Biochemical Journal

Research article

RANKL induces NFATc1 acetylation and stability via histone acetyltransferases during osteoclast differentiation

Jung Ha Kim , Kabsun Kim , Bang Ung Youn , Hye Mi Jin , Ji-Young Kim , Jang Bae Moon , Aeran Ko , Sang-Beom Seo , Kwang-Youl Lee , Nacksung Kim

Abstract

NFATc1 (nuclear factor of activated T-cells c1), a key transcription factor, plays a role in regulating expression of osteoclast-specific downstream target genes such as TRAP (tartrate-resistant acid phosphatase) and OSCAR (osteoclast-associated receptor). It has been shown that RANKL [receptor activator of NF-κB (nuclear factor κB) ligand] induces NFATc1 expression during osteoclastogenesis at a transcriptional level. In the present study, we demonstrate that RANKL increases NFATc1 protein levels by post-translational modification. RANKL stimulates NFATc1 acetylation via HATs (histone acetyltransferases), such as p300 and PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor], thereby stabilizing NFATc1 proteins. PCAF physically interacts with NFATc1 and directly induces NFATc1 acetylation and stability, subsequently increasing the transcriptional activity of NFATc1. In addition, RANKL-mediated NFATc1 acetylation is increased by the HDAC (histone deacetylase) inhibitors sodium butyrate and scriptaid. Overexpression of HDAC5 reduces RANKL- or PCAF-mediated NFATc1 acetylation, stability and transactivation activity, suggesting that the balance between HAT and HDAC activities might play a role in the regulation of NFATc1 levels. Furthermore, RANKL and p300 induce PCAF acetylation and stability, thereby enhancing the transcriptional activity of NFATc1. Down-regulation of PCAF by siRNA (small interfering RNA) decreases NFATc1 acetylation and stability, as well as RANKL-induced osteoclastogenesis. Taken together, the results of the present study demonstrate that RANKL induces HAT-mediated NFATc1 acetylation and stability, and subsequently increases the transcriptional activity of NFATc1 during osteoclast differentiation.

  • cytokine
  • nuclear factor of activated T-cells (NFAT)
  • osteoclastogenesis
  • post-translational modification
  • receptor activator of NF-κB (nuclear factor κB) ligand (RANKL)
  • transcription factor

INTRODUCTION

Bone is a highly dynamic tissue that is strictly maintained by a delicate balance between bone formation and bone resorption under the regulation of systemic factors [1,2]. Two major cell types, osteoclasts and osteoblasts, play an essential role in bone remodelling, as well as skeletal development and calcium homoeostasis [3,4]. Excessive bone resorption by osteoclasts under various pathological conditions leads to osteoporosis. Importantly, osteoporotic fractures are a cause of significant mortality and morbidity in the elderly population, and represent a substantial economic burden to society [5]. Understanding the mechanisms of osteoclast formation is important for the development of new therapeutic strategies against bone diseases.

Osteoclasts are differentiated from haemopoietic cells in the presence of M-CSF (macrophage colony stimulating factor) and RANKL {RANK [receptor activator of NF-κB (nuclear factor κB)] ligand} [1]. Osteoclast precursor cells undergo differentiation to TRAP (tartrate-resistant acid phosphatase)-positive mononuclear cells and fuse to form multinucleated cells that resorb bone [3,4,6].

RANKL, a TNF (tumour necrosis factor) family member, supports osteoclast differentiation, survival and activation. RANKL activates and/or induces various transcription factors including NF-κB, c-Fos and NFAT (nuclear factor of activated T-cells) c1, which act as positive modulators of osteoclast differentiation [4,6]. During osteoclastogenesis, RANKL binding to its receptor up-regulates c-Fos expression. The binding of c-Fos to the NFATc1 promoter region induces NFATc1 gene expression [7,8]. NFATc1 induces the expression of target genes by binding to the NFAT-binding sites in the promoter regions of genes such as TRAP, cathepsin K, OSCAR (osteoclast-associated receptor), the d2 isoform of vacuolar ATPase V0 domain (Atp6v0d2) and DC-STAMP (dendritic cell-specific transmembrane protein), which play important roles in osteoclast differentiation and function [3,911]. Therefore NFATc1 is thought be a key regulator of RANKL-induced osteoclast differentiation, fusion and activation.

