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Parathyroid Hormone Regulates Histone Deacetylase (HDAC) 4 through Protein Kinase A-mediated Phosphorylation and Dephosphorylation in Osteoblastic Cells *

Parathyroid Hormone Regulates Histone Deacetylase (HDAC) 4 through Protein Kinase A-mediated... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 31, pp. 21340 –21350, August 1, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Parathyroid Hormone Regulates Histone Deacetylase (HDAC) 4 through Protein Kinase A-mediated Phosphorylation and Dephosphorylation in Osteoblastic Cells Received for publication, January 17, 2014, and in revised form, May 23, 2014 Published, JBC Papers in Press, June 5, 2014, DOI 10.1074/jbc.M114.550699 ‡§ ‡ ‡ ‡1 Emi Shimizu , Teruyo Nakatani , Zhiming He , and Nicola C. Partridge ‡ § From the Departments of Basic Science and Craniofacial Biology and Endodontics, New York University College of Dentistry, New York, New York 10010 Background: PTH regulates HDAC4 to control its dissociation from Runx2. Results: PTH regulates phosphorylation and dephosphorylation of HDAC4 through PKA and partial degradation of HDAC4. Conclusion: PTH controls MMP-13 transcription through PKA-dependent phosphorylation and dephosphorylation of HDAC4. Significance: The results provide insight into how PTH participates in modification of HDAC4, which is crucial for under- standing hormonal regulation of gene expression. Histone deacetylases (HDACs) are crucial regulators of gene chromatin remodeling, and post-translational modifications of expression in transcriptional co-repressor complexes. Previ- histones, which includes the dynamic acetylation and deacety- ously, we reported that HDAC4 was a basal repressor of matrix lation of epsilon-amino groups of lysine residues present in the metalloproteinase-13 (MMP-13) transcription and parathyroid tails of core histones. Histone deacetylases (HDACs) are cru- hormone (PTH) regulates HDAC4 to control MMP-13 pro- cial regulators of gene expression in transcriptional co-repres- moter activity through dissociation from Runx2. Here, we show sor complexes and control gene expression important for that PTH induces the protein kinase A (PKA)-dependent phos- diverse cellular functions. The class I HDACs (HDAC1, -2, -3, phorylation of HDAC4 in the nucleus of the rat osteoblastic cell and -8) have homology to the yeast global transcriptional reg- line, UMR 106–01. We demonstrate that PKA-dependent ulator Rpd3 and are widely expressed. In contrast, the class II phosphorylated HDAC4 is released from Runx2 bound to the HDACs (HDAC4, -5, -6, -7, -9, and -10) show homology to MMP-13 promoter in these cells. Point mutation of Ser-740 in yeast Hda1 and are expressed in cell type-restricted patterns. rHDAC4 prevents the release of HDAC4 from Runx2 on the Among class II HDACs, HDAC4, -5, -7, and -9 form a subclass MMP-13 promoter and also prevents the PTH stimulation of known as class IIa, whereas HDAC6 and 10 constitute class IIb. MMP-13 transcription. Thus, PTH-induced phosphorylation of The class IIa histone deacetylases can be expressed in a tissue- rHDAC4 at Ser-740 is crucial for regulating MMP-13 transcrip- specific fashion, and are regulated by nuclear-cytoplasmic shut- tion in osteoblasts. PTH causes degradation of HDAC4, and this tling (1). product appears in the cytoplasm. The cytoplasmic degradation HDAC3 binds class II HDACs (2, 3) and form large multi- of HDAC4 is blocked by PKA and lysosomal inhibitors, but is protein complexes. These multi-protein complexes are not affected by proteasome, caspase-3, or serine and aspartic recruited to specific DNA sequences and chromatin substrates protease inhibitors. In addition, the phosphatase inhibitor, oka- by transcription factors (4). Genetic deletion of HDAC3 and daic acid, prevents degradation indicating that dephosphory- HDAC4 revealed the roles of these proteins in chondrocyte lation is associated with degradation. These mechanisms regu- maturation (5). Several class II HDACs appear to have a role in lating HDAC4 and their roles in such processes are crucial for skeletal formation (6). Notably, Hdac4-null mice display pre- bone and chondrocyte development. Our data support a link mature ossification of developing bones due to constitutive between PTH regulating HDAC4 phosphorylation by PKA, traf- Runx2 expression. Thus, HDAC4 regulates chondrocyte hyper- ficking, partial degradation, and the control of MMP-13 tran- trophy and endochondral bone formation by inhibiting the scription through association with Runx2. activity of Runx2 (7). In osteoblasts, HDAC4 and HDAC5 par- ticipate in TGF signaling pathways that suppress Runx2 activ- ity (8). Moreover, HDAC4 and HDAC5 deacetylate Runx2 and lead to Smurf-mediated degradation of Runx2 (9). Thus, Epigenetic modification plays an important role in the con- HDAC4 is considered an important target for osteoblast or trol of cell fate during mammalian development. Among these, chondrocyte differentiation. the most studied so far is DNA methylation, ATP-dependent Dephosphorylation by phosphatases and phosphorylation by serine/threonine kinases controls nuclear import and export * This work was supported, in whole or in part, by National Institutes of Health of HDAC4 and influences HDAC4 repressive activity (10). Grant DK47420 (to N. C. P.). To whom correspondence should be addressed: Department of Basic Sci- ence and Craniofacial Biology, New York University College of Dentistry, The abbreviations used are: HDAC, histone deacetylase; PTH, parathyroid th 345 East 24 St, New York, New York 10010. Tel.: 212-992-7145; Fax: 212- hormone; PTHrP, parathyroid hormone-related peptide; MEF, myocyte 995-4204; E-mail: [email protected]. enhancer factor; CIP, calf intestinal alkaline phosphatase. This is an Open Access article under the CC BY license. 21340 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation 14-3-3 proteins shuttle class II HDACs to the cytoplasm (11). mine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum. These proteins have a phosphoserine/threonine-binding motif Antibodies—Anti-HDAC4 (against 10 N-terminal amino through which they interact with phosphorylated HDACs (12). Subcellular localization of HDAC4, HDAC5, and HDAC7 is acids), anti-GFP, and anti--actin were purchased from Cell regulated by calcium-calmodulin-dependent kinase IV (CaMK Signaling Technology. Anti-HDAC4 (H92, against amino acids 530–631), anti-Runx2 (M-70), anti-Cdk2 (M2), and anti-tubu- IV) (13) or PKD phosphorylation and association with 14-3-3 lin (TU-2) were purchased from Santa Cruz Biotechnology. proteins for export to the cytoplasm (14, 15). Backs et al. (16, Western Blot—UMR 106-01 cells were treated with or with- 17) showed that CaMK II signals specifically to HDAC4 but not out rat PTH (1–34, 10 M) for the indicated times. The cells HDAC5 by binding to a unique kinase-docking site contained were washed twice in PBS, pH 7.4 and pelleted by centrifuga- in HDAC4. HDAC4 can subsequently re-enter the nucleus after tion at 2000 rpm for 5 min at 4 °C. The pellets were resuspended dephosphorylation and dissociation from 14-3-3 (11). in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Parathyroid hormone (PTH) is an 84-amino acid peptide PMSF, 1 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, and hormone, which functions as an essential regulator of calcium protease inhibitors) and incubated for 15 min at 4 °C. Amounts homeostasis and as a mediator of bone remodeling (18). PTH of total protein were determined by the Bradford dye binding acts via the PTH/PTH-related protein 1 receptor (a G protein- (Bio-Rad) method. The preparation of cytoplasmic and nuclear coupled receptor) on osteoblast membranes (19), and both its extracts from cells was by the NE-PER nuclear and cytoplasmic anabolic and catabolic effects on bone appear to be primarily extraction reagents (Thermo Scientific). To examine the inter- mediated by the cAMP/PKA pathways (20). Parathyroid hor- action between HDAC4 and Runx2 using immunoprecipita- mone-related peptide (PTHrP) or forskolin are reported to tion, the GFP-HDAC4 or mutant HDAC4 expression plasmids cause dephosphorylation of hHDAC4 at Ser-246 by PP2A were transfected into UMR 106-01 cells. The total lysates were through PKA resulting in an increase in the nuclear localization precleared by incubating with Protein A/G-agarose beads of HDAC4, inhibition of myocyte enhancer factor 2 (MEF2) (Santa Cruz Biotechnology). After the cleared supernatants had transcriptional activity, and suppression of collagen X expres- been incubated overnight with 2 g/ml antibody at 4 °C, the sion in chondrocytes (21). agarose beads were washed three times with PBS. Proteins were A further level of regulation of HDAC4 has been shown to be separated using SDS-PAGE and were transferred to polyvi- through its partial degradation. This has been previously shown nylidene difluoride membranes. Proteins were detected by to be due to cleavage by caspase (22), or through SUMOylation SuperSignal West Dura Extended Duration Substrate (Thermo and proteasome degradation (23). Most recently, Backs et al. Scientific) according to the manufacturer’s instructions. Quan- (24) showed that PKA induces cleavage of HDAC4 to produce titation was obtained using ImageJ. an N-terminal fragment, which acts as a CaMKII-insensitive Dephosphorylating Proteins in Vitro with Calf Intestinal repressor that selectively inhibits MEF2. The cleavage of Alkaline Phosphatase (CIP)—CIP can be used to release phos- HDAC4 is associated with a PKA activated-serine