components17 may identify novel therapeutic targets for the treatment of cardiovascular diseases. Moreover, these studies may shed new light on the pathological actions of TGF-β in other tissues and the effort directed at TGF-β antagonism.
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Acknowledgments |
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Sources of Funding
D.D. is supported by NIH National Center for Research Resources grant P-20 RR-15581 and National Heart, Lung, and Blood Institute grant HL63914, and I.L.O.B. is supported by NIH grant RO1 HD035028.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Related Article:
Circ. Res. 2008 102: 185-192.
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