[ Harold Singer first developed interest in vascular smooth muscle regulation and function during his Ph.D. training in Pharmacology with Michael Peach and postdoctoral fellowship in Physiology with Richard Murphy, both at the University of Virginia. He was a founding faculty member in the Geisinger Clinic's Weis Center for Research where he initiated studies on protein kinase C and CaM kinase II signalling in smooth muscle. In 1998 he was recruited to Albany Medical College where he is Professor and Director of the interdisciplinary Center for Cardiovascular Sciences. His lab continues to focus on Ca2+-signalling pathways regulating smooth muscle phenotype and function.]
Abstract Vascular smooth muscle (VSM) undergoes a phenotypic switch in response to injury, a process that contributes to pathophysiological vascular wall remodelling. VSM phenotype switching is a consequence of changes in gene expression, including an array of ion channels and pumps affecting spatiotemporal features of intracellular Ca2+ signals. Ca2+ signalling promotes vascular wall remodelling by regulating cell proliferation, motility, and/or VSM gene transcription, although the mechanisms are not clear. In this review, the functions of multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in VSM phenotype switching and synthetic phenotype function are considered. CaMKII isozymes have complex structural and autoregulatory properties. Vascular injury in vivo results in rapid changes in CaMKII isoform expression with reduced expression of CaMKIIγ and upregulation of CaMKIIδ in medial wall VSM. SiRNA-mediated suppression of CaMKIIδ or gene deletion attenuates VSM proliferation and consequent neointimal formation. In vitro studies support functions for CaMKII in the regulation of cell proliferation, motility and gene expression via phosphorylation of CREB1 and HDACIIa/MEF2 complexes. These studies support the concept, and provide potential mechanisms, whereby Ca2+ signalling through CaMKIIδ promotes VSM phenotype transitions and vascular remodelling.
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Ca2+/calmodulin-dependent protein kinase II
cyclic AMP response element binding protein 1
epidermal growth factor
extracellular signal regulated kinase 1/2
green fluorescent protein
myocyte enhancing factor 2
platelet-derived growth factor
sarco/endoplasmic reticulum Ca2+-ATPase
small interfering RNA
stromal interaction molecule 1
total internal reflection fluorescence
transient receptor potential channel
vascular smooth muscle
Diverse actions of Ca2+ signals are mediated through Ca2+/calmodulin activated serine/threonine protein kinases, including multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Soderling & Stull, 2001). Four differentially expressed but highly homologous CaMKII isoforms are encoded by separate genes (α, β, δ, γ) and assemble into large dodecameric holoenzymes (Rosenberg et al. 2006). CaMKII structure and function have been most thoroughly studied in brain, where holoenzymes composed of α and β isoforms are very abundant and involved in regulating postsynaptic signalling complexes, neurotransmission and memory (Hudmon & Schulman, 2002). Substantial progress has also been made defining functions of CaMKII in cardiac Ca2+ homeostasis and pathophysiology where it contributes to heart failure secondary to chronic pressure overload (Erickson et al. 2011). The following brief review highlights progress made in our understanding of CaMKII structure and function in vascular smooth muscle (VSM), focusing on studies that indicate roles for the kinase in regulating VSM cell growth, motility, and gene transcription. Based on these studies we propose a model whereby Ca2+ signalling events mediated specifically by CaMKIIδ isoforms, promote synthetic phenotype VSM function and post-developmental vascular wall remodelling in response to injury or vascular disease.
CaMKII isozymes and activation in VSM
Major CaMKII isoforms in VSM have been identified as mixtures of alternatively spliced products from δ and γ genes (Schworer et al. 1993; Singer et al. 1997; Gangopadhyay et al. 2003). Because holoenzymes are heteromultimers, there is the potential for extensive structural heterogeneity which can direct kinase localization and protein interactions, and therefore function. For example, an alternatively spliced exon in δ, α and γ isoforms encoding an 11 amino acid motif targets holoenzymes to the nucleus (Heist et al. 1998). Variably expressed domains in some β- (Urquidi & Ashcroft, 1995) and γ-subunits (Gangopadhyay et al. 2003) have been implicated in SH3 binding interactions which could link Ca2+ signals to intracellular signalling pathways involving non-receptor tyrosine kinases and ERK1/2 activation (Marganski et al. 2005).
