CaMKIIδ-dependent Inhibition of cAMP-response Element-binding Protein Activity in Vascular Smooth Muscle*

Background: CaMKII is implicated in both positive and negative regulation of cAMP-response element-binding protein (CREB) activity. Results: CaMKIIδ increases CREB-Ser142 phosphorylation and inhibits binding of CREB to Sik1 and Rgs2 promoters in vascular smooth muscle (VSM). Conclusion: In VSM CaMKIIδ negatively regulates CREB activity and target gene transcription by phosphorylating CREB-Ser142. Significance: Negative regulation of CREB by CaMKIIδ is a mechanism mediating Ca2+ signal-dependent regulation of gene transcription in VSM. One transcription factor mediator of Ca2+-signals is cAMP response element-binding protein (CREB). CREB expression and/or activity negatively correlates with vascular smooth muscle (VSM) cell proliferation and migration. Multifunctional Ca2+/calmodulin-dependent protein kinases, including CaMKII, have been demonstrated to regulate CREB activity through both positive and negative phosphorylation events in vitro, but the function of CaMKII as a proximal regulator of CREB in intact cell systems, including VSM, is not clear. In this study, we used gain- and loss-of-function approaches to determine the function of CaMKIIδ in regulating CREB phosphorylation, localization, and activity in VSM. Overexpression of constitutively active CaMKIIδ specifically increased CREB phosphorylation on Ser142 and silencing CaMKIIδ expression by siRNA or blocking endogenous CaMKII activity with KN93 abolished thrombin- or ionomycin-induced CREB phosphorylation on Ser142 without affecting Ser133 phosphorylation. CREB-Ser142 phosphorylation correlated with transient nucleocytoplasmic translocation of CREB. Thrombin-induced CREB promoter activity, CREB binding to Sik1 and Rgs2 promoters, and Sik1/Rgs2 transcription were enhanced by a kinase-negative CaMKIIδ2 (K43A) mutant and inhibited by a constitutively active (T287D) mutant. Taken together, these studies establish negative regulation of CREB activity by endogenous CaMKIIδ-dependent CREB-Ser142 phosphorylation and suggest a potential mechanism for CaMKIIδ/CREB signaling in modulating proliferation and migration in VSM cells.

Vascular smooth muscle (VSM) 2 cells comprise the medial layer of blood vessels providing mechanical support and a means for adjusting vascular wall tone and diameter. However, VSM cells are not terminally differentiated and can switch to a proliferative/synthetic phenotype in response to changes in local environment that accompany a number of chronic vascular diseases as well as acute and chronic vascular injuries (1,2). Identifying transcriptional and post-transcriptional regulatory processes governing VSM phenotype plasticity are therefore of considerable basic interest and clinical relevance.
One key signal in VSM is free intracellular Ca 2ϩ ([Ca 2ϩ ] i ), which is well known to regulate differentiated contractile function, as well as synthetic phenotype proliferation and migration (3). [Ca 2ϩ ] i signals also regulate a number of transcription factors, including type II HDAC-MEF2, calcineurin/nuclear factor of activated T cells, and cyclic AMP response element-binding protein (CREB) (4 -6). CREB, in addition to its defining regulation by cAMP-dependent protein kinase (PKA), has long been known to be regulated by [Ca 2ϩ ] i signals via multifunctional Ca 2ϩ /calmodulin-dependent protein kinases including CaMKIV and CaMKII (7). Expression of CREB is down-regulated in several vascular diseases, such as hypertension, dyslipidemia, and atherosclerosis, as well as in response to vascular injury (8), suggesting a vasculoprotective effect of CREB (8). Conversely, CREB activation has also been reported to be positively related to thrombin-induced proliferation (9) and TNF␣-induced migration in the smooth muscle cells (10).
