doi: 10.1161/01.RES.0000216596.73005.3c
© 2006 American Heart Association, Inc.
Reviews |
Excitation–Transcription Coupling in Arterial Smooth Muscle
From Biomedical Sciences (D.K.B.), Veterinary School of Medicine, University of Missouri, Columbia, Mo; and the Department of Molecular Physiology and Biological Physics (B.R.W., G.K.O.), Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Va.
Correspondence to Gary K. Owens, Department of Molecular Physiology and Biological Physics, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, PO Box 800736, Charlottesville, VA 22908-0736. E-mail gko{at}virginia.edu
This Review is part of a thematic series on New Paradigms of Transcriptional Control of Myocardial and Vascular Growth, which includes the following articles:
Redox-Dependent Transcriptional Regulation
Control of Cardiac Growth by Histone Acetylation/Deacetylation
Excitation–Transcription Coupling in Arterial Smooth Muscle
Gordon F. Tomaselli Editor
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Abstract |
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 Top Abstract IntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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The primary function of the vascular smooth muscle cell (SMC) is contraction for which SMCs express a selective repertoire of genes (eg, SM
-actin, SM myosin heavy chain [SMMHC], myocardin) that ultimately define the SMC from other muscle cell types. Moreover, the SMC exhibits extensive phenotypic diversity and plasticity, which play an important role during normal development, repair of vascular injury, and in vascular disease states. Diverse signals modulate ion channel activity in the sarcolemma of SMCs, resulting in altered intracellular calcium (Ca) signaling, activation of multiple intracellular signaling cascades, and SMC contraction or relaxation, a process known as "excitation–contraction coupling" (EC-coupling). Over the past 5 years, exciting new studies have shown that the same signals that regulate EC-coupling in SMCs are also capable of regulating SMC-selective gene expression programs, a new paradigm coined "excitation–transcription coupling" (ET-coupling). This article reviews recent progress in our understanding of the mechanisms by which ET-coupling selectively coordinates the expression of distinct gene subsets in SMCs by disparate transcription factors, including CREB, NFAT, and myocardin, via selective kinases. For example, L-type voltage-gated Ca2+ channels modulate SMC differentiation marker gene expression, eg, SM -actin and SMMHC, via Rho kinase and myocardin and also regulate c-fos gene expression independently via CaMK. In addition, we discuss the potential role of IK channels and TRPC in ET-coupling as potential mediators of SMC phenotypic modulation, ie, negatively regulate SMC differentiation marker genes, in vascular disease.
Key Words: calcium • ion channel • sproliferation • smooth muscle • transcription
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Introduction |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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The vascular smooth muscle cell (SMC) in adult animals performs a specialized form of contraction that requires expression of a unique repertoire of genes, including SM
-actin, smooth muscle myosin heavy chain (SMMHC), SM22, calponin, desmin, and smoothelin—genes we refer to as SMC differentiation marker genes. This repertoire of genes or "transcriptome" is typically used to describe the "contractile" phenotype or mature SMC. There is clear evidence that vascular SMCs are highly plastic and can alter the expression profiles of SMC differentiation marker genes during development, wound repair, and, in several disease states, a process termed phenotypic modulation or switching (reviewed in Owens, Kumar, and Wamhoff1). For example, during early stages of vasculogenesis, SMCs are highly migratory and undergo very rapid cell proliferation. During vascular development, immature SMCs also exhibit very high rates of synthesis of extracellular matrix components including collagen, elastin, proteoglycans, cadherins, and integrins that comprise a major portion of the blood vessel mass. In contrast, in adult blood vessels the mature SMC shows an exceedingly low rate of proliferation/turnover, is largely nonmigratory, shows a very low rate of synthesis of extracellular matrix components, and is primarily committed to performing its contractile function. To this end, the mature, fully differentiated SMC expresses a repertoire of appropriate receptors, ion channels, signal transduction molecules, calcium regulatory proteins, and contractile proteins necessary for the unique contractile properties of the SMC. Importantly, different SMC subtypes express certain common differentiation marker genes such as SM -actin and SMMHC and also unique genes that contribute to their specialized functional properties and mechanisms of excitation–contraction (EC) coupling.2–4
The importance of SMC phenotypic switching and SMC plasticity for vascular repair undoubtedly has evolved in higher organisms because it conferred a survival advantage, although paradoxically these same properties likely contribute to disease susceptibility. For example, the plasticity of the SMC that enables it to rapidly undergo phenotypic switching in response to vessel injury also makes it susceptible to environmental cues that contribute to development of atherosclerosis, post-angioplasty restenosis, and other SMC-related diseases (see Owens et al1). It is also important to recognize that there is a continuous spectrum of SMC phenotype switching that can occur as a function of changing environmental cues. For example, after vascular injury, the SMC rapidly undergoes phenotypic modulation, which results in suppression of genes that define the quiescent/contractile phenotype, including SM -actin, SMMHC, and SM22 while rapidly upregulating genes required for proliferation, including matrix metalloproteinases, and migration to lay down a new collagen matrix and repair the vessel wall. This response is required for vascular repair and is beneficial, assuming SMCs revert back to the more mature phenotype by re-inducing SMC differentiation marker genes once the injury/plaque has been stabilized. Unchecked, however, this response can often result in luminal narrowing neointimal hyperplasia that, in many contexts, can be detrimental. Thus, SMCs within adult mammals are highly plastic cells that are capable of rather profound alterations in their phenotype, ie, expression profiles of SMC differentiation marker genes, in response to changes in local environmental cues important for their differentiation and function.1
The purpose of this review is to highlight recent advancements in understanding how environmental cues that regulate EC-coupling are also involved in regulating SMC-selective gene expression and phenotypic modulation, mechanisms described herein as excitation–transcription (ET) coupling. We define ET-coupling as the process by which common signaling pathways that regulate EC-coupling also translate into transcriptional gene regulatory events in arterial smooth muscle. A particularly exciting aspect of ET-coupling is that it represents a potential integrative mechanism whereby short-term regulation of calcium signaling and contraction are transduced into long-term regulation of SMC growth, differentiation, and remodeling. We acknowledge that there are many different SMC subtypes, eg, arterial, venous, and visceral, and that EC-coupling varies depending on the function of each SMC subset. Moreover, whereas there are certainly critical differences in the transcriptome within different SMC subtypes, eg, including ion channels and receptors, there are also a very large number of common differentiation markers genes shared by all SMC subsets that are the central focus of the present review, including SM -actin and smooth muscle myosin heavy chain.1,2 Thus, the next two sections of this review briefly review fundamental principles of EC-coupling and transcriptional regulation of SMC differentiation marker genes that are integral to our current knowledge of SMC ET-coupling in arterial SMCs. The final sections focus on specific mechanisms of ET-coupling that regulate arterial SMC gene expression and SMC phenotypic switching (Figure).
