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Previous Article | Next Article 
Molecular and Cellular Biology, October 2004, p. 8374-8385, Vol. 24, No. 19
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.19.8374-8385.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas,1 Myogen, Inc., Westminster, Colorado2
Received 17 May 2004/ Returned for modification 9 June 2004/ Accepted 6 July 2004
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ABSTRACT |
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INTRODUCTION |
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There are two classes of HDACs that can be distinguished by their structures and expression patterns. Class I HDACs (HDAC1, HDAC2, and HDAC3) are expressed ubiquitously and are composed mainly of a catalytic domain (13). In contrast, class II HDACs (HDAC4, HDAC5, HDAC7, and HDAC9) display more restricted expression patterns and contain an N-terminal extension, which mediates interactions with other transcriptional cofactors and confers responsiveness to calcium-dependent signaling (12, 25, 33). Signaling by calcium/calmodulin-dependent protein kinase (CaMK) results in phosphorylation of the N termini of class II HDACs, which govern their intracellular localization and interactions with other factors (29, 32). Phosphorylation of signal-responsive serine residues creates docking sites for the 14-3-3 family of chaperone proteins, which promote shuttling of HDACs from the nucleus to the cytoplasm in a CRM1-dependent fashion (14, 21, 30, 31, 48).
CaMK signaling to class II HDACs governs the activity of the myocyte enhancer factor-2 (MEF2) transcription factor, which plays central roles in the control of muscle-specific and stress-responsive gene expression (32). Class II HDACs interact with MEF2 through a short motif near their N termini; this interaction represses the expression of MEF2 target genes. Phosphorylation of class II HDACs, in response to CaMK signaling, results in their dissociation from MEF2 with consequent potentiation of MEF2 activity. Thus, class II HDACs provide a calcium-sensitive switch to control large sets of genes regulated by MEF2.
Recently, we reported that class II HDACs act as signal-responsive repressors of cardiac hypertrophy, which is triggered by calcium-sensitive signals (28, 49). Hypertrophy of cardiomyocytes is accompanied by an increase in cell size, assembly of sarcomeres, and activation of a fetal gene program (8, 27). We have shown that signal-resistant HDAC mutants block cardiomyocyte hypertrophy in response to diverse agonists and that mice lacking HDAC9 are sensitized to hypertrophic stimuli (6, 49). These findings suggest that HDAC phosphorylation is an essential step in coupling stress signals to the hypertrophic gene program. Induction of cardiac hypertrophy is accompanied by the posttranslational activation of MEF2, which is presumed to occur, at least in part, as a consequence of the dissociation and nuclear export of class II HDACs (38). CaMK can also promote skeletal myogenesis by relieving HDAC repression of MEF2 activity (26, 29).
Numerous signaling pathways have been implicated in cardiac hypertrophy (11, 27). Because of the critical role of HDAC phosphorylation in regulating myocyte differentiation and hypertrophy, there has been intense interest in identifying the kinase(s) responsible for class II HDAC nuclear export and inactivation. To further define the signaling pathways leading to the phosphorylation of class II HDACs, we examined the potential of multiple kinase pathways to stimulate HDAC5 nuclear export. Here we show that the protein kinase C (PKC) pathway promotes nuclear export of HDAC5 by stimulating phosphorylation of the 14-3-3 docking sites. Signal-resistant HDAC5 blocks cardiomyocyte hypertrophy stimulated by PKC activators. Conversely, PKC inhibition selectively blocks HDAC5 export and hypertrophy in response to a subset of hypertrophic agonists. In addition, PKCµ/protein kinase D (PKD), which acts as a downstream effector kinase of PKC, stimulates the nuclear export of HDAC5. These findings establish class II HDACs as critical effectors in the pathway whereby PKC signaling leads to cardiac hypertrophy.
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MATERIALS AND METHODS |
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or PKC
were provided by J. W. Son (Inha University, Inchon, Korea). A plasmid encoding constitutively active PKC
was provided by P. Ping (University of California Los Angels), and P. Goldspink (University of Illinois, Chicago, Ill.) and S. Shaw (National Cancer Institute, Bethesda, Md.) provided vectors for PKCßII and PKC
, respectively. Generation of phospho-HDAC-specific antibodies. Research Genetics, Huntsville, Ala., generated antibodies against human HDAC5 phosphorylated at serine 259. Rabbits were immunized with a keyhole limpet hemocyanin-conjugated peptide corresponding to amino acids 251 to 269 of HDAC5 and containing a phosphate group at position 259, as indicated by [p] in the following sequence: (DFPLRKTAS[p]EPNLKVRSRI). Phosphospecific antibodies were purified from crude rabbit serum by sequential negative and positive affinity purification with nonphosphorylated and phosphorylated peptide, respectively.
Cell culture and transfection assays.