protease. phate groups from phosphorylated tyrosine, serine, and threo- We recently showed that HDAC4 repressed MMP-13 tran- nine residues in proteins. UMR 106-01 cells stimulated with or scription under basal conditions and parathyroid hormone without PTH were washed twice by PBS. Nuclear extracts were (PTH) regulates HDAC4 to control MMP-13 promoter activity divided into two aliquots (90 g). One aliquot was treated for 60 through dissociation from Runx2 (25). Here, we report that min at 37 °C with 10 units of CIP, and one aliquot was mock- PTH stimulates phosphorylation of rHDAC4 at Ser-740 in the treated. The reaction was stopped with 50 mM EDTA. The sam- nucleus of osteoblastic cells. Phosphorylated Ser-740 rHDAC4 ples were then resolved by SDS-PAGE. is associated with release from Runx2 on the MMP-13 pro- In Vitro Phosphorylation Assay—The phosphorylation reac- moter and activation of the gene. HDAC4 is then partially tion was performed in 20 mM Tris-HCl (pH 7.5), 5 mM MgCl ,1 degraded in the cytoplasm after PTH treatment, which is M EDTA, with 50 g of nuclear extracts from UMR 106–01 blocked by PKA, phosphatase, and lysosomal inhibitors. This is cells treated with or without PTH for 30, 120, 240 min, and the first observation of this complete system of regulation of purified catalytic subunit of PKA (1.0 or 5.0 g) at 30 °C for 30 HDAC4. min. The samples were then resolved by SDS-PAGE. Luciferase Activities—For transient transfections, UMR EXPERIMENTAL PROCEDURES 106-01 cells were seeded in 12-well plates overnight and then Materials—Parathyroid hormone (rat PTH 1–34), prosta- transfected with indicated plasmids using GeneJammer (Strat- glandin E , okadaic acid, and NH Cl were purchased from Sigma- agene) according to the manufacturer’s protocol. After 2 days, 2 4 Aldrich. H89, GF109203, MG132, lactacystin, AcDEVDCHO, the cells were treated with PTH (10 M) for 6 h. Lysates were KN-62, KN-92, KN-93, Gö6976, 3.4 DCl, AEBSF, pepstatin A, analyzed immediately for luciferase activity using the luciferase and purified catalytic subunit of PKA were purchased from assay reagent (Promega) and an OptiCompII luminometer EMD Millipore. (MGM Instruments, Inc., Hamden, CT). To make the mutation Cell Culture—The UMR 106-01 cells were cultured in Eagle’s constructs of GFP-HDAC4, a site-directed mutagenesis kit minimal essential medium (EMEM) supplemented with 25 mM (Agilent Technologies) was used, and detailed procedures were Hepes, pH 7.4, 1% nonessential amino acids, 100 units/ml pen- in accordance with the manufacturer’s instructions. icillin, 100 g/ml streptomycin, 5% fetal bovine serum. Saos-2 Chromatin Immunoprecipitation Assays (ChIP Assays)— cells were cultured in -MEM supplemented with 1% L-gluta- UMR 106-01 cells were incubated for 10 min at room temper- AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21341 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 1. PTH induces the phosphorylation of HDAC4 in the nucleus of osteoblastic cells. A, nuclear and cytoplasmic extracts from control or PTH treated (5, 10, 15, 20, 25, 30 min) UMR 106-01 cells were subjected to immunoblotting with anti-HDAC4, anti-Cdk, and anti-tubulin antibodies. Anti-Cdk2 was used for the nucleus as a loading control; anti-tubulin was used for the cytoplasm. B, UMR 106-01 cells were preincubated with protein kinase A inhibitor H89 (50 M), CaMK inhibitors KN92 (5 M), KN62 (5 M), KN93 (5 M), protein kinase C inhibitor GF109203 (5 M), or PKD inhibitor Gö6976 (5 M) for 30 min, and then treated with or without PTH (1–34, 10 M) for 30 min. Nuclear and cytoplasmic extracts were subjected to immunoblotting with anti-HDAC4, anti-Cdk, and anti-tubulin antibodies. Anti-Cdk2 was used for the nucleus and anti-tubulin was used for the cytoplasm as loading controls. C, nuclear extracts isolated from control and PTH-treated UMR 106-01 cells were incubated with buffer (Mock) or with 10 units CIP for 60 min at 37 °C. The samples were used for Western blot analysis using anti-HDAC4 and anti-Cdk antibodies. Anti-Cdk2 was used as a loading control. D, nuclear extracts isolated from control and PTH-treated UMR 106-01 cells were incubated with buffer (Mock) or with purified catalytic subunit of PKA (1.5 or 5 g) for 30 min at 30 °C. The samples were used for Western blot analysis using anti-HDAC4 and anti-Cdk2 antibodies. Anti-Cdk2 was used as a loading control. ature with medium containing 0.8% formaldehyde. Cells were buffer of 1% SDS, 0.1 M NaHCO . For re-ChIP assays, the then washed in ice-cold PBS containing protease inhibitors and immunoprecipitated complexes obtained by ChIP with anti- 1mM phenylmethylsulfonyl fluoride and resuspended in SDS Runx2 antibody were eluted in ChIP elution buffer and then lysis buffer (1% SDS, 10 mM EDTA, pH 8.0, 25 mM Tris-HCl, pH diluted 5-fold and subjected to the ChIP procedure with anti- 8.1, containing protease inhibitors and 1 mM PMSF) for 10 min GFP. The cross-linking reaction was reversed with 5 M NaCl by on ice. Samples were sonicated to reduce the DNA length to 6 h incubation at 65 °C. The samples were digested with pro- 0.5–1 kbp, cellular debris was removed by centrifugation, and teinase K (10 mg/ml), 40 mM Tris-HCl pH 6.5, 10 mM EDTA at the supernatant was diluted 10-fold in dilution buffer (0.01% 42 °C for 1 h, and DNA was recovered by phenol/chloroform SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH extractions. The DNA was precipitated with two volumes of 8.1, 167 mM NaCl supplemented with protease inhibitors). For ethanol using glycogen as carrier. The input lysates were also PCR analysis, aliquots (1:100) of total chromatin DNA before processed as above. The DNA was resuspended in water and immunoprecipitation were saved (input). Prior to chromatin used for quantitative PCR. The sequences of the oligonu- immunoprecipitations, the samples were precleared with 80 l cleotides of the rat MMP-13 promoter (the distal RD and of a 25% (v/v) suspension of DNA-coated protein A/G-agarose proximal AP-1 sites, 204/34) were as follows: forward for 30 min at 4 °C. The supernatant was recovered and used primer 5-CAGATGCGTTTTGATATGCC-3, reverse directly for immunoprecipitation experiments with appropri- primer 5-AATAGTGATGAGTCACCACTT-3. The PCR ate antibody overnight at 4 °C. Immune complexes were mixed reactions and program are described in detail in a previous with 60 l of a 25% precoated protein A/G-agarose suspension publication (25). followed by incubation for1hat4 °C. Beads were collected and Statistical Analysis—All results are expressed as means sequentially washed with 1 ml of each of the following buffers: S.E. of triplicate measurements with all experiments being low salt wash buffer, high salt wash buffer, and LiCl wash buffer. repeated at least three times. Statistical analyses were carried The beads were then washed twice using 1 ml of TE buffer. The out using Student’s t test or one-way ANOVA using the Tukey immunocomplexes were eluted two times by adding elution HSD test. 21342 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 2. PTH causes phosphorylation of a specific site of HDAC4 in the nucleus. A, structure and PKA-dependent phosphorylation sites of human and rat HDAC4 proteins. MEF2 (MEF2C interaction domain): human (118 –313), rat (227– 422), NLS (nuclear localization signal): human (244 –279), rat (353–388), HDA1 (HDA1-related domain): human (655–1084), rat (763–1187), and NES (nuclear export signal): human (1051–1084), rat (1154 –1187). B, nuclear extracts from control or PTH-treated (0, 30, 60 min) UMR 106-01 cells were subjected to immunoblotting with anti-phosphoSer632 hHDAC4, anti-phosphoSer246 hHDAC4, 8 6 and anti-Cdk antibodies. The nuclear extracts from control or PTH (human, 1–34, 10 M, 30 min) or prostaglandin E (10 M, 30 min) treated Saos-2 cells were subjected to immunoblotting with anti-HDAC4, anti-phosphoSer632 hHDAC4, and anti-Cdk antibodies. Anti-Cdk2 was used for the nucleus as a loading control. C, UMR 106-01 cells were transfected with GFP rHDAC4 construct. Cells were stimulated with PTH for 30 and 60 min. The nuclear extracts from control or PTH treated (0, 30, 60 min) UMR 106-01 cells were subjected to immunoblotting with anti-phosphoSer632 hHDAC4, anti-phosphoSer246 hHDAC4, GFP, and anti-Cdk antibodies. Anti-Cdk2 was used for the nucleus as a loading control. D, UMR 106 – 01 cells were preincubated with protein kinase A inhibitor H89 (50 M) for 30 min, and then treated with or without PTH (1–34, 10 M) for 30 min. Nuclear extracts were subjected to immunoblotting with anti-phosphoSer632 hHDAC4 and anti-Cdk antibodies. Anti-Cdk2 was used as a loading control. RESULTS previously shown that HDAC4 suppresses MMP-13 transcrip- PTH Affects the Modification of HDAC4 in the Nucleus tion through its association with Runx2; PTH regulates this through Protein Kinase A—We focused on understanding the repression in osteoblastic cells and this involves PKA (25). effects of PTH on phosphorylation of HDAC4 because it was Phosphorylation and de-phosphorylation control nuclear AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21343 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 3. Ser-740 site of HDAC4 is important for dissociation from Runx2. A, mutation sites of GFP-rHDAC4; Serine residues mutated to alanine. B, UMR 106 – 01 cells were transfected with GFP-rHDAC4 or mutant GFP-rHDAC4 constructs. Cells were stimulated with PTH (1–34, 10 M) for 30 min. Total cellular lysates isolated from UMR 106-01 cells were immunoprecipitated with anti-Runx2 or control rabbit IgG antibodies. Samples were immunoblotted with anti-GFP antibody. The