VSMCs (vascular smooth muscle cells) dedifferentiate from the contractile to the synthetic phenotype in response to acute vascular diseases such as restenosis and chronic vascular diseases such as atherosclerosis, and contribute to growth of the neointima. We demonstrated previously that balloon catheter injury of rat carotid arteries resulted in increased expression of CaMKII (Ca2+/calmodulin-dependent protein kinase) IIδ2 in the medial wall and the expanding neointima [House and Singer (2008) Arterioscler. Thromb. Vasc. Biol. 28, 441–447]. These findings led us to hypothesize that increased expression of CaMKIIδ2 is a positive mediator of synthetic VSMCs. HDAC (histone deacetylase) 4 and HDAC5 function as transcriptional co-repressors and are regulated in a CaMKII-dependent manner. In the present paper, we report that endogenous HDAC4 and HDAC5 in VSMCs are activated in a Ca2+- and CaMKIIδ2-dependent manner. We show further that AngII (angiotensin II)- and PDGF (platelet-derived growth factor)-dependent phosphorylation of HDAC4 and HDAC5 is reduced when CaMKIIδ2 expression is suppressed or CaMKIIδ2 activity is attenuated. The transcriptional activator MEF2 (myocyte-enhancer factor 2) is an important determinant of VSMC phenotype and is regulated in an HDAC-dependent manner. In the present paper, we report that stimulation of VSMCs with ionomycin or AngII potentiates MEF2's ability to bind DNA and increases the expression of established MEF2 target genes Nur77 (nuclear receptor 77) (NR4A1) and MCP1 (monocyte chemotactic protein 1) (CCL2). Suppression of CaMKIIδ2 attenuates increased MEF2 DNA-binding activity and up-regulation of Nur77 and MCP1. Finally, we show that HDAC5 is regulated by HDAC4 in VSMCs. Suppression of HDAC4 expression and activity prevents AngII- and PDGF-dependent phosphorylation of HDAC5. Taken together, these results illustrate a mechanism by which CaMKIIδ2 mediates MEF2-dependent gene transcription in VSMCs through regulation of HDAC4 and HDAC5.
VSMCs (vascular smooth muscle cells) are not terminally differentiated and can undergo dramatic changes in phenotype following acute and chronic vascular injuries . This phenotype switch is characterized by loss of expression of transcription factors such as myocardin and contractile proteins including α-SMA (α-smooth muscle actin) and SMMHC (smooth muscle myosin heavy chain) that are required for differentiated smooth muscle contractile function. Concomitant with the reduced expression of these proteins is the up-regulation of genes and proteins associated with the proliferative/synthetic state such as KLF4 (Krüppel-like factor 4), versican and PDGFR (platelet-derived growth factor receptor) [1,2]. This phenotype switch also includes changes in a repertoire of Ca2+ signalling proteins including down-regulation of L-type voltage-gated Ca2+ channels and ryanodine receptors [3,4], up-regulation of store-operated STIM1 (stromal interaction molecule 1)/Orai1 channels , and changes in relative expression of various TRP (transient receptor potential) channels  and SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) pump isoforms . Changes in expression of Ca2+ signalling proteins can affect the kinetics and localization of Ca2+ signals and result in differential coupling of Ca2+ signals to transcriptional events, as summarized in reviews on excitation–transcription coupling in VSMCs [7,8].
Consistent with this concept, our laboratory has documented that the multifunctional serine/threonine protein kinase CaMKII (Ca2+/calmodulin-dependent protein kinase II) isoform expression pattern is altered in VSMCs after vascular injury . Studies in intact rat carotid arteries revealed that CaMKIIγ, which has been associated with modulation of contractile function  predominates in differentiated medial smooth muscle, but its expression decreases rapidly after balloon catheter injury and is accompanied by a significant up-regulation of the CaMKIIδ2 isoform in both injured medial wall and neointimal VSMCs . Suppression of injury-induced CaMKIIδ2 up-regulation with shRNA (short hairpin RNA) inhibited VSMC proliferation and attenuated neointimal formation . The function of CaMKIIδ in the response to vascular injury was confirmed recently using a carotid ligation injury model in a CaMKIIδ-null mouse . These results and previous in vitro studies are consistent with a function for CaMKIIδ isoforms in modulating VSMC proliferation  and migration . The rapid kinetics of the reciprocal CaMKII isoform switch in response to injury also raises the possibility that either the loss of CaMKIIγ and/or the gain of CaMKIIδ may be involved in regulating the transition of differentiated VSMCs to the synthetic phenotype, potentially by differential coupling of these isoforms to specific transcriptional processes.
