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(Circulation Research. 2003;93:1034.)
© 2003 American Heart Association, Inc.

Reviews

Regulation of Gene Expression by Cyclic GMP

Renate B. Pilz, Darren E. Casteel

From the Department of Medicine and Cancer Center, University of California at San Diego, La Jolla, Calif.

Correspondence to Renate B. Pilz, Department of Medicine and Cancer Center, University of California at San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0652. E-mail rpilz{at}ucsd.edu

Rudi F. Busse Editor This Review is part of a thematic series on Cyclic GMP–Generating Enzymes and Cyclic GMP–Dependent Signaling, which includes the following articles:

Regulation of Nitric Oxide–Sensitive Guanylyl Cyclase

Cyclic GMP Phosphodiesterases and Regulation of Smooth Muscle Function

Structure, Regulation, and Function of Mammalian Membrane Guanylyl Cyclase Receptors, With a Focus on Guanylyl Cyclase-A

Cyclic GMP–Dependent Protein Kinases and the Cardiovascular System: Insights From Genetically Modified Mice

Regulation of Gene Expression by Cyclic GMP

Explaining the Phenomenon of Nitrate Tolerance

	Abstract

Top Abstract Introduction (Patho)Physiological Processes... Mechanisms of Transcriptional... Posttranscriptional Regulation... Conclusions References

 
Cyclic GMP, produced in response to nitric oxide and natriuretic peptides, is a key regulator of vascular smooth muscle cell contractility, growth, and differentiation, and is implicated in opposing the pathophysiology of hypertension, cardiac hypertrophy, atherosclerosis, and vascular injury/restenosis. cGMP regulates gene expression both positively and negatively at transcriptional as well as at posttranscriptional levels. cGMP-regulated transcription factors include the cAMP-response element binding protein CREB, the serum response factor SRF, and the nuclear factor of activated T cells NF/AT. cGMP can regulate CREB directly, through phosphorylation by cGMP-dependent protein kinase, or indirectly, through activation of mitogen-activated protein kinase pathways; regulation of SRF and NF/AT by cGMP is indirect, through modulation of RhoA and calcineurin signaling, respectively. Downregulation of the RNA-binding protein HuR by cGMP leads to destabilization of guanylate cyclase mRNA, but this posttranscriptional mechanism may affect many more cGMP-regulated genes. In this review, we discuss the role of cGMP-regulated gene expression in (patho)physiological processes most relevant to the cardiovascular system, such as regulation of vascular tone, cardiac hypertrophy, phenotypic modulation of vascular smooth muscle cells, and regulation of cell proliferation and apoptosis.

Key Words: cyclic GMP • transcription • mRNA stability • translation • gene expression

	Introduction

Top Abstract Introduction (Patho)Physiological Processes... Mechanisms of Transcriptional... Posttranscriptional Regulation... Conclusions References

 
Cyclic GMP is generated by cytoplasmic soluble guanylate cyclases (sGCs), which are activated by nitric oxide (NO), and by receptor guanylate cyclases (rGCs), which are activated by natriuretic peptides [atrial natriuretic peptide (ANP) or B- and C-type natriuretic peptides (BNP and CNP); Figure 1].^1–3 cGMP effector proteins include cGMP-dependent protein kinase (PKG) I and II, cyclic nucleotide-regulated ion channels, and phosphodiesterases (PDEs), which hydrolyze cGMP and/or cAMP.¹ PKG is the major intracellular cGMP target in many cell types, but high cGMP concentrations can cross-activate cAMP-dependent protein kinases (PKA).^3,4 PKG I is highly expressed in platelets, smooth muscle, glomerular mesangial cells, cardiomyocytes, and many endothelial and neuronal cells.^3,5–9 PKG II is encoded by a different gene with more limited expression³; throughout this article PKG will refer to PKG I. The importance of cGMP signaling for the cardiovascular system has been demonstrated in knockout mice lacking either PKG, neuronal, or endothelial NO synthase isoforms (nNOS and eNOS), ANP, or its rGC-A receptor.^2,10,11

View larger version (63K): [in this window] [in a new window]   Figure 1. Cyclic GMP signaling pathway. Cyclic GMP is synthesized by soluble guanylate cyclases in response to NO or by receptor guanylate cyclases, which are activated by natriuretic peptides. Depending on the cell type, cGMP has several intracellular targets in addition to cGMP-dependent protein kinases (PKG).

NO can regulate gene expression by multiple, including cGMP-independent mechanisms, as recently reviewed.^12,13 We will concentrate on cGMP-mediated regulation of gene expression defined by the following criteria: the effects should be observed with activators of sGCs or rGCs at physiologically relevant concentrations; the effect of NO or natriuretic peptides should be blocked by sGC inhibition or an appropriate rGC antagonist, respectively; and effects should be mimicked by membrane-permeable cGMP analogues. At a minimum, we will consider changes in mRNA levels induced by an activator of sGC or rGC that are mimicked by a membrane-permeable cGMP analogue. cGMP-mediated regulation of gene expression can be attributed to the action of PKG, if it is prevented by specific PKG inhibitors in PKG-expressing cells and enhanced by overexpression of PKG; the effect should be absent in PKG-deficient cells and restored by transfection of PKG. The PKG inhibitor KT5823 cannot be used as a sole criterion, because its specificity and effectiveness in intact cells has been seriously questioned.¹⁴

We will discuss specific genes regulated by cGMP, and review mechanisms of transcriptional and posttranscriptional regulation by cGMP.