COS cells were maintained in Dulbecco minimal essential medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin-streptomycin. COS cells were transfected with Fugene 6 (Roche Molecular Biochemicals) as specified by the manufacturer. For HDAC localization experiments, cells were treated 16 to 24 h after transfection with 100 nM PMA, 1 µM ionomycin, 1 µM 8-Br-cAMP, 1 µM pCPT-GMP, or 1 µM anisomycin. Where noted, specific protein kinase inhibitors were added 30 min prior to the addition of any chemical stimulus. Green fluorescent protein (GFP)-HDAC5 was visualized by standard fluorescent microscopic techniques. For indirect immunofluorescence of FLAG-HDAC5, COS cells were seeded on glass coverslips, transfected, and treated as above. After specific treatment, cells were fixed with 10% buffered formalin and exposed to FLAG M2 antibody (1:200 dilution; Sigma) in phosphate-buffered saline (PBS) containing 3% bovine serum albumin and 0.1% Nonidet P-40. Secondary fluorescein-conjugated antibody (Vector Laboratories) was also used at a concentration of 1:200. Cardiomyocytes were stained for sarcomeres and atrial natriuretic factor (ANF) by indirect immunofluorescence detection as above, with antibodies directed against sarcomeric -actinin (Sigma) and ANF (Peninsula Laboratories), respectively.
Cardiac myocyte culture and adenovirus infection. Neonatal rat cardiac myocytes (NRVMs) were isolated from 1- to 2-day-old Sprague-Dawley rats as previously described (2, 35). For adenovirus production, cDNAs encoding LacZ or FLAG-tagged HDAC5 containing alanines in place of serines 259 and 498 (S259/498A) were subcloned into the pACCMV vector and cotransfected with pJM17 into 293 cells. Primary lysates were used to reinfect 293 cells, and viral plaques were obtained using the agar overlay method. Complementary DNA for full-length human HDAC5 (encoding 1,122 amino acids) was fused to sequences encoding enhanced GFP (Clontech) in pcDNA3.1+ (Invitrogen). The resultant construct encodes GFP fused in frame to the amino terminus of HDAC5. A construct encoding GFP fused to HDAC5 (S259/498A) was generated in the same manner. GFP-HDAC5 cDNAs were subcloned into pACCMV for adenovirus production. Clonal populations of adenoviruses were amplified and subjected to titer determination.
Coimmunoprecipitation assays. FLAG-HDAC5 expression plasmid was transfected into COS cells treated as described above. Treated cells were harvested in 50 mM Tris (pH 7.4)-150 mM NaCl--1 mM EDTA—1% Triton X-100 and further disrupted by passage through a 22-gauge needle, and cell debris removed by centrifugation. FLAG-HDAC5 was immunoprecipitated with M2-agarose conjugate (Sigma) and thoroughly washed with lysis buffer. Bound proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis was performed using FLAG M2 (Sigma) or 14-3-3 antibody (Santa Cruz Biotechnology). For studies with NRVMs, whole-cell protein extracts were prepared from cells expressing GFP-HDAC5 by using the same buffer supplemented with protease inhibitor cocktail (Complete; Roche), 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM sodium fluoride, 10 mM ß-glycerol phosphate, 1 mM sodium molybdate, 1 mM sodium orthovanadate). Lysates were sonicated briefly and clarified by centrifugation. For immunoprecipitation, protein lysates were exposed to HDAC5-specific antiserum (29) and protein G-Sepharose beads (Amersham Biosciences). Immunoprecipitates were washed five times with lysis buffer, resolved by SDS-PAGE, and immunoblotted with mouse monoclonal antibodies specific for either GFP (BD Biosciences; 1:2,500 dilution) or 14-3-3 (Santa Cruz [H-8]; 1:1,000 dilution). Associated PKD was detected by immunoblotting with rabbit polyclonal antibodies against either PKD-1 or PKD-1 phosphorylated at serine 916 (Cell Signaling Technologies), employed at 1:1,000 dilutions.
In vitro kinase assays.
FLAG-HDAC5 was immunoprecipitated with anti-FLAG M2 antibody, as described above. Bound FLAG-HDAC5 was washed and equilibrated with kinase buffer (25 mM Tris [pH 7.4], 10 mM MgCl2, 1 mM dithiothreitol). Following equilibration, kinase reaction mix was added (kinase buffer plus 0.1 mM ATP and 50 µCi of [
-32P]-ATP). Kinase reactions were carried out at 30°C for 30 min and terminated by the addition of an equal volume of 2x SDS-PAGE loading buffer. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography.
GFP-HDAC5 localization studies.
For analysis of GFP-HDAC5 in NRVMs, cells were plated in the presence of adenovirus (multiplicity of infection, 
50 to 100) on gelatin-coated 96-well dishes (Costar; 104 cells/well) containing DMEM plus 10% FBS, 2 mM L-glutamine, and penicillin-streptomycin. After overnight culture, the cells were washed with serum-free medium and maintained in 100 µl of DMEM supplemented with 0.1% Neutridoma-SP (Roche Applied Science), which contains albumin, insulin, transferrin, and other defined organic and inorganic compounds. Following culture in serum-free medium (3 h), the cells were exposed to kinase inhibitors (for 30 min) prior to stimulation with the agonist for 2.5 h. The cells were washed with PBS and fixed with 10% formalin in PBS containing Hoechst dye 33342 (H-3570; Molecular Probes). Images were captured at a magnification of x40, using a fluorescence microscope (Nikon Eclipse TS100) equipped with a digital camera (Photometrics CoolSNAP HQ) and MetaMorph imaging software. The relative abundance of GFP-HDAC5 in the nucleus versus the cytoplasm was quantified by using the high-content imaging system (Cellomics, Inc., Pittsburgh, Pa.), which demarcates nuclei based on Hoechst fluorescence and defines a cytoplasmic ring based on these nuclear dimensions. Values for HDAC5 localization represent averages from a minimum of 400 randomly captured cells per experimental condition.