Several phosphorylation sites in CREB have been demonstrated to regulate transcriptional activity. Ser 133 is the primary target of PKA and phosphorylation is required for CREB activation, whereas phosphorylation of Ser 142 by CaMKII has been reported to inhibit CREB activity by interfering with CREB dimerization and protein interactions to form an active promoter complex (11). In vitro studies using purified brain CaMKIV and CaMKII have clearly demonstrated that CaMKIV phosphorylates CREB on Ser 133 , whereas CaMKII has equal affinity for Ser 133 and Ser 142 (12). These results were confirmed using transient expression assays and constitutively active CaMKIV and CaMKII constructs. In the case of CaMKII overexpression, inhibitory phosphorylation on Ser 142 overrides the activating phosphorylation on Ser 133 (11,12). Yet in intact neural cells, CaMKII activation has been positively associated with CREB activation (13)(14)(15). Based on these studies, it appears that endogenous CaMKII could be either a positive or negative regulator of CREB, perhaps in a tissue or context-specific manner. Despite the widespread importance of CREB as a transcription factor, there is very little information regarding the importance of negative CREB regulation mediated by CaMKII-dependent Ser 142 phosphorylation in any intact cellular system.
Pathways regulating CREB activity in response to Ca 2ϩ -dependent stimuli in VSM are incompletely understood, and what little is known has been inferred via indirect pharmacological approaches that suggest a net positive function for CaM kinases in regulating CREB (16 -18). In this study, we demonstrate for the first time in VSM cells that stimulation with Ca 2ϩ -dependent stimuli thrombin and ionomycin resulted in transient CREB-Ser 142 phosphorylation that preceded the activating phosphorylation on Ser 133 . Multiple gain-and loss-of-function approaches indicated a specific function for endogenous CaMKII␦ in regulating VSM CREB-Ser 142 phosphorylation, nucleocytoplasmic translocation, promoter activity, and endogenous target gene CREB binding and transcription. Taken together, these studies definitively establish negative regulation of CREB activity by endogenous CaMKII␦-dependent phosphorylation of CREB-Ser 142 and suggest a potential mechanism for CaMKII/CREB signaling in modulating proliferation and migration in VSM cells.

EXPERIMENTAL PROCEDURES
Materials-The antibody specific for phospho-Ser 142 -CREB was purchased from GeneScript and the antibodies against total CREB, phospho-Ser 133 -CREB, histone H3, and ␣-tubulin were purchased from Cell Signaling. The antibodies against ␤-actin and GAPDH were purchased from Sigma. Polyclonal antibodies against pan-CaMKII, CaMKII␦ 2 , and phospho-Thr 287 -CaMKII were raised in rabbits as described previously (19,20). Adenoviruses expressing kinase-negative CaMKII␦ 2 (K43A), constitutively active CaMKII␦ 2 (T287D), and GFP were described previously (21,22). The plasmid expressing luciferase under a promoter construct containing four CREB binding site repeats was purchased from Affymetrix. Thrombin was purchased from Sigma; and KN93 and ionomycin were purchased from Calbiochem. The cell culture media and all immunoblotting supplies were purchased from Invitrogen and Bio-Rad Laboratories, respectively.
VSM Cell Dispersion and Culture-Primary cultures of VSM cells were obtained from rat aorta as previously described (23). In brief, Sprague-Dawley rats (150 -200 g) were sacrificed using CO 2 , and following brief digestion with collagenase the medial layer of the aorta containing VSM cells was freed from the adventitial layer and intima containing endothelial cells was disrupted mechanically. Medial layer VSM cells were isolated by enzymatic digestion with collagenase and elastase and cultured in DMEM/F-12, supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin. Cells were passaged when ϳ80% confluence was reached. Experiments were performed with VSM cells from passages 3 to 7. The biochemical and growth properties of these and similar VSM cell cultures have been extensively characterized (5, 19 -23). All animal protocols were reviewed and approved by the Albany Medical Center Institutional Animal Care and Use Committee.