	(Patho)Physiological Processes Involving cGMP Regulation of Gene Expression

Top Abstract Introduction (Patho)Physiological Processes... Mechanisms of Transcriptional... Posttranscriptional Regulation... Conclusions References

 
At the present time, cGMP-mediated increases or decreases in mRNA expression fulfilling the minimal criteria described above have been reported for >50 different genes, but in most cases the mechanisms of cGMP regulation have not been explored. cDNA microarray analyses of gene expression in rat aorta or cardiomyocytes after exposure to NO donors have revealed hundreds of genes changed by NO.^15,16 Similarly, hearts of eNOS-, nNOS-, and ANP-deficient mice have been compared with hearts of wild-type mice and microarray analyses have demonstrated divergent transcriptional programs, some of which may be related to alterations in cGMP signaling.^17,18 Although microarray analyses are providing large amounts of information regarding changes in gene expression in response to specific signals or gene alterations, only detailed analysis of single genes is able to distinguish direct versus indirect effects and determine the physiological significance of these changes. Table 1 summarizes cGMP regulation of specific genes that are discussed in the following sections.

View this table: [in this window] [in a new window]   Table 1. Regulation of Specific Genes by cGMP

Regulation of Vascular Tone
NO regulates vascular tone through stimulation of sGC, and ANP/BNP through stimulation of rGC-A, with cGMP activation of PKG causing smooth muscle relaxation via multiple mechanisms, including lowering of intracellular Ca²⁺ and inhibition of RhoA-dependent Ca²⁺ sensitization of contraction.^3,19 However, cGMP also positively regulates RhoA expression in vascular smooth muscle cells (VSMCs).²⁰ Prolonged exposure to nitrates leads to downregulation of sGC and PKG expression associated with nitrate tolerance,^21,22 and prolonged elevation of circulating ANP leads to downregulation of rGC.²³ These negative feedback loops involve cGMP inhibition of sGC, PKG, and rGC expression.

Soluble Guanylate Cyclase (sGC)
Low concentrations of NO donors, ANP, or cGMP analogues decrease mRNA and protein levels of both sGC-₁ and -ß₁ subunits in VSMCs by >90% within 24 hours.²¹ cGMP accelerates the decay of sGC-₁ and ß₁ mRNA; the effect is mediated by cGMP-induced downregulation of the RNA-stabilizing protein HuR (described later).^21,24

PKG
Continuous exposure to NO-releasing agents, cGMP, or cAMP analogues suppresses PKG mRNA and protein levels in VSMCs by decreasing transcription without affecting mRNA stability.²² Similarly, reduced PKG levels are found in cGMP-treated cardiomyocytes, and in the aortas of transgenic mice overexpressing eNOS in the endothelium.^6,25 Two SP-1 binding sites appear to be required for basal PKG promoter activity, and SP-1 and SP-3 DNA binding activities are reduced in cyclic nucleotide-treated cells.²⁶

Receptor Guanylate Cyclase A (rGC-A)
Ligand-dependent downregulation of rGC-A is associated with cGMP-mediated reduction in rGC-A mRNA levels and promoter activity in VSMCs.²³ Correspondingly, rGC-A expression is upregulated in ANP-deficient mice.²⁷ The effect of cGMP on the rGC-A promoter is not mimicked by cAMP; it is mediated by a negative cis-acting element localized approximately 1400 nucleotides upstream of the transcription start site, but the trans-acting factors remain to be identified.²³

RhoA
Long-term exposure of primary VSMCs to NO donors or cGMP analogues increases RhoA expression through increased rhoA transcription as well increased RhoA protein stability due to PKG phosphorylation.²⁰ The PKG-mediated transcriptional effect requires an intact CRE in the RhoA promoter and is associated with increased phosphorylation of CREB and ATF-1.²⁰ Inhibition of NO synthesis in rats decreases RhoA mRNA and protein expression in aorta and pulmonary artery, suggesting that basal release of NO is necessary to maintain RhoA expression and function in VSMCs.²⁰

Cardiac Hypertrophy
Cardiac hypertrophy involves induction of embryonic genes, including ANP, BNP, and skeletal -actin, by hypertrophic stimuli such as ET-1, angiotensin II (Ang II), phenylephrine, and fibroblast growth factor (FGF).²⁸ Hypertrophy-promoting signaling pathways include the small GTPases Ras, RhoA, and Rac, mitogen-activated protein kinases (MAP kinases), and Ca²⁺/calcineurin.²⁸ Mice deficient in ANP or rGC-A develop increased ventricular mass out of proportion to the mild changes in blood pressure,² and mice deficient in nNOS develop cardiac hypertrophy with normal systemic blood pressure,¹¹ suggesting that NO/cGMP negatively regulate cardiac hypertrophy. PKG-deficient mice may not live long enough to develop cardiac hypertrophy, but PKG overexpression enhances the antihypertrophic effects of NO in phenylephrine-stimulated cardiomyocytes in vitro, and prevents phenylephrine-induced increase in prepro-ANP mRNA.⁶ Similarly, natriuretic peptides and cGMP analogues suppress the phenylephrine-induced hypertrophic pattern of gene expression including skeletal -actin and ß-myosin heavy chain gene expression.²⁹ Because RhoA signaling is required for phenylephrine-induced hypertrophic gene expression, and cardiac-specific overexpression of serum response factor (SRF) results in hypertrophy, it is possible that some of PKGs antihypertrophic effects could be through inhibition of RhoA signaling to SRF.^5,28 NO/cGMP activation of PKG also inhibits Ca²⁺-dependent activation of the hypertrophic calcineurin-NF/AT pathway, preventing NF/AT induction of BNP mRNA and promoter activity.³⁰ cGMP inhibition of SRF- and NF/AT-dependent transcription is further discussed below. Cardiac expression profiling of genes stimulated by the hypertrophic peptide ET-1 in the presence or absence of NO demonstrates downregulation of muscle LIM protein (MLP) by NO; this effect is at least partially through cGMP/PKG, and may explain some of PKGs antihypertrophic effects, because MLP expression is necessary and sufficient for ET-1–induced hypertrophy.¹⁶ In addition, ET-1 synthesis is negatively regulated by cGMP (see later).