RNA analysis.
NRVMs were plated on gelatin-coated 10-cm dishes (2 x 106 cells/dish). Following the indicated treatments, RNA was isolated from cardiomyocytes by using Trizol reagent (Gibco/BRL). Total RNA (2 µg) was vacuum blotted onto nitrocellulose membranes (Bio-Rad) by using a 96-well format dot blotter (Bio-Rad). Membranes were blocked in 4x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 1% SDS, 5x Denhardt's reagent, 0.05% sodium pyrophosphate, and 100 µg of sonicated salmon sperm DNA per ml (4 h at 50°C) and incubated with 32P-end-labeled oligonucleotide probes (106 cpm/ml; 14 h at 50°C). The sequences of the oligonucleotides were as follows: ANF, 5'-AATGTGACCAAGCTGCGTGACACACCACAAGGGCTTAGGATCTTTTGCGATCTGCTCAAG-3; brain natriuretic protein (BNP), 5'-TGAACTATGTGCCATCTTGGAATTTCGAAGTCTCTCCT-3'; -SK-actin 5'-TGGAGCAAAACAGAATGGCTGGCTTTAATGCTTCAAGTTTTCCATTTCCTTTCCACAGGG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GGAACATGTAGACCATGTAGTTGAGGTCAATGAAG-3'. Blots were washed twice with 0.5x SSC containing 0.1% SDS (10 min at 50°C) and analyzed by autoradiography.
Mammalian two-hybrid analysis. A mammalian expression vector encoding the GAL4 DNA binding domain fused to the amino terminus of human HDAC5 (amino acids 2 to 664) was generated in the pM1 expression vector (42). GAL4-HDAC5 fusions harboring alanine in place of serine 259 and/or 498 were constructed in an analogous manner. A construct encoding the herpesvirus VP16 transcriptional activation domain fused to the amino terminus of 14-3-3 sigma was generated by employing pVP16 (Clontech). COS cells were transiently transfected with vectors for GAL4-HDAC5, VP16-14-3-3, and a luciferase reporter gene under the control of five copies of a GAL4 DNA binding site (5XUAS-luciferase) in the absence or presence of a construct for constitutively active PKD-1. At 48 h posttransfection, the cells were harvested and luciferase levels were quantified by using the luciferase assay kit (Promega).
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RESULTS |
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Phosphorylation of serines 259 and 498 in HDAC5 creates docking sites for 14-3-3 proteins, which escort HDAC5 to the cytoplasm (14, 30). To further confirm that PMA promoted the phosphorylation of these sites, we analyzed the interaction of HDAC5 with 14-3-3 in coimmunoprecipitation assays. As shown in Fig. 1D, the association of 14-3-3 with HDAC5 was enhanced in the presence of PMA. In contrast, HDAC5 S259/498A failed to respond to PMA and did not associate with 14-3-3. We conclude that PKC signaling leads to the phosphorylation of serines 259 and 498 of HDAC5 and consequent nuclear export through a 14-3-3-dependent mechanism.
Since non-PKC targets of PMA have been identified, we used a panel of expression vectors encoding distinct PKCs to confirm that PKC signaling is sufficient to trigger nuclear export of HDAC5. As shown in Fig. 2A, overexpression of the calcium-dependent PKC and PKCß had no effect on the subcellular localization of HDAC5. In contrast, calcium-independent forms of PKC, PKC, PKC, and PKC, each triggered nuclear export of HDAC5, albeit to different degrees. In these experiments with CV-1 cells, PKC was the most efficacious HDAC5 export kinase. PKC-mediated nuclear export of HDAC5 was dependent on phosphorylation of the 14-3-3 target sites at serines 259 and 498 and was blocked by the CRM1 antagonist, leptomycin B (Fig. 2B).
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1-adrenergic agonist PE, a hypertrophic agonist that promotes nuclear export of HDAC5 (6). In unstimulated cells, 65 to 80% of HDAC5 was found in the nucleus, reflecting a low level of basal nuclear export of the protein. Conversely, HDAC5 S259/498A was found exclusively in the nuclear compartment. As shown in Fig. 3B, PE triggered the nuclear export of HDAC5 in a concentration-dependent manner. HDAC5 S259/498A was resistant to PE-mediated nuclear export (Fig. 3D and data not shown). The results of Cellomics analyses were confirmed by visual inspection of the cells (Fig. 3D).