quantitative intensity was obtained using ImageJ. *, p 0.05 versus respective control. C, UMR 106 – 01 cells were transfected with GFP rHDAC4 or mutant GFP rHDAC4 constructs (S355A, S576A, S740A, and T815A). Total cell lysates were immunoblotted with anti-GFP, anti-Runx2, and anti-Cdk antibodies. Anti-Cdk2 was used as a loading control. export and import of HDAC4 (10). To begin deciphering the without PTH. These data indicate that PKA activation may be effects of PTH on HDAC4, we examined the nuclear fractions required for import of HDAC4 to the nucleus as well as the in UMR 106-01 cells after 5–30 min of exposure to PTH. A phosphorylation in the nucleus after PTH treatment, whereas slower-migrating upper band was detected 5 min after PTH PKC and PKD activation may be related to export of HDAC4 to treatment only in the nucleus, which was apparent for up to 240 the cytoplasm. The faster-migrating band in the cytoplasm also min (Fig. 1, A and C). A faster migrating band of HDAC4 was appears to require PKA activation in response to PTH for its detectable in the cytoplasmic fraction after 15 min of PTH stim- generation. ulation which we think is a partial degradation product (Fig. 1, Protein Kinase A Induces the Nuclear Phosphorylation of A and B). We next examined whether PKA affected the appear- HDAC4—PTH causes activation of protein kinase A, which ance of a slower-migrating band of HDAC4 in the nucleus. We phosphorylates proteins on serine/threonine residues in its used the following inhibitors, protein kinase A (H89), protein specific recognition sequence in osteoblastic cells (26). We kinase C (PKC) (GF109203), CaMK (KN62, 92, and 93) and hypothesized that the slower migrating band of HDAC4 in the PKD (Gö6976). As shown in Fig. 1B, H89 prevented the appear- nucleus might be due to phosphorylation by protein kinase A. ance of the slowly migrating band of HDAC4 in the nucleus in To investigate this hypothesis, we performed dephosphory- response to PTH but CaMK inhibitors had no effect, eliminat- lation assays using nuclear extracts from UMR 106-01 cells. ing this pathway in PTH action in the nucleus. GF109203 and The nuclear extracts were incubated with calf intestinal alkaline Gö6976 clearly increased the appearance of HDAC4 in the phosphatase (CIP) for 60 min at 37 °C. The samples were nucleus in the absence and presence of PTH treatment. H89 detected by Western blot analysis. The PTH-induced upper increased accumulation of HDAC4 in the cytoplasm with or band of HDAC4 was decreased by CIP treatment in comparison 21344 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation with the mock treated samples to a molecular mass identical to UMR 106–01 cells were incubated with the purified catalytic the control cells (Fig. 1C). For a more direct confirmation of subunit of PKA for 30 min at 30 °C. After incubation, the sam- whether HDAC4 is phosphorylated by PKA, we performed in ples were detected by Western blot analysis. The upper band of vitro phosphorylation assays. The nuclear extracts from control HDAC4 was increased by this enzymatic activity in comparison AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21345 PTH-induced HDAC4 Phosphorylation and Dephosphorylation with the mock-treated cells. This upper band migrated identi- constructs (S355A, S576A, and T815A) was decreased after cally to that from PTH-treated samples (Fig. 1D). These data PTH stimulation whereas the GFP-rHDAC4-S740A mutant indicate that PTH causes phosphorylation of HDAC4 by acti- remained associated with Runx2 (Fig. 3B). As shown in Fig. 3C, vation of PKA in the nucleus. these protein levels were equally detected when we performed PTH Stimulates Phosphorylation at a Specific HDAC4 Site in Western blots. This result suggests that Ser-740 in rat HDAC4 the Nucleus—Next, we investigated which residues of HDAC4 is a crucial phosphorylation site for dissociation from Runx2. are phosphorylated by PTH in the nucleus. Examination of the To investigate further whether Ser-740 in rHDAC4 is func- database of PKA-dependent phosphorylation sites (Phos- tionally related to dissociation from Runx2 on the runt domain pho.ELM and KinasePhos), as shown in Fig. 2A, illustrated of the MMP-13 promoter, we performed luciferase promoter there were three serine (355, 576 and 740aa) and one threonine assays using the GFP-rHDAC4 constructs. As shown in Fig. 4A, (815aa) residues in rat HDAC4. Interestingly, all sites in the rat wild type GFP-rHDAC4 significantly inhibited MMP-13 tran- HDAC4 were homologous to human sequences and r355 scriptional activity after PTH stimulation similarly to previous (h246), r576 (h467), and r740 (h632) were also CaMK-depen- published data of ours (25). All phosphorylation site mutations, dent phosphorylation sites (Fig. 2A). Ser-246, -467, and -632 in except the S740A mutation inhibited the PTH stimulation to a hHDAC4 are binding sites for 14-3-3 proteins and play a role in similar extent. The S740A mutation in rHDAC4 significantly trafficking HDAC4 from nucleus to cytoplasm (13, 27). We inhibited the promoter activity more than the wild type examined whether phosphorylation of these sites of HDAC4 rHDAC4. To further explore the potential role of HDAC4 was stimulated by PTH using available HDAC4 phosphoryla- interaction with Runx2, we investigated the presence of GFP- tion antibodies. As shown in Fig. 2B, HDAC4 and its phosphor- rHDAC4 or GFP-rHDAC4 mutant proteins with Runx2 at the ylation using anti phospho-Ser-632 (r740) HDAC4 were Runx2 binding site of the MMP-13 promoter by ChIP re-ChIP increased at 30 min after PTH stimulation, whereas phosphor- assays of UMR 106–01 cells with or without PTH stimulation. ylation using anti-phospho-Ser-246 (r355) was not detected in As shown in Fig. 4B, the association of Runx2-GFP-rHDAC4 the nucleus of UMR 106-01 cells. Interestingly, phospho-Ser- and Runx2-GFP-rHDAC4 mutations (S355A, S576A, and 632 HDAC4 was stimulated at 30 min with PTH or prostaglan- T815A) with the MMP-13 promoter was decreased after PTH din E (10 M) treatment in the human osteoblastic cell line, stimulation using primers encompassing the RD and AP-1 sites. The association of Runx2-GFP-rHDAC4-S740A mutation Saos-2 cells. Although total HDAC4 was increased for up to 240 min in UMR cells, phosphorylation at r740 HDAC4 was tran- actually increased with PTH treatment, indicating that the hor- siently increased at 30 min (Figs. 1B and 2, C and D). We have mone-stimulated phosphorylation of this residue is required previously shown that PTH induces the transcription and total for dissociation of HDAC4 from Runx2 on the MMP-13 expression of HDAC4 at later times in these cells (25). Next, we promoter. overexpressed GFP-rHDAC4 constructs in UMR 106-01 cells. Protein Kinase A and Lysosomal Inhibitors Prevent Partial Phosphorylation using anti-phospho-Ser-632 was increased on HDAC4 Degradation—We had seen a faster-migrating band of GFP-HDAC4 and endogenous HDAC4 with PTH treatment HDAC4 in cytoplasmic and total cell lysates which appears to whereas phosphorylation using anti-phospho-Ser-246 on GFP- be a partial degradation product (Fig. 1, A and B) similar to that HDAC4 was decreased (Fig. 2C). In addition the phosphoryla- seen by Backs et al. (24). HDAC6 also slightly decreased after tion at Ser-740 was abolished in the presence of H89, indicating PTH treatment (Fig. 5A). We next examined whether PKA or this is dependent on PKA. Thus, HDAC4 phosphorylation sites other pathways affected the appearance of the faster migrating were differently modified by PTH stimulation. We hypothe- band and potential degradation of HDAC4 in the cytoplasm. sized that a specific phosphorylation site of HDAC4 may be We used protein kinase A (H89) and protein kinase C inhibitors related to dissociation from Runx2 on the MMP-13 promoter. (GF109203), proteasome inhibitors (MG132, lactacystin), and Mutation of Ser-740 in rHDAC4 Prevents Dissociation from caspase-3 inhibitor (Ac-DEVD-CHO) (Fig. 5B). H89 was the Runx2—Next, to confirm our hypothesis, we performed immu- only agent to block HDAC4 degradation (see also Fig. 1B), indi- noprecipitation assays of Runx2 using wild type and mutation cating that HDAC4 degradation occurs via non-proteosomal or constructs of rHDAC4. As shown in Fig. 3A, we made single non-caspase pathways and requires the PKA pathway in PTH point mutation constructs of rat HDAC4 (S355A, S576A, action. S740A, and T815A) which are activated by PKA, CaMK, or Since trafficking and partial degradation of HDAC4 are asso- PKD-dependent pathways. Immunoprecipitated endogenous ciated with phosphorylation, we used the phosphatase inhibi- Runx2 bound to GFP-rHDAC4 (wild type) and four GFP-rH- tor, okadaic acid. The faster migrating band almost disappeared DAC4 mutant constructs under basal conditions. Runx2 bind- with PTH treatment in the presence of this inhibitor (Fig. 5C), ing with GFP-rHDAC4 (wild type) and GFP-rHDAC4 mutant indicating that HDAC4 degradation occurs through serine/ FIGURE 4. GFP-rHDAC4-S740A mutation abolished dissociation from Runx2 on the MMP-13 promoter. A, UMR 106 – 01 cells were transiently transfected with various vectors (100 ng of pGL2 vector, 100 ng of -148 rat MMP-13 promoter-Luc, 100 ng of GFP vector, 100 ng of GFP rHDAC4, and mutant GFP rHDAC4 constructs), and the luciferase activities were measured with (P) or without (C)6hof10 M PTH (1–34) treatment. The luciferase activities were normalized to total protein. Error bars represent S.E. of three independent experiments. *, p 0.05 versus respective control. B, UMR 106-01 cells were transfected with GFP rHDAC4 or mutant GFP rHDAC4 constructs (S355A, S576A, S740A, and T815A). The cells were treated with control or PTH (10 M) for 30 min, then prepared for ChIP assays. After immunoprecipitation of the cross-linked lysates with anti-Runx2 or with rabbit IgG as a negative control, the immune complexes were collected then diluted with ChIP dilution buffer and re-subjected to ChIP assays with anti-GFP or rabbit IgG. The DNA was subjected to PCR with primers that amplify the distal RD and proximal AP-1 sites of the endogenous rat MMP-13 promoter. Input DNA (1:100) is a positive control for the assay. Data are shownas -fold change to control after normalizing to input and IgG. Error bars represent  S.E. of three independent experiments. *, p  0.05 versus respective control. 