A potential transcriptional target for CaMKII is MEF2 (myocyte-enhancer factor 2), a MADS-box family of DNA-binding transcription factors with essential roles in a wide range of cell types, including skeletal muscle, cardiomyocytes, neurons and haemopoietic cells [14–18]. Studies in transgenic mice demonstrate that MEF2 regulates smooth muscle expression of HRC (histidine-rich calcium-binding protein) and myocardin, which in turn positively regulates the transcription of most smooth-muscle-specific genes studied to date [19,20]. In contrast with these data indicating a role in the differentiated phenotype, two early studies demonstrated that MEF2 expression and activity are up-regulated in the neointima of rat carotid arteries after balloon catheter injury and are required for synthetic-phenotype VSMC proliferation in culture [21,22]. A similar increase in MEF2 activity has been reported in mouse neointima using a MEF2 reporter mouse .
MEF2 can act as either an activator or a repressor depending on its interaction with co-activators or co-repressors respectively. HDAC (histone deacetylase) 4 and HDAC5 are class IIa HDACs that directly interact with MEF2 to promote its repressive activity . Insight into the mechanisms coupling CaMKII activation to MEF2 activation are gained from studies in cardiomyocytes where the MEF2 co-repressors HDAC4 and HDAC5 have been shown to be substrates for CaMKII phosphorylation, leading to nuclear export and cytoplasmic sequestration through interaction with 14-3-3 proteins [25–27]. Studies in VSMCs evaluating CaMKII-dependent regulation of type IIa HDACs and MEF2 are limited and somewhat conflicting. An early study suggested that CaMKIIδ2 acts by phosphorylating 14-3-3 proteins in the cytosol, disrupting their ability to interact with and localize HDAC4 to the cytosol, thereby promoting interaction with nuclear MEF2 to repress transcription . Conversely, treatment of VSMCs by the growth factor PDGF (platelet-derived growth factor)-BB was shown to result in HDAC4 phosphorylation and cytoplasmic sequestration, with activation of MEF2 and subsequent c-Jun expression in cultured VSMCs . Similarly, CaMKII was implicated in AngII (angiotensin II)-dependent VSMC hypertrophy through phosphorylation of HDAC4 to derepress the MEF2 complex . A study by Pang et al.  indicated that CaMKIIδ2 forms a functional complex with the scaffolding protein GIT1 [G-protein-coupled receptor kinase-interacting ArfGAP1 (ADP-ribosylation factor GTPase-activating protein 1)] and HDAC5, mediating AngII-dependent phosphorylation of HDAC5 and activation of MEF2 . Conversely, Xu et al.  indicated that AngII-dependent phosphorylation of HDAC5 and subsequent increases in MEF2 activity are PKC (protein kinase C)/PKD (protein kinase D)-dependent and do not involve CaMKII . Thus the conditions and mechanisms by which CaMKII regulates HDACs in VSMCs, and the relative contribution of this pathway in regulating MEF2 activity, remain unclear.
The aim of the present study was to systematically evaluate the relative contribution of CaMKIIδ in the regulation of endogenous HDAC4, HDAC5 and MEF2 in VSMCs in response to two relevant stimuli, AngII and PDGF, which activate Ca2+ signalling via distinct receptor-coupled mechanisms. Using gain- and loss-of-function molecular approaches, we established CaMKIIδ isoform-dependent regulation of endogenous HDAC4 and HDAC5 in response to both stimuli in VSMCs and demonstrated that HDAC4 is required for coupling of the Ca2+ signalling pathways to HDAC5. Downstream coupling of this regulatory mechanism to activation of MEF2 and transcription of the known MEF2 target genes Nur77 (nuclear receptor 77) (NR4A1) and MCP1 (monocyte chemotactic protein 1) (CCL2) was stimulus-dependent. AngII-stimulated activation of MEF2 and induction of Nur77 and MCP1 was strongly dependent upon CaMKIIδ, whereas PDGF stimulation of these transcription readouts was relatively independent of CaMKIIδ, suggesting that there are additional redundant MEF2 activation pathways invoked by this growth factor.