Gene expression can be controlled largely by regulating expression of transcription factors, which themselves are tightly regulated by post-translational modifications, such as phosphorylation, ubiquitination and acetylation [12]. The acetylation or deacetylation of substrate proteins is regulated by the balance of activities between HATs (histone acetyltransferases) and HDACs (histone deacetylases) [13]. In contrast with HATs, HDAC families inhibit the acetylation of histone and non-histone proteins. Protein acetylation is a reversible post-translational modification known to be a ubiquitous means for controlling diverse cellular processes, including transcription factor activity [12]. Acetylation can also stabilize proteins because acetylation of lysine residues prevents ubiquitination of the same residues [14,15].

PCAF {p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein]-associated factor} was originally identified as a p300/CBP-binding protein and can form a complex with more than 20 associated polypeptides [16]. The p300/CBP family is one of a major group of HATs that has been extensively characterized. Like p300 and CBP, PCAF acts as a transcriptional co-activator able to acetylate histones and non-histone proteins, such as p53 [17], NF-κB [18], and Smad2, -3, and -7 [19,20]. PCAF itself is auto-acetylated at lysine residues and acetylated by p300, resulting in an increase in its HAT activity [21].

Regulation of NFATc1 is important for the differentiation of osteoclast precursors into multinuclear osteoclasts, but its mechanisms of post-translational regulation have not been known. In the present study, we investigated the post-translational modifications of NFATc1 regulated by RANKL during osteoclastogenesis. We demonstrate that RANKL induces HAT-mediated NFATc1 acetylation, which is important for the stability and transcriptional activity of NFATc1.

EXPERIMENTAL

Reagents and plasmids

Garcinol and sodium butyrate were purchased from Sigma–Aldrich. Scriptaid and nullscript were purchased from Biomol-Enzo Life Sciences. The reporter constructs and expression constructs encoding FLAG- or HA (haemagglutinin)-tagged NFATc1, HA–p300 and FLAG-tagged PCAF have been described previously [9,22,23]. Expression constructs encoding HDAC5 were kindly provided by Dr H. Kook (Chonnam National University Medical School, Gwangju, South Korea). For PCAF-knockdown experiments, oligonucleotides for siRNA (small interfering RNA) were generated by targeting 19 bp sequences of the human PCAF gene (5′-TCGCCGTGAAGAAAGCGCA-3′) or murine Pcaf gene (5′-TCGCCGTGAAGAAGGCGCA-3′) into the pSuper-retro vector (Oligoengine).

Cell cultures

All of the cell culture media and supplements were obtained from HyClone. HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and RANK-expressing HEK-293 cells [24] were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum). Murine BMMs (bone marrow-derived macrophages) were prepared from bone marrow cells as described previously [10]. The experimental protocol was approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. In brief, bone marrow cells were cultured in α-minimal essential medium containing 10% FBS with M-CSF (30 ng/ml) for 3 days. Floating cells were removed and adherent cells (BMMs) were harvested as osteoclast precursors. To generate osteoclasts, BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for the times indicated. Cultured cells were fixed and stained for TRAP as described previously [9]. TRAP-positive MNCs (multinuclear cells) [TRAP(+) MNCs], containing more than three nuclei, were counted.

In vitro HAT assay

Full-length NFATc1 was subcloned into the GST (glutathione transferase) fusion vector pGEX-6P. Recombinant GST–NFATc1 and GST–p300 proteins were expressed in BL21 (DE3) Escherichia coli cells and purified using glutathione beads (Amersham Biosciences). Purification of FLAG–PCAF was carried out as described previously [25]. Acetyltransferase assays were performed as described previously with modification [25]. For assays, GST–NFATc1 proteins were incubated with FLAG–PCAF or GST–p300 proteins and 14[C]acetyl CoA (50 μCi/μl, 1000 pmol/μl, PerkinElmer) for 1 h at 30°C. Reaction products were separated by SDS/PAGE (6% gel) and analysed using a phosphorimager.