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EC-Coupling in SMCs: Sparks and SOCE |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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In vivo, arterial SMCs exist in a partially constricted state in which the average intracellular Ca2+ concentration is maintained several orders of magnitude lower than the extracellular fluid, eg,
100 to 300 nmol in pressurized microvessels.5 Membrane potential, through activation of voltage-gated Ca2+ channels (VGCC), is a primary determinant of myoplasmic Ca2+ and vascular tone.5 Due to the steep relationship between global myoplasmic Ca2+ and vascular tone, myoplasmic Ca2+, and thus membrane potential, must be tightly regulated to maintain proper vascular resistance. The underlying principle of EC-coupling in SMCs is that changes in membrane potential affect actin–myosin interaction. For example, depolarization-induced Ca2+ influx through L-type VGCC increases global myoplasmic Ca2+ and activation of the contractile apparatus both directly and via Ca2+ release from the sarcoplasmic reticulum (SR), ie, Ca2+-induced Ca2+ release or CICR. Conversely, relaxation results from lowering global myoplasmic Ca2+ levels via hyperpolarization of the cell, primarily by activation of K+ channels, to limit Ca2+ influx via VGCC in combination with increased extrusion/uptake by the plasmalemmal and endoplasmic Ca2+ ATPases, respectively. In vivo, membrane potential is determined by the net sum of all ionic conductances across the cell membrane. Regulation of membrane potential in arterial SMCs is further complicated by channel interactions, for example, membrane depolarization and Ca2+ influx directly activate K+ channels to limit depolarization, VGCC activation and contraction in a negative feedback manner.6 Furthermore, intracellular Ca2+ is not homogeneously distributed but rather exhibits dynamic, spatially localized events such as Ca2+ sparks, puffs, and waves that are highly dependent on the intracellular ultrastructure and the spatial relationship of various ion channels and ion pumps on the SMC sarcolemma and sarcoplasmic reticulum.4 In addition, Ca2+ regulation varies with vessel caliber and the nature of the vascular bed. Obviously, a comprehensive discussion of each of these aspects of SMC Ca2+ regulation in various SMC subsets is beyond the scope of this review; therefore, we limit our discussion to two concepts of Ca2+ regulation that have been shown to play a role in ET-coupling, Ca2+ sparks and SOCE (Figure).
Sparks
Much of our knowledge of Ca2+ regulation in SMCs was derived from measures of global, averaged cytosolic Ca2+ levels. The emergence of high-resolution, high-speed Ca2+ imaging techniques in live SMCs, such as laser scanning confocal microscopy, has led to the realization that spatially localized Ca2+ signaling events play a major role in both EC- and ET-coupling in SMCs. Spatial and functional coupling of Ca2+ regulatory proteins and structures are critical to subcellular Ca2+ signaling. For example, tight spatial coupling (<100 nm in distance) between the superficial sarcoplasmic reticulum (junctional SR) and the sarcolemma produces a spatially and temporally discrete Ca2+ signaling complex. Spontaneous transient outward K+ currents (STOCs), first described by Benham and Bolton,7 are produced by activation of large-conductance, Ca2+-activated K+ (BKCa) channels by the spontaneous bolus release of Ca2+ from this junctional SR via ryanodine receptors (RyR). The first direct visualization of "Ca2+ sparks" in vascular SMCs was provided by Nelson and colleagues, demonstrating that a local increase in subsarcolemmal Ca2+, a Ca2+"spark," released by RyRs on the SR also activates BKCa channels in close proximity, producing membrane hyperpolarization and favoring relaxation.8 This concept has been expanded into a functional unit involving the L-type voltage-gated Ca2+ channel, BKCa channel and RyR.9 Downregulation of functional BKCa channels was shown to be associated with a decreased frequency of sparks in hypertension and disruption of the ß-subunit of BKCa resulted in hypertension.10 As discussed, the concept of sparks will prove critical in ET-coupling in SMCs and our understanding of how a SMC differentiates informational Ca2+ content required for EC-coupling from ET-coupling.
SOCE
Store-operated Ca2+ entry (SOCE), ie, capacitative Ca2+ entry (CCE), is yet another mechanism of EC-coupling and Ca2+ regulation that emphasizes tight Ca2+ regulation in the SMC through interaction with the SR. It has been shown in multiple cell types, including SMCs, that mitogen or growth factor depletion of the sarcoplasmic reticulum by IP3-mediated Ca2+ release results in the subsequent activation of transient receptor potential (TRP) cation channels that triggers Ca2+ entry into the cell, likely via TRPC.11 SOCE has been proposed to increase myoplasmic and nuclear Ca2+ required for the regulation of contraction,12 cell proliferation,13 and apoptosis. SOCE is also proposed to be responsible for replenishing Ca2+ within the SR. Although the gating of TRP channels and other voltage-independent channels are not altered by membrane potential per se, changes in membrane potential have a dramatic effect on the magnitude of Ca2+ flux through these channels by changing the electrochemical driving force for Ca2+ entry. For example, hyperpolarization due to activation of K+ channels displaces membrane potential (Vm) farther away from the equilibrium potential for Ca2+ (ECa), resulting in increased Ca2+ entry via TRP channels, whereas depolarization has the opposite effect. Thus, SMC excitation can significantly modulate Ca2+ entry through voltage-independent Ca2+ channels. However, the robustness and physiological relevance of SOCE in EC-coupling varies significantly with species, vascular bed, and phenotypic state of the SMC. For example, a standard protocol for determining SOCE is to deplete the SR with CPA or thapsigargin in the presence of an L-type VGCC blocker. When this approach was applied to intact pulmonary arterial rings, a contraction could be elicited and potentiated by overexpressing TRPC1.14 Regardless of the physiological relevance of TRPC/SOCE in contraction, there is evidence that SOCE plays a role in SMC proliferation as select TRPC isoforms are upregulated after mitogen-induced proliferation13,15 and after vascular injury in vitro.16 Thus, SOCE may play a critical role in ET-coupling and regulation of growth related genes in SMCs.