MATERIALS AND METHODS
Polyclonal antibodies against HDAC4 and HDAC7 were purchased from Santa Cruz Biotechnology. Polyclonal antibody against HDAC5 was purchased from Signalway Antibody. Antibody against phosphorylated HDAC4 (Ser632) was purchased from Abnova. Antibody against phosphorylated HDAC5 (Ser498) was purchased from Abcam. Antibodies against CaMKIIδ2 and pan-CaMKII were produced as described previously [32,33]. shDELTA2 was produced as described previously . Smart pools for HDAC4 (SiHDAC4) and CaMKIIδ2 (SiCaMKIIδ2) were purchased from Dharmacon. Adenoviral constructs expressing kinase-negative CaMKIIδ2 and GFP (green fluorescent protein) have been described previously . Expression plasmid encoding GFP (pMAXGFP) was purchased from Lonza. Expression plasmid encoding mutant HDAC4 (S278A/S478A/S632A) was purchased from Addgene. All tissue culture media were purchased from Gibco-BRL (Life Technologies) unless specifically stated. Tissue culture supplies (dishes, pipettes, etc) were purchased from Fisher Scientific. SDS/PAGE and Western blotting supplies were purchased from Bio-Rad Laboratories unless stated otherwise. All other chemicals were purchased from Sigma.
VSMCs were obtained from the medial layer of the thoracic aorta of 200–300 g Sprague–Dawley rats as described previously . Briefly, the adventitial and endothelial layer of intact thoracic aortas was removed; the medial layer was then enzymatically dispersed and cultured in DMEM (Dulbecco's modified Eagle's medium)/Ham's F12 and 10% (v/v) FBS (fetal bovine serum) (Hyclone). The VSMCs were maintained at subconfluent densities by passaging twice a week using standard cell culture techniques and conditions (37°C with 5% CO2). All experiments were performed on cells passaged three to ten times.
Cell lysates and immunoblotting
Cells were lysed (0.5 ml/60-mm-diameter dish or 1 ml/100-mm-diameter dish) in a modified RIPA buffer [10 mM Tris/HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% (v/v) Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10% (v/v) glycerol, 1 mM DTT (dithiothreitol), 0.1 mM PMSF and 0.2 unit/ml aprotinin]. Samples were resolved using standard SDS/PAGE procedures and transferred on to NitroPure nitrocellulose membranes (GE Healthcare) before immunoblotting. Standard immunoblotting using 5% (w/v) non-fat dried milk powder or 3% (w/v) BSA was performed. Incubation with the primary antibody was either 1 h at room temperature (22°C) or overnight at 4°C as needed (indicated in the Figure legends). Nitrocellulose membranes were incubated in enhanced chemiluminescent substrate (GE Healthcare) and visualized using either standard X-ray film (E.M. Parker) or a Fuji LAS 4000.
Cell lysates were made using RIPA buffer as described above. The cell lysates were cleared by centrifugation at 14000 g for 10 min. After transferring to a clean tube, primary antibody was added and incubated overnight with rocking. Washed Protein A–agarose beads (Thermo Scientific) were then added to the antibody/lysate mixture and incubated for an additional 90 min. The antibody–Protein A–agarose bead complex was then washed three times with RIPA buffer and resolved by SDS/PAGE and immunoblotting as described above.
Immunoflourescence and confocal microscopy
VSMCs were seeded on to collagen (PureCol)-coated glass coverslips previously coated with collagen. The cells were fixed in 4% (w/v) paraformeldehyde and permeabilized with brief exposure to a Triton X-100-containing buffer. After incubation with primary antibody, the cells were incubated with a secondary antibody that fluoresces at specific wavelengths (Molecular Probes). For indirect immunofluorescence, the cells were imaged using a Leica DM IRB inverted fluorescent microscope (Leica Microsystems). For confocal microscopy, the cells were imaged with a Zeiss LSM 510 META confocal microscope.