One consequence of CaMKII holoenzyme structure is cooperative, inter-subunit, intra-holoenzyme autophosphorylation on thr287 (thr286 in α-isoforms) upon binding of Ca2+/CaM. This results in a thousand-fold decrease in Ca2+/CaM dissociation rate, effectively ‘trapping’ the activator complex, and generation of an ‘autonomous’ form of the kinase that has partial activity even in the absence of bound Ca2+/CaM (Meyer et al. 1992). Additional autophosphorylation on thr306/307 can block Ca2+/CaM binding and limit total Ca2+/CaM-stimulated activity following a Ca2+ transient. These unique autoregulatory properties provide CaMKII with the capacity to accumulate activity in a Ca2+ oscillation frequency dependent manner (Dosemeci & Albers, 1996), a property with as yet unexplored functional consequences in VSM. Autophosphorylation also affects CaMKII localization and protein interactions in postsynaptic densities, providing yet another mechanism for modulating function of the kinase (McNeill & Colbran, 1995).
Thr287 phosphorylation can be detected with an epitope-specific antibody and autonomous activity in cell extracts can be assayed in vitro, providing two indices of CaMKII activation that are useful experimentally. Using the latter approach, an apparent EC50 of 692 nm free intracellular [Ca2+] was required for in situ activation of CaMKII in permeabilized cultured VSM cells, agreeing well with in vitro studies under conditions of saturating calmodulin (Abraham et al. 1996). Despite this relatively low apparent affinity for Ca2+/calmodulin, physiological stimuli efficiently activate the kinase in both intact cultured VSM and intact arterial segments (Abraham et al. 1996; Rokolya & Singer, 2000), suggesting that CaMKII is positioned to sense transiently high [Ca2+]i localized near sources of Ca2+ entry or release.
Considering the loose consensus sequence for CaMKII phosphorylation (R/K-X-X-S/T; (Soderling & Stull, 2001) and diverse potential substrates, as well as functional implications of structural and autoregulatory complexities mentioned above, understanding the mechanisms of CaMKII action in a complex cellular process such as differentiated smooth muscle contraction is problematic. Pharmacological inhibition of CaMKII activity (Rokolya & Singer, 2000) or siRNA mediated suppression of CaMKIIδ isoforms (Kim et al. 2000) results in net inhibition of tonic contractile responses in arterial smooth muscle, although the targets are not yet clear. A discussion of CaMKII function and potential targets in differentiated VSM can be found in recent reviews (House et al. 2008b; Kim et al. 2008). Future studies in arterial tissues from genetically engineered mice lacking genes for either CaMKIIδ (Backs et al. 2009) or γ (Backs et al. 2010) hold promise for more completely characterizing CaMKII isoform-dependent functions and substrates in differentiated VSM.
VSM phenotype switching: Ca2+ signalling
VSM cells are not terminally differentiated and can transition to a ‘synthetic’ phenotype characterized functionally by proliferation, motility, and capacity for extracellular matrix remodelling. The synthetic phenotype typifies VSM in cell culture, and more importantly, those VSM cells contributing to myoproliferative vascular diseases, including atherosclerosis and restenosis. Most studies in this field have focused on identification of factors that promote the process (e.g. growth factors and cytokines, oxidative stress, mechanical stress, endothelial factors) or on transcriptional regulation of contractile phenotype-specific genes and synthetic phenotype genes. The literature in this field is extensive and the reader is referred to a recent review for detailed information (Owens et al. 2004).