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PKC signaling induces cardiac hypertrophy via HDAC phosphorylation. PKC activation is sufficient, and in some cases necessary, for cardiomyocyte hypertrophy (see Discussion). The above results implicate PKC-dependent nuclear export of HDAC5 or other class II HDACs in the development of cardiomyocyte hypertrophy. To address this possibility, we examined whether hypertrophy in response to PKC activation required phosphorylation and nuclear export of class II HDACs. NRVMs were infected with adenoviruses encoding the signal-resistant HDAC5 S259/498A mutant protein or LacZ as a control. As shown in Fig. 4A, expression of the HDAC5 S259/498A mutant in primary cardiomyocytes prevented sarcomere assembly and cell enlargement in response to PE or PMA.
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-skeletal actin. Agonist-dependent elevation of ANF expression can be examined by immunostaining cardiomyocytes with ANF-specific antibodies. As shown in Fig. 4B, prominent perinuclear ANF protein expression was observed in NRVMs treated with PE or PMA. Agonist-dependent induction of ANF expression was unaffected by ectopic expression of LacZ but was markedly reduced in the presence of signal-resistant HDAC5. In addition, nonphosphorylatable HDAC5 blocked PE- and PMA-mediated induction of ANF transcripts, as well as those for BNP and -skeletal actin (Fig. 4C). Together, these results suggest that PKC signaling triggers cardiac hypertrophy in part by stimulating the nuclear export of class II HDACs. Differential sensitivity of HDAC5 nuclear export to PKC inhibitors. We next examined whether nuclear export of HDAC5 in response to other hypertrophic signals was also dependent on PKC signaling. ET-1 and FBS, which stimulate hypertrophy, also effectively promote nuclear export of HDAC5 in cardiac myocytes (Fig. 5A) (B. C. Harrison and T. A. McKinsey, submitted for publication). However, in contrast to its inhibitory effect on PE-dependent HDAC5 nuclear export, Bis I had no effect on the nuclear export of HDAC5 in response to ET-1 or FBS (Fig. 5B). These findings suggested that PE triggers different kinase pathways than ET-1 and FBS to promote the nuclear export of HDAC5.
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and PKCß isozymes, efficiently blocked the nuclear export of HDAC5 triggered by PE, ET-1 or FBS (Fig. 5D and E). The differential effects of the above inhibitors on HDAC5 nuclear export were paralleled by their effects on the association of 14-3-3 with HDAC5, an indicator of HDAC5 phosphorylation. Gö6976, but not Bis I, blocked the association of HDAC5 and 14-3-3 in response to both PE and ET-1 (Fig. 5F).
The ability of Gö6976, but not Bis I or Gö6983, to block HDAC5 nuclear export in response to multiple agonists was seemingly paradoxical, since the last two compounds block PKC and PKCß as effectively as Gö6976 does. However, this inhibitor profile was similar to that used by others to distinguish the actions of PKC or PKCß from PKD/PKCµ (51), which is sensitive to Gö6976 but not to Bis I or Gö6983 (Table 1) (15).
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To further explore the potential role of PKD as an HDAC5 nuclear export kinase, we performed coimmunoprecipitation and in vitro kinase assays. Coimmunoprecipitation of HDAC5 and PKD followed by an in vitro kinase assay confirmed that PKD directly phosphorylated HDAC5. As shown in Fig. 7A, cotransfection of wild-type PKD resulted in little phosphorylation of HDAC5, and treatment of cells with PMA increased the degree of HDAC5 phosphorylation coincident with PKD binding. Binding and phosphorylation of HDAC5 by activated PKD S/E did not require PMA, although PMA treatment enhanced the phosphorylation of HDAC5, perhaps owing to the presence of endogenous PKD in immune complexes. No phosphorylation of HDAC5 was observed with the catalytically inactive mutant PKD K/W. Interestingly, however, PKD K/W bound to HDAC5 even in the absence of PMA.
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Finally, we assessed the ability of PKD to phosphorylate HDAC5 in cells by employing an antibody that specifically recognizes HDAC5 that has been phosphorylated at serine 259. HDAC5 was basally phosphorylated at serine 259, and phosphorylation at this site was dramatically increased by coexpression of constitutively active PKD S/E (Fig. 7C). HDAC5 S259/498A was not recognized by the phosphoantibody, establishing the specificity of the reagent. The L254/493G substitution abolished basal phosphorylation at serine 259 and reduced PKD-directed phosphorylation of this site. We predict that this level of HDAC5 L254/493G phosphorylation is insufficient to trigger HDAC5 nuclear export. In addition, it should be noted that the antibody does not detect phosphorylation of serine 498 in HDAC5, which is also required for efficient nuclear export of the protein. Together, these data suggest that PKD is capable of directly phosphorylating HDAC5.