21346 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 5. PKA, phosphatase, and lysosomal inhibition blocks PTH-induced HDAC4 degradation. A, total cellular lysates (30 g) isolated from 10 M PTH and control-treated UMR 106 – 01 cells for 5, 15, 30, and 60 min were used for Western blot analysis using anti-HDAC 4, 6, or Cdk2 antibodies. Anti-Cdk2 was used as the loading control. B, total cellular lysates isolated from UMR 106-01 cells, which had been preincubated with protein kinase A inhibitor H89 (50 M) for 30 min, protein kinase C inhibitor GF109203 (5 M) for 30 min, proteasome inhibitor MG132 (5 M), lactacystin (5 M), or caspase-3 inhibitor AcDEVD-CHO (100 M) for 60 min, and then with or without PTH (10 M) stimulation for 30 min. Total cellular lysates were used for Western blot analysis using anti-HDAC4. Anti-Cdk2 was used as a loading control. C, total cellular lysates or nuclear/cytoplasmic extracts isolated from UMR 106-01 cells, which had been preincubated with phosphatase inhibitor, okadaic acid (OKA, 50 nM) for 60 min and then with or without PTH (10 M) stimulation for 30 min. Total cellular lysates or nuclear/cytoplasmic extracts were used for Western blot analysis using anti-HDAC4. Anti--actin was used for total cell lysates or cytoplasm as a loading control. Anti-Cdk2 was used as a nuclear loading control. D, nuclear and cytoplasmic extracts isolated from UMR 106-01 cells, which had been preincubated with phosphatase inhibitor, okadaic acid (OKA, 50 nM) for 60 min and then with or without PTH (10 M) stimulation for 30 min. The nuclear and cytoplasm extracts were subjected to immunoblotting with anti-phosphoSer632 hHDAC4, anti-phosphoSer246 hHDAC4, anti-GFP, anti--actin, and anti-Cdk antibodies. Anti-Cdk2 was used for the nucleus and anti--actin was used for cytoplasm as loading controls. E, total cellular lysates isolated from UMR 106-01 cells were preincubated with NH Cl (20 mM) for 16 h and then with PTH (10 M) stimulation for 30 min. Total cellular lysates were used for Western blot analysis using anti-HDAC4. Anti--actin was used for loading control. F, UMR 106 – 01 cells were preincubated with vehicle, serine protease inhibitors, 3.4 DCl (50M) or AEBSF (200M), aspartic protease inhibitor pepstatin A (10M) for 90 min and then with or without PTH (10 M) stimulation for 30 min. Total cellular lysates were used for Western blot analysis using anti-HDAC4. Anti--actin was used as a loading control. threonine dephosphorylation. There was also less HDAC4 pho-Ser-632 (r740) HDAC4 was increased at 30 min after PTH in the cytoplasm. Interestingly, the phosphatase inhibitor stimulation as seen in Fig. 2, and okadaic acid additively increased phospho-Ser-246 (r355) in the nucleus which had increased phospho-Ser-632 (r740) with PTH treatment in the been seen to be reduced with PTH treatment (Fig. 5D). Phos- nucleus. In the cytoplasm, phosphorylation at both sites was AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21347 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 6. Model of PKA-dependent phosphorylated regulation of HDAC4. PKA phosphorylates HDAC4 in the nucleus, especially inducing phosphoryla- tion of HDAC4 at serine 740, which results in HDAC4 being released from Runx2 bound to the Runt domain site of the MMP-13 promoter. PKA phosphorylation also induces dephosphorylation of HDAC4 at serine 355 in the nucleus. After transport of HDAC4 into the cytoplasm, HDAC4 is partially degraded through the lysosomal pathway. decreased with PTH and okadaic acid. Thus, dephosphory- to the MMP-13 promoter and exported to the cytoplasm and is lation likely by PP2A is also involved with partial degradation of then dephosphorylated in the cytoplasm and partially degraded HDAC4 and its trafficking from the nucleus in osteoblastic through a lysosomal pathway (Fig. 6). cells. PTH appears to cause the site-selective dephosphory- Work by several laboratories showed that HDAC4 phosphor- lation or phosphorylation of HDAC4 in the nucleus. ylation, binding to 14-3-3 proteins and HDAC4 export to the The intracellular systems of protein degradation are classi- cytoplasm is through phosphorylation by the CaMKII/IV path- fied as lysosomal and non-lysosomal. The lysosomal process way. HDAC4 is also required for hetero-oligomerization with involves uptake into lysosomes, and the non-lysosomal step HDAC5 and its regulation by CaMKII (16, 17). We demonstrate generally involves tagging of proteins prior to degradation by here that PTH induces HDAC4 phosphorylation, especially at the proteasome. Because proteasome inhibitors did not affect Ser-740 in rHDAC4 through PKA activation in the nucleus, but the degradation of HDAC4, we hypothesized that HDAC4 was not by CaMKII/IV as detected by use of inhibitors and antibod- degraded by the lysosome. To assess a relationship with the ies. In addition, protein expression of HDAC5 is very low and lysosome, we examined the effects of the lysosomal inhibitor, PTH does not stimulate phosphorylation of HDAC5 and NH Cl, on HDAC4 mobility in response to PTH. Preincubating HDAC7 in UMR 106-01 cells and rat primary osteoblasts (data cells with NH Cl (Fig. 5E) blocked PTH-induced degradation of not shown). PTHrP or forskolin repress chick chondrocyte HDAC4. Chloroquine and bafilomycine A1 also blocked its hypertrophy through PKA-dependent dephosphorylation of degradation (data not shown). In addition, we used serine pro- hHDAC4 at phospho-S246 by PP2A, which increases the tease inhibitors (3.4 DCl, AEBSF) and an aspartic protease nuclear localization of HDAC4 and inhibits MEF2 function inhibitor (Pepstatin A). AEBSF and Pepstatin A had no effect on (21). Consistent with their data, we found PTH decreases PTH-induced HDAC4 degradation whereas 3.4 DCl increases phosphorylation of Ser-355 in GFP-rHDAC4 (equivalent to the faster migrating band (Fig. 5F). These results indicate that hHDAC4 246S), but we could not detect endogenous phosphor- HDAC4 degradation is associated with lysosomal pathways but ylated Ser-355 rHDAC4 in UMR 106-01 cells, indicating that not serine or aspartic proteases. this requires overexpression of HDAC4. We showed that DISCUSSION dephosphorylation of Ser-355 rHDAC4 is not related to dis- sociation from Runx2 or regulation of the transcriptional The results of this study show the effects of PTH on the activity of MMP-13 (Fig. 4, A and B). However, the phospha- phosphorylation and processing of HDAC4 in rat osteoblastic tase inhibitor, okadaic acid, enhances phosphorylation of Ser- cells. We focused on HDAC4 function, because HDAC4 plays 355 rHDAC4 in the nucleus and this phosphorylation was an important role to suppress chondrocyte and osteoblast dif- reduced after PTH stimulation whereas phosphorylation of ferentiation through association with Runx2 and particularly Ser-740 rHDAC4 was increased by PTH and okadaic acid. suppresses MMP-13 gene expression in vitro and in vivo (7, 25). Phosphorylation of Ser-355 rHDAC4 and 740 rHDAC4 may be We conclude that PKA-dependent phosphorylated HDAC4, especially at Ser-740 in rHDAC4, is released from Runx2 bound regulated inversely by PTH through PP2A and PKA, control- 21348 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation ling HDAC4 trafficking out of the nucleus and subsequent par- regulation of HDAC4. It demonstrates a role for HDAC4 in tial degradation. Therefore, we conclude that transient PTH- hormonal regulation of gene expression in osteoblasts. induced phosphorylation of Ser-740 rHDAC4 (Ser-632 in REFERENCES hHDAC4) via PKA activation plays a role to cause the induction 1. Verdin, E., Dequiedt, F., and Kasler, H. G. (2003) Class II histone deacety- of MMP-13 gene expression in osteoblasts. Moreover, PKA- lases: versatile regulators. Trends Genet. 19, 286–293 activated phosphorylation and dephosphorylation of HDAC4 2. Fischle, W., Dequiedt, F., Hendzel, M. J., Guenther, M. G., Lazar, M. A., may regulate terminal chondrocyte differentiation through the Voelter, W., and Verdin, E. (2002) Enzymatic activity associated with class MMP-13 gene in hypertrophic chondrocytes. II HDACs is dependent on a multiprotein complex containing HDAC3 Prior work has shown that HDAC4 suppresses MMP-13 and SMRT/N-CoR. Mol. Cell 9, 45–57 transcription and PTH controls HDAC4 functions via the dis- 3. Fischle, W., Dequiedt, F., Fillion, M., Hendzel, M. 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(2007) SIK1 is a class II HDAC kinase ysis of HDAC4. J. Cell Biol. 195, 403–415 that promotes survival of skeletal myocytes. Nat. Med. 13, 597–603 21350 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Parathyroid Hormone Regulates Histone Deacetylase (HDAC) 4 through Protein Kinase A-mediated Phosphorylation and Dephosphorylation in Osteoblastic Cells *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2014 Elsevier Inc.