Luciferase Reporter Assay-VSM cells (1 ϫ 10 6 ) were electroporated with plasmids expressing CREB luciferase reporter (2 g) and TK-Renilla (0.1 g) and incubated for a total of 72 h prior to cell lysis. Adenoviruses encoding kinase-negative CaMKII␦ 2 (K43A) mutant, which acts as a dominant-negative with respect to kinase activity, or constitutively active CaMKII␦ 2 (T287A) mutant (22) were transduced for 48 and 24 h prior to cell lysis, respectively. CREB luciferase reporter activity was stimulated by thrombin (0.5 nM) or forskolin (5 M) for 6 h prior to lysis. The level of promoter activity was determined by measuring firefly luciferase signal intensity normalized over the internal control TK-Renilla luciferase signal intensity using protocols provided in the Dual Luciferase Assay System (Promega).
Cell Stimulation and Western Blot-VSM cells were washed twice with pre-warmed Hanks' balanced salt solution and placed on a warm plate (37°C) for 30 min prior to stimulation with either 50 nM thrombin or 0.5 M ionomycin. At appropriate times cells were lysed by addition of 3ϫ Laemmli sample buffer. Lysates were heated at 95°C for 5 min and then resolved using standard 10% SDS-PAGE. Resolved proteins were electrophoretically transferred onto nitrocellulose membranes (GE Healthcare), the membranes blocked with 5% nonfat milk or 5% BSA in Tris-buffered saline supplemented with 1% Tween 20 (TBST) for 1 h, followed by primary antibody incubation for 1 h or overnight at 4°C and secondary antibody incubation for 1 h. After washing three times with TBST, the membranes were incubated with SuperSignal chemiluminescent substrate (Thermo) for 5 min. Gel images shown were generated by the Fuji LAS4000 and quantified with Multi Gauge software.
siRNA Electroporation-VMS cells (1 ϫ 10 6 ) were harvested and electroporated with 2 g of siRNA targeting CaMKII␦ gene products (Smart pool siRNA Thermo Fisher, catalog number L-080171-00-0010) or non-targeting siRNA (Thermo Fisher) using the Amaxa Nucleofector system (Lonza) and the VSM cell-specific D33 program (Lonza Amaxa). After electroporation, the cells were resuspended in 4 ml of cell culture media and reseeded onto four 60-mm culture dishes. Using previously established conditions (5), the cells were used 48 h following electroporation.
Chromatin Immunoprecipitation (ChIP)-To study the interaction between CREB and gene promoters, a ChIP assay was performed using the ChIP-IT Express Kit from Active Motif (53008). 5-6 ϫ 10 6 VSM cells were used for each sample. Following cross-linking and recovery of chromatin following kit protocols, chromatin was sheared in 200 l of kit shearing buffer using a Bioruptor sonicator at medium power for a total time of 16 min (30 s on and 30 s off for 16 cycles). 50 l of chromatin lysate was immunoprecipitated overnight at 4°C with anti-CREB antibody and isolated with magnetic protein G beads. Following cross-link reversal and incubation with proteinase K per kit protocols, immunoprecipitated DNA was analyzed by quantitative PCR. Promoter regions were predicted using PromoSer software (24) and CREB-binding elements were predicted with PROMO 3.0 software (25). Primers flanking a predicted CREB binding site ( Ϫ1020 CTGACGTCA Ϫ1012 ) in the rat Sik1/Kid2 promoter were (5Ј-3Ј): forward, Ϫ1040 GCG-GCTGCTGAGCCCGGT Ϫ1023 -3Ј and reverse, Ϫ962 GGCGGT-TGCCAAGAGACTGGAG Ϫ941 . Primers flanking an Rgs2 predicted CREB-binding element ( Ϫ221 GCCTACGTCA Ϫ212 ) were: forward, Ϫ243 TGTGTGCGCACGAGGATGCGG Ϫ223 and reverse, Ϫ124 ACCTCTAGAGGGCGCGGATTG Ϫ104 .