PKD is a cardiac HDAC5 kinase. We next examined whether PKD could serve as an HDAC kinase in cardiomyocytes. Cells were infected with adenovirus encoding FLAG-HDAC5 and treated with PMA. Increased HDAC5 phosphorylation was observed in an in vitro kinase assay performed with FLAG-HDAC5 immunoprecipitated from PMA-treated cells (Fig. 8A). Incubation of the cells with Bis I before the addition of PMA blocked the phosphorylation of HDAC5. However, addition of Bis I directly to the kinase reaction mixture had no effect, while Gö6976 blocked the phosphorylation of HDAC5. These results suggest that PKD binds HDAC5 in cardiac myocytes and that Bis I blocks the PMA-induced activation of the kinase, while Gö6976 is able to directly inhibit HDAC5-bound PKD.
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DISCUSSION |
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Convergence of multiple protein kinases on class II HDACs. The results of this study show that PKC signaling leads to the phosphorylation of the same sites in HDAC5 that were previously shown to be phosphorylated by CaMK (29). Thus, the PKC and CaMK pathways converge at the same regulatory sites on HDAC5 (and presumably other class II HDACs). Both CaMK and PKC have been implicated in cardiac hypertrophy. Cardiac-specific expression of CaMK induces hypertrophy, at least in part through direct phosphorylation of class II HDACs, leading to their dissociation from and subsequent activation of MEF2 (25, 38). However, there is also evidence suggesting that kinases other than CaMK can serve as HDAC kinases. We have observed, for example, an HDAC kinase activity from cardiac extracts that is enhanced by hypertrophic stimuli but is not inhibited by inhibitors of CaMK or a variety of other kinases (49). In addition, agonist-dependent nuclear export of HDAC5 in cardiac myocytes is resistant to the CaMK inhibitors KN93 (Fig. 3C) and KN62 (data not shown).
While CaMK and some isoforms of PKC are calcium dependent, they each display distinct cofactor requirements and calcium requirements, as well as different subcellular distributions (17, 43). Thus, some hypertrophic stimuli may selectively activate one or the other of these kinases while others could activate both. In addition, it is possible that CaMK cross talks with the PKC-PKD pathway.
PKC and cardiac hypertrophy.
The PKC family includes at least 12 different isoforms, many but not all of which are expressed at appreciable levels in the myocardium. PKC family members can be subdivided based on their activation profiles. Conventional PKCs including PKC, PKCßI, PKCßII, and PKC require calcium in addition to phospholipids for activation, while the atypical PKCs including PKC, PKC, PKC
, and PKC are calcium independent.
Numerous studies have implicated PKC signaling in cardiac hypertrophy. PMA activates the fetal gene program and increases cardiomyocyte size (1, 10). PKC, the most predominant PKC isoform in the heart, is sufficient to promote cardiac hypertrophy when overexpressed in neonatal rat cardiac myocytes (4). PKC activation has also been shown to precede hypertrophy in a mouse model of L-type voltage-dependent calcium channel overexpression (36). Recently, it was shown that cardiac function was maintained in PKC-null mice following aortic banding, a surgical model of pressure overload induced heart failure (5). Cardiac-specific overexpression of PKCß in mice also leads to cardiac hypertrophy and sudden death (3), although genetic ablation of PKCß does not abolish hypertrophy in response to pressure overload or PE infusion (41). In addition, a peptide modulator of PKC which leads to membrane translocation and activation of PKC elicits cardiac hypertrophy in vivo while maintaining normal heart function (34).
Our results demonstrate that direct activation of PKC by PMA is sufficient to induce the nuclear export of HDAC5 and that hypertrophic agonists such as PE stimulate the nuclear export of HDAC5 in cardiac myocytes through a signaling pathway that depends on PKC activation. Signal-resistant forms of HDAC5 are also capable of blocking hypertrophy caused by PE and PMA. These findings demonstrate that HDAC5 is a target for PKC signaling in cardiomyocytes and demonstrate that the nuclear export of HDAC5 or other class II HDACs by PKC is a critical step in the signaling pathway leading to cardiac hypertrophy.
PKD regulation of class II HDACs during cardiac hypertrophy. We also show here that PKD can serve as an HDAC kinase. The amino acid sequence surrounding the signal-responsive serines of HDAC5 conforms to the PKD consensus phosphorylation site (29, 37). PKD is activated through direct phosphorylation by PKC on two serine residues in its activation loop (18). PKD activation can be inhibited by Bis I or Gö6983, which block PKC activity (18, 24). Calcium-independent PKCs have been most strongly implicated in the control of PKD (39). Thus, our demonstration that calcium-independent, but not calcium-dependent, PKCs trigger the nuclear export of HDAC5 (Fig. 2) further suggests a role for PKD in the regulation of HDAC5. According to the model (Fig. 9), PKD-directed phosphorylation of HDAC5 occurs in the nucleus. While we have not established this to be the case in the present study, it should be noted that PKD has previously been shown to translocate to the nucleus in response to G-protein-coupled receptor signaling (40).
Interestingly, HDAC5 nuclear export mediated by ET-1 or FBS is not blocked by Bis I or Gö6983 but is suppressed by Gö6976, a direct inhibitor of PKD. This suggests that there may be PKC-independent pathways stimulated by ET-1 and FBS that are responsible for PKD activation and HDAC5 export (Fig. 9). In this regard, PKD is also activated by direct binding of the ß subunits of heterotrimeric G proteins to the pleckstrin homology domain (19). In addition, tyrosine phosphorylation of the pleckstrin homology domain by the Abl kinase precedes the phosphorylation of PKD by PKC in response to oxidative stress (44). PKC-independent activation of PKD was recently demonstrated in osteoblasts, although the alternative mechanism was not revealed (22).