VSM phenotype switching is accompanied by changes in expression of Ca2+ signalling proteins including voltage-gated Ca2+ channels, potassium channels, IP3 and ryanodine Ca2+ release receptors, TRPC non-selective cation channels and SERCA isoforms (Wamhoff et al. 2006; House et al. 2008a). In addition, STIM1 (Potier et al. 2009; Bisaillon et al. 2010) and Orai1 (Zhang et al. 2011) components of store-operated Ca2+ entry have recently been shown to be up-regulated in VSM in response to vascular injury or cell culture and positively regulate cell proliferation, migration and vascular remodelling. General challenges are to understand how these changes affect the localization and kinetics of Ca2+ signals and how these Ca2+ signals are translated into regulation of VSM phenotype switching or synthetic phenotype-specific functions such as proliferation or migration.
CaMKII promotes vascular remodelling
As a ubiquitous and abundant Ca2+ signal mediator, changes in CaMKII isoform expression, localization and activity could be integral to VSM phenotype modulation. Studies from this laboratory originally demonstrated rapid changes in CaMKII isoform expression upon acute transition of VSM cells from a differentiated to synthetic phenotype in primary culture (House et al. 2007) and in vivo in response to carotid artery balloon catheter injury in rats (House & Singer, 2008). Up-regulation of the CaMKIIδ2 isoform and reciprocal down-regulation of CaMKIIγ isoforms in both systems were found to coincide with, or precede, the onset of VSM cell proliferation. Treatment with siRNA to prevent upregulation of the δ2 isoform attenuated subsequent VSM proliferation in vitro and nearly completely prevented neointima formation in vivo in response to injury (Fig. 1). CaMKIIδ expression was recently reported to be induced in the mouse carotid artery following ligation injury; and in mice with global CaMKIIδ gene deletion, medial wall VSM proliferation and ultimately neointima formation was strongly inhibited (Li et al. 2011). CaMKII has also been implicated in VSM hypertrophy in response to angiotensin II both in vitro and in vivo (Li et al. 2010), but it was not possible from that study to discern CaMKII isoform-specific effects. Thus, both in vitro and in vivo studies indicate functions for CaMKIIδ in modulating VSM cell proliferation and vascular wall remodelling. As discussed in the following sections, general mechanisms include direct effects of CaMKIIδ in the regulation of VSM cell proliferation and motility, and indirect effects affecting VSM phenotype more globally through regulation of gene transcription.
The mechanistic basis for the specific requirement of the CaMKIIδ isoform in vascular remodelling is not yet known. One interesting possibility is that the δ-isoform may be spatially distributed to respond to Ca2+ signals from specific sources and/or couple to specific protein substrates involved in regulating synthetic phenotype functions. In cultured synthetic phenotype VSM cells the δ-isoform distributes in a largely perinuclear compartment with concentrations in peripheral membrane domains undergoing stimulus-induced remodelling (Mercure et al. 2008). Although comparative studies of CaMKII δvs. γ isoform distribution in differentiated VSM have not been reported, specific γ-isoform variants were shown to associate with intermediate filaments in differentiated VSM and to translocate to membrane dense bodies upon contractile stimulation (Marganski et al. 2005).
CaMKII and VSM proliferation
While there is evidence implicating Ca2+ signalling and CaMKII as regulators of cell proliferation (Lipskaia & Lompre, 2004; Skelding et al. 2011), the mechanisms remain obscure and the concept is not widely integrated into models of cell cycle regulation. Using siRNA technology to suppress expression of δ-isoforms, or expressing a kinase-negative CaMKIIδ2 mutant which inhibits CaMKII activity, inhibited VSM proliferation rates in vitro with effects pinpointed to the G2/M phase transition by cell cycle analysis (House et al. 2007). More recently, it was demonstrated that cultured aortic VSM cells from CaMKIIδ null mice proliferate significantly slower than cells from control animals and substantial evidence was provided to show that CaMKIIδ regulated p53- and p21-dependent control of the G1/S phase transition (Li et al. 2011). One factor that could explain the differences in apparent mechanisms between the studies is relatively acute down-regulation of endogenous CaMKII by siRNA compared to genetic deletion, which may result in compensatory changes during development that more broadly affect the VSM phenotype and cell cycle control. Additional studies are required to resolve this.