Cell Fractionation-Cell fractionation was performed on VSM cells to separate nuclear and cytosolic fractions using a cell fractionation kit and protocols (Active Motif). In brief, cells were washed with pre-cooled PBS containing phosphatase inhibitors (5 ml), collected with a cell scraper and isolated by centrifugation at 500 ϫ g for 5 min at 4°C. After the resuspension in a kit hypotonic lysis buffer (500 l), the cells were incubated on ice for 15 min. Detergent (25 l) was added followed by centrifugation at 14,000 ϫ g for 30 s at 4°C to pellet a crude fraction containing nuclei. The crude supernatant is considered the "cytosolic" fraction and the washed pellet the "nuclear" fraction. The nuclear marker histone H3 and cytosolic marker tubulin were used to verify the fractionation. Proteins in the fractions were by Western blotting.
Statistical Analysis-Quantitative data are presented as mean Ϯ S.E. Student's unpaired t test was used in the comparisons between two groups of data and one-way ANOVA for simple comparisons between multiple groups. If found significant by ANOVA, Dunnett's Multiple Comparison or Bonferroni post hoc tests were used to identify between group differences. Two-way ANOVA with a Bonferroni post hoc test was used in Fig. 3 to compare time points across siRNA and siControl groups. Statistics were performed using the GraphPad Prism4 program. "n" indicates the number of independent experiments and results were considered to be significantly different if p Ͻ 0.05. Statistical significance is presented, *, **, and *** indicate p Ͻ 0.05, p Ͻ 0.01, and p Ͻ 0.001, respectively.

RESULTS
CREB Phosphorylation in VSM-Thrombin has been proven to increase [Ca 2ϩ ] i via activation of membrane PAR receptors (reviewed in Ref. 26), resulting in activation of both CaMKII (27) and CREB (9). CREB Ser 133 and Ser 142 are two candidate phosphorylation sites for regulation by Ca 2ϩ -dependent pathways with the former leading to CREB activation and the latter to CREB inhibition (12,28). Addition of the Ca 2ϩ ionophore, ionomycin, was used to selectively induce Ca 2ϩ influx and stimulate activation of CaMKII␦ in VSM cells. CaMKII␦ activation was indexed by immunoblotting lysates with an antibody recognizing phospho-Thr 287 , the activation-dependent autophosphorylation site (29) (Fig. 1A). Ionomycin (0.5 M) stimulated CREB phosphorylation 2-fold on both Ser 133 and Ser 142 , suggesting either or both phosphorylation events could be mediated by CaMKII␦ (Fig. 1A). Addition of the physiological stimulus thrombin (50 nM) biphasically activated CaMKII with peaks between 30 -60 s and 30 min, consistent with a previous report (27) (Fig. 1B). Thrombin also increased CREB phosphorylation on both Ser 133 and Ser 142 , but with distinct time courses. CREB phosphorylation on the inhibitory site Ser 142 peaked in 2 min, slightly lagging the CaMKII activation time course, whereas phosphorylation on the activating site Ser 133 lagged considerably with a peak between 5 and 10 min following stimulation. Thus, in VSM cells, activation of CaMKII in response to thrombin correlated temporally with CREB-Ser 142 phosphorylation compared with CREB-Ser 133 phosphorylation.
CaMKII␦ Phosphorylates CREB on Ser 142 in VSM-In vitro studies using purified CaMKII from brain have demonstrated that both CREB Ser 133 and Ser 142 are substrates for the kinase (12). The function of CaMKII in regulating endogenous CREB in VSM has only been inferred through pharmacological approaches that do not distinguish between CaMKII and CaMKIV (30). The principal isoform of CaMKII expressed in VSM cells is CaMKII␦ 2 (19), or CaMKII␦ C by alternative nomenclature (31). Transduction of VSM cells with an increasing dose of adenovirus expressing a constitutively active CaMKII␦ 2 (T287D) mutant (22) significantly enhanced CREB phosphorylation on Ser 142 in a dose-dependent manner (Fig. 2,  A and B). However, overexpression of the constitutively active mutant did not affect CREB phosphorylation on Ser 133 at any of the levels tested. This gain of function experiment indicates a strong specificity of CaMKII␦ for the inhibitory CREB Ser 142 site in intact VSM cells.