Little is known about the role of PKD in cardiac hypertrophy. PKD is activated by PMA and PE in a Bis I-sensitive manner in NRVMs (16). Aldosterone also activates PKD in cardiomyocytes in culture and in mice (46). Furthermore, Gö6976 inhibits fetal gene expression in cardiomyocytes activated by aldosterone. In none of these cases were the intracellular targets of PKD responsible for hypertrophy identified. Our results demonstrate that PKD can serve as an HDAC kinase and suggest that PKD regulates cardiac hypertrophy, at least in part through phosphorylation and subsequent nuclear export of class II HDACs. However, this does not rule out the possible involvement of other kinases, including CaMK, in the regulation of the HDAC subcellular distribution and hypertrophy. It is interesting that although PKD is also referred to as PKCµ, its catalytic domain is most homologous to that of CaMK and is only distantly related to the catalytic domains of PKCs (24).
Implications for other cell types. Given the expression of class II HDACs in a variety of cell types and the importance of calcium signaling in modulating cellular phenotypes, it is intriguing to speculate that class II HDACs have evolved to use the same regulatory phosphorylation sites to respond to different physiological stimuli and regulate distinct sets of genes in different cell types. In this regard, HDAC7 was recently shown to undergo nuclear export in T lymphocytes in response to PMA (9). Similarly, HDAC4 and HDAC5 are exported from the nucleus in hippocampal neurons in response to spontaneous activity and HDAC5 is similarly controlled in cerebellar granule neurons subjected to depolarizing potassium (7, 23). Although CaMK inhibition disrupts HDAC5 nuclear export in these cells, it has little effect on HDAC4 export, suggesting that CaMK-independent mechanisms regulate class II HDAC localization in neurons.
PKC signaling influences a wide range of cellular processes, including proliferation, differentiation, and oncogenesis. The specific response to PKC activation is dependent on cell identity and on other intracellular signals. In a postnatal cardiomyocyte, PKC signaling can evoke hypertrophy, while in other cell types it may control other cellular responses. The results of this study reveal a specific mechanism whereby PKC and PKD signaling can be coupled to transcriptional machinery via class II HDACs. Because these HDACs interact with a variety of positive and negative transcriptional regulators, they provide the potential to convey "generic" upstream signals from the cell membrane to cell-type-specific target genes. Pharmacological modulation of the upstream kinases and phosphatases that ultimately govern the phosphorylation of class II HDACs offers interesting possibilities for modulation of cellular phenotypes during development and disease.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the NIH and the Donald W. Reynolds Center for Clinical Cardiovascular Research to E.N.O. and from the American Heart Association to R.V.
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Antos, C. L., T. A. McKinsey, M. Dreitz, L. M. Hollingsworth, C. L. Zhang, K. Schreiber, H. Rindt, R. J. Gorczynski, and E. N. Olson. 2003. Dose-dependent blockade to cardiomyocyte hypertrophy by histone deacetylase inhibitors. J. Biol. Chem. 278:28930-28937.
3. Bowman, J. C., S. F. Steinberg, T. Jiang, D. L. Geenen, G. I. Fishman, and P. M. Buttrick. 1997. Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates. J. Clin. Investig. 100:2189-2195.[Medline]
4. Braz, J. C., O. F. Bueno, L. J. De Windt, and J. D. Molkentin. 2002. PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2). J. Cell Biol. 156:905-919.
5. Braz, J. C., K. Gregory, A. Pathak, W. Zhao, B. Sahin, R. Klevitsky, T. F. Kimball, J. N. Lorenz, A. C. Nairn, S. B. Liggett, I. Bodi, S. Wang, A. Schwartz, E. G. Lakatta, A. A. DePaoli-Roach, J. Robbins, T. E. Hewett, J. A. Bibb, M. V. Westfall, E. G. Kranias, and J. D. Molkentin. 2004. PKC-alpha regulates cardiac contractility and propensity toward heart failure. Nat. Med. 10:248-254.[CrossRef][Medline]
6. Bush, E., J. Fielitz, L. Melvin, M. Martinez-Arnold, T. A. McKinsey, R. Plichta, and E. N. Olson. 2004. A small molecular activator of cardiac hypertrophy uncovered in a chemical screen for modifiers of the calcineurin signaling pathway. Proc. Natl. Acad. Sci. USA 101:2870-2875.