Retroviral infection

To generate retroviral stock, retroviral vectors were transfected into the packaging cell line Plat E (a gift from Dr T. Kitamura, Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan) using FuGENE™ 6 (Roche Applied Sciences). Viral supernatant was collected from cultured medium 24–48 h after transfection. BMMs were incubated with viral supernatant for 8 h in the presence of polybrene (10 μg/ml). After removing the viral supernatant, BMMs were incubated with RANKL for the times indicated tmes.

Semi-quantitative RT (reverse transcription)–PCR

Semi-quantitative RT–PCR was performed as described previously [9]. Endogenous NFATc1 mRNA was amplified with a sense primer obtained from the NFATc1-coding sequence (5′-TCTGGGAGATGGAAGCAAAGACTG-3′) and an antisense primer obtained from the NFATc1 3′UTR (untranslated region) sequence (5′-AGGGCTATCACGTGGTGTGAAGAG-3′). The exogenous NFATc1 mRNA was amplified with a sense primer obtained from the NFATc1-coding sequence (5′-GTGCTGTCTGGCCATAACTTTCTG-3′) and an antisense primer designed against the vector sequence (5′-TATGCAGTCGTCGAGGAATTG-3′).

Immunoprecipitation and Western blot analysis

Cells from transfected HEK-293Ts, BMMs or osteoclasts were harvested after washing with ice-cold PBS and then lysed in extraction buffer [50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P40 and 0.01% protease inhibitor cocktail]. Samples immunoprecipitated with the indicated antibodies and whole-cell lysates were subjected to SDS/PAGE (10% gel) and Western blot analysis. Primary antibodies included anti-FLAG, anti-actin (Sigma–Aldrich), anti-HA (Roche Applied Sciences), anti-(acetyl lysine) (Cell Signaling Technology), anti-PCAF and anti-NFATc1 (BD Biosciences). HRP (horseradish peroxidase)-conjugated secondary antibodies (Amersham Biosciences) were used and blots were developed with ECL (enhanced chemiluminescence) solution (Amersham Biosciences). Signals were detected and analysed using a LAS3000 luminescent image analyser (Fuji Photo Film).

Transfection and reporter assay

For transfection of reporter plasmids, HEK-293T cells were plated into 24-well plates (2×104 cells/well) 24 h prior to transfection. Plasmid DNA was mixed with FuGENE™ 6 (Roche Applied Sciences) and transfected into the cells according to the manufacturer's protocol. After 48 h of transfection, the cells were washed twice with PBS and then lysed in passive lysis buffer (Promega). Luciferase activity was measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions.

Statistical analyses

Statistical analyses were performed using the two-tailed Student's t test to analyse differences between groups. P<0.05 was considered statistically significant. Results represent the means and S.D. for three independent replicate experiments.

RESULTS

RANKL increases NFATc1 protein levels via a transcription-independent mechanism

RANKL induces NFATc1 expression during osteoclastogenesis via transcriptional regulation. To investigate whether RANKL regulates NFATc1 expression at the post-translational level, RANK-expressing HEK-293 cells [24] were transfected with FLAG-tagged NFATc1 in the absence or presence of RANKL, and the protein levels of NFATc1 were compared by Western blot analysis. The level of exogenously expressed NFATc1 was increased by RANKL in a dose-dependent manner (Figure 1A). This result was confirmed by retroviral overexpression of NFATc1 in BMMs with RANKL stimulation. Treatment with RANKL strongly induced NFATc1 protein levels in BMMs (Figure 1B), whereas RANKL did not affect the protein level of retrovirally overexpressed c-Src in BMMs (Figure 1C). To rule out the possibility of RANKL-mediated transcriptional induction of exogenously overexpressed NFATc1 gene, we determined the mRNA levels of endogenous and exogenous NFATc1 using RT–PCR. RANKL induced the mRNA expression of endogenous NFATc1, but did not affect the mRNA expression level of exogenous NFATc1 in BMMs (Figure 1D). We also observed that the protein level of retrovirally overexpressed NFATc1 in BMMs was increased by RANKL stimulation in a dose-dependent manner (Figure 1E). These results suggest that RANKL can induce NFATc1 expression levels during osteoclastogenesis by transcriptional activation, as well as by post-translational regulation.