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0021-9258
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10.1074/jbc.m114.550699
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 31, pp. 21340 –21350, August 1, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Parathyroid Hormone Regulates Histone Deacetylase (HDAC) 4 through Protein Kinase A-mediated Phosphorylation and Dephosphorylation in Osteoblastic Cells Received for publication, January 17, 2014, and in revised form, May 23, 2014 Published, JBC Papers in Press, June 5, 2014, DOI 10.1074/jbc.M114.550699 ‡§ ‡ ‡ ‡1 Emi Shimizu , Teruyo Nakatani , Zhiming He , and Nicola C. Partridge ‡ § From the Departments of Basic Science and Craniofacial Biology and Endodontics, New York University College of Dentistry, New York, New York 10010 Background: PTH regulates HDAC4 to control its dissociation from Runx2. Results: PTH regulates phosphorylation and dephosphorylation of HDAC4 through PKA and partial degradation of HDAC4. Conclusion: PTH controls MMP-13 transcription through PKA-dependent phosphorylation and dephosphorylation of HDAC4. Significance: The results provide insight into how PTH participates in modification of HDAC4, which is crucial for under- standing hormonal regulation of gene expression. Histone deacetylases (HDACs) are crucial regulators of gene chromatin remodeling, and post-translational modifications of expression in transcriptional co-repressor complexes. Previ- histones, which includes the dynamic acetylation and deacety- ously, we reported that HDAC4 was a basal repressor of matrix lation of epsilon-amino groups of lysine residues present in the metalloproteinase-13 (MMP-13) transcription and parathyroid tails of core histones. Histone deacetylases (HDACs) are cru- hormone (PTH) regulates HDAC4 to control MMP-13 pro- cial regulators of gene expression in transcriptional co-repres- moter activity through dissociation from Runx2. Here, we show sor complexes and control gene expression important for that PTH induces the protein kinase A (PKA)-dependent phos- diverse cellular functions. The class I HDACs (HDAC1, -2, -3, phorylation of HDAC4 in the nucleus of the rat osteoblastic cell and -8) have homology to the yeast global transcriptional reg- line, UMR 106–01. We demonstrate that PKA-dependent ulator Rpd3 and are widely expressed. In contrast, the class II phosphorylated HDAC4 is released from Runx2 bound to the HDACs (HDAC4, -5, -6, -7, -9, and -10) show homology to MMP-13 promoter in these cells. Point mutation of Ser-740 in yeast Hda1 and are expressed in cell type-restricted patterns. rHDAC4 prevents the release of HDAC4 from Runx2 on the Among class II HDACs, HDAC4, -5, -7, and -9 form a subclass MMP-13 promoter and also prevents the PTH stimulation of known as class IIa, whereas HDAC6 and 10 constitute class IIb. MMP-13 transcription. Thus, PTH-induced phosphorylation of The class IIa histone deacetylases can be expressed in a tissue- rHDAC4 at Ser-740 is crucial for regulating MMP-13 transcrip- specific fashion, and are regulated by nuclear-cytoplasmic shut- tion in osteoblasts. PTH causes degradation of HDAC4, and this tling (1). product appears in the cytoplasm. The cytoplasmic degradation HDAC3 binds class II HDACs (2, 3) and form large multi- of HDAC4 is blocked by PKA and lysosomal inhibitors, but is protein complexes. These multi-protein complexes are not affected by proteasome, caspase-3, or serine and aspartic recruited to specific DNA sequences and chromatin substrates protease inhibitors. In addition, the phosphatase inhibitor, oka- by transcription factors (4). Genetic deletion of HDAC3 and daic acid, prevents degradation indicating that dephosphory- HDAC4 revealed the roles of these proteins in chondrocyte lation is associated with degradation. These mechanisms regu- maturation (5). Several class II HDACs appear to have a role in lating HDAC4 and their roles in such processes are crucial for skeletal formation (6). Notably, Hdac4-null mice display pre- bone and chondrocyte development. Our data support a link mature ossification of developing bones due to constitutive between PTH regulating HDAC4 phosphorylation by PKA, traf- Runx2 expression. Thus, HDAC4 regulates chondrocyte hyper- ficking, partial degradation, and the control of MMP-13 tran- trophy and endochondral bone formation by inhibiting the scription through association with Runx2. activity of Runx2 (7). In osteoblasts, HDAC4 and HDAC5 par- ticipate in TGF signaling pathways that suppress Runx2 activ- ity (8). Moreover, HDAC4 and HDAC5 deacetylate Runx2 and lead to Smurf-mediated degradation of Runx2 (9). Thus, Epigenetic modification plays an important role in the con- HDAC4 is considered an important target for osteoblast or trol of cell fate during mammalian development. Among these, chondrocyte differentiation. the most studied so far is DNA methylation, ATP-dependent Dephosphorylation by phosphatases and phosphorylation by serine/threonine kinases controls nuclear import and export * This work was supported, in whole or in part, by National Institutes of Health of HDAC4 and influences HDAC4 repressive activity (10). Grant DK47420 (to N. C. P.). To whom correspondence should be addressed: Department of Basic Sci- ence and Craniofacial Biology, New York University College of Dentistry, The abbreviations used are: HDAC, histone deacetylase; PTH, parathyroid th 345 East 24 St, New York, New York 10010. Tel.: 212-992-7145; Fax: 212- hormone; PTHrP, parathyroid hormone-related peptide; MEF, myocyte 995-4204; E-mail: [email protected]. enhancer factor; CIP, calf intestinal alkaline phosphatase. This is an Open Access article under the CC BY license. 21340 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation 14-3-3 proteins shuttle class II HDACs to the cytoplasm (11). mine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum. These proteins have a phosphoserine/threonine-binding motif Antibodies—Anti-HDAC4 (against 10 N-terminal amino through which they interact with phosphorylated HDACs (12). Subcellular localization of HDAC4, HDAC5, and HDAC7 is acids), anti-GFP, and anti--actin were purchased from Cell regulated by calcium-calmodulin-dependent kinase IV (CaMK Signaling Technology. Anti-HDAC4 (H92, against amino acids 530–631), anti-Runx2 (M-70), anti-Cdk2 (M2), and anti-tubu- IV) (13) or PKD phosphorylation and association with 14-3-3 lin (TU-2) were purchased from Santa Cruz Biotechnology. proteins for export to the cytoplasm (14, 15). Backs et al. (16, Western Blot—UMR 106-01 cells were treated with or with- 17) showed that CaMK II signals specifically to HDAC4 but not out rat PTH (1–34, 10 M) for the indicated times. The cells HDAC5 by binding to a unique kinase-docking site contained were washed twice in PBS, pH 7.4 and pelleted by centrifuga- in HDAC4. HDAC4 can subsequently re-enter the nucleus after tion at 2000 rpm for 5 min at 4 °C. The pellets were resuspended dephosphorylation and dissociation from 14-3-3 (11). in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Parathyroid hormone (PTH) is an 84-amino acid peptide PMSF, 1 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, and hormone, which functions as an essential regulator of calcium protease inhibitors) and incubated for 15 min at 4 °C. Amounts homeostasis and as a mediator of bone remodeling (18). PTH of total protein were determined by the Bradford dye binding acts via the PTH/PTH-related protein 1 receptor (a G protein- (Bio-Rad) method. The preparation of cytoplasmic and nuclear coupled receptor) on osteoblast membranes (19), and both its extracts from cells was by the NE-PER nuclear and cytoplasmic anabolic and catabolic effects on bone appear to be primarily extraction reagents (Thermo Scientific). To examine the inter- mediated by the cAMP/PKA pathways (20). Parathyroid hor- action between HDAC4 and Runx2 using immunoprecipita- mone-related peptide (PTHrP) or forskolin are reported to tion, the GFP-HDAC4 or mutant HDAC4 expression plasmids cause dephosphorylation of hHDAC4 at Ser-246 by PP2A were transfected into UMR 106-01 cells. The total lysates were through PKA resulting in an increase in the nuclear localization precleared by incubating with Protein A/G-agarose beads of HDAC4, inhibition of myocyte enhancer factor 2 (MEF2) (Santa Cruz Biotechnology). After the cleared supernatants had transcriptional activity, and suppression of collagen X expres- been incubated overnight with 2 g/ml antibody at 4 °C, the sion in chondrocytes (21). agarose beads were washed three times with PBS. Proteins were A further level of regulation of HDAC4 has been shown to be separated using SDS-PAGE and were transferred to polyvi- through its partial degradation. This has been previously shown nylidene difluoride membranes. Proteins were detected by to be due to cleavage by caspase (22), or through SUMOylation SuperSignal West Dura Extended Duration Substrate (Thermo and proteasome degradation (23). Most recently, Backs et al. Scientific) according to the manufacturer’s instructions. Quan- (24) showed that PKA induces cleavage of HDAC4 to produce titation was obtained using ImageJ. an N-terminal fragment, which acts as a CaMKII-insensitive Dephosphorylating Proteins in Vitro with Calf Intestinal repressor that selectively inhibits MEF2. The