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Transcriptional Regulation of SMC Differentiation Marker Genes |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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To date, expression of the majority of SMC differentiation marker genes that define the contractile SMC phenotype, including SM
-actin, SMMHC, SM22, telokin, desmin, and h1-calponin, have been shown to be dependent on at least one CArG element located within the promoter-enhancer region of the gene.1,17 Whereas the transcriptomes of SMC subsets vary, ie, differential expression of various ion channels and receptors, the CArG-dependent major SMC marker genes that have been most thoroughly studied at the transcriptional level are expressed in all SMC subtypes, a topic recently reviewed by Yoshida and Owens.2 The CArG element has the general sequence motif of CC(A/T-rich)6GG and binds serum response factor (SRF), a MADS (MCM1, Agamous, Deficiens, SRF) box transcription factor.17 The general paradigm is that expression of CArG-dependent SMC differentiation genes requires SRF binding to CArG boxes to drive transcription. In vivo studies using promoter-reporter constructs in transgenic mice have shown that these CArG elements are required to recapitulate normal endogenous gene expression patterns of SMMHC, SM -actin, and SM22.1 The CArG element was first identified in the promoter-enhancer region of the early-immediate growth response gene, c-fos.18 This presents an interesting paradox for regulation of vascular SMC phenotype in that the ubiquitous transcription factor, SRF, can stimulate both SMC differentiation and growth. In this review, we discuss differential regulation of SMC gene subsets in the context of c-fos compared with CArG-dependent SMC differentiation markers, eg, SM -actin, SMMHC, SM22, etc. Initial insight into this paradox came from the observation that the c-fos promoter contains only one CArG element, whereas as CArG-dependent SMC differentiation markers, eg, SM -actin, typically contain two CArGs in the promoter, often with a single CArG in the first intronic region.1 Moreover, the SM -actin CArGs have a single G or C substitution within the central A/T-rich region of the CArG box, which substantially lowers SRF binding affinity.17 Our laboratory has provided evidence that this degenerate SMC CArG box serves to limit SRF binding, thereby restricting expression of SMC markers to cells that express high levels of SRF and/or cells like SMCs that have evolved mechanisms to enhance SRF binding to degenerate CArGs, ie, a SMC-selective transcriptional SRF cofactor.19,20
One of the most significant advances in the field of SMC differentiation in recent years was the discovery of myocardin by Olson and colleagues.21 Myocardin is exclusively expressed in cardiac myocytes and SMCs. It is a highly potent SRF coactivator that lacks intrinsic DNA binding activity but associates with SRF and stimulates CArG-dependent SMC differentiation marker gene expression up to several orders of magnitude.20,22 This activity is highly selective in that myocardin does not enhance activation of the c-fos gene, which is also CArG-SRF–dependent. Conventional knockout of myocardin is associated with embryonic lethality at E10.5 days and failure to form SMCs.23 However, results of recent collaborative studies by our laboratory and that of Olson showed that autonomous myocardin is not absolutely required for SMC development, in that lacZ tagged homozygous KO embryonic stem cells (ESC) developed into SMCs in the context of chimeric KO mice generated by injection of myocardin null ESCs into the inner cell mass of blastocysts from wild-type C57bl6 mice.24 There is also evidence that interaction of myocardin with degenerate CArG elements within the promoter regions of SMC marker genes plays an important role in phenotypic switching of vascular SMC in vivo in response to vascular injury.19 Surprisingly, substitution of the SM -actin degenerate CArG elements with c-fos CArG elements retained appropriate SMC-restricted tissue specificity.19 However, these substitutions prevented vascular injury-induced downregulation of the mutant transgene under conditions in which SRF was increased but myocardin was decreased. Thus, this illustrates the importance of the subtle, but highly conserved, base pair mutations in the SM -actin CArG to SRF and myocardin-dependent regulation of SM -actin during phenotypic switching in vivo.
Of interest, both regulation of SMC gene expression26,27 and SMC calcium sensitivity of contraction3 are regulated by the RhoA/ROK pathway. For example, the RhoA/ROK pathway has been shown to regulate the translocation of SRF from the cytosol to the nucleus in multiple cell types, including SMCs.28 Overexpression of the constitutively active form of RhoA stimulates transcription of SM -actin, SM22 and SMMHC.27 In contrast, administration of the C3 transferase, which ADP ribosylates and irreversibly inhibits RhoA, or Y72632, a selective ROK inhibitor, decreased expression of multiple CArG-dependent SMC genes. Importantly, these effects were selective in that no effects were seen on the c-fos gene promoter that contains only one CArG element. The effects of RhoA have also been shown to be mediated through regulation of the actin cytoskeleton and specifically through alterations in the concentration of monomeric or G-actin.27 Treatment of SMCs with agents that increased formation of filamentous (F)-actin and decreased G-actin profoundly increased SMC gene transcription, whereas treatment with agents that increased G-actin markedly inhibited SMC gene expression. Recent studies by Miralles et al29 showed Rho-actin signaling also regulated the subcellular localization of the myocardin-related SRF coactivator MAL or MRTF-A in NIH 3T3 fibroblasts. MRTFA was predominantly localized in the cytoplasm in serum-starved 3T3 cells but accumulated in the nucleus after serum stimulation. Solway and associates have provided very interesting results showing that serum-induced translocation of SRF into the nucleus in cultured tracheal SMC was Rho kinase–dependent.28,30 Thus, agonist-induced activation of RhoA and subsequent translocation of SRF and/or myocardin/MRTFs by actin shuttling may play an important role in activation of CArG-dependent SMC genes. Recent work by Kuwahara et al31 showed that both MRTF A and B are shuttled to and from the nucleus by actin treadmilling in the SMC, a mechanism that may couple the contractile apparatus to SMC gene expression.