Figure 1 RANKL increases the stability of NFATc1

(A) RANK-expressing HEK-293 cells were transfected with FLAG–NFATc1 and stimulated with various concentrations of RANKL. Lysates were immunoblotted with anti-FLAG and anti-actin antibodies. (B and C) BMMs were transduced with FLAG–NFATc1 (B) or FLAG–c-Src (C) and cultured with RANKL for the times indicated. Lysates were immunoblotted with anti-FLAG and anti-actin antibodies. (D) BMMs were transduced with FLAG–NFATc1 and cultured with RANKL for the times indicated. Total RNA was collected from each time point and analysed by RT–PCR using specific primers for endogenous NFATc1 (Endo-NFATc1), exogenous NFATc1 (Exo-NFATc1) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). (E) BMMs were transduced with FLAG–NFATc1 and cultured with various concentrations of RANKL. Lysates were immunoblotted with anti-FLAG and anti-actin antibodies. Relative intensities of the bands on each gel, measured by densitometry, are shown below each lane. d, days.

RANKL induces NFATc1 acetylation via PCAF

Recently, we found that NFATc1 can be degraded by the ubiquitin–proteasome-mediated pathway [23]. Since acetylation can protect proteins from ubiquitination-mediated degradation [26,27], we examined whether RANKL induces NFATc1 acetylation, thereby stabilizing NFATc1 proteins. RANK-expressing HEK-293 cells were transfected with FLAG-tagged NFATc1, and stimulated with RANKL in the absence or presence of garcinol, a potent inhibitor of HATs such as p300 and PCAF [28]. Whole-cell extracts were immunoprecipitated with anti-(acetyl lysine), and acetylated forms of NFATc1 were detected by Western blotting with an anti-FLAG antibody. As shown in Figure 2(A), acetylation of NFATc1 was strongly induced by RANKL, and RANKL-mediated NFATc1 acetylation was blocked by garcinol. To confirm that RANKL induces acetylation of endogeneous NFATc1 in osteoclasts, we cultured BMMs with M-CSF and RANKL for 2 days in the absence or presence of garcinol and analysed osteoclast samples. As shown in Figure 2(B), acetylation of endogenous NFATc1 was detected in osteoclasts, and garcinol strongly attenuated RANKL-mediated NFATc1 acetylation.

Figure 2 RANKL induces NFATc1 acetylation via PCAF

(A) RANK-expressing HEK-293 cells were transfected with FLAG–NFATc1 and stimulated with RANKL in the absence or presence of garcinol (10 μM). Garcinol was added 6 h before cell lysis. (B) Pre-osteoclasts were derived from BMMs by treatment with M-CSF and RANKL for 2 days in the absence or presence of 10 μM garcinol, added 6 h prior to cell lysis. Lysates were immunoprecipitated with an anti-(acetyl lysine) antibody. Immunoprecipitated samples (top panel) and whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of NFATc1 and actin. (C) RANK-expressing HEK-293 cells were co-transfected with HA–NFATc1 and FLAG–PCAF in the absence or presence of RANKL. Lysates were immunoprecipitated with an anti-(acetyl lysine) antibody. Immunoprecipitated samples (top panel) or whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of NFATc1, PCAF and actin. (D) NFATc1 acetylation assays were performed with or without increasing concentrations of PCAF. An autoradiogram of the acetylation assay with recombinant PCAF and GST–NFATc1, as well as Coomassie Blue staining, are shown. (E) HEK-293T cells were co-transfected with HA–NFATc1 and FLAG–PCAF as indicated. Lysates were immunoprecipitated with an anti-FLAG antibody. (F) Lysates from pre-osteoclasts were immunoprecipitated with IgG or anti-PCAF antibodies. (E and F) Immunoprecipitated samples (top panels) and whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of NFATc1 and PCAF. Relative intensities of the bands on each gel, measured by densitometry, are shown below each lane. Ac-Lys, acetyl lysine; d2, day 2; IP, immunoprecipitation; pOC, pre-osteoclast; WCE, whole-cell extract.