Cell transfections and adenoviral infections
For introduction of siRNA (small interfering RNA), VSMCs were transfected using the Amaxa Nucleofector™ system (Lonza Group) according to the prescribed specifications for VSMCs. For introduction of expression plasmids into the VSMCs, the FuGENE™ HD (Roche Applied Science) transfection reagent was used according to manufacturer's specifications. For adenoviral infection, VSMCs at 70% confluence were infected at a MOI (multiplicity of infection) of 50 with purified adenovirus expressing shDELTA2, shGFP or kinase-negative CaMKIIδ2 in DMEM/Ham's F12 containing 10% (v/v) FBS. All adenovirus stocks were propagated by adding small amounts of virus to HEK (human embryonic kidney)-293 cells. When cells were approximately 50% lysed, cells and medium were collected, subjected to three freeze–thaw cycles, divided into aliquots of 50 μl and stored at −80°C.
MEF2 DNA-binding assay
MEF2 DNA binding was measured using a DNA-binding assay (TransAm MEF2 Assay, Active Motif). Briefly, nuclear extracts were prepared from 100-mm-diameter dishes of confluent VSMCs using Active Motif's nuclear extract kit according to the manufacturer's specifications. The nuclear extracts were then incubated in 96-well plates coated with oligonucleotide DNA corresponding to the MEF2 consensus binding site. Bound (active) MEF2 was detected by incubation with anti-MEF2 antibody, an HRP (horseradish peroxidase)-conjugated secondary antibody and spectrophotometric analyses.
Total RNA was extracted from cells after 1 h of treatment with 0.5 μM Iono (ionomycin) or 100 nm AngII with TRIzol® (Invitrogen) according to the manufacturer's specifications. The first-strand cDNA was synthesized using a QuantiTect RT (reverse transcription) kit (Qiagen). The quantitative RT–PCR was performed on a Mx3000P QPCR System using Brilliant III Ultra-Fast SYBR® Green (Agilent). The primers for rat target genes and internal controls were as follows: GAPDH (glyceraldehyde-3-phosphate dehydrogenase) forward, 5′-TCGTCTCATAGACAAGATGGT-3′, and reverse, 5′-GTAGTTGAGGTCAATGAAGGG-3′; Nur77 forward, 5′-AGTCCGCCTTTCTGGAGCTCTTTA-3′, and reverse, 5′-CAGGCAAAGGCAGGAACATCAACA-3′; MCP1 forward, 5′-TGTTCACAGTTGCTGCCTGT-3′, and reverse, 5′-ACCTGCTGCTGGTGATTCTCTT-3′
All data are expressed as means±S.E.M. Mean values of groups were compared by ANOVA with post-hoc comparisons using the Newman–Keuls test. For all comparisons, P<0.05 was considered statistically significant.
To determine the extent that an isolated Ca2+-dependent pathway mediates HDAC4 and HDAC5 regulation in VSMCs, we stimulated cultured VSMCs with the Ca2+ ionophore Iono, over a time course of 60 min. Iono-dependent phosphorylation of HDAC4 and HDAC5 on Ser632 and Ser498 respectively could be detected as early as 30 s after stimulation, peaked after 5 min of stimulation, and decreased to basal levels by 60 min (Figure 1A). Cell fractionation of VSMCs after a similar time course of Iono stimulation revealed that the levels of HDAC4 present in the nucleus decreased compared with unstimulated cells as early as 10 min after Iono stimulation and remained low through 40 min of Iono stimulation. After 60 min of Iono stimulation, the levels of HDAC4 found in the nuclear fraction returned to levels found in unstimulated cells (Figure 1B, left-hand panel). Conversely, stimulation of VSMCs with Iono resulted in an increase in HDAC4 levels found in the cytosol compared with unstimulated cells as early as 10 min after stimulation. After 60 min of Iono stimulation, HDAC4 levels in the cytosol were similar to those found in unstimulated cells (Figure 1B, right-hand panel). These results are consistent with a previous finding  that phosphorylation of class IIa HDACs such as HDAC4 confines their intracellular location to the VSMC cytosolic compartment.