Thus far, we have discussed the regulation CArG-dependent SMC genes in the context of SRF and myocardin as activators of transcription. However, SMC differentiation genes can be rapidly switched off, an underlying event of phenotypic switching in several vascular diseases. Recent evidence indicates that SMC phenotypic switching results from a combination of loss of activators as well as by stimulation of active suppressor pathways or repressors. For example, Liu et al showed that the Kruppel-like transcription factor KLF4 suppressed SM -actin promoter activity by interacting with a transforming growth factor-ß control element (TCE) adjacent to CArGB in the SM -actin promoter.32 Moreover, follow-up studies showed that KLF4 suppressed myocardin and prevented myocardin/SRF from associating with CArG regions in SMC genes.33 There appear, however, to be redundant pathways for transcriptionally repressing SMC genes. For example, there is evidence that PDGF BB-induced suppression of SMC genes also involves SP1,34 competition for myocardin binding to SRF by phosphorylated ELK-1,35 and ets-1.36 Finally, a substantial body of evidence demonstrates that Ca2+-activated transcription factors, such as CREB, play a role in regulating expression of c-fos in SMCs.37 However, their role in regulating SMC-selective gene expression and phenotypic switching is relatively unexplored and thus the remaining topic of discussion in this review.
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ET-Coupling in SMCs |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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Although ET-coupling has been extensively studied in neurons, immune cells and other cell types, ET-coupling in smooth muscle is truly in its infancy. A number of recent studies from our laboratory26 and others37–39 have begun to establish general paradigms for ET-coupling in SMCs, including the following: (1) ET-coupling is highly dependent on precise subcellular control of Ca2+ levels; (2) specific excitation signals can selectively activate distinct transcription factors, eg, NFAT, CREB, and/or SRF; (3) activation of distinct gene subsets, ie, c-fos and CArG-dependent SMC genes, can share a common initial excitation signal but selective secondary activation pathways, eg, CaMK and RhoA/ROK; and (4) there exist a multitude of potential players in EC-coupling whose role in ET-coupling have yet to be defined. Given space constraints, we have chosen to review key studies that link EC-coupling to ET-coupling, with the final end point being a transcriptional event. We believe this is an important distinction as there are several studies showing that EC-coupling regulates transcription factors, but few that reveal a transcriptional outcome or change in the expression of a specific gene, ie, ET-coupling.
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ET-Coupling: CREB and CaMK |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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Ca2+-calmodulin-dependent enzymes are involved in both SMC contraction and activation of transcription factors. Calmodulin (CaM) is activated by Ca2+ to form Ca2+-CaM, which activates myosin light chain kinase to phosphorylate the 20 kDa light chain of myosin and induce contraction. Ca2+-CaM is also a regulator of the phosphatase calcineurin, which regulates the transcription factor NFAT (nuclear factor of activator T-cells), and calcium–calmodulin-dependent protein kinase (CaMK), which regulates the transcription factor CREB (cAMP response element binding protein). CREB transcriptional activation occurs through its binding to the CREB-binding protein (CBP), a coactivator that links many proteins to transcriptional machinery or by binding to the Ca2+/cAMP response element (CRE) in the promoter enhancer of genes.40 Of interest, c-fos contains one CRE element 3' to the CArG element whereas CArG-dependent SMC differentiation marker genes do not contain consensus CRE elements. Phosphorylation of CREB (pCREB) at serine 133 enables CREB to modulate gene transcription through CRE elements.41 CREB can be phosphorylated not only at serine 133 but also at serine 142, which is thought to be a negative regulatory site.42 Phosphorylation of CREB is mediated by various kinases with the cAMP/PKA pathway being the principle modulator. However, in the case of Ca2+-dependent activation, CREB phosphorylation is thought to be regulated by the following signaling cascade: Ca2+ binds to calmodulin (Ca2+-CaM), Ca2+-CaM freely diffuses into the nucleus through the nuclear pore complex and activates nuclear CaMK, resulting in CREB phosphorylation.
Perhaps the greatest impact and advancements in the field of SMC ET-coupling have been made by seminal studies by Nelson and colleagues, who tested the hypothesis that signals which regulate EC-coupling could translate to ET-coupling in SMCs.37,39,43 They showed that depolarization-induced constriction with 60 mmol/L KCl activated L-type VGCCs and Ca2+ influx resulting in increased pCREB and c-fos expression in pressurized intact cerebral microvessels.37 Moreover, this response was dependent on CaMK activity as deduced using the generic Ca2+-calmodulin kinase inhibitor KN-93, which inhibits CaMKII and CaMKIV. Although these were the first studies to show ET-coupling in SMCs, these results were not initially surprising given that L-type VGCCs mediate CREB phosphorylation and c-fos activation in several excitable cell types, including neurons. However, what was unique about this study is that they further tested whether altering regulation of subsarcolemmal Ca2+ would alter ET-coupling. RyRs on the superficial SR generate Ca2+ sparks, activating BKCa channels, hyperpolarizing the membrane and inactivating L-type VGCCs to limit Ca2+ influx. If L-type VGCCs played the dominant role in regulating ET-coupling of pCREB and c-fos, then inhibiting Ca2+ sparks would favor Ca2+ influx via VGCC and increased ET-coupling. In these studies, blocking ryanodine receptors with ryanodine ablated Ca2+ sparks and increased phosphorylation of CREB and translocation of pCREB to nuclei and c-fos activation, a process that could be prevented by nisoldipine, an L-type VGCC blocker.37
Both native and culture vascular SMCs express CaMKII and CaMKIV.37,44 In SMC tissues and cultured cells, CaMKII is predominantly cytosolic and implicated in the modulation of myosin light chain kinase sensitivity to Ca2+ and in regulation of cell migration, and it is involved in the control of Ca2+ channels, sarcoplasmic reticulum Ca2+-ATPase activity, and MAP kinase activation. In neurons, CaMKIV is thought to be predominantly located in the nucleus and thus may play a role in ET-coupling as opposed to EC-coupling, although there is limited direct support of this in SMCs. The functions of CaMKII and CaMKIV on CREB signaling are opposite. That is, CaMKII phosphorylates CREB on Ser 142 which is a negative regulatory site, whereas CaMKIV phosphorylates CREB at Ser 133, the activation phosphorylation site.42 Thus, it seems likely that activation of nuclear CaMKIV phosphorylates nuclear CREB driving subsequent CRE-dependent transcription of c-fos. However, when nuclear import is blocked in SMCs by inhibiting the RAN GTPase with wheat germ agglutinin or an inactivating Ran mutant (T24N Ran), pCREB accumulated in the cytoplasm after depolarization and increased intracellular Ca2+.43 Interpretation of these results is decidedly complex but suggests that nuclear export of CREB may be required for phosphorylation, which raises new questions regarding the function of CREB export, as it is predominantly localized to the nucleus, and the role of intracellular Ca2+ dynamics in regulating CREB localization.