VSMC Differentiation/Phenotypic Modulation
In response to vascular injury and during in vitro culture, VSMCs change their state of differentiation from a highly differentiated, "contractile" phenotype to a dedifferentiated "synthetic" phenotype; during this process, they acquire the capacity to proliferate, migrate, and produce extracellular matrix proteins such as osteopontin and thrombospondin, thus contributing to neointima formation after vascular injury.³¹ Dedifferentiation to the synthetic phenotype is associated with loss of PKG expression and transcriptional downregulation of contractile proteins such as smooth muscle myosin heavy chain-2 (SM-MHC-2), SM--actin, and SM calponin.³²

Transfecting synthetic VSMCs with constitutively active or wild-type PKG (plus cGMP stimulation) restores a more contractile phenotype with fusiform morphology, increased expression of SM-MHC-2, SM--actin, and calponin protein, and decreased expression of osteopontin, thrombospondin, and FGF receptors-1/2.^31–34 FGF receptor protein and mRNA levels are dramatically and proportionally decreased in PKG-transfected cells compared with control-transfected cells, but osteopontin and thrombospondin mRNA levels decrease only minimally, suggesting PKG-induced changes in mRNA translation or protein stability.^33,34 Whether the PKG-mediated induction of contractile protein expression occurs at the transcriptional level is unclear, although one preliminary report suggests that PKG activation may activate the SM-MHC promoter.³⁵ In addition, SM-MHC-2 mRNA and protein expression is increased in CNP peptide-overexpressing VSMCs in vitro and in vivo after angioplasty,³⁶ and downregulation of the SM-MHC promoter by platelet-derived growth factor is prevented by NO.³⁷ These data suggest that PKG plays an important role during VSMC phenotypic modulation, positively and negatively regulating gene expression. A preliminary report of cDNA microarray analyses comparing PKG-transfected and control-transfected, late passage VSMCs suggests that >100 transcripts may be up- or downregulated more than 3-fold by cGMP/PKG.³⁸

The mechanism(s) responsible for PKG downregulation in dedifferentiating VSMCs remains unknown, but PKG mRNA is downregulated when differentiated VSMCs are chronically exposed to NO, cyclic nucleotides, or various growth factors in vitro.^22,31 PKG levels may transiently decrease after vascular injury in vivo, and appear to decrease in proliferating neointimal VSMCs coincidentally with transcriptional downregulation of contractile marker expression and increased synthesis of osteopontin.^39–42 Increased growth factor and cytokine production at the site of vascular injury could increase local cGMP through iNOS induction and may explain PKG downregulation.³¹ Reexpression of constitutively active PKG (or wild type PKG with cGMP stimulation) inhibits VSMC migration, enhances apoptosis, reduces proliferation, and decreases neointima formation after vascular injury.^32,39 These results suggest that PKG modulates the VSMC phenotype in vivo and are consistent with the finding that adenoviral overexpression of CNP or soluble guanylate cyclase reduces neointima formation, whereas eNOS-deficient mice demonstrate a hyperplastic response after vascular injury.^36,43,44

Regulation of Cell Proliferation
Depending on the cell type, cGMP can have pro- or antiproliferative effects. In VSMCs, mesangial cells, and various fibroblasts, cGMP inhibits proliferation, and the effect is mostly mediated by PKG, although it may involve PKA cross-activation under some conditions.^4,45–54 The antiproliferative effect correlates with cGMP inhibition of growth factor-induced extracellular signal-regulated kinase (Erk-1/2) activity (online Table, available in the online data supplement at http://www.circresaha.org; also discussed later), increased expression of MAP kinase phosphatase-1 (MKP-1),^46,47,53 modulation of cell cycle-associated genes, and reduction of ET-1 synthesis.^52,55

In contrast to the antiproliferative effects in VSMCs and fibroblasts, cGMP increases proliferation of endothelial cells.^36,56–58 The proproliferative effect of cGMP in endothelial cells correlates with increased Erk-1/2 activity, and may be related to increased production of vascular endothelial growth factor (VEGF).^56–58

MAP Kinase Phosphatase-1 (MKP-1)
MKP-1 dephosphorylates and inactivates the MAP kinases Erk-1/2, p38, and c-Jun N-terminal kinase (JNK) (Figure 2).⁵⁹ MKP-1 mRNA expression is increased by NO-releasing agents, ANP, and cGMP analogues in endothelial and smooth muscle cells^59–61; induction by cGMP can occur in the absence of Erk-1/2 stimulation.⁵³ cGMP, via PKG, induces MKP-1 mRNA sufficiently to inhibit growth factor-induced Erk-1/2 activity, and MKP-1 induction contributes to the antiproliferative effect of cGMP-elevating agents.^53,60

View larger version (28K): [in this window] [in a new window]   Figure 2. Regulation of MAP kinase pathways by cGMP. Three MAP kinases comprise the extracellular signal-regulated kinases Erk-1/2, p38 isoforms , ß, and , and the c-Jun amino terminal kinases JNK-1/2/3. Upstream kinases include the MAP kinase kinases MEK-1/2 and MEK-3/6 for Erk and p38, respectively, and the JNK kinases JKK-1/2 for JNK-1/2/3. All three pathways are activated by multiple extracellular stimuli, including growth factors, cytokines, and stress (such as oxidative stress and heat shock), and all three types of MAP kinases are inactivated by MAP kinase phosphatase-1 (MKP-1). Some transcription factors targeted by MAP kinases are shown; a more complete list has been reviewed.92 cGMP can either activate or inhibit each pathway, depending on the cell type and growth conditions (online Table). Inhibition of Raf-1 by cGMP/PKG53 and PKG-dependent activation of MEK-1/2,106,133 MEK-3,139 or MEKK-171 have been reported; however, it is not clear whether PKG directly activates these enzymes (indicated by "?"). GEFs, guanine nucleotide exchange factors.