CaMKII and VSM motility
Stimulation of cultured VSM with platelet-derived growth factor (PDGF) (Pauly et al. 1995), scratch wounding a monolayer (Zhang et al. 2003; Mercure et al. 2008), or simply adhering VSM cells to culture dishes in the absence of added stimulus (Lu et al. 2005), results in activation of CaMKII. Pharmacological inhibition of activity (Pauly et al. 1995) or siRNA mediated depletion of CaMKIIδ inhibits VSM cell migration in vitro (Mercure et al. 2008), suggesting a net positive role for the kinase. Activated CaMKII in motile VSM cells promotes polarization, as assessed by Golgi position relative to the leading edge, and can be localized to lamellipodia based on indirect immunofluorescence of autophosphorylated CaMKII or fractionation of leading edges and analysis of autophosphorylated kinase by Western blotting (Mercure et al. 2008). Thus, CaMKII is activated within the motile VSM cell and positioned to coordinate complex cellular events involved in this process (Fig. 2). The sources and nature of Ca2+ signals activating CaMKII in migrating VSM are unknown at this time.
Substrates mediating CaMKII function in VSM cell migration have not been identified; however, the studies in differentiated muscle mentioned earlier suggest Ca2+ regulatory proteins or cytoskeletal proteins are possible targets. In addition to direct effects, CaMKII could affect motility indirectly through interaction with other intracellular signalling molecules. CaMKII inputs into activation of ERK1/2 in VSM in response to Ca2+-dependent stimuli (Abraham et al. 1997; Muthalif et al. 1998), an effect dependent upon the activity of src family kinases, PYK2 and transactivation of EGF receptor tyrosine kinase (Ginnan & Singer, 2002). Rac activation and subsequent Golgi polarization in scratch-wounded VSM monolayers was also found to be dependent upon expression of CaMKIIδ (Mercure et al. 2008). Focal adhesion proteins are known targets of regulation by Rac, tyrosine kinases, and ERK1/2 signalling pathways in adhering or migrating cells (Webb et al. 2004) and therefore represent potential targets of CaMKII. Recently, GFP-tagged CaMKIIδ was localized by TIRF microscopy in fibroblasts and evidence was provided that focal adhesion protein tyrosine phosphorylation was negatively regulated by the kinase (Easley et al. 2008), consistent with this idea.
CaMKII in VSM gene transcription
Ca2+ signalling pathways have been implicated in the regulation of gene transcription in a number of systems (Dolmetsch, 2003). In VSM, a general model has emerged whereby Ca2+ signals from L-type Ca2+ channels, IP3 receptors, and ryanodine receptors regulate both contractile activity and transcription of contractile proteins in differentiated VSM. Conversely, signals resulting from activation of intermediate conductance K+ channels, IP3 receptors, and TRPC channels, which are upregulated in synthetic phenotype cells, promote transcription of growth and proliferation related genes (Barlow et al. 2006; Wamhoff et al. 2006). Proposed Ca2+ signal mediators in this model of excitation–transcription coupling include Rho/ROCK, calcineurin, and multifunctional Ca2+/camodulin-activated kinases (CaMKs). Potential target transcriptional regulators include SRF, NFAT, CREB1 and MEF2. Exactly how endogenous CaMKII isozymes fit into this model of VSM gene transcriptional remains poorly understood.
One potential transcription factor target of CaM kinases is the cyclic AMP response element (CRE) binding protein, CREB1. Down-regulation of CREB expression has been associated with several vascular related diseases, suggesting important roles in regulating normal VSM function (Schauer et al. 2010). CREB activation has been reported to differentially couple to gene transcription in VSM dependent on a Ca2+-entry pathway (Pulver-Kaste et al. 2006), and in the case of thrombin stimulation, CREB1 activation was positively associated with cell proliferation (Tokunou et al. 2001). An activating phosphorylation site in CREB1 (ser133) can be phosphorylated by both protein kinase A and multifunctional CaM kinases. While it is clear that CaMKIV positively regulates CREB by this mechanism, the role of CaMKII remains unclear as in vitro studies indicate an alternative inhibitory site (ser142) as a CaMKII substrate (Sun et al. 1994; Wu et al. 1998). Most studies to date in intact cells have inferred function of CaM kinases through pharmacological approaches or through expression of constituently active mutants, both leading to non-specific actions. Additional studies are needed in VSM to clearly distinguish functions of endogenous CaMKII isoforms and CaMKIV in the positive and negative regulation of CREB1.