cleavage of Alkaline Phosphatase (CIP)—CIP can be used to release phos- HDAC4 is associated with a PKA activated-serine protease. phate groups from phosphorylated tyrosine, serine, and threo- We recently showed that HDAC4 repressed MMP-13 tran- nine residues in proteins. UMR 106-01 cells stimulated with or scription under basal conditions and parathyroid hormone without PTH were washed twice by PBS. Nuclear extracts were (PTH) regulates HDAC4 to control MMP-13 promoter activity divided into two aliquots (90 g). One aliquot was treated for 60 through dissociation from Runx2 (25). Here, we report that min at 37 °C with 10 units of CIP, and one aliquot was mock- PTH stimulates phosphorylation of rHDAC4 at Ser-740 in the treated. The reaction was stopped with 50 mM EDTA. The sam- nucleus of osteoblastic cells. Phosphorylated Ser-740 rHDAC4 ples were then resolved by SDS-PAGE. is associated with release from Runx2 on the MMP-13 pro- In Vitro Phosphorylation Assay—The phosphorylation reac- moter and activation of the gene. HDAC4 is then partially tion was performed in 20 mM Tris-HCl (pH 7.5), 5 mM MgCl ,1 degraded in the cytoplasm after PTH treatment, which is M EDTA, with 50 g of nuclear extracts from UMR 106–01 blocked by PKA, phosphatase, and lysosomal inhibitors. This is cells treated with or without PTH for 30, 120, 240 min, and the first observation of this complete system of regulation of purified catalytic subunit of PKA (1.0 or 5.0 g) at 30 °C for 30 HDAC4. min. The samples were then resolved by SDS-PAGE. Luciferase Activities—For transient transfections, UMR EXPERIMENTAL PROCEDURES 106-01 cells were seeded in 12-well plates overnight and then Materials—Parathyroid hormone (rat PTH 1–34), prosta- transfected with indicated plasmids using GeneJammer (Strat- glandin E , okadaic acid, and NH Cl were purchased from Sigma- agene) according to the manufacturer’s protocol. After 2 days, 2 4 Aldrich. H89, GF109203, MG132, lactacystin, AcDEVDCHO, the cells were treated with PTH (10 M) for 6 h. Lysates were KN-62, KN-92, KN-93, Gö6976, 3.4 DCl, AEBSF, pepstatin A, analyzed immediately for luciferase activity using the luciferase and purified catalytic subunit of PKA were purchased from assay reagent (Promega) and an OptiCompII luminometer EMD Millipore. (MGM Instruments, Inc., Hamden, CT). To make the mutation Cell Culture—The UMR 106-01 cells were cultured in Eagle’s constructs of GFP-HDAC4, a site-directed mutagenesis kit minimal essential medium (EMEM) supplemented with 25 mM (Agilent Technologies) was used, and detailed procedures were Hepes, pH 7.4, 1% nonessential amino acids, 100 units/ml pen- in accordance with the manufacturer’s instructions. icillin, 100 g/ml streptomycin, 5% fetal bovine serum. Saos-2 Chromatin Immunoprecipitation Assays (ChIP Assays)— cells were cultured in -MEM supplemented with 1% L-gluta- UMR 106-01 cells were incubated for 10 min at room temper- AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21341 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 1. PTH induces the phosphorylation of HDAC4 in the nucleus of osteoblastic cells. A, nuclear and cytoplasmic extracts from control or PTH treated (5, 10, 15, 20, 25, 30 min) UMR 106-01 cells were subjected to immunoblotting with anti-HDAC4, anti-Cdk, and anti-tubulin antibodies. Anti-Cdk2 was used for the nucleus as a loading control; anti-tubulin was used for the cytoplasm. B, UMR 106-01 cells were preincubated with protein kinase A inhibitor H89 (50 M), CaMK inhibitors KN92 (5 M), KN62 (5 M), KN93 (5 M), protein kinase C inhibitor GF109203 (5 M), or PKD inhibitor Gö6976 (5 M) for 30 min, and then treated with or without PTH (1–34, 10 M) for 30 min. Nuclear and cytoplasmic extracts were subjected to immunoblotting with anti-HDAC4, anti-Cdk, and anti-tubulin antibodies. Anti-Cdk2 was used for the nucleus and anti-tubulin was used for the cytoplasm as loading controls. C, nuclear extracts isolated from control and PTH-treated UMR 106-01 cells were incubated with buffer (Mock) or with 10 units CIP for 60 min at 37 °C. The samples were used for Western blot analysis using anti-HDAC4 and anti-Cdk antibodies. Anti-Cdk2 was used as a loading control. D, nuclear extracts isolated from control and PTH-treated UMR 106-01 cells were incubated with buffer (Mock) or with purified catalytic subunit of PKA (1.5 or 5 g) for 30 min at 30 °C. The samples were used for Western blot analysis using anti-HDAC4 and anti-Cdk2 antibodies. Anti-Cdk2 was used as a loading control. ature with medium containing 0.8% formaldehyde. Cells were buffer of 1% SDS, 0.1 M NaHCO . For re-ChIP assays, the then washed in ice-cold PBS containing protease inhibitors and immunoprecipitated complexes obtained by ChIP with anti- 1mM phenylmethylsulfonyl fluoride and resuspended in SDS Runx2 antibody were eluted in ChIP elution buffer and then lysis buffer (1% SDS, 10 mM EDTA, pH 8.0, 25 mM Tris-HCl, pH diluted 5-fold and subjected to the ChIP procedure with anti- 8.1, containing protease inhibitors and 1 mM PMSF) for 10 min GFP. The cross-linking reaction was reversed with 5 M NaCl by on ice. Samples were sonicated to reduce the DNA length to 6 h incubation at 65 °C. The samples were digested with pro- 0.5–1 kbp, cellular debris was removed by centrifugation, and teinase K (10 mg/ml), 40 mM Tris-HCl pH 6.5, 10 mM EDTA at the supernatant was diluted 10-fold in dilution buffer (0.01% 42 °C for 1 h, and DNA was recovered by phenol/chloroform SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH extractions. The DNA was precipitated with two volumes of 8.1, 167 mM NaCl supplemented with protease inhibitors). For ethanol using glycogen as carrier. The input lysates were also PCR analysis, aliquots (1:100) of total chromatin DNA before processed as above. The DNA was resuspended in water and immunoprecipitation were saved (input). Prior to chromatin used for quantitative PCR. The sequences of the oligonu- immunoprecipitations, the samples were precleared with 80 l cleotides of the rat MMP-13 promoter (the distal RD and of a 25% (v/v) suspension of DNA-coated protein A/G-agarose proximal AP-1 sites, 204/34) were as follows: forward for 30 min at 4 °C. The supernatant was recovered and used primer 5-CAGATGCGTTTTGATATGCC-3, reverse directly for immunoprecipitation experiments with appropri- primer 5-AATAGTGATGAGTCACCACTT-3. The PCR ate antibody overnight at 4 °C. Immune complexes were mixed reactions and program are described in detail in a previous with 60 l of a 25% precoated protein A/G-agarose suspension publication (25). followed by incubation for1hat4 °C. Beads were collected and Statistical Analysis—All results are expressed as means sequentially washed with 1 ml of each of the following buffers: S.E. of triplicate measurements with all experiments being low salt wash buffer, high salt wash buffer, and LiCl wash buffer. repeated at least three times. Statistical analyses were carried The beads were then washed twice using 1 ml of TE buffer. The out using Student’s t test or one-way ANOVA using the Tukey immunocomplexes were eluted two times by adding elution HSD test. 21342 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 2. PTH causes phosphorylation of a specific site of HDAC4 in the nucleus. A, structure and PKA-dependent phosphorylation sites of human and rat HDAC4 proteins. MEF2 (MEF2C interaction domain): human (118 –313), rat (227– 422), NLS (nuclear localization signal): human (244 –279), rat (353–388), HDA1 (HDA1-related domain): human (655–1084), rat (763–1187), and NES (nuclear export signal): human (1051–1084), rat (1154 –1187). B, nuclear extracts from control or PTH-treated (0, 30, 60 min) UMR 106-01 cells were subjected to immunoblotting with anti-phosphoSer632 hHDAC4, anti-phosphoSer246 hHDAC4, 8 6 and anti-Cdk antibodies. The nuclear extracts from control or PTH (human, 1–34, 10 M, 30 min) or prostaglandin E (10 M, 30 min) treated Saos-2 cells were subjected to immunoblotting with anti-HDAC4, anti-phosphoSer632 hHDAC4, and anti-Cdk antibodies. Anti-Cdk2 was used for the nucleus as a loading control. C, UMR 106-01 cells were transfected with GFP rHDAC4 construct. Cells were stimulated with PTH for 30 and 60 min. The nuclear extracts from control or PTH treated (0, 30, 60 min) UMR 106-01 cells were subjected to immunoblotting with anti-phosphoSer632 hHDAC4, anti-phosphoSer246 hHDAC4, GFP, and anti-Cdk antibodies. Anti-Cdk2 was used for the nucleus as a loading control. D, UMR 106 – 01 cells were preincubated with protein kinase A inhibitor H89 (50 M) for 30 min, and then treated with or without PTH (1–34, 10 M) for 30 min. Nuclear extracts were subjected to immunoblotting with anti-phosphoSer632 hHDAC4 and anti-Cdk antibodies. Anti-Cdk2 was used as a loading control. RESULTS previously shown that HDAC4 suppresses MMP-13 transcrip- PTH Affects the Modification of HDAC4 in the Nucleus tion through its association with Runx2; PTH regulates this through Protein Kinase A—We focused on understanding the repression in osteoblastic cells and this involves PKA (25). effects of PTH on phosphorylation of HDAC4 because it was Phosphorylation and de-phosphorylation control nuclear AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21343 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 3. Ser-740 site of HDAC4 is important for dissociation