One interesting issue that arises from these results is whether Ca2+-mediated processes, ie, non-VGCC–mediated, can regulate c-fos expression. SOCE (store operated Ca2+ entry) has been observed in SMCs, although in the normal contractile SMC phenotype this observation may be vascular bed and species specific. Studies by Pulver et al39 showed that experimental manipulation of Ca2+ handling in intact cerebral blood vessels to elicit SOCE resulted in increased nuclear accumulation of phosphorylated CREB and c-fos message. This response was independent of L-type VGCCs as nisoldipine did not block the effect. Although the signaling mechanisms/kinases involved in CREB phosphorylation were not explored, it is intriguing to presume that this response is mediated by CaMK. However, although details of this mechanism need to be resolved, what has become apparent is that events like Ca2+"sparks" and SOCE exemplify the localized interrelationship of Ca2+ and intracellular ultrastructure underlying ET-coupling in SMCs in intact blood vessels.
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ET-Coupling: NFAT |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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A very interesting story that has evolved in ET-coupling is the regulation of the transcription factor NFAT in SMCs. NFAT represents a family of Ca2+-dependent transcription factors comprising four well characterized isoforms, designated NFAT1 (NFATc2/p), NFAT2 (NFATc1/c), NFAT3 (NFATc4), and NFAT4 (NFATc3/x).45,46 Most NFAT isoforms are constitutively expressed and exist in the cytsol as transcriptionally inactive phosphoproteins. The NFATs are activated by calcineurin, a Ca2+/CaM-dependent, serine/threonine-specific phosphatase 2B that is activated during EC-/ET-coupling by fluctuations in myoplasmic Ca2+. Dephosphorylation of NFAT by calcineurin unmasks nuclear localization signals and transcriptionally active NFAT is translocated to the nucleus.45 Stimuli that provoke a sustained moderate elevation in global Ca2+ have consistently been shown to effectively promote nuclear localization of NFAT in nonexcitable cells. It has been shown that SMCs express NFAT1, NFAT2, NFAT3, and NFAT4, although NFAT4 appears to be the predominant isoform expressed in intact blood vessels.38 Insight that NFAT may be regulated in SMCs was first described in the A7r5 aortic smooth muscle cell line whereby treatment with PDGF-BB induced transcriptional activation of an NFAT-specific promoter reporter construct.47 These studies were extended into intact blood vessels by Nelson colleagues, and it was shown that PDGF-BB elicited nuclear translocation of NFAT4 via L-type VGCCs but depolarization alone, ie, activation of VGCCs, did not result in nuclear tranlocation of NFAT4, indicating that Ca2+ influx is necessary but not sufficient.38 These results are in direct contrast to CREB regulation in which VGCC activation alone is sufficient to activate CREB and translocate p-CREB to the nucleus. Moreover, where VGCCs are required for PDGF-BB-induced translocation of NFAT to the nucleus, PDGF-BB induced translocation of pCREB does not require VGCCs.43 Taken together, these results suggest that specific Ca2+-dependent transcription factors are differentially regulated by specific Ca2+ signaling pathways. The signaling specificity of Ca2+-dependent activation of NFAT4 in SMCs is further refined by the SR. SR Ca2+ release is required for NFAT4 dephosphorylation by calcineurin; however, the mode of SR Ca2+ release appears to be critical. That is, activation of NFAT4 by Gq/11-coupled receptors requires IP3-mediated SR Ca2+ release and Ca2+ influx via VGCCs, whereas Ca2+ release in the form of sparks by ryanodine receptors in the SR may exert an inhibitory effect on NFAT4 nuclear accumulation.38 A proposed role of sparks is to maintain myoplasmic Ca2+ levels at low concentrations. As such, these results emphasize the importance of global increases in Ca2+ for NFAT4 activation in SMCs. Of interest, in physiologically pressurized cerebral arteries (100 mm Hg), NFAT4 is localized to the nucleus and this response is abrogated by blocking VGCCs.48
It is clear that NFAT plays some role in SMC development. For example, work by Crabtree and colleagues49 showed that mice with disruptions to the NFAT3 and NFAT4 genes die at embryonic day 11 with a generalized defect in vessel assembly and mice with a mutation to inactivate the calcineurin B gene show a similar phenotype. Further evidence that NFAT can regulate SMC differentiation marker gene expression was shown by Wada et al,50 whereby overexpression of NFAT2 activated the SMMHC promoter through interaction with GATA-6 and pharmacological blockage of calcineurin decreased SMMHC promoter activity. There is a putative NFAT binding site in the SMMHC promoter enhancer (GGAAAA), at –519/–514 relative to the transcriptional start site as well as a GATA-6 binding site at –810/–805. However, there is no evidence NFAT2 binds to this NFAT cis element in intact chromatin in SMCs nor has it been shown that loss of NFAT2 function results in altered SMMHC promoter activity. Moreover, there is no evidence that ET-coupling plays any role in regulating this process as these studies were performed in unstimulated, quiescent cells. It is likely, however, that NFATs do play a critical role in SMC differentiation and hence expression of SMC differentiation marker genes as overexpression of NFAT2 or calcineurin in a neural crest cell model of SMC differentiation increased the induction of SMC populations as determined by increased cell numbers expressing SMMHC and SM -actin.51
Although the preceding studies clearly implicate the NFAT family of transcription factors and Ca2+ signaling in SMC differentiation and potentially SMC differentiation marker gene expression, evidence defining the specific ET-coupling pathways that regulate SMC differentiation marker gene expression, both SRF-CArG–dependent and CArG-independent genes, at the molecular level is limited (Figure). Of interest, Nelson and colleagues recently published an interesting result suggesting that NFAT4 positively regulates SM -actin through a NFAT cis element that is very near the intronic CArG of the SM -actin promoter,52 supporting a role of NFATs in positively regulating SMC phenotype. Although SRF was shown to interact with NFAT4 in vitro, it still remains to be determined whether this interaction exists in vivo in intact chromatin and whether NFAT4 affects activity of the full-length SM -actin promoter as only a truncated, 56-bp insert containing the NFAT cis element was used in these studies.