To determine whether endogenous CaMKII␦ regulates CREB phosphorylation on Ser 133 or Ser 142 , we used siRNAs specifically targeting CaMKII␦ isoforms to reduce CaMKII␦ expression by ϳ80 -90% (Fig. 3A). Consistent with the results in Fig. 1, thrombin stimulated CREB phosphorylation at Ser 142 (Fig. 3, A and B) and Ser 133 (Fig. 3, A and C) in the presence of control non-targeting siRNA. Suppression of CaMKII␦ expression abolished thrombin-induced CREB phosphorylation on Ser 142 (Fig. 3B), but had no significant effect on thrombin-stimulated CREB phosphorylation on Ser 133 (Fig. 3C). As an alternative approach, we pharmacologically inhibited CaMKII activation in VSM cells using KN93, a selective CaM kinase inhibitor (32). Pretreatment with KN93 (30 M) for 30 min inhibited thrombin-or ionomycin-induced CaMKII␦ activation (Fig. 4, A and B) and strongly inhibited CREB phosphorylation on Ser 142 induced by either stimulus (Fig. 4C). Taken together with the gain-of-function results (Fig. 2), these loss-of-function results indicate that endogenous CaMKII␦ mediates CREB phosphorylation on Ser 142 , but not Ser 133 in VSM cells.
Additional studies using the MEK1 inhibitor U0126 (5 M) indicated that essentially complete inhibition of ERK1/2 activation (Fig. 5, A and B) resulted in a strong inhibition of thrombin-and ionomycin-stimulated CREB-Ser 133 phosphorylation (Fig. 5C), confirming a previous study (9). Residual Ser 133 phosphorylation in this experiment suggests that another unidentified pathway(s) may also contribute to CREB activation in response to these Ca 2ϩ -dependent stimuli.
Thrombin Stimulates Nucleocytoplasmic Translocation of Phospho-Ser 142 -CREB in VSM-Stevenson et al. (33) observed membrane depolarization induced CREB translocation in VSM cells and Garat et al. (34) reported nuclear export of CREB in response to sustained PDGF in pulmonary artery smooth muscle cells. The mechanisms signaling nuclear CREB export are not known. Western blotting of nuclear and cytosolic fractions was carried out to assess localization of CREB. Effective fractionation was verified by nuclear histone H3 and cytoplasmic tubulin content, respectively (Fig. 5A). Under unstimulated conditions, total CREB and resting levels of phospho-Ser 142 -CREB were detected in both nuclear and cytosolic fractions. Thrombin stimulation transiently decreased nuclear phospho-Ser 142 -CREB content by over 50% (Fig. 6B), with concomitant increases in the cytosolic fraction (Fig. 6C). Phospho-Ser 133 -CREB increased but remained localized in the nuclear fraction by 5-10 min (Fig. 6, A and D). Interesting, total CREB also translocated from the nucleus with a time course identical to phospho-Ser 142 -CREB (Fig. 6, A and B). CaMKII␦ was detected in nuclear fractions and transiently activated with a peak at 2 min, corresponding temporally with nuclear export of phospho-Ser 142 and total CREB.