Next, we tested whether HAT proteins, such as p300 and PCAF, can induce NFATc1 acetylation and stability. When RANK-expressing HEK-293 cells were co-transfected with NFATc1 and PCAF, PCAF strongly induced NFATc1 acetylation and stability. In addition, RANKL stimulation further increased NFATc1 acetylation and an accumulation of NFATc1 protein (Figure 2C). Similar results were obtained from p300 (results not shown).

In order to investigate whether PCAF or p300 can directly induce acetylation of NFATc1, we performed an in vitro HAT assay using GST–NFATc1 proteins and purified p300 or PCAF proteins. Addition of increasing amounts of PCAF led to a dose-dependent increase in PCAF-mediated NFATc1 acetylation and auto-acetylation of PCAF (Figure 2D). In contrast, acetylated NFATc1 induced in vitro by p300 was barely detectable (Supplementary Figure S1 at http://www.BiochemJ.org/bj/436/bj4360253add.htm). These results suggest that PCAF is a more significant direct inducer of NFATc1 acetylation than p300.

To examine the interaction of NFATc1 and PCAF, HEK-293T cells were co-transfected with HA–NFATc1 and FLAG–PCAF, and whole-cell extracts were immunoprecipitated with an anti-FLAG antibody followed by Western blot analysis. As shown in Figure 2(E), NFATc1 can interact with PCAF. The interaction between endogenous NFATc1 and PCAF was also confirmed using osteoclast samples (Figure 2F). Taken together, the results suggest that RANKL induced NFATc1 acetylation primarily via PCAF, thereby increasing the stability of NFATc1.

PCAF increases NFATc1 stability and transcriptional activity

Since PCAF interacts with NFATc1 and directly induces acetylation of NFATc1, we investigated the effect of PCAF on the stability of NFATc1 protein. When we examined NFATc1 protein levels by Western blot analysis, the level of NFATc1 protein was increased by PCAF in a dose-dependent manner (Figure 3A). In order to determine the half-life of NFATc1, HEK-293T cells were transfected with NFATc1 and treated with CHX (cycloheximide). The half-life of NFATc1 was approximately 6.5 h, whereas overexpression of PCAF extended it to more than 12 h (Figures 3B and 3C). Next, we examined whether NFATc1 acetylation affects its transactivation activity using a luciferase reporter assay. The NFATc1-mediated transactivation of target genes TRAP and OSCAR was strongly increased by PCAF in a dose-dependent manner (Figures 3D and 3E). These results suggest that PCAF increases the stability of NFATc1 protein via acetylation, thereby increasing the transcriptional activity of NFATc1.

Figure 3 PCAF increases NFATc1 stability and its transcriptional activity

(A) HEK-293T cells were co-transfected with HA–NFATc1 and various concentrations of FLAG–PCAF. Lysates were immunoblotted with anti-FLAG, anti-HA and anti-actin antibodies. Relative intensities of the bands on each gel, measured by densitometry, are shown below each lane. (B and C) HEK-293T cells were co-transfected with FLAG–NFATc1 or FLAG–NFATc1 as well as PCAF, as indicated. Cells were treated with CHX (1 μg/ml) and cultured for the times indicated. (B) Lysates were immunoblotted with anti-FLAG and anti-actin antibodies. (C) Relative intensities of NFATc1 proteins, as determined by densitometry, are shown. (D and E) HEK-293T cells were co-transfected with a TRAP luciferase reporter (D) or an OSCAR luciferase reporter (E) together with NFATc1 and PCAF, as indicated. Results represent the means±S.D. for three independent replicate experiments. *P<0.01 and **P<0.001. (h), hours; Luc, luciferase.