Ca2+-dependent regulation of HDAC4 and HDAC5 in VSMCs
Regulation of HDAC4 and HDAC5 has been closely linked to regulation of MEF2 activity . To determine the extent to which the ability of MEF2 to bind DNA is regulated by Ca2+-dependent stimuli in VSMCs, cultured cells were stimulated with Iono over a 60 min time course and the ability of MEF2 to bind its DNA consensus sequence was measured. MEF2 DNA binding increased nearly 3-fold after 10 min of Iono stimulation compared with unstimulated cells (Figure 1C). These results illustrate a tight relationship between HDAC4 and HDAC5 phosphorylation, their intracellular location and their putative ability to mediate MEF2 DNA binding.
Along with the ability to induce HDAC4 and HDAC5 phosphorylation (Figure 1), Iono stimulation is well known to result in a robust activation of CaMKII in VSMCs [36,37]. Peak activation of CaMKII was detected within 30 s of Iono stimulation in the present study, as reflected by autophosphorylation on phospho-Thr287 (Figure 1A). To investigate the potential link between CaMKIIδ2 activation and HDAC4/HDAC5 phosphorylation, we adenovirally overexpressed a constitutively active form of CaMKIIδ2 (T287D)  in VSMCs. Relatively low expression levels of constitutively active CaMKIIδ2 resulted in HDAC4 and HDAC5 phosphorylation (Figures 2A and 2B). Importantly, no apparent increase in PKD activity was detected (results not shown) as PKD has been shown to mediate HDAC4 and HDAC5 phosphorylation in VSMCs . Similarly, overexpression of constitutively active CaMKIIδ2 increased MEF2 DNA binding, consistent with the increasing levels of HDAC4 and HDAC5 phosphorylation detected (Figure 2C). To assess further the contribution of CaMKIIδ-dependent processes in Ca2+-dependent MEF2 DNA binding and gene transcription, CaMKIIδ2 was suppressed with a CaMKIIδ2-specific shRNA sequence introduced by adenoviral infection  (Figure 3A) or siRNA duplexes introduced by electroporation (Figure 3B). In both cases, suppression of CaMKIIδ2 attenuated Iono (Ca2+)-dependent increases in MEF2 DNA binding in VSMCs.
CaMKIIδ2 increases HDAC phosphorylation and MEF2 DNA binding
CaMKIIδ2 mediates Ca2+-dependent gene transcription
Transcriptional targets for MEF2 include the immediate-response gene Nur77 [40,41] and the chemokine MCP1 . To determine whether CaMKIIδ2-dependent MEF2 regulation extends to a transcriptional readout in VSMCs, we measured Nur77 and MCP1 mRNA induction in response to Iono (Figures 3C and 3D). Suppression of CaMKIIδ2 protein levels significantly attenuated Iono-dependent increases in Nur77 and MCP1 mRNA levels in VSMCs. Taken together, these results describe an Iono (Ca2+)-dependent activation of CaMKIIδ2 in VSMCs that results in phosphorylation-dependent nucleocytoplasmic shuttling of HDAC4 and HDAC5 and mediates the expression of the MEF2-dependent transcription factors Nur77 and MCP1.
To investigate the potential role for CaMKIIδ2 in mediating relevant transcriptional pathways in VSMCs, we repeated the above experiments after stimulation with AngII and PDGF. Stimulation with AngII and PDGF has been reported to increase HDAC4 and HDAC5 phosphorylation in multiple cell types [23,26,31]. In cultured VSMCs, AngII- and PDGF-stimulated increases in HDAC4 and HDAC5 phosphorylation were observed after 30 s, and this phosphorylation was sustained for up to 3 h (Figures 4A and 4B, and results not shown). Cell fractionation studies indicated that AngII and PDGF stimulation induced a rapid loss of HDAC4 in the nuclear fraction compared with unstimulated cells and a concomitant gain in HDAC4 in the cytoplasmic fraction (Figure 4C). Although the extent of HDAC4 phosphorylation was similar using both AngII and PDGF (Figure 4B, left-hand panel), it appeared that AngII-dependent increases in HDAC5 phosphorylation at earlier time points were more robust than PDGF-dependent responses (Figure 4B, right-hand panel). The implications of these results are not clear, but may indicate the differential ability of AngII to activate kinases that are specific to HDAC5 alone.