CaMKII␦ Negatively Regulates CREB Reporter Activity and CREB-regulated Genes-The strong correlation between nuclear export of CREB and Ser 142 phosphorylation by CaMKII␦ suggests one potential mechanism for negative regulation of CREB activity by CaMKII in VSM. To evaluate the function of CaMKII␦ in regulating CREB activity in VSM, cells were transfected with a luciferase reporter plasmid containing 4 tandem copies of the CREB binding domain. Forskolin, which is a potent activator of the PKA pathway and strongly stimulates CREB activity (7), was used as a positive control. As expected, forskolin (5 M) strongly increased CREB reporter activity nearly 6-fold, whereas thrombin significantly activated reporter activity by less than 2-fold (Fig. 7, A and B). Adenoviral overexpression of a kinase-negative CaMKII␦ 2 (K43A) mutant, which exerts a dominant-negative effect on endogenous CaMKII activity in situ (19), significantly enhanced subsequent throm-   bin-induced CREB reporter activity by 3-fold over unstimulated levels (Fig. 7A). Conversely, adenoviral transduction and expression of a constitutively active CaMKII␦ 2 (T287D) mutant significantly inhibited thrombin-induced CREB reporter activity to nearly basal levels (Fig. 7B).
To investigate whether CaMKII␦-dependent negative regulation of CREB activity extends to endogenous transcriptional targets for CREB in VSM cells, we examined the effect of CaMKII␦ 2 mutants on mRNA expression of Sik1 (salt-induced kinase 1; Fig. 7C) and Rgs2 (regulator of G-protein signaling 2; Fig. 7D). SIK1 is endogenously expressed in VSM and contributes to hypertension through regulation of the plasma membrane Na ϩ ,K ϩ -ATPase (35). In addition, SIK1 has been identified as a transcriptional target for CREB in skeletal muscle cells and to interact with and regulate the type 2 HDAC-MEF2 pathway (36). Rgs2 contains a conserved CREB binding site in its promoter (37) and functions in an angiotensin II-dependent feedback loop to regulate blood pressure (38). Similar to the luciferase CREB reporter data above, forskolin treatment dramatically increased mRNA levels of Sik1 and Rgs2 by 30 -40-fold, whereas thrombin treatment induced Sik1 and Rgs2 mRNA expression by less than 2-fold. Inhibition of CaMKII␦ activity by overexpressing kinase-negative CaMKII␦ 2 significantly enhanced the induction of Sik1 and Rgs2 mRNA expression in response to thrombin. Adenoviral introduction of constitutively active CaMKII␦ 2 abolished thrombin-induced Sik1 and Rgs2 mRNA induction. In contrast to previous reports (16 -18), these data suggest that Ca 2ϩ signaling mediated by CaMKII␦ inhibits CREB activity in VSM cells.
To directly test the effects CaMKII␦ activity on CREB binding to Sik1 and Rgs2 promoters, chromatin immunoprecipitation (ChIP) assays were performed using CREB antibody to isolate CREB/CREB-binding element complexes (Fig. 8). Control experiments in unstimulated cells confirmed CREB binding to the targeted binding elements in the Sik1 and Rgs2 promoters (Fig. 8A). Thrombin stimulation for 10 min increased CREB binding to the promoters in 3 separate experiments. Because the fold-inductions with thrombin were variable between the experiments (2-35-fold for Sik1; 3-75-fold for Rgs2), effects of overexpressing constitutively active and kinase-negative CaMKII␦ 2 were normalized to the thrombin signal. Expression of constitutively active CaMKII␦ 2 (Fig. 8B) was reproducibly effective at blocking thrombin-induced CREB binding on both the Sik1 (Fig. 8C) and Rgs2 (Fig. 8D) promoters, whereas overexpression of kinase-negative CaMKII␦ 2 reproducibly enhanced CREB binding.

DISCUSSION
Increasing evidence indicates that Ca 2ϩ signaling via CaMKII␦ positively regulates VSM cell proliferation and migration and contributes to vascular remodeling in response to injury or hypertensive disease (38 -40). Here, we present evidence that Ca 2ϩ signaling via CaMKII␦ mediates thrombininduced CREB phosphorylation selectively on Ser 142 , correlating with CREB translocation from the nucleus. One outcome of thrombin-induced CaMKII-dependent signaling in VSM is negative regulation of CREB binding to CREB regulatory elements in specific target genes (in this case Sik1 and Rgs2) and negative regulation of CREB-induced transcriptional activity as indicated by a luciferase reporter assay and transcription of Sik1 and Rgs2. We believe the results of this study provide a definitive answer to the function of CaMKII␦ in regulating CREB activity in VSM cells and correct any potential misinterpretations of experiments that rely solely on pharmacological inhibitors of CaMKII or extrapolations of results from other systems such as neural tissues, which have suggested positive regulation of CREB activity by CaMKII (17,18,30).