HDAC5 deacetylates NFATc1

Given our observation that NFATc1 can be acetylated, we examined whether HDAC(s) are involved in post-translational modification of NFATc1. Treatment of HEK-293T cells with the HDAC inhibitors sodium butyrate and scriptaid strongly enhanced acetylation of exogenously overexpressed NFATc1, whereas nullscript, a negative control of scriptaid, had no significant effect on NFATc1 acetylation (Figure 4A). We also observed that post-translational modification of endogenous NFATc1 in osteoclasts could be regulated by HDAC(s) (Figure 4B). Next, in order to identify which HDAC(s) are responsible for deacetylation of NFATc1, we analysed the effect of several HDACs on deacetylation of PCAF-induced NFATc1 acetylation. Overexpression of HDAC1, 2 or 4 had no detectable effect on NFATc1 acetylation, whereas HDAC5 and HDAC6 reduced the level of PCAF-mediated NFATc1 acetylation (Figure 4C and results not shown). Among the HDACs tested, HDAC5 had the strongest effect on deacetylation of exogenously overexpressed NFATc1 in HEK-293T cells (Figure 4C). In addition, retroviral overexpression of HDAC5 in osteoclasts reduced the acetylation level of endogenous NFATc1, and this reduction in acetylation was attenuated by sodium butyrate and scriptaid, but not by nullscript (Figure 4D). Immunoprecipitation and Western blot analysis revealed that HDAC5 physically interacts with NFATc1 (Figure 4E).

Figure 4 HDAC5 deacetylates NFATc1

(A) HEK-293T cells were transfected with HA–NFATc1. (B) Pre-osteoclasts were derived from BMMs by treatment with M-CSF and RANKL for 2 days. (A and B) Cells were treated with the HDAC inhibitors sodium butyrate (1 mM) or scriptaid (0.5 μg/ml), or nullscript (a negative control of scriptaid, 0.5 μg/ml). Lysates were immunoprecipitated with an anti-(acetyl lysine) antibody. Immunoprecipitated samples (top panel) or whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of NFATc1 and actin. (C) HEK-293T cells were co-transfected with HA–NFATc1 and FLAG–PCAF, together with empty vector or HA–HDAC5. Lysates were immunoprecipitated with an anti-(acetyl lysine) antibody. Immunoprecipitated samples (top panel) and whole-cell extracts (bottom panels) were subjected to Western blot analysis for the detection of NFATc1, PCAF, HDAC5 and actin. (D) BMMs were transduced with control or HDAC5-expressing retrovirus and cultured with M-CSF and RANKL for 2 days. Cells were treated with the HDAC inhibitors sodium butyrate or scriptaid, or nullscript for 2 h before cell lysis. Lysates from pre-osteoclasts were immunoprecipitated with an anti-(acetyl lysin) antibody. Immunoprecipitated samples (top panel) and whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of NFATc1 and actin. (E) HEK-293T cells were co-transfected with FLAG–NFATc1 and HA–HDAC5 as indicated. Lysates were immunoprecipitated with an anti-HA antibody. Immunoprecipitated samples (top panel) and whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of NFATc1, HDAC5 and actin. (F) HEK-293T cells were co-transfected with FLAG–NFATc1, HDAC5 and PCAF as indicated. Cells were treated with CHX (1 μg/ml) and cultured for the times indicated. Lysates were immunoblotted with an anti-FLAG antibody for detection of NFATc1 expression. (G) HEK-293T cells were co-transfected with an OSCAR luciferase reporter and/or NFATc1, PCAF and HDAC5 in the absence or presence of HDAC inhibitors as indicated. Relative intensities of the bands on each gel, measured by densitometry, are shown below each lane. Results represent the means±S.D. for three independent replicate experiments. #P<0.05, *P<0.01, and ns is not significant. Ac-Lys, acetyl lysine; d2, day 2; IP, immunoprecipitation; NaB, sodium butyrate; pOC, pre-osteoclast; WCE, whole-cell extract.

In order to investigate the effect of HDAC5 on NFATc1 stability, HEK-293T cells were co-transfected with NFATc1, PCAF and HDAC5, and treated with CHX. Overexpression of PCAF strongly increased NFATc1 stability, and this increase was attenuated by overexpression of HDAC5 (Figure 4F). Next, we examined whether HDAC5 can regulate the transcriptional activity of NFATc1 using a luciferase reporter assay. Co-transfection of NFATc1 and PCAF with a reporter plasmid containing luciferase driven by the OSCAR promoter region resulted in an increase in luciferase activity (Figure 4G). However, co-expression of HDAC5 decreased the induction of luciferase activity by NFATc1 and PCAF, and this HDAC5-mediated reduction was blocked by sodium butyrate and scriptaid, but not by nullscript (Figure 4G). Consistent with these results, retroviral overexpression of HDAC5 in BMMs impaired their RANKL-induced differentiation into osteoclasts (Supplementary Figure S2 at http://www.BiochemJ.org/bj/436/bj4360253add.htm). Taken together, our results suggest that HDAC5 can induce NFATc1 deacetylation, thereby down-regulating the transcriptional activity of NFATc1 and diminishing RANKL-induced osteoclast differentiation.