7. Chawla, S., P. Vanhoutte, F. J. Arnold, C. L. Huang, and H. Bading. 2003. Neuronal activity-dependent nucleocytoplasmic shuttling of HDAC4 and HDAC5. J. Neurochem. 85:151-159.[Medline]
8. Chien, K. R. 1999. Stress pathways and heart failure. Cell 98:555-558.[CrossRef][Medline]
9. Dequiedt, F., H. Kasler, W. Fischle, V. Kiermer, M. Weinstein, B. G. Herndier, and E. Verdin. 2003. HDAC7, a thymus-specific class II histone deacetylase, regulates Nur77 transcription and TCR-mediated apoptosis. Immunity 18:687-698.[CrossRef][Medline]
10. Dunnmon, P. M., K. Iwaki, S. A. Henderson, A. Sen, and K. R. Chien. 1990. Phorbol esters induce immediate-early genes and activate cardiac gene transcription in neonatal rat myocardial cells. J. Mol. Cell. Cardiol. 22:901-910.[CrossRef][Medline]
11. Frey, N., and E. N. Olson. 2003. Cardiac hypertrophy: the good, the bad, and the ugly. Annu. Rev. Physiol. 65:45-79.[CrossRef][Medline]
12. Grozinger, C. M., C. A. Hassig, and S. L. Schreiber. 1999. Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc. Natl. Acad. Sci. USA 96:4868-4873.
13. Grozinger, C. M., and S. L. Schreiber. 2002. Deacetylase enzymes: biological functions and the use of small-molecule inhibitors. Chem. Biol. 9:3-16.[CrossRef][Medline]
14. Grozinger, C. M., and S. L. Schreiber. 2000. Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-3-3-dependent cellular localization. Proc. Natl. Acad. Sci. USA 97:7835-7840.
15. Gschwendt, M., S. Dieterich, J. Rennecke, W. Kittstein, H. J. Mueller, and F. J. Johannes. 1996. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase C isoenzymes. FEBS Lett. 392:77-80.[CrossRef][Medline]
16. Haworth, R. S., M. W. Goss, E. Rozengurt, and M. Avkiran. 2000. Expression and activity of protein kinase D/protein kinase C mu in myocardium: evidence for alpha1-adrenergic receptor- and protein kinase C-mediated regulation. J. Mol. Cell. Cardiol. 32:1013-1023.[CrossRef][Medline]
17. Hook, S. S., and A. R. Means. 2001. Ca2+/CaM-dependent kinases: from activation to function. Annu. Rev. Pharmacol. Toxicol. 41:471-505.[CrossRef][Medline]
18. Iglesias, T., R. T. Waldron, and E. Rozengurt. 1998. Identification of in vivo phosphorylation sites required for protein kinase D activation. J. Biol. Chem. 273:27662-27667.
19. Jamora, C., N. Yamanouye, J. Van Lint, J. Laudenslager, J. R. Vandenheede, D. J. Faulkner, and V. Malhotra. 1999. Gbetagamma-mediated regulation of Golgi organization is through the direct activation of protein kinase D. Cell 98:59-68.[CrossRef][Medline]
20. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science 293:1074-1080.
21. Kao, H. Y., A. Verdel, C. C. Tsai, C. Simon, H. Juguilon, and S. Khochbin. 2001. Mechanism for nucleocytoplasmic shuttling of histone deacetylase 7. J. Biol. Chem. 276:47496-47507.
22. Lemonnier, J., C. Ghayor, J. Guicheux, and J. Caverzasio. 2004. Protein kinase C-independent activation of protein kinase D is involved in BMP-2- induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation. J. Biol. Chem. 279:259-264.
23. Linseman, D. A., C. M. Bartley, S. S. Le, T. A. Laessig, R. J. Bouchard, M. K. Meintzer, M. Li, and K. A. Heidenreich. 2003. Inactivation of the myocyte enhancer factor-2 repressor histone deacetylase-5 by endogenous Ca2+/calmodulin-dependent kinase II promotes depolarization-mediated cerebellar granule neuron survival. J. Biol. Chem. 278:41472-41481.
24. Lint, J. V., A. Rykx, T. Vantus, and J. R. Vandenheede. 2002. Getting to know protein kinase D. Int. J. Biochem. Cell Biol. 34:577-581.[CrossRef][Medline]
25. Lu, J., T. A. McKinsey, R. L. Nicol, and E. N. Olson. 2000. Signal-dependent activation of the MEF2 transcription factor by dissociation from histone deacetylases. Proc. Natl. Acad. Sci. USA 97:4070-4075.
26. Lu, J., T. A. McKinsey, C. L. Zhang, and E. N. Olson. 2000. Regulation of skeletal myogenesis by association of the MEF2 transcription factor with class II histone deacetylases. Mol. Cell 6:233-244.[CrossRef][Medline]
27. MacLellan, W. R., and M. D. Schneider. 2000. Genetic dissection of cardiac growth control pathways. Annu. Rev. Physiol. 62:289-319.[CrossRef][Medline]
28. McKinsey, T. A., and E. N. Olson. 2004. Cardiac histone acetylation—therapeutic opportunities abound. Trends Genet. 20:206-213.[CrossRef][Medline]
29. McKinsey, T. A., C. L. Zhang, J. Lu, and E. N. Olson. 2000. Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408:106-111.[CrossRef][Medline]
30. McKinsey, T. A., C. L. Zhang, and E. N. Olson. 2000. Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5. Proc. Natl. Acad. Sci. USA 97:14400-14405.