Early studies demonstrated that purified brain CaMKII phosphorylated recombinant CREB at both Ser 133 and Ser 142 in several cultured cell lines (12,41). Moreover, Wu and McMurray (11) reported that CaMKII-phosphorylated CREB at both Ser 133 and Ser 142 in human neuroblastoma and monkey kidney cells. Our experiments, using an adenovirally transduced constitutively active CaMKII␦ 2 mutant and multiple molecular and pharmacological loss-of-function approaches demonstrate that in intact VSM cells stimulated by thrombin or a Ca 2ϩ ionophore, CaMKII selectively phosphorylates endogenous CREB on Ser 142 . Lack of CREB Ser 133 phosphorylation downstream of CaMKII in VSM could be due to isoform-specific effects with CaMKII␦ 2 the predominant isoform expressed in VSM cells, compared with CaMKII␣ and -␤ isoforms, which are predominant in neural tissue. Alternatively, unknown factors that are properties of intact VSM cells might restrict CaMKII␦ access to CREB-Ser 133 .
Most of the positive regulation of CREB via Ser 133 phosphorylation in this study could be attributed to ERK1/2-dependent signaling as reflected by inhibition of responses by the MEK inhibitor U0126, confirming previous results (9). Although the mechanisms underlying the residual Ser 133 phosphorylation are not known, we do not expect that Ser 133 phosphorylation is mediated by CaMKIV in VSM cells, because we are unable to reliably detect either CaMKIV mRNA or protein expression. 3 Phosphorylation of CREB on Ser 142 has been shown to attenuate CREB activity, even if Ser 133 is phosphorylated (11). Consistent with this concept, we observed that expression of constitutively active CaMKII␦ 2 nearly abolished the thrombin-induced activation of a CREB reporter, whereas, thrombin-stimulated CREB reporter activity was further enhanced by inhibiting CaMKII activity using dominant-negative CaMKII␦ 2 . Wu and McMurray (11) presented evidence that CREB Ser 142 phosphorylation in response to CaMKII activation prevented CREB dimerization and interaction with the CREBbinding protein. There are a few reports of CREB translocation from the nucleus that would also result in decreased activity (33,34). Membrane depolarization in VSM cells induced elevated intracellular Ca 2ϩ and mediated acute nuclear export of CREB (33) and prolonged stimulation with PDGF-BB-induced nuclear export of CREB and subsequently degradation through the ubiquitination pathway (34). Our studies of CREB localization in VSM cells following acute stimulation with thrombin demonstrate a strong correlation between CaMKII-dependent Ser 142 phosphorylation and nuclear export, a mechanism that could also explain depolarization-induced nuclear export. Even with the relatively brief period of Ser 142 phosphorylation, the extent of CREB nuclear export is substantial (greater than 50% of total CREB in the nucleus) suggesting a functionally important mode of CREB regulation. However, basal levels of CREB Ser 142 phosphorylation appear in the nucleus in unstimulated cells, suggesting that mechanisms in addition to Ser 142 phosphorylation may be required for nuclear export.