Cyclins and Cyclin Inhibitors
cGMP decreases expression of cell cycle-promoting genes, such as cyclin A, D1, and E.^48,50,62 In addition, cGMP may increase expression of cell cycle inhibitors, such as p21^Waf1/Cip1 and p16^INK4a.^36,49 Under some conditions, cGMP-induced changes in expression of cell cycle–associated genes appear to be dependent on inhibition of Erk-1/2, but this is not always the case.^47,49,50

Endothelin-1 (ET-1)
ET-1 is a potent vasoconstrictor and mitogenic peptide produced by proteolytic cleavage from an inactive precursor and secreted by endothelial cells.⁵⁵ Basal prepro-ET-1 mRNA levels and peptide synthesis appear to be decreased by cGMP, because incubation of endothelial cells with NOS inhibitors, NO scavenger molecules, or sGC inhibitors increase prepro-ET-1 synthesis and ET-1 peptide secretion, and this effect is reversed by cGMP analogues.⁵⁵ Thrombin- or Ang II–mediated increases in prepro-ET-1 mRNA levels are also inhibited by natriuretic peptides and cGMP analogues.⁵²

Vascular Endothelial Growth Factor (VEGF)
VEGF mediates angiogenesis and vascular permeability; in normoxic VSMCs, hepatoma, and glioma cells, NO donors increase basal VEGF mRNA levels in a cGMP-dependent fashion.^63,64 An in vivo model of embolic stroke suggests a positive role for cGMP in angiogenesis, which is dependent on VEGF.⁵⁶ However, VEGF expression is induced by hypoxia, and treating hypoxic VSMCs or endothelial cells with NO or cGMP decreases VEGF mRNA,⁶⁵ and NOS inhibition increases VEGF mRNA in balloon-injured arterial walls, suggesting that endogenous NO may suppress VEGF expression under these conditions.⁶⁶

Apoptosis
Pro- and antiapoptotic effects of cGMP have been described in different cell types. In VSMCs, cardiomyocytes, and endothelial cells, NO, natriuretic peptides, and cGMP analogues increase apoptosis, and the effect appears to be PKG mediated.^45,67–69 In cardiomyocytes, cGMP sharply decreases mRNA expression of the antiapoptotic Bcl-2 homologue Mcl-1.⁶⁷ In VSMCs and endothelial cells, the effect of cGMP occurs in low serum–containing medium and is counteracted by Ang II and ET-1, respectively.^68,69 The mechanism(s) of the proapoptotic effects of cGMP in vascular cells remains to be determined, but cGMP may increase apoptosis through PKG-dependent activation of JNK and/or phosphorylation and inactivation of ß-catenin.^70–72

In neuronal cells, hepatocytes, and lymphocytes, cGMP has antiapoptotic effects that appear to be mediated by Bcl-2 or Bcl-2–associated proteins.^73–75 In neuronal cells, prolonged inhibition of NOS or sGC induces apoptosis that is prevented by cGMP analogues; the protective effect of cGMP is associated with increased CREB phosphorylation and increased mRNA and protein expression of the apoptosis inhibitor Bcl-2.⁷³ During growth factor deprivation, cGMP/PKG protect neuronal cells from apoptosis by increasing expression of the oxidative stress-related proteins thioredoxin and thioredoxin peroxidase (Tpx-1), which leads to increased Bcl-2 expression.⁹

Mitochondrial Biogenesis
NO, in a cGMP-dependent fashion, triggers mitochondrial biogenesis in many different cell types, and mitochondrial biogenesis induced by cold exposure is reduced in eNOS-deficient mice.⁷⁶ The cGMP effect is mediated by induction of peroxisome proliferator-activated receptor- coactivator-1 (PGC-1), a master regulatory factor of mitochondrial biogenesis in brown adipose tissue and in cardiac and skeletal muscle; this coactivator increases expression of other transcription factors that regulate nuclear and mitochondrial genes encoding mitochondrial proteins.⁷⁶

Other cGMP-Regulated Genes
cGMP regulates expression of the inflammatory cytokine TNF-, and the cytokine-inducible genes iNOS and COX-2 involved in inflammatory processes.^77–80 Both positive and negative effects of cGMP have been described for each of these genes, in some cases leading to biphasic regulation, eg, early transcriptional upregulation followed by downregulation due to mRNA destabilization; the latter could be related to HuR downregulation by cGMP (discussed later).^81–83

Tumor Necrosis Factor- (TNF-)
In cardiomyocytes, NO increases TNF- mRNA in a cGMP- and PKG-dependent fashion, and this effect correlates with increased nuclear factor-B (NF-B) activity and is mimicked by cGMP, but not cAMP analogues.⁷⁷ In VSMCs and resting macrophages, cGMP analogues induce basal TNF- mRNA and enhance IL-1–induced TNF- mRNA.^84,85 However, in LPS-stimulated macrophages, induction of TNF- mRNA and protein is attenuated by cGMP due to mRNA destabilization.⁸⁶