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ET-Coupling: SMC Differentiation Marker Genes |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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Although the aforementioned studies defined a fundamental physiological pathway linking Ca2+ to c-fos gene expression, eg, VGCC
[Ca2+]iCaMKP-CREBc-fos, very little is known about the role of intercellular Ca2+ in mediating changes in the expression of a subset of genes that typify the differentiated SMC. This distinction is important in that unlike markers defining the differentiated SMC phenotype, eg, SM -actin, SMMHC or SM22, c-fos is expressed in all cell types and thus is not a marker for any terminally differentiated cell. Moreover, the expression patterns of markers that typify the differentiated SMC can be highly variable because the SMC is prone to rapid phenotypic switching in response to vascular injury or pathological stimuli, as previously discussed.
We recently observed that depolarization-induced Ca2+ influx through L-type VGCCs in cultured aortic SMCs resulted in an increase in the mRNA levels of SM -actin, SMMHC, SM22.26 VGCC-induced activation of these SMC differentiation marker genes was CArG-dependent and required myocardin as shown by deletion promoter analyses of CArG regions in the SM -actin promoter and siRNA to myocardin, respectively. VGCC activation also resulted in increased c-fos promoter activity as well as mRNA. These results present a unique dichotomy: How can two disparate gene subsets (SMC differentiation genes versus growth-response/proliferation genes) share a common pathway of activation, VGCC-mediated activation? RhoA/ROK signaling plays an integral role in regulating SRF-dependent activation of CArG-dependent SMC differentiation marker gene expression, but not of c-fos.26 Thus, RhoA/ROK serves as a point of divergence between SMC differentiation marker gene regulation and c-fos gene regulation in VGCC-mediated ET-coupling. Blocking ROK activity with Y27632 prevented VGCC-mediated activation of SM -actin, SMMHC and SM22 but had no effect on c-fos activation. In contrast, inhibition of CaMKs with KN93 had no effect on VGCC-mediated induction of SM -actin, SMMHC and SM22 but blocked c-fos induction. Thus, what has evolved from these studies and work by Nelson and colleagues is a signaling pathway whereby ET-coupling via L-type VGCCs selectively regulates myocardin/SRF/CArG-dependent SMC gene expression via RhoA/ROK, whereas CArG-dependent growth response genes, such as c-fos, are regulated by CaMK (Figure). What remains to be determined is whether non-CArG–mediated SMC-selective gene expression is regulated by ET-coupling. The non-CArG–dependent gene ACLP (aortic-carboxy-like peptidase), which is upregulated by SMCs after vascular injury, is not regulated by VGCCs and RhoA/ROK.26 However, ACLP, unlike SM -actin and SMMHC, is presumably a marker of the proliferative SMC cell phenotype and hence may be regulated by yet undefined ET-coupling pathways that promote expression of genes that define the synthetic phenotype.53 Moreover, it is not known whether Ca2+ sparks and SOCE, which regulate CREB and c-fos expression, also regulate SRF and CArG-dependent SMC gene expression.
It is interesting to speculate that signals which regulate short term contraction also regulate long term SMC differentiation marker gene expression. As discussed, actin polymerization is critical for SRF and MRTF shuttling to and from the nucleus and likely plays a critical role in CArG-dependent gene regulation.28,30,31 In practice, treatment of SMCs to destabilize actin polymerization would alter contraction, but simultaneously alter actin treadmilling of SRF/MRTF to the nucleus, a process that is required for CArG-dependent SMC gene expression.27 Although contraction and transcription are likely coupled, addressing this experimentally is difficult as uncoupling contraction will undoubtedly require uncoupling the contractile apparatus. In addition, turnover of SMC differentiation marker genes in mature blood vessels is very low, such that normal homeostatic input signals including transient alterations in circulating vasoactive compounds would not be expected to elicit altered transcriptional responses. Rather, it seems more likely that ET-coupling would require either chronic elevations in a humoral factor such as angiotensin II, or alternatively "cellular integration" or "memory." ET-coupling may share common regulatory controls involved in neural control of memory, which include RGS proteins shown to be highly expressed in vascular SMCs, including far higher levels in arteries versus veins.54 There is also the possibility that epigenetic control of chromatin structure contributes to integrative ET-coupling. Our laboratory has provided novel evidence showing that SMCs exhibit unique patterns of histone modifications at gene loci encoding SMC marker genes that not only distinguish them from non-SMCs but which also appear to play a key role in phenotypic switching in response to vascular injury.55 However, as yet, very few studies have directly investigated the role of epigenetic modifications in ET-coupling.
To begin to address these complex issues, we recently developed a unique embryonic stem cell embryoid body (EB) model of SMC differentiation that permits not only isolation of purified populations of fully contractile embryonic stem cell (ESC)-derived SMCs, but also the opportunity to directly dissect the role of specific regulatory genes and pathways in ET-coupling.56,57 For example, using KO embryonic stem cells one can ask not only whether SMC differentiation still occurs within the embryoid body but also whether specific deletion of a gene postulated in EC-/ET-coupling alters SMC gene expression or contractile function. Of major significance, we recently clearly established the validity of this ESC-EB model for predicting SMC phenotypes in vivo in that in collaborative studies with Eric Olson’s group we found that myocardin null ESCs were capable of differentiating into SMCs both in the in vitro ESC-EB system as well as in vivo in chimeric KO mice generated by injection of KO ESCs into the inner cell mass of wild type mouse blastocysts.24 Moreover, in separate studies in this system we tested whether L-type VGCCs regulate SMC differentiation marker expression in the 28-day EB.26 Chronic 24-hour exposure of the EB to nifedipine blocked spontaneous contraction of ESC-derived SMC but also suppressed expression of SM -actin and SMMHC. These findings suggest that basal Ca2+ influx required for phasic contraction is also required to maintain contractile gene expression. However, whether contraction, per se, is required for ET-coupling remains to be determined, as well as whether alterations in chromatin structure contribute to how sustained contractile responses couple to transcriptional regulation of SMC contractile proteins.