Although CaMKII␦ 2 is the most abundant isoform in VSM and is localized predominantly in the cytosol, we did detect CaMKII␦ in VSM nuclei and activation of the nuclear kinase following thrombin stimulation. This may be explained by co-3 Z. Wang   expression of small amounts of the alternative splice variant CaMKII␦ 3 (or CaMKII␦ B ) that was first cloned from VSM cells (19). CaMKII␦ 3 contains a nuclear targeting domain and when co-expressed with CaMKII␦ 2 targets the kinase to the nucleus in proportion to composition of hetromultimeric holoenzymes (42). The small fraction of total and active CaMKII␦ in the nuclear fraction of VSM cells is consistent with the relative expression of the ␦ 2 isoform compared with ␦ 3 in VSM cells (19). How much negative CREB regulation is due directly to CaMKII␦-mediated phosphorylation of Ser 142 in the nucleus versus indirectly by CREB-Ser 142 phosphorylation and trapping in the cytosol remains to be determined. We would predict that in other systems such as intact differentiated VSM (19) or heart (43)(44) where the nuclear targeting isoform CaMKII␦ 3 is expressed more abundantly, this mode of regulating CREB may be even more predominant. Alternatively, conditions of prolonged CaMKII activation occurring under conditions with high frequency Ca 2ϩ transients (45) or oxidizing conditions (46 -48), may be predicted to promote nuclear CREB export and potentially degradation (34). Sik1 and Rgs2 have been reported to be regulated by CREB in multiple cell types, including VSM cells (35)(36)(37). In this study, ChIP assays confirmed thrombin-induced CREB binding to an element in the RGS2 promoter previously identified using mutagenesis approaches (36). We also confirmed CREB binding to a predicted element in the Sik1 promoter (37). Moreover, we extended knowledge of CREB target regulation by demonstrating CaMKII␦ negatively regulated thrombin-induced CREB binding to Sik1 and Rgs2 promoters, as well as transcription of the respective mRNAs. It has been reported that SIK1 regulates both the activity and expression of Na,K-ATPase in the renal proximal tubule, and in turn influences sodium and fluid reabsorption (49), which may provide a pathway affecting blood pressure regulation. Xie et al. (37) identified a CREB binding site in the RGS2 promoter sequence and demonstrated that angiotensin II-induced CREB activation upregulated Rgs2 expression in VSM cells with effects on blood pressure homeostasis. CaMKII is also activated in response to angiotensin II stimulation in VSM (5) and is a likely component of angiotensin II-dependent regulation of CREB activity and RGS2 expression.
Our laboratory first reported that CaMKII␦ 2 promoted VSM cell migration and proliferation in vitro (50) and exerted a positive effect on neointima formation in vivo (38). Recent studies using global CaMKII␦ gene knock-out mice have confirmed positive roles of CaMKII␦ 2 in regulating the VSM cell proliferation and migration (39,40). Aside from direct regulation of proteins involved in cell cycle control and cell motility, we have reported that CaMKII␦ 2 phosphorylates HDAC4/5, resulting in nucleocytoplasmic shuttling of the HDACs and subsequent de-repression of MEF2 transcriptional activity in VSM cells (5). CREB is a well known downstream effector of the cAMP-PKA signaling pathway and has been shown to exert negative regulation of VSM cell migration and proliferation (34,51). Negative regulation of CREB by CaMKII␦ as defined in the present study, and subsequent relief of its inhibitory effects could be another mechanism whereby CaMKII affects the phenotype of VSM and contributes to increased VSM cell proliferation and migration.
In summary, we have shown that activation of CaMKII␦ 2 in response to Ca 2ϩ mobilizing stimuli results in CREB-Ser 142 phosphorylation and inhibition of CREB activity and transcriptional responses in vascular smooth muscle. In this system, CaMKII has no effect on the activating CREB-Ser 133 phosphorylation event. For the first time we demonstrate a strong correlation between CREB-Ser 142 phosphorylation and nuclear export of CREB suggesting one potential mechanism whereby CaMKII negatively regulates CREB activity. Further studies are required to determine the mechanism of CaMKII-dependent nuclear export of CREB and function of the CaMKII␦ 2 -CREB pathway in VSM cell phenotype control and vascular remodeling contributing to occlusive vascular diseases.