Inducible NO Synthase (iNOS)
In cardiomyocytes, VSMCs, and mesangial cells, cytokine-induced transcription of iNOS is enhanced by NO donors, ANP/BNP, or cGMP analogues, and reduced by NOS inhibitors, whereas in the absence of cytokines, cGMP-elevating agents have very little or no effect on basal iNOS expression.^78,79,81,84 In addition, in some cell types, cGMP destabilizes iNOS mRNA leading to downregulation of cytokine-induced iNOS mRNA at later time points.^81,87

Inducible Cyclooxygenase-2 (COX-2)
Like iNOS, COX-2 is transcriptionally induced in response to inflammatory stimuli, and the effects of NO on cytokine-induced COX-2 and iNOS expression are similar.⁸⁰ NO is necessary for cytokine induction of COX-2 mRNA in mesangial cells, and NO donors, ANP, and cGMP analogues synergistically enhance the effect of IL-1ß on COX-2 mRNA at early time points.^82,88 However, at later time points, NO donors decrease cytokine-induced COX-2 mRNA levels, reminiscent of the biphasic effects of NO on iNOS expression in these cells.^81,82 Destabilization of COX-2 mRNA by ANP and cGMP analogues has been described in LPS-stimulated macrophages.⁸⁷

In whole animal models, NO positively affects COX-2 expression, and its importance for prostaglandin synthesis has been confirmed in iNOS-deficient mice.⁸⁰ Induction of COX-2 mRNA by salt depletion and/or angiotensin-converting enzyme inhibition in renal cortex is blocked by NOS inhibitors, and this effect is relieved by cGMP analogues; correspondingly, NO donors and cGMP analogues increase basal COX-2 mRNA levels in renal tubule cells.⁸⁹

Plasminogen Activator Inhibitor-1 (PAI-1)
PAI-1 is produced by VSMCs and endothelial cells, and elevated levels are found in atherosclerotic and balloon-injured vessels.⁹⁰ NO donors, natriuretic peptides, and cGMP analogues reduce Ang II–induced PAI-1 mRNA levels without affecting mRNA stability.^90,91

	Mechanisms of Transcriptional Regulation by cGMP

Top Abstract Introduction (Patho)Physiological Processes... Mechanisms of Transcriptional... Posttranscriptional Regulation... Conclusions References

 
Overview
cGMP can regulate transcription factors directly by inducing phosphorylation or by increasing expression of short-lived proteins. Transcription factors controlled by cGMP-dependent phosphorylation include the cAMP response-element (CRE)-binding protein CREB, activating transcription factor-1 (ATF-1), and the multifunctional transcription factor TFII-I (summarized in Table 2). Transcription factors whose expression is regulated by cGMP include the AP-1 family proteins c-Fos and JunB, the early growth response gene Egr-1, and the growth arrest–specific homeobox gene GAX (Table 2). In addition, cGMP can regulate transcription factors indirectly, through modulation of upstream signal transduction pathways. Examples include cGMP regulation of an inhibitor of NF-B, and inhibition of calcineurin signaling to NF/AT and of RhoA signaling to SRF (Table 2). Through activation or inhibition of MAP kinase pathways, cGMP can regulate the activity of multiple transcription factors, including ternary complex factor (TCF), CREB, ATF-2, and c-Jun (online Table and Figure 2).⁹²

View this table: [in this window] [in a new window]   Table 2. Transcription Factors Regulated by cGMP

Transcription Factors Directly Controlled by cGMP-Dependent Phosphorylation
CREB and ATF-1
The transcription factor CREB is activated by a diverse array of stimuli through phosphorylation of Ser¹³³ in its kinase-inducible domain; CREB is critical for proliferation, differentiation, and survival of many different cell types, and absence of CREB causes dwarfism and cardiac myopathy.⁹³ Increased intracellular cGMP leads to increased CREB Ser¹³³ phosphorylation in VSMCs, neuronal cells, and PKG-transfected Baby Hamster Kidney (BHK) cells, but not in PKG-deficient BHK cells.^20,73,94,95 cGMP-induced CREB phosphorylation in BHK cells occurs at physiologically relevant PKG levels and is independent of changes in intracellular Ca²⁺, activation of MAP kinases (Erk-1/2 and p38), or cross-activation of PKA, thus ruling out the effect of other known CREB kinases.⁹⁵ PKG can directly phosphorylate CREB on Ser¹³³ in vitro; the kinetics are slower than those of PKA, but comparable to those of Ca²⁺/calmodulin-dependent protein kinase IV.⁹⁵ Correspondingly, PKG is less effective than PKA, but similar to Ca²⁺/calmodulin-dependent protein kinase, in stimulating cAMP-response-element (CRE)–dependent transcription in intact cells.^95–98 In BHK cells and in some neuronal cells, cGMP-mediated transactivation of the c-fos promoter is dependent on CREB phosphorylation and nuclear translocation of PKG, as demonstrated by the ineffectiveness of extranuclear PKG constructs.^95,99 Nuclear translocation of PKG has been demonstrated in neuronal cells, neutrophils, macrophages, and some embryonal smooth muscle cells.^95,99–102 However, other investigators found no evidence of PKG nuclear translocation in primary VSMCs, HEK293, and CV-1 cells, or observed nuclear PKG only in a minority of the cell population.^97,103,104 Based on recent findings, we have speculated that PKG may be retained in extranuclear compartments by binding to cell type–specific anchoring proteins.¹⁰⁵ In primary VSMCs, cGMP/PKG-induced CREB phosphorylation may be indirect, eg, via activation of MAP kinases (Figure 2 and see later discussion).^20,106 The closely related CREB family member ATF-1 is also phosphorylated in response to PKG activation by cGMP, and ATF-1 appears to mediate transcriptional activation of the RhoA promoter by PKG.^20,95