One interesting observation from our recent studies was that activation of VGCCs increased myocardin expression in SMCs.26 An insightful commentary on this by Joseph M. Miano59 proposed several vexing questions, a few of which we reiterate and expand on. First, how does membrane depolarization induce myocardin expression? Second, does elevated myocardin expression elicit contraction? That is, is myocardin more than a transcription factor, possibly a protein that interacts with the contractile apparatus? Third, are the two paralogs of myocardin (MRTF-A and MRTF-B) similarly responsive to Ca2+ transients and can they substitute for myocardin in membrane depolarization-induced gene expression? Fourth, what is the mechanism underlying membrane depolarization-induced SRF binding to CArG boxes, how does SRF-myocardin direct increases in CArG-dependent gene expression, does myocardin remodel chromatin as with CBP to increase DNA-binding factor accessibility?
Unfortunately, to date, mechanisms that regulate myocardin expression at the transcriptional level are unknown as the promoter-enhancer for this gene has not been identified and validated for recapitulation of endogenous expression patterns using transgenic mice. Thus, evidence that myocardin is regulated at the transcriptional level is limited to measuring changes in endogenous myocardin mRNA levels. However, depolarization and angiotensin II induce myocardin expression, whereas acute vascular injury suppresses myocardin.19,26,60 In contrast to this limited knowledge, more mechanistic studies have shed insight into how myocardin regulates CArG-dependent SMC gene expression at the chromatin-promoter level. Several findings give insight into how ET-coupling may play a role in this process. First, p300 and CBP (CREB-binding protein), which are among the most characterized histone acetyl-transferases (HATs), are extremely sensitive to changes in nuclear Ca2+61 and have been shown to be involved in the recruitment of transcription factors that regulate muscle contractile protein expression. Second, studies by Qui and Li62 showed that stimulation of the SM22 promoter by the coactivator CBP (CREB-binding protein) was dependent on histone acetyltransferase activity and that CBP binding to intact chromatin was associated with SRF. CBP contains a signal-regulated transcriptional activation domain that is controlled by nuclear calcium and calcium/calmodulin-dependent (CaM) protein kinase IV and by cAMP.63 Third, exciting new work by Olson and colleagues revealed that myocardin induces the acetylation of nucleosomal histones surrounding SRF binding sites (CArG regions) in SMC differentiation marker genes.64 Myocardin HAT activity was dependent on p300 binding to the TAD region (transcriptional activating domain) of myocardin. Thus, although purely speculative at this point, Ca2+-dependent activation of p300 and/or CBP may be involved in regulating myocardin-activated SMC gene expression. However, results by our own group contradict this hypothesis as inhibition of CaMKs in cultured SMCs had no effect on VGCC-mediated activation of myocardin/SRF/CArG-dependent SMC gene expression.26 Thus, future studies are needed to advance our understanding of how myocardin and myocardin induced-activation of CArG-dependent gene expression are regulated by ET-coupling in SMCs by CBP, p300, and HATs in general, as well as other histone modifying enzymes that control chromatin structure and the "permissiveness" of SMC marker gene loci for transcriptional activation.55
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ET-Coupling: SMC Phenotypic Switching, IKCa, and TRPC |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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Thus far we have considered how Ca2+ influx via VGCC positively regulates CArG-dependent gene expression, yet also promotes early growth response gene activation, ie, c-fos. An outstanding question, however, is whether there are opposing ET-coupling events that negatively regulate SMC differentiation marker gene expression, ie, promote SMC phenotypic modulation and suppression of SMC differentiation marker genes, perhaps in circumstances such as vascular injury. The most compelling data that such events exist in response to vascular injury relate to the expression patterns of intermediate conductance Ca2+-activated K channels (IKCa1) and transient receptor potential channels (TRPC), as discussed previously.
Although mature, quiescent, native arterial SMCs do not typically express IKCa1, Neylon et al65 demonstrated that proliferating rat aortic SMCs had enhanced charybdotoxin-sensitive potassium channels, which they concluded to be IKCa1 channels. In addition, a reciprocal expression between IKCa1 and the BKCa channel has been observed in intimal versus medial SMCs after injury.66 Of particular interest, both restenosis after rat carotid balloon injury66 and SMC proliferation in vitro67 were attenuated with the specific IKCa1 channel blocker, TRAM-34, implicating that functional upregulation of IKCa contributes to SMC proliferation. Increased IKCa expression in proliferating or injured vascular SMCs is due, at least in part, to downregulation of repressor element 1-silencing transcription factor (REST),67 thus providing another transcription factor that may be involved in SMC phenotype modulation. The expression profile of TRP family proteins also varies with SMC phenotype. That is, the potent SMC mitogen PDGF-BB has been shown to upregulate TRPC6,13 and both TRPC1 and TRPC6 are upregulated with an organ culture model of vascular injury.16 The role of TRPC in regulating genes involved in SMC proliferation is intriguing especially given that TRPC-mediated SOCE has been shown to regulate c-fos gene expression in SMCs, as discussed previously.39 Taken together, these studies provide preliminary evidence for a role of IKCa1 and TRPC channels as a regulator of vascular SMC phenotypic modulation. However, it is unknown whether IKCa1 or TRPC channel activity influence cell phenotype by regulating SMC differentiation marker gene expression directly or whether IKCa and TRPC expression are required for SMC proliferation independent of SMC differentiation marker gene expression. It is also unknown whether IKCa and TRPC activation suppress myocardin/MRTFs or activate transcription factor pathways that are involved in directly suppressing CArG-dependent SMC genes. Conversely, it is possible that IKCa and TRPC are upregulated by a common factor, such as PDGF-BB (which suppresses SMC differentiation marker genes), and hence their role is completely independent of regulating SMC differentiation marker gene expression but required for other SMC injury responses, such as migration and or proliferation. Similarly, Miguel-Velado et al recently demonstrated the importance of voltage-dependent K channel (Kv) isoform switching in phenotype remodeling of uterine arterial SMC.68 Taken together, these studies emphasize the complex interaction between specific ion channel expression profiles, Ca signaling, and selective gene expression in SMCs, which will require much more investigation to fully understand.