Agonist-dependent regulation of HDAC4 and HDAC5 in VSMCs
To determine the ability of CaMKIIδ2 to mediate AngII- and PDGF-dependent phosphorylation of HDAC4 and HDAC5, we suppressed CaMKIIδ2 protein levels using the shRNA approach described above. Silencing CaMKIIδ2 expression attenuated AngII- and PDGF-dependent phosphorylation of HDAC4 and HDAC5 (Figures 5A and 5B). Similarly, overexpression of kinase-negative CaMKIIδ2  reduced AngII- and PDGF-dependent phosphorylation of HDAC4 and HDAC5 (Figures 5C and 5D). The results of this set of experiments are consistent with the results obtained using Iono (Figures 1 and 2) and support further a role for CaMKIIδ2 in regulating HDAC4 and HDAC5 in VSMCs.
CaMKIIδ2 mediates HDAC4 and HDAC5 phosphorylation
In contrast with the transient MEF2 DNA binding induced by Iono (Figure 1C), AngII and PDGF stimulation of VSMCs resulted in a sustained increase in MEF2 DNA binding over a 60 min time period (Figure 6A). Suppression of CaMKIIδ2 blocked the increase in MEF2 DNA binding after 10 min of AngII stimulation (Figure 6B). Interestingly, loss of CaMKIIδ2 did not attenuate MEF2 DNA binding induced by 2 h of AngII treatment (results not shown) or PDGF-dependent increases in MEF2 DNA binding under any conditions (Figure 6B). Consistent with results obtained with Iono stimulation (Figure 3), suppression of CaMKIIδ2 by shRNA significantly attenuated AngII-dependent increases in Nur77 and MCP1 mRNA levels in VSMCs (Figures 6C and 6D). Loss of CaMKIIδ2 was ineffective in suppressing PDGF-dependent increases in Nur77 or MCP1 (Figure 6C, and results not shown). Taken together, these results indicate that a CaMKII/MEF2-dependent pathway selectively mediates Ca2+-and AngII-dependent increases in Nur77 and MCP1 induction in VSMCs.
CaMKIIδ2 mediates agonist-dependent MEF2 activity
We next wanted to gain an insight into the mechanism by which CaMKII regulates this HDAC/MEF2 signalling pathway. An earlier study in cardiomyocytes using exogenously expressed proteins revealed that CaMKII interacts physically with HDAC4 at a site on HDAC4 identified as amino acids 585–608 . To determine whether a similar interaction occurs in VSMCs with endogenous proteins, CaMKIIδ2 was immunoprecipitated from cell lysates of unstimulated and AngII-stimulated cells. Immunoblot analysis of these CaMKIIδ2 immunoprecipitates revealed the presence of both HDAC4 and HDAC5 (Figure 7A). Similarly, CaMKIIδ2 and HDAC5 were detected in HDAC4 immunoprecipitates of cell lysates from unstimulated VSMCs (Figure 7B). Interestingly, stimulation with AngII or Iono resulted in a reduction of CaMKIIδ2 that co-immunoprecipitates with HDAC4 (Figures 7B and 7C). These results indicate that CaMKIIδ2 interacts directly with HDAC4 and HDAC5 in VSMCs through a protein–protein interaction and suggest that this interaction is regulated in a stimulation-dependent manner.
CaMKIIδ2 interacts physically with HDAC4 and HDAC5
It was demonstrated that the CaMKII interaction site on HDAC4 is not conserved on HDAC5 and that CaMKII-dependent regulation of HDAC5 is dependent on complex formation and heterodimerization with HDAC4 . To evaluate the role of HDAC4 in HDAC5 regulation in VSMCs, we suppressed HDAC4 expression in VSMCs using siRNA (Figure 8A). Attenuation of HDAC4 levels in VSMCs resulted in a significant loss of both AngII- and PDGF-dependent phosphorylation of HDAC5 (Figure 8A, right-hand panel). Previous work showed that a mutant of HDAC4 (S246A/S267A/S632A) acts as a dominant repressor owing to its inability to be phosphorylated and shuttled from the nucleus . Overexpression of dominant-repressive HDAC4 attenuated AngII- and PDGF-dependent increases in HDAC5 phosphorylation (Figure 8B). These results confirm a key role for HDAC4 in mediating HDAC5 phosphorylation in response to stimuli relevant to VSMC function.