TFII-I
TFII-I is a transcriptional regulator for many genes, including c-fos and so-called endoplasmic reticulum stress-response genes; TFII-I interacts with multiple transcription factors including SRF, TCF, ATF-6, c-Myc, and NF-B, and with histone deacetylases.^107–109 We found that TFII-I physically interacts with PKG Iß, with these two proteins coimmunoprecipitating in C2C12 myoblasts.¹⁰⁵ PKG phosphorylates TFII-I in vitro and in vivo; PKG Iß activation enhances the transactivation potential of wild type, but not phosphorylation-deficient mutant TFII-I on an SRF/TCF-dependent reporter gene, and PKG cooperates with TFII-I to transactivate the fos promoter.¹⁰⁵

Short-Lived Transcription Factors With Expression Regulated by cGMP
AP-1 (Fos/Jun)
Fos and Jun proteins dimerize to form the AP-1 transcription factor complex and are important for cell cycle progression and apoptosis; resting cells typically express low levels of fos and jun mRNA, but transcription of AP-1–related genes is rapidly induced in response to many stimuli.⁷² NO-releasing agents, natriuretic peptides, and cGMP analogues increase c-fos and junB mRNA expression in cultured cells and primary tissues,^110–112 and NOS inhibitors can reduce c-fos expression.¹¹² Cardiac expression profiling from eNOS-deficient mice demonstrate decreased c-fos mRNA expression compared with wild-type mice.¹⁷ In cardiac fibroblasts, cGMP-elevating agents modestly enhance c-fos induction by Ang II.⁵¹ As a consequence of NO/cGMP-induced c-fos and junB mRNA expression, AP-1 DNA binding activity increases resulting in increased transcription of AP-1–dependent reporter genes and endogenous genes.^110,111,113 In pulmonary endothelial cells, NO-releasing agents and cGMP analogues increase AP-1 DNA binding activity only if cells are costimulated with TNF-.¹¹⁴

Although high concentrations of NO can decrease DNA binding of recombinant Fos and Jun in vitro through S-nitrosylation and/or S-glutathionylation, the in vivo significance of this effect is unknown.^115,116 We demonstrated that NO stimulation of the fos promoter in different cell types is strictly cGMP-dependent and requires PKG activity.^95,117 PKG targets several transcriptional elements in the fos promoter: the CRE and the fos AP-1 site, which both bind CREB-related proteins, and the serum response element (SRE), which binds multiple transcription factors including SRF, TCF, C/EBP-ß, and TFII-I.^95,96,105 cGMP/PKG transactivation of the fos promoter requires CREB-related proteins, but is independent of AP-1 and C/EBP-related transcription factors and can occur independently of MAP kinase activation by cGMP.⁹⁵

Egr-1
Transcription of egr-1 is induced by many growth factors and stress stimuli with similar kinetics as c-fos.¹¹⁸ Egr-1 plays an important role in cell growth, differentiation, and apoptosis, and high, sustained expression of Egr-1 has been observed in atherosclerotic lesions.¹¹⁸ In pheochromocytoma cells, NO-releasing agents and natriuretic peptides increase egr-1 promoter activity, mRNA, and protein expression, and increase transcription from an Egr-1-responsive reporter.^110,119 Moreover, cGMP analogues increase serum-stimulated Egr-1 DNA binding activity; this effect is enhanced by overexpression of PKG and occurs in the absence of significant changes in MAP kinase activity.⁸ In contrast, NO and cGMP analogues appear to have no effect on Ang II–induced egr-1 mRNA expression in cardiac fibroblasts.⁵¹

The Growth Arrest–Specific Homeobox Gene (GAX)
GAX is found predominantly in cardiovascular tissues; it is expressed in quiescent VSMCs and rapidly downregulated after mitogen-stimulation or after vascular injury; overexpression of GAX induces growth arrest.^118,120 CNP and cGMP analogues increase GAX mRNA expression in serum-starved primary VSMCs and largely prevent the decrease in GAX mRNA in Ang II–stimulated cells;¹²⁰ this could be important for VSMC phenotypic modulation.¹¹⁸

Transcription Factors Indirectly Regulated by cGMP
NF-B
NF-B is a transcriptional activator of genes involved in inflammation, including cytokines and adhesion molecules.¹²¹ NF-B subunits are inactive when bound to cytoplasmic IB proteins, but multiple stimuli can induce phosphorylation and degradation of IB, thereby allowing activation and nuclear translocation of NF-B.¹²¹ NO can increase or decrease NF-B activity depending on the NO concentration, redox milieu, cell type, and co-stimulus, and in most cases, the effects of high NO concentrations on NF-B appear to be cGMP-independent.^12,13,80 However, in cardiomyocytes, NO-donors increase NF-B (p50/p65) DNA binding activity and induce expression of an NF-B–responsive gene in a cGMP/PKG-dependent fashion.⁷⁷ In these cells, cGMP induces phosphorylation and degradation of IB-, and direct phosphorylation of IB- by PKG occurs in vitro.⁷⁷ NF-B may also be activated by cGMP through direct PKG phosphorylation of p50 and p65 subunits.¹²² In contrast, cytokine-induced NF-B activation in human endothelial cells is inhibited by NO/cGMP and IB- is stabilized.^123,124 Natriuretic peptides and cGMP analogues also inhibit NF-B activation during hepatic ischemia/reperfusion injury through stabilization of IB; this correlates with cGMP induction of heat shock protein 70 and increased HSP70 association with IB.¹²⁵