from Runx2. A, mutation sites of GFP-rHDAC4; Serine residues mutated to alanine. B, UMR 106 – 01 cells were transfected with GFP-rHDAC4 or mutant GFP-rHDAC4 constructs. Cells were stimulated with PTH (1–34, 10 M) for 30 min. Total cellular lysates isolated from UMR 106-01 cells were immunoprecipitated with anti-Runx2 or control rabbit IgG antibodies. Samples were immunoblotted with anti-GFP antibody. The quantitative intensity was obtained using ImageJ. *, p 0.05 versus respective control. C, UMR 106 – 01 cells were transfected with GFP rHDAC4 or mutant GFP rHDAC4 constructs (S355A, S576A, S740A, and T815A). Total cell lysates were immunoblotted with anti-GFP, anti-Runx2, and anti-Cdk antibodies. Anti-Cdk2 was used as a loading control. export and import of HDAC4 (10). To begin deciphering the without PTH. These data indicate that PKA activation may be effects of PTH on HDAC4, we examined the nuclear fractions required for import of HDAC4 to the nucleus as well as the in UMR 106-01 cells after 5–30 min of exposure to PTH. A phosphorylation in the nucleus after PTH treatment, whereas slower-migrating upper band was detected 5 min after PTH PKC and PKD activation may be related to export of HDAC4 to treatment only in the nucleus, which was apparent for up to 240 the cytoplasm. The faster-migrating band in the cytoplasm also min (Fig. 1, A and C). A faster migrating band of HDAC4 was appears to require PKA activation in response to PTH for its detectable in the cytoplasmic fraction after 15 min of PTH stim- generation. ulation which we think is a partial degradation product (Fig. 1, Protein Kinase A Induces the Nuclear Phosphorylation of A and B). We next examined whether PKA affected the appear- HDAC4—PTH causes activation of protein kinase A, which ance of a slower-migrating band of HDAC4 in the nucleus. We phosphorylates proteins on serine/threonine residues in its used the following inhibitors, protein kinase A (H89), protein specific recognition sequence in osteoblastic cells (26). We kinase C (PKC) (GF109203), CaMK (KN62, 92, and 93) and hypothesized that the slower migrating band of HDAC4 in the PKD (Gö6976). As shown in Fig. 1B, H89 prevented the appear- nucleus might be due to phosphorylation by protein kinase A. ance of the slowly migrating band of HDAC4 in the nucleus in To investigate this hypothesis, we performed dephosphory- response to PTH but CaMK inhibitors had no effect, eliminat- lation assays using nuclear extracts from UMR 106-01 cells. ing this pathway in PTH action in the nucleus. GF109203 and The nuclear extracts were incubated with calf intestinal alkaline Gö6976 clearly increased the appearance of HDAC4 in the phosphatase (CIP) for 60 min at 37 °C. The samples were nucleus in the absence and presence of PTH treatment. H89 detected by Western blot analysis. The PTH-induced upper increased accumulation of HDAC4 in the cytoplasm with or band of HDAC4 was decreased by CIP treatment in comparison 21344 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation with the mock treated samples to a molecular mass identical to UMR 106–01 cells were incubated with the purified catalytic the control cells (Fig. 1C). For a more direct confirmation of subunit of PKA for 30 min at 30 °C. After incubation, the sam- whether HDAC4 is phosphorylated by PKA, we performed in ples were detected by Western blot analysis. The upper band of vitro phosphorylation assays. The nuclear extracts from control HDAC4 was increased by this enzymatic activity in comparison AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21345 PTH-induced HDAC4 Phosphorylation and Dephosphorylation with the mock-treated cells. This upper band migrated identi- constructs (S355A, S576A, and T815A) was decreased after cally to that from PTH-treated samples (Fig. 1D). These data PTH stimulation whereas the GFP-rHDAC4-S740A mutant indicate that PTH causes phosphorylation of HDAC4 by acti- remained associated with Runx2 (Fig. 3B). As shown in Fig. 3C, vation of PKA in the nucleus. these protein levels were equally detected when we performed PTH Stimulates Phosphorylation at a Specific HDAC4 Site in Western blots. This result suggests that Ser-740 in rat HDAC4 the Nucleus—Next, we investigated which residues of HDAC4 is a crucial phosphorylation site for dissociation from Runx2. are phosphorylated by PTH in the nucleus. Examination of the To investigate further whether Ser-740 in rHDAC4 is func- database of PKA-dependent phosphorylation sites (Phos- tionally related to dissociation from Runx2 on the runt domain pho.ELM and KinasePhos), as shown in Fig. 2A, illustrated of the MMP-13 promoter, we performed luciferase promoter there were three serine (355, 576 and 740aa) and one threonine assays using the GFP-rHDAC4 constructs. As shown in Fig. 4A, (815aa) residues in rat HDAC4. Interestingly, all sites in the rat wild type GFP-rHDAC4 significantly inhibited MMP-13 tran- HDAC4 were homologous to human sequences and r355 scriptional activity after PTH stimulation similarly to previous (h246), r576 (h467), and r740 (h632) were also CaMK-depen- published data of ours (25). All phosphorylation site mutations, dent phosphorylation sites (Fig. 2A). Ser-246, -467, and -632 in except the S740A mutation inhibited the PTH stimulation to a hHDAC4 are binding sites for 14-3-3 proteins and play a role in similar extent. The S740A mutation in rHDAC4 significantly trafficking HDAC4 from nucleus to cytoplasm (13, 27). We inhibited the promoter activity more than the wild type examined whether phosphorylation of these sites of HDAC4 rHDAC4. To further explore the potential role of HDAC4 was stimulated by PTH using available HDAC4 phosphoryla- interaction with Runx2, we investigated the presence of GFP- tion antibodies. As shown in Fig. 2B, HDAC4 and its phosphor- rHDAC4 or GFP-rHDAC4 mutant proteins with Runx2 at the ylation using anti phospho-Ser-632 (r740) HDAC4 were Runx2 binding site of the MMP-13 promoter by ChIP re-ChIP increased at 30 min after PTH stimulation, whereas phosphor- assays of UMR 106–01 cells with or without PTH stimulation. ylation using anti-phospho-Ser-246 (r355) was not detected in As shown in Fig. 4B, the association of Runx2-GFP-rHDAC4 the nucleus of UMR 106-01 cells. Interestingly, phospho-Ser- and Runx2-GFP-rHDAC4 mutations (S355A, S576A, and 632 HDAC4 was stimulated at 30 min with PTH or prostaglan- T815A) with the MMP-13 promoter was decreased after PTH din E (10 M) treatment in the human osteoblastic cell line, stimulation using primers encompassing the RD and AP-1 sites. The association of Runx2-GFP-rHDAC4-S740A mutation Saos-2 cells. Although total HDAC4 was increased for up to 240 min in UMR cells, phosphorylation at r740 HDAC4 was tran- actually increased with PTH treatment, indicating that the hor- siently increased at 30 min (Figs. 1B and 2, C and D). We have mone-stimulated phosphorylation of this residue is required previously shown that PTH induces the transcription and total for dissociation of HDAC4 from Runx2 on the MMP-13 expression of HDAC4 at later times in these cells (25). Next, we promoter. overexpressed GFP-rHDAC4 constructs in UMR 106-01 cells. Protein Kinase A and Lysosomal Inhibitors Prevent Partial Phosphorylation using anti-phospho-Ser-632 was increased on HDAC4 Degradation—We had seen a faster-migrating band of GFP-HDAC4 and endogenous HDAC4 with PTH treatment HDAC4 in cytoplasmic and total cell lysates which appears to whereas phosphorylation using anti-phospho-Ser-246 on GFP- be a partial degradation product (Fig. 1, A and B) similar to that HDAC4 was decreased (Fig. 2C). In addition the phosphoryla- seen by Backs et al. (24). HDAC6 also slightly decreased after tion at Ser-740 was abolished in the presence of H89, indicating PTH treatment (Fig. 5A). We next examined whether PKA or this is dependent on PKA. Thus, HDAC4 phosphorylation sites other pathways affected the appearance of the faster migrating were differently modified by PTH stimulation. We hypothe- band and potential degradation of HDAC4 in the cytoplasm. sized that a specific phosphorylation site of HDAC4 may be We used protein kinase A (H89) and protein kinase C inhibitors related to dissociation from Runx2 on the MMP-13 promoter. (GF109203), proteasome inhibitors (MG132, lactacystin), and Mutation of Ser-740 in rHDAC4 Prevents Dissociation from caspase-3 inhibitor (Ac-DEVD-CHO) (Fig. 5B). H89 was the Runx2—Next, to confirm our hypothesis, we performed immu- only agent to block HDAC4 degradation (see also Fig. 1B), indi- noprecipitation assays of Runx2 using wild type and mutation cating that HDAC4 degradation occurs via non-proteosomal or constructs of rHDAC4. As shown in Fig. 3A, we made single non-caspase pathways and requires the PKA pathway in PTH point mutation constructs of rat HDAC4 (S355A, S576A, action. S740A, and T815A) which are activated by PKA, CaMK, or Since trafficking and partial degradation of HDAC4 are asso- PKD-dependent pathways. Immunoprecipitated endogenous ciated with phosphorylation, we used the phosphatase inhibi- Runx2 bound to GFP-rHDAC4 (wild type) and four GFP-rH- tor, okadaic acid. The faster migrating band almost disappeared DAC4 mutant constructs under basal conditions. Runx2 bind- with PTH treatment in the presence of this inhibitor (Fig. 5C), ing with GFP-rHDAC4 (wild