HDAC4 mediates agonist-dependent phosphorylation of HDAC5
Given the importance of MEF2 in VSMC development  and regulation in response to injury , understanding the factors that mediate its activity are critical. Previous studies in VSMCs have implicated CaMKII and class IIa HDACs, HDAC4 and HDAC5 in particular, as being important regulators of MEF2 activity [23,29,30]. Examination of these studies reveals that there are several important questions remaining as to how CaMKII and HDACs co-ordinately regulate MEF2 function. Some of these questions include: how are HDACs regulated in response to different agonists; which HDAC(s) are targeted by CaMKII in vivo; and what MEF2 target genes are regulated in a CaMKII- and HDAC-dependent manner? In the present study, we unambiguously established that HDAC4 and HDAC5 are regulated in response to increases in intracellular Ca2+ concentration and stimulation with AngII and PDGF in VSMCs. We also provided evidence that CaMKIIδ2 is capable of mediating AngII- and PDGF-dependent increases in HDAC4 and HDAC5 phosphorylation and Ca2+- and AngII-dependent increases in MEF2 DNA-binding activity. We showed further that these CaMKII-dependent increases in MEF2 DNA-binding activity can be linked to CaMKII-dependent increases in expression of the MEF2 target genes Nur77 and MCP1. Finally, we demonstrated that HDAC5 phosphorylation is mediated by HDAC4 in VSMCs, suggesting that CaMKIIδ2-dependent regulation of HDAC5 involves HDAC4.
Previous studies have shown that stimulation with AngII [29,30] and PDGF  results in HDAC4 and HDAC5 phosphorylation in VSMCs. In the present study, we not only confirm these results, but also demonstrate that stimulation with Iono, which causes rapid increases in intracellular [Ca2+], results in transient HDAC4 and HDAC5 phosphorylation, and rapid and transient nucleocytoplasmic shuttling of HDAC4. These findings reveal the dynamic nature of HDAC regulation in vivo and provide a means of understanding the differential regulation of HDAC4 and HDAC5 by CaMKII compared with other HDAC kinases such as PKD . It is well established that CaMKIIδ2 is activated within 30 s of Iono or agonist stimulation of VSMCs. Coupling this with Iono-dependent increases in MEF2 DNA-binding activity and gene expression (Nur77 and MCP1) provides further evidence that transient activation of CaMKII is capable of mediating important downstream VSMC functions.
The rapidity of agonist-stimulated CaMKII-dependent HDAC4/HDAC5 phosphorylation in VSMCs suggests that CaMKII is physically proximal to the HDACs, either in the nucleus or in the cytosol. In a previous study, we reported that CaMKIIδ2 is capable of being transported into the nucleus . Perhaps there is some CaMKIIδ2 or even a small amount of nuclear-targeting CaMKIIδ3  in VSMCs which is responsible for the initial phosphorylation events necessary for HDAC4 and HDAC5 nucleocytosolic shuttling. The alternative is that CaMKII phosphorylates cytosolic HDACs. There is one report that suggests that CaMKII interacts with HDACs through 14-3-3 proteins in the cytosol and prevents HDAC re-entry into the nucleus . HDAC regulation also involves reciprocal regulation by phosphatases such as PP2A (protein phosphatase 2A)  and MYPT1 (myosin phosphatase 1)  in the cytosol. Given the evidence that CaMKII is capable of regulating phosphatase activity , it is possible that CaMKII may regulate HDAC function in the cytosol in multiple ways.
Previous studies in cardiomyocytes using overexpressed HDAC4/HDAC5 reporters and mutants suggested that a physical complex of CaMKIIδ2 with HDAC4 and between HDAC4 and HDAC5 was required to couple CaMKII to HDAC5 regulation . We were interested in testing this mechanism in VSMCs by evaluating endogenous proteins, in part, because the major CaMKIIδ isoform expressed in these cells is the δ2 isoform, which is largely cytosolic . The physiological implications of this coupling are not clear, but provide a selective means that increases in intracellular Ca2+ concentration and consequent CaMKII activation may directly mediate transcriptional events. For example, agonists such as AngII that result in rapid and robust increases in intracellular Ca2+ concentrations may be more likely to utilize the CaMKII/HDAC4 signalling axis and co-opt HDAC5 to enhance the transcriptional response, whereas stimuli that induce relatively low levels of intracellular Ca2+ concentrations may bypass HDAC4 and signal primarily through HDAC5. This level of signalling specificity with regard to HDAC4 and HDAC5 has not been carefully studied in any system and may provide new insights into transcriptional regulation of immediate-early-response genes in VSMCs.