Calcineurin-Dependent Transcription Factors
Calcineurin is a Ca²⁺-dependent phosphatase that dephosphorylates and thereby activates the transcription factors NF/AT, myocyte enhancer factor-2 (MEF-2), and Elk-1. In cardiomyocytes, PKG activation inhibits calcineurin-dependent NF/AT nuclear translocation, phenylephrine-induced BNP promoter activity, and cell enlargement by interfering with Ca²⁺ entry.³⁰ MEF-2–dependent transcription is also inhibited by cGMP/PKG.³⁰

Serum-Response Factor (SRF)
Transcriptional activation of muscle-specific genes requires cooperation between SRF and other coactivators, such as myocardin or the myocardin-related protein MAL, and is regulated by RhoA-induced actin polymerization.^126,127 RhoA activation induces MAL translocation from the cytoplasm to the nucleus and stimulates SRF-dependent transcription.¹²⁷ Growth factor-induced transcriptional activation of immediate-early genes such as c-fos and egr-1 also involves SRF, but on these promoters SRF cooperates with TCF; the SRF-TCF complex is regulated by MAP kinases, but is insensitive to RhoA signaling.^92,128

NO/cGMP/PKG signaling can inhibit RhoA functions such as Ca²⁺ sensitization of VSMC contractility and RhoA-dependent actin polymerization.¹⁹ We showed that PKG inhibits SRF-dependent transcription by interfering with RhoA signaling in cardiomyocytes and VSMCs; PKG acts both upstream of RhoA, inhibiting serum- and G₁₃-induced Rho activation, and downstream of RhoA, inhibiting steps distal to the Rho targets ROK, PKN, and PRK-2.⁵ PKG also inhibits serum induction of vinculin mRNA, thus regulating an endogenous SRF target gene.⁵

During differentiation of SM cells, RhoA appears to play a dual role: (1) in undifferentiated cells, high RhoA activity delays differentiation by restricting SRF to the cytoplasm, whereas RhoA downregulation induces nuclear translocation of SRF and promotes myogenesis¹²⁹; (2) in differentiated cells, SRF is associated with active, hyperacetylated chromatin, and RhoA activation by G protein–coupled receptors stimulates SRF-dependent transcription from many muscle-specific promoters.¹³⁰ Thus, PKG inhibition of RhoA in undifferentiated cells could provide a differentiation-promoting signal, while PKG inhibition of RhoA in differentiated cells may serve to restrict SM-specific promoter activity under conditions where RhoA is activated.

Regulation of Gene Transcription Through cGMP Modulation of MAP Kinase Pathways
Pathways that activate the MAP kinases Erk-1/2, p38, or JNK regulate gene expression through direct or indirect phosphorylation of multiple transcription factors, including TCF, CREB, ATF-2, and c-Jun (Figure 2).⁹² Increased intracellular cGMP can lead to increased or decreased activity of all three MAP kinase pathways depending on cell type and growth conditions (online Table), and changes in MAP kinase activity may explain the effect of cGMP on many genes involved in cell proliferation, differentiation, or apoptosis. This has been documented in some cases, eg, the cGMP-mediated increase of metalloproteinase-13 and heme oxygenase-1 expression in endothelial cells is dependent on Erk-1/2 activation.^131,132

In neonatal cardiomyocytes, ANF and cGMP analogues increase Erk-1/2 activity but have little effect on p38 MAP kinase or JNK activity.¹³³ In adult cardiomyocytes, NO increases both Erk-1/2 and p38 activity, but Erk-1/2 activation is cGMP-independent, whereas p38 activation is mediated by cGMP and PKG, with cAMP analogues having an opposing effect.¹³⁴

In serum-starved aortic VSMCs, Erk-1/2 and JNK activities are stimulated by cGMP analogues in a PKG-dependent fashion.¹⁰⁶ However, platelet-derived growth factor–, epithelial growth factor–, and Ang II–stimulated Erk-1/2 activities in early passage VSMCs and in aortic strips are inhibited by NO, ANP/CNP, with several studies demonstrating that this effect is not due to cross-activation of PKA.^46,47,135 In fact, endothelium-derived NO appears to tonically inhibit the effect of endogenous Ang II on Erk-1/2 activity in the aorta.¹³⁵ Thus, cGMP-elevating agents may augment basal, but inhibit mitogen-stimulated Erk activity in primary VSMCs. Inhibition of mitogen-stimulated Erk activity has also been observed in mesangial cells and fibroblast-like cells.^53,136,137

In endothelial cells, NO donors stimulate basal Erk-1/2 activity in a cGMP-dependent fashion; this effect is mimicked by cGMP analogues and in some cases by overexpression of constitutively active PKG.^{57,58,131,132} Moreover, the mitogenic effect of VEGF on endothelial cells appears to be mediated by NO and cGMP, as VEGF-induced Erk-1/2 activation and proliferation is blocked by NOS and sGC inhibitors.^57,58