CaMKII has been reported to regulate myocyte enhancing factor 2 (MEF2), a MADS-box family of DNA binding transcription factors in striated muscle (Zhang et al. 2007). Studies in transgenic mice demonstrate that MEF2 regulates smooth muscle expression of myocardin, which in turn positively regulates the transcription of smooth muscle specific genes (Creemers et al. 2006). Conversely, MEF2 expression and activity are upregulated in the neointima of rat carotid arteries after balloon catheter injury and is required for synthetic-phenotype VSM cell proliferation in culture (Firulli et al. 1996). CaMKII does not directly regulate MEF2, but instead targets class IIA histone deacetylases which complex with and repress MEF2 on target gene promotors. CaMKII-dependent phosphorylation of HDAC4 and HDAC5 result in 14-3-3 protein binding and nucleocytoplasmic translocation, thereby de-repressing MEF2 activity (Backs et al. 2006). Specifically in VSM, PDGF-BB (Gordon et al. 2009) and angiotensin II (Li et al. 2010) stimulation of VSM has been shown to result in CaMKII-dependent HDAC4 phosphorylation with activation of MEF2. A study by Pang et al. (2008) indicated that CaMKIIδ2 forms a functional complex with the scaffolding protein GIT1 and HDAC5, mediating angiotensin II-dependent phosphorylation of HDAC5 and activation of MEF2. On the other hand, Xu et al. (2007) indicated that angiotensin-dependent phosphorylation of HDAC5 and subsequent increases in MEF2 activity are PKC/PKD dependent and do not involve CaMKII. Additional studies are required to more carefully define the function of endogenous CaMKII isozymes in regulation of HDACs and the relative contribution of this pathway in regulating MEF2 activity and transcriptional control during VSM phenotype switching.
Summary and model
Substantial evidence indicates that sources, dynamics and mediators of Ca2+ signals change as a function of VSM phenotype and that activation of specific Ca2+ signalling pathways is stimulus dependent. This raises the question as to how these signals are differentially ‘interpreted’ to regulate synthetic VSM phenotype function and gene expression patterns that promote the synthetic phenotype? As a multi-functional serine/threonine kinase, CaMKII is predicted to have diverse actions and participate in the coordination of complex cellular functions. Available evidence summarized in this review points to a model whereby induction of CaMKIIδ gene products in response to injury promotes acquisition of the synthetic VSM phenotype and regulates proliferation and motility leading to vascular wall remodelling (Fig. 3).
Most of the information on CaMKII function in VSM which was discussed in this review results from studies in large conduit vessels and the VSM cells derived from these vessels. The results from those studies and the model presented may not be fully representative of CaMKII function in resistance vessels or veins. Molecular and genetic approaches to manipulate CaMKII activity or expression levels are now available to test the functions of the kinase. With these tools, rapid progress can be expected in more completely defining the functions of specific CaMKII isozymes in regulating VSM physiology and pathophysiology. At a mechanistic level, important areas for future investigations of CaMKII in VSM include: how specific structural and autoregulatory features of this complex protein kinase affect various differentiated and synthetic functions; mechanisms involved in regulating phenotype-specific expression of CaMKII isozymes; and identification of sources and localization of activating Ca2+ signals.
The author is grateful for discussions with his collaborators Drs Roman Ginnan, Dee Van Riper, John Schwarz and Mohamed Trebak, who helped shape the content of this review. The author's research is supported by grants from the National Institutes of Health: HL049426, and HL092510.