The role of MEF2 in VSMCs is poorly understood, but is probably multifactorial, reflecting its known functions in regulating cell-type-specific genes and as an integrator of cellular signals . On the basis of its regulation of myocardin expression in cardiac and smooth muscle, and its control of contractile and metabolic genes in striated muscle, the prediction would be for MEF2 to promote the differentiated phenotype of VSMCs [15,18]. Although compelling, this hypothesis remains to be tested in vivo through targeted MEF2 deletions. There is evidence, however, that MEF2 also has a role in regulating the synthetic VSMC phenotype: MEF2 protein and activity are strongly up-regulated in the neointima of rodent models of vascular injury, promote proliferation and indirectly inhibit myocardin activity [21,23]. Indeed, a dominant-negative MEF2 will reduce the size of the neointima following vascular injury . Our findings are consistent with this model in that CaMKIIδ2 is up-regulated following balloon injury and its subsequent phosphorylation of HDAC4/HDAC5 could contribute to increased MEF2 activity in this system. Still, the mechanism by which MEF2 regulates the synthetic phenotype is poorly understood, and the current data suggest multiple possible effectors. As stated above, MCP-1 is regulated by MEF2 and promotes macrophage infiltration and growth of a vascular lesion . MEF2 may play a role in VSMC migration from the media to the neointima because, in other cell types, MEF2 has been linked to migration and tissue invasion by its regulation of matrix metalloproteases, and chemokine ligands and receptors [50,51].
MEF2 is a well-studied activator of Nur77 induction in many cell types, including VSMCs [40,52] (Figure 6). In the present study, the primary purpose of assaying Nur77 was as a ‘physiological’ readout of MEF2 transcriptional activity. Previous studies have shown that, in contrast with MEF2 , Nur77 inhibits VSMC proliferation in vitro and neointimal formation in vivo [53,54]. Reconciling these apparently opposite functions of MEF2 and its transcriptional target, Nur77, in neointimal formation into a cohesive model as well as understanding the functional significance of CaMKII-dependent regulation of Nur77 will require additional studies. Our model proposes a regulatory axis from CaMKII to MEF2 through regulation of HDAC4/HDAC5 nuclear localization. However, many transcription factors are targets of the class II HDACs, including SRF (serum-response factor), which has been linked to CaMKII and HDAC4  in other cell types. Thus MEF2 is unlikely to be the only transcription factor targeted by CaMKII through HDAC4/HDAC5 in VSMCs.
These studies provide a mechanism by which CaMKIIδ2 is capable of mediating VSMC transcriptional activity through the regulation of class IIa HDACs, including a specific HDAC4/HDAC5/MEF2 pathway. On the basis of the results of the present study, it is apparent that a CaMKII/HDAC4,5/MEF2 signal axis will function in a stimulus- and cell-context-dependent manner. How ultimately such a pathway factors into functional responses, including specific transcriptional events, VSMC phenotype switching or VSMC function (proliferation or migration), will require additional studies.
Ca2+/calmodulin-dependent protein kinase II
Dulbecco's modified Eagle's medium
fetal bovine serum
green fluorescent protein
monocyte chemotactic protein 1
myocyte-enhancer factor 2
multiplicity of infection
nuclear receptor 77
platelet-derived growth factor
protein kinase D
short hairpin RNA
small interfering RNA
vascular smooth muscle cell
Roman Ginnan and Li Yan Sun conducted all of experimental work, data analysis and preparation of Figures. The paper was written by Roman Ginnan with assistance from John Schwarz and Harold Singer. The overall study was conceived by Harold Singer with help from Roman Ginnan and John Schwarz.
We thank Virginia Foster and Scott Eichen for technical assistance in completing this project. We are also thankful to Wendy Vienneau for assistance in preparing the paper for submission.
This work was supported by the National Heart, Lung, and Blood Institute [grant numbers RO1-HL-092510 and RO1-HL-49426 (to H.A.S.)].