31. McKinsey, T. A., C. L. Zhang, and E. N. Olson. 2001. Identification of a signal-responsive nuclear export sequence in class II histone deacetylases. Mol. Cell. Biol. 21:6312-6321.
32. McKinsey, T. A., C. L. Zhang, and E. N. Olson. 2002. MEF2: a calcium- dependent regulator of cell division, differentiation and death. Trends Biochem. Sci. 27:40-47.[CrossRef][Medline]
33. Miska, E. A., C. Karlsson, E. Langley, S. J. Nielsen, J. Pines, and T. Kouzarides. 1999. HDAC4 deacetylase associates with and represses the MEF2 transcription factor. EMBO J. 18:5099-5107.[CrossRef][Medline]
34. Mochly-Rosen, D., G. Wu, H. Hahn, H. Osinska, T. Liron, J. N. Lorenz, A. Yatani, J. Robbins, and G. W. Dorn, Jr. 2000. Cardiotrophic effects of protein kinase C epsilon: analysis by in vivo modulation of PKCepsilon translocation. Circ. Res. 86:1173-1179.
35. Molkentin, J. D., J. R. Lu, C. L. Antos, B. Markham, J. Richardson, J. Robbins, S. R. Grant, and E. N. Olson. 1998. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93:215-228.[CrossRef][Medline]
36. Muth, J. N., I. Bodi, W. Lewis, G. Varadi, and A. Schwartz. 2001. A Ca2+-dependent transgenic model of cardiac hypertrophy: a role for protein kinase Calpha. Circulation 103:140-147.
37. Nishikawa, K., A. Toker, F. J. Johannes, Z. Songyang, and L. C. Cantley. 1997. Determination of the specific substrate sequence motifs of protein kinase C isozymes. J. Biol. Chem. 272:952-960.
38. Passier, R., H. Zeng, N. Frey, F. J. Naya, R. L. Nicol, T. A. McKinsey, P. Overbeek, J. A. Richardson, S. R. Grant, and E. N. Olson. 2000. CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo. J. Clin. Investig. 105:1395-1406.[Medline]
39. Rey, O., J. R. Reeve, E. Zhukova, J. Sinnett-Smith, and E. Rozengurt. 2004. G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase c epsilon. J. Biol. Chem. 279:34361-34372.
40. Rey, O., E. Zhukova, J. Sinnett-Smith, and E. Rozengurt. 2003. Vasopressin- induced intracellular redistribution of protein kinase D in intestinal epithelial cells. J. Cell. Physiol. 196:483-492.[CrossRef][Medline]
41. Roman, B. B., D. L. Geenen, M. Leitges, and P. M. Buttrick. 2001. PKC-beta is not necessary for cardiac hypertrophy. Am. J. Physiol. Ser. H 280:H2264-H2270.
42. Sadowski, I., B. Bell, P. Broad, and M. Hollis. 1992. GAL4 fusion vectors for expression in yeast or mammalian cells. Gene 118:137-141.[CrossRef][Medline]
43. Shirai, Y., and N. Saito. 2002. Activation mechanisms of protein kinase C: maturation, catalytic activation, and targeting. J. Biochem. (Tokyo). 132:663-668.
44. Storz, P., H. Doppler, and A. Toker. 2004. Protein kinase C selectively regulates protein kinase D-dependent activation of NF-
B in oxidative stress signaling. Mol. Cell. Biol. 24:2614-2626.
45. Storz, P., and A. Toker. 2003. Protein kinase D mediates a stress-induced NF- kappaB activation and survival pathway. EMBO J. 22:109-120.[CrossRef][Medline]
46. Tsybouleva, N., L. Zhang, S. Chen, R. Patel, S. Lutucuta, S. Nemoto, G. DeFreitas, M. Entman, B. A. Carabello, R. Roberts, and A. J. Marian. 2004. Aldosterone, through novel signaling proteins, is a fundamental molecular bridge between the genetic defect and the cardiac phenotype of hypertrophic cardiomyopathy. Circulation 109:1284-1291.
47. Wang, L., M. Rolfe, and C. G. Proud. 2003. Ca2+-independent protein kinase C activity is required for alpha1-adrenergic-receptor-mediated regulation of ribosomal protein S6 kinases in adult cardiomyocytes. Biochem. J. 373:603-611.[CrossRef][Medline]
48. Wang, A. H., and X. J. Yang. 2001. Histone deacetylase 4 possesses intrinsic nuclear import and export signals. Mol. Cell. Biol. 21:5992-6005.
49. Zhang, C. L., T. A. McKinsey, S. Chang, C. L. Antos, J. A. Hill, and E. N. Olson. 2002. Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy. Cell 110:479-488.[CrossRef][Medline]
50. Zhang, L., S. Maier, N. D. Dalton, S. Miyamoto, J. Ross, Jr., D. M. Bers, and J. H. Brown. 2003. The deltaC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ. Res. 92:912-919.
51. Zugaza, J. L., J. Sinnett-Smith, J. Van Lint, and E. Rozengurt. 1996. Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway. EMBO J. 15:6220-6230.[Medline]
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