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ET-Coupling: The Nucleus? |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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An obviously critical issue in consideration of ET-coupling is how alterations in cytosolic calcium are "transmitted" across the nuclear membrane, and whether the process involves changes in nuclear calcium. Thus far, a dominant theme for ET-coupling is that it is primarily mediated by changes in cytosolic rather than nuclear calcium, which in turn alters the activity and/or nuclear-cytoplasmic transport of transcriptional activators and repressors. However, there is unequivocal evidence in non-SMC cell types that nuclear Ca2+ plays a role in the regulation of Ca2+-activated transcription factors and gene expression.61,69,70 One of the most notable examples is the demonstration of the requirement of nuclear Ca2+ and cytoplasmic Ca2+ for CREB-dependent transcription in neuroblastoma cells. In brief, cells were injected with dextran-coupled BAPTA, a Ca2+ buffer that is too large to pass through nuclear pores and thus can be used to clamp Ca2+ independently in the nucleus or cytoplasm.71 Injection of dextran-BAPTA into the nucleus prevented CREB phosphorylation, suggesting that either the kinetics or the amplitude of the increase in nuclear Ca2+ is important in CREB activation. Other results suggest that CREB phosphorylation on SER 133 can also occur in the absence of elevated cytoplasmic Ca2+ and nuclear Ca2+. Phosphorylation of CREB occurred despite buffering cytoplasmic Ca2+ with EGTA, which also prevents elevations in nuclear Ca2+, but allows Ca2+ elevation close to the plasma membrane.72,73 Taken together, these results imply that a membrane-bound Ca2+ sensor can activate a signaling pathway that leads to phosphorylation of CREB, but additional Ca2+-dependent steps that occur in the nucleus are required. Two other transcription factors of potential relevance to ET-coupling in SMCs are NFAT (as discussed) and Elk-1, an ETS family transcription factor that binds and regulates SRF in multiple cell types, including SMCs. A decrease in nuclear Ca2+ deactivates nuclear calcineurin allowing one of multiple NFAT kinases to rephosphorylate NFAT, resulting in transport of pNFAT out of the nucleus and inactivation of NFAT-dependent transcription.70,74 Elk-1 phosphorylation and MAPK (mitogen-activated protein kinase) translocation to the nucleus, which appear to be required or Elk-1 transcriptional activity, require an increase in nuclear Ca2+.75 Of particular interest, Elk-1 has recently been shown by Olson and colleagues to displace myocardin from SRF in response to PDGF-BB signaling,35 a signaling event that also activates multiple Ca2+ events. An important caveat, however, is that our knowledge of nuclear Ca2+ regulation and transcriptional activation described herein is taken solely from studies in neurons and non-SMCs. Thus, a key question is whether these results can be extrapolated to ET-coupling in SMCs. Virtually nothing is known about these mechanisms in SMCs and similar mechanisms undoubtedly play a role in ET-coupling to SMC gene expression and specifically CArG-dependent SMC differentiation. An attractive hypothesis is that Ca2+ in the nucleus of SMCs is tightly buffered and regulated similar to Ca2+ in the subsarcolemmal space and that this regulation dictates gene regulation in response to Ca2+-activated transcription factors. Although limited, there is evidence that SMC nuclear Ca2+ regulation can be altered in response to physiological or pathophysiological stimuli, eg, vascular disease, such that the amplitude and duration of nuclear Ca2+ responses to contractile agonists are severely altered.76,77 Whether this alters gene expression remains to be determined.
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Conclusions and Perspectives |
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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In conclusion, mechanisms for ET coupling in vascular SMC, and regulation of SMC-selective gene expression and phenotype, are beginning to evolve. The Figure summarizes major advances thus far. First, L-type VGCCs differentially regulate SRF/CArG-dependent SMC differentiation marker gene expression and c-fos gene expression via RhoA/ROK/myocardin and CaMK, respectively. Second, both "sparks" (which activate BKCa channels) and store-operated Ca2+ entry (SOCE, likely mediated by TRPC) regulate c-fos expression, although whether they regulate SMC differentiation marker genes is unknown. Third, the calcineurin/NFAT axis is tightly regulated in SMCs yet the precise function of NFAT signaling in regulating SMC differentiation marker gene expression or growth response genes is unknown. Fourth, we hypothesize that "proatherogenic" stimuli, eg, growth factors or hemodynamic forces that promote SMC phenotypic modulation, upregulate pathways involved in controlling proliferation or growth response gene expression (IKCa, TRPC, SOCE) and suppress SMC differentiation marker gene expression mediated by L-type VGCCs and "sparks," although the latter is untested (Figure). Admittedly, we have greatly oversimplified very complex mechanisms. Several outstanding questions remain. How do SMCs integrate short-term calcium responses into long term changes in gene expression and how do these events differ in various vascular beds, eg, conduit vessels versus microvessels? Moreover, how do SMCs regulate nuclear Ca2+ to control Ca2+-activated transcription factors? Does Ca2+ signal to chromatin modifying enzymes, ie, p300 or CBP, to modify histones and regulate transcription factor access to genes? Do IKCa and TRPC activation suppress myocardin/MRTFs or activate transcription factor pathways, eg, KLF4, that are involved in directly suppressing CArG-dependent SMC gene? Of course, understanding how ET-coupling operates in the SMC is only one aspect of the multitude of hemodynamic and humoral regulated signaling pathways, cis-regulatory elements and transcription factors that collaborate to ensure homeostasis in the SMC. An ultimate goal, therefore, will be to bring to bear all of the latest technology, including functional genomics and mouse models of conditional mutagenesis, to understand ET-coupling in the context of such regulatory networks with the goal of developing novel ways of manipulating SMC homeostasis in the face of disease.
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Acknowledgments |
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Funding to B.R.W. to investigate SMC Ca2+ regulation, SMC gene transcription, and SMC ET-coupling in health and disease over the past 8 years was supported by the American Heart Association (AHA pre-doc with Dr Michael Sturek), American Physiological Society (post-doc with Dr Gary K. Owens), and the AHA (New Investigator), respectively. Other support for this work includes NIH HL52490 and HL071574 to Doug Bowles and NIH PO1 HL19242, RO1 HL38854, and R37 HL57353 to Gary Owens. There are no conflicts of interest to disclose.
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Footnotes |
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Original received November 3, 2005; revision received February 6, 2006; accepted February 24, 2006.
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TopAbstractIntroductionEC-Coupling in SMCs: Sparks...Transcriptional Regulation of...ET-Coupling in SMCsET-Coupling: CREB and CaMKET-Coupling: NFATET-Coupling: SMC Differentiation...ET-Coupling: SMC Phenotypic...ET-Coupling: The Nucleus?Conclusions and PerspectivesReferences |
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