type) and GFP-rHDAC4 mutant indicating that HDAC4 degradation occurs through serine/ FIGURE 4. GFP-rHDAC4-S740A mutation abolished dissociation from Runx2 on the MMP-13 promoter. A, UMR 106 – 01 cells were transiently transfected with various vectors (100 ng of pGL2 vector, 100 ng of -148 rat MMP-13 promoter-Luc, 100 ng of GFP vector, 100 ng of GFP rHDAC4, and mutant GFP rHDAC4 constructs), and the luciferase activities were measured with (P) or without (C)6hof10 M PTH (1–34) treatment. The luciferase activities were normalized to total protein. Error bars represent S.E. of three independent experiments. *, p 0.05 versus respective control. B, UMR 106-01 cells were transfected with GFP rHDAC4 or mutant GFP rHDAC4 constructs (S355A, S576A, S740A, and T815A). The cells were treated with control or PTH (10 M) for 30 min, then prepared for ChIP assays. After immunoprecipitation of the cross-linked lysates with anti-Runx2 or with rabbit IgG as a negative control, the immune complexes were collected then diluted with ChIP dilution buffer and re-subjected to ChIP assays with anti-GFP or rabbit IgG. The DNA was subjected to PCR with primers that amplify the distal RD and proximal AP-1 sites of the endogenous rat MMP-13 promoter. Input DNA (1:100) is a positive control for the assay. Data are shownas -fold change to control after normalizing to input and IgG. Error bars represent  S.E. of three independent experiments. *, p  0.05 versus respective control. 21346 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 5. PKA, phosphatase, and lysosomal inhibition blocks PTH-induced HDAC4 degradation. A, total cellular lysates (30 g) isolated from 10 M PTH and control-treated UMR 106 – 01 cells for 5, 15, 30, and 60 min were used for Western blot analysis using anti-HDAC 4, 6, or Cdk2 antibodies. Anti-Cdk2 was used as the loading control. B, total cellular lysates isolated from UMR 106-01 cells, which had been preincubated with protein kinase A inhibitor H89 (50 M) for 30 min, protein kinase C inhibitor GF109203 (5 M) for 30 min, proteasome inhibitor MG132 (5 M), lactacystin (5 M), or caspase-3 inhibitor AcDEVD-CHO (100 M) for 60 min, and then with or without PTH (10 M) stimulation for 30 min. Total cellular lysates were used for Western blot analysis using anti-HDAC4. Anti-Cdk2 was used as a loading control. C, total cellular lysates or nuclear/cytoplasmic extracts isolated from UMR 106-01 cells, which had been preincubated with phosphatase inhibitor, okadaic acid (OKA, 50 nM) for 60 min and then with or without PTH (10 M) stimulation for 30 min. Total cellular lysates or nuclear/cytoplasmic extracts were used for Western blot analysis using anti-HDAC4. Anti--actin was used for total cell lysates or cytoplasm as a loading control. Anti-Cdk2 was used as a nuclear loading control. D, nuclear and cytoplasmic extracts isolated from UMR 106-01 cells, which had been preincubated with phosphatase inhibitor, okadaic acid (OKA, 50 nM) for 60 min and then with or without PTH (10 M) stimulation for 30 min. The nuclear and cytoplasm extracts were subjected to immunoblotting with anti-phosphoSer632 hHDAC4, anti-phosphoSer246 hHDAC4, anti-GFP, anti--actin, and anti-Cdk antibodies. Anti-Cdk2 was used for the nucleus and anti--actin was used for cytoplasm as loading controls. E, total cellular lysates isolated from UMR 106-01 cells were preincubated with NH Cl (20 mM) for 16 h and then with PTH (10 M) stimulation for 30 min. Total cellular lysates were used for Western blot analysis using anti-HDAC4. Anti--actin was used for loading control. F, UMR 106 – 01 cells were preincubated with vehicle, serine protease inhibitors, 3.4 DCl (50M) or AEBSF (200M), aspartic protease inhibitor pepstatin A (10M) for 90 min and then with or without PTH (10 M) stimulation for 30 min. Total cellular lysates were used for Western blot analysis using anti-HDAC4. Anti--actin was used as a loading control. threonine dephosphorylation. There was also less HDAC4 pho-Ser-632 (r740) HDAC4 was increased at 30 min after PTH in the cytoplasm. Interestingly, the phosphatase inhibitor stimulation as seen in Fig. 2, and okadaic acid additively increased phospho-Ser-246 (r355) in the nucleus which had increased phospho-Ser-632 (r740) with PTH treatment in the been seen to be reduced with PTH treatment (Fig. 5D). Phos- nucleus. In the cytoplasm, phosphorylation at both sites was AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21347 PTH-induced HDAC4 Phosphorylation and Dephosphorylation FIGURE 6. Model of PKA-dependent phosphorylated regulation of HDAC4. PKA phosphorylates HDAC4 in the nucleus, especially inducing phosphoryla- tion of HDAC4 at serine 740, which results in HDAC4 being released from Runx2 bound to the Runt domain site of the MMP-13 promoter. PKA phosphorylation also induces dephosphorylation of HDAC4 at serine 355 in the nucleus. After transport of HDAC4 into the cytoplasm, HDAC4 is partially degraded through the lysosomal pathway. decreased with PTH and okadaic acid. Thus, dephosphory- to the MMP-13 promoter and exported to the cytoplasm and is lation likely by PP2A is also involved with partial degradation of then dephosphorylated in the cytoplasm and partially degraded HDAC4 and its trafficking from the nucleus in osteoblastic through a lysosomal pathway (Fig. 6). cells. PTH appears to cause the site-selective dephosphory- Work by several laboratories showed that HDAC4 phosphor- lation or phosphorylation of HDAC4 in the nucleus. ylation, binding to 14-3-3 proteins and HDAC4 export to the The intracellular systems of protein degradation are classi- cytoplasm is through phosphorylation by the CaMKII/IV path- fied as lysosomal and non-lysosomal. The lysosomal process way. HDAC4 is also required for hetero-oligomerization with involves uptake into lysosomes, and the non-lysosomal step HDAC5 and its regulation by CaMKII (16, 17). We demonstrate generally involves tagging of proteins prior to degradation by here that PTH induces HDAC4 phosphorylation, especially at the proteasome. Because proteasome inhibitors did not affect Ser-740 in rHDAC4 through PKA activation in the nucleus, but the degradation of HDAC4, we hypothesized that HDAC4 was not by CaMKII/IV as detected by use of inhibitors and antibod- degraded by the lysosome. To assess a relationship with the ies. In addition, protein expression of HDAC5 is very low and lysosome, we examined the effects of the lysosomal inhibitor, PTH does not stimulate phosphorylation of HDAC5 and NH Cl, on HDAC4 mobility in response to PTH. Preincubating HDAC7 in UMR 106-01 cells and rat primary osteoblasts (data cells with NH Cl (Fig. 5E) blocked PTH-induced degradation of not shown). PTHrP or forskolin repress chick chondrocyte HDAC4. Chloroquine and bafilomycine A1 also blocked its hypertrophy through PKA-dependent dephosphorylation of degradation (data not shown). In addition, we used serine pro- hHDAC4 at phospho-S246 by PP2A, which increases the tease inhibitors (3.4 DCl, AEBSF) and an aspartic protease nuclear localization of HDAC4 and inhibits MEF2 function inhibitor (Pepstatin A). AEBSF and Pepstatin A had no effect on (21). Consistent with their data, we found PTH decreases PTH-induced HDAC4 degradation whereas 3.4 DCl increases phosphorylation of Ser-355 in GFP-rHDAC4 (equivalent to the faster migrating band (Fig. 5F). These results indicate that hHDAC4 246S), but we could not detect endogenous phosphor- HDAC4 degradation is associated with lysosomal pathways but ylated Ser-355 rHDAC4 in UMR 106-01 cells, indicating that not serine or aspartic proteases. this requires overexpression of HDAC4. We showed that DISCUSSION dephosphorylation of Ser-355 rHDAC4 is not related to dis- sociation from Runx2 or regulation of the transcriptional The results of this study show the effects of PTH on the activity of MMP-13 (Fig. 4, A and B). However, the phospha- phosphorylation and processing of HDAC4 in rat osteoblastic tase inhibitor, okadaic acid, enhances phosphorylation of Ser- cells. We focused on HDAC4 function, because HDAC4 plays 355 rHDAC4 in the nucleus and this phosphorylation was an important role to suppress chondrocyte and osteoblast dif- reduced after PTH stimulation whereas phosphorylation of ferentiation through association with Runx2 and particularly Ser-740 rHDAC4 was increased by PTH and okadaic acid. suppresses MMP-13 gene expression in vitro and in vivo (7, 25). Phosphorylation of Ser-355 rHDAC4 and 740 rHDAC4 may be We conclude that PKA-dependent phosphorylated HDAC4, especially at Ser-740 in rHDAC4, is released from Runx2 bound regulated inversely by PTH through PP2A and PKA, control- 21348 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014 PTH-induced HDAC4 Phosphorylation and Dephosphorylation ling HDAC4 trafficking out of the nucleus and subsequent par- regulation of HDAC4. It demonstrates a role for HDAC4 in tial degradation. 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(2007) SIK1 is a class II HDAC kinase ysis of HDAC4. J. Cell Biol. 195, 403–415 that promotes survival of skeletal myocytes. Nat. Med. 13, 597–603 21350 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Aug 1, 2014

Keywords: Histone Deacetylase (HDAC); Osteoblast; Parathyroid Hormone; Phosphorylation; Protein Kinase A (PKA)

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