In the classic MAP kinase pathway, growth factor receptors activate Ras, Raf kinases, MEK-1/2, and Erk-1/2 (Figure 2).⁹² Although NO can directly activate Ras in vitro through S-nitrosylation of C¹¹⁸, the in vivo significance of this reaction remains to be confirmed.¹³⁸ In growth factor–stimulated VSMCs and BHK cells, NO/cGMP have no effect on Ras activity.^46,53 One group suggested that PKG can directly activate Raf-1 through phosphorylation of the kinase in a soluble Ras/Raf-1/PKG complex present in VEGF-stimulated human umbilical vein endothelial cells (HUVECs); however, specificity of the PKG antibody used was not demonstrated,⁵⁷ and HUVECs express very little or no detectable PKG by Western blotting,⁷ making it doubtful that specific coimmunoprecipitation of PKG with Raf-1 and Ras would be detectable. Other investigators were unable to find Raf-1 activation by cGMP in cardiomyocytes where cGMP activated MEK-1/2 and Erk¹³³; MEK-1/2 activation by cGMP/PKG has been confirmed in VSMCs.¹⁰⁶ PKG directly phosphorylates Ser⁴³ of Raf-1 in vitro and in vivo; this phosphorylation uncouples the Ras-Raf interaction and thereby prevents mitogen-induced Raf-1 activation.^46,53 A phosphorylation-deficient Raf-1(A⁴³) mutant is insensitive to cGMP/PKG inhibition and protects cells from the inhibition of mitogen-induced Erk activity by cGMP.⁵³ Thus, Erk activation by cGMP in resting cells may or may not include activation of Ras and Raf kinases, but occurs at the level of MEK-1/2. On the other hand, Erk inhibition by cGMP/PKG in mitogen-stimulated cells may be explained by PKG phosphorylation of Raf-1 and by cGMP/PKG induction of the MAP kinase inhibitor MPK-1 (Figure 2 and Table 1).⁵³ In addition, cross-activation of PKA under conditions of high intracellular cGMP can lead to Erk pathway inhibition.^4,54 Cell type-specific activation of p38 or JNK by cGMP appears to involve activation of the upstream kinases MEK-3 or MEKK-1 by PKG, respectively.^71,139

	Posttranscriptional Regulation of Gene Expression by cGMP

Top Abstract Introduction (Patho)Physiological Processes... Mechanisms of Transcriptional... Posttranscriptional Regulation... Conclusions References

 
Regulation of gene expression at the posttranscriptional level includes regulation of pre-mRNA splicing, mRNA stability, and translation. PKG phosphorylates splicing factor 1 (SF1) in vitro and in vivo, and thereby inhibits prespliceosome assembly; however, whether this phosphorylation results in alternative splice site selection or otherwise regulates RNA processing of specific genes is presently unknown.¹⁰⁰

Elevation of intracellular cGMP can decrease mRNA levels of sGC-₁ and -ß₁ subunits, iNOS, COX-2, TNF-, and TGF-ß₃ via message destabilization in VSMCs, mesangial cells, and cardiac fibroblasts.^{21,24,81,86,87,140} Stability of sGC-₁ mRNA is regulated by the ubiquitous mRNA binding protein HuR, which binds to AU-rich elements in the 3'-untranslated region (UTR) and increases mRNA half-life; cGMP-elevating agents decrease expression and RNA binding of HuR, thereby destabilizing sGC-₁ mRNA.²⁴ The cGMP-induced mRNA destabilization requires transcription of an unknown factor(s)^24,140; this could be an RNA-destabilizing protein or a factor involved in downregulation of HuR. HuR regulates mRNA stability of many different genes also regulated by cGMP, including iNOS, COX-2, TNF-, VEGF, and cyclins A and D1.^83,141 Therefore, cGMP downregulation of HuR could potentially explain the cGMP downregulation of these mRNAs observed under some conditions. cGMP can also increase mRNA stability, as illustrated by the example of the potassium chloride cotransporter-3.¹⁴²

Translational regulation by cGMP has been most thoroughly studied for the asialoglycoprotein receptor (ASGR), a hepatocellular surface lectin.^143,144 Increased intracellular cGMP shifts ASGR mRNA into a translationally active polysomal pool, whereas decreased intracellular cGMP causes a negative trans-acting factor (called COPI) to associate with the 5'-UTR of the ASGR mRNA, thereby preventing ribosomal scanning to the site of translational initiation.^143,144 cGMP induced phosphorylation of COPI in intact cells is prevented by PKG inhibition and correlates with increased ASGR synthesis; PKG-dependent phosphorylation of COPI may regulate association of the COPI/RNA complex.¹⁴³ Synthesis of insulin receptor subunits and ß appears to be regulated by cGMP via a similar mechanism.¹⁴³

	Conclusions

Top Abstract Introduction (Patho)Physiological Processes... Mechanisms of Transcriptional... Posttranscriptional Regulation... Conclusions References

 
Although transcriptional regulation by cAMP has been intensely studied for decades, the regulation of gene expression by cGMP has only more recently been recognized. Many (patho)physiological processes in the cardiovascular system involve cGMP-induced changes in gene expression: (1) cGMP downregulates genes involved in the modulation of vascular tone, including sGC, PKG and rGC-A; (2) cGMP negatively regulates cardiac hypertrophy by inhibiting the expression of prohypertrophic genes such as muscle LIM protein; (3) cGMP/PKG modulate the VSMC phenotype and increase expression of differentiation-associated genes such as SM-myosin heavy chain-2 and SM--actin; (4) cGMP regulation of cell proliferation correlates with the modulation of cell cycle–associated genes and growth factor synthesis; and (5) cGMP’s effects on apoptosis appear to be mediated, at least in part, through regulation of Bcl-2-related genes. Gene expression profiling will likely contribute to the rapidly growing list of cGMP-regulated genes; however, the mechanisms of transcriptional and translational regulation by cGMP are only beginning to be understood. Some effects of cGMP on gene expression involve cross-talk with other signaling pathways, such MAP kinase, calcineurin, and RhoA pathways; other effects of cGMP may be directly attributed to PKG phosphorylation of specific transcription factors, such as CREB and TFII-I.

	Acknowledgments

 
Acknowledgments

The authors were supported by NIH grant R01-GM55586.

	Footnotes

 
Original received May 23, 2003; revision received October 7, 2003; accepted October 8, 2003.

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