RANKL induces PCAF acetylation and stability

To investigate whether RANKL regulates PCAF expression at the post-translational level, RANK-expressing HEK-293 cells were transfected with FLAG-tagged PCAF in the absence or presence of RANKL, and the protein levels of PCAF were compared by Western blot analysis. The level of exogenously expressed PCAF was increased by RANKL in a dose-dependent manner (Figure 5A).

Figure 5 RANKL induces PCAF acetylation and stability

(A) RANK-expressing HEK-293 cells were transfected with FLAG–PCAF and stimulated with increasing concentrations of RANKL, as indicated. Lysates were immunoblotted with anti-FLAG and anti-actin antibodies. (B) RANK-expressing HEK-293 cells were transfected with FLAG–PCAF and cultured in the absence or presence of RANKL. (C) Pre-osteoclasts were derived from BMMs by treatment with M-CSF and RANKL for 2 days in the absence or presence of garcinol (10 μM). Garcinol was added 6 h before cell lysis. (B and C) Lysates were immunoprecipitated with an anti-(acetyl lysine) antibody. Immunoprecipitated samples (top panel) and whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of PCAF and actin. (D) RANK-expressing HEK-293 cells were co-transfected with a TRAP luciferase reporter and/or NFATc1 and PCAF in the presence or absence of RANKL, as indicated. Relative intensities of the bands on each gel, measured by densitometry, are shown below each lane. Results represent the means±S.D. for three independent replicate experiments. *P<0.01 and **P<0.001. Ac-Lys, acetyl lysine; d2, day 2; IP, immunoprecipitation; pOC, pre-osteoclast; WCE, whole-cell extract.

To examine whether RANKL induces PCAF acetylation, RANK-expressing HEK-293 cells were transfected with FLAG-tagged PCAF, and cultured in the absence or presence of RANKL. Whole-cell extracts were immunoprecipitated with an anti-(acetyl lysine) antibody, and acetylated forms of PCAF were detected by Western blotting with an anti-FLAG antibody. As shown in Figure 5(B), acetylation of PCAF was strongly induced by RANKL. RANKL-induced PCAF acetylation was also detected in osteoclasts, and PCAF acetylation was attenuated by garcinol treatment (Figure 5C).

Next, we examined whether PCAF acetylation affects the transactivation activity of NFATc1 using a luciferase reporter assay. NFATc1 increased TRAP promoter activity and its activity was further enhanced by RANKL treatment or PCAF overexpression (Figure 5D). Moreover, the effects of RANKL and PCAF on NFATc1 transactivation activity were additive. Collectively, our results suggest that RANKL induces PCAF acetylation and stability, and subsequently increases the transcriptional activity of NFATc1.

p300 stabilizes PCAF, which acetylates and stabilizes NFATc1

Next, we examined the effect of p300 on NFATc1 acetylation and stability. HEK-293T cells were co-transfected with HA–NFATc1, FLAG–PCAF and HA–p300, and whole-cell extracts were immunoprecipitated with an anti-(acetyl lysine) antibody followed by Western blot analysis. PCAF strongly induced NFATc1 acetylation, whereas p300 slightly induced NFATc1 acetylation. Notably, p300 strongly enhanced PCAF-mediated NFATc1 acetylation (Figure 6A), suggesting that p300 might enhance PCAF activity. Therefore we investigated whether p300 acetylates PCAF. As expected, p300 induced PCAF acetylation and increased PCAF protein levels (Figure 6B).

Figure 6 Effect of p300 on NFATc1 acetylation and stability

(A) HEK-293T cells were co-transfected with HA–NFATc1, as well as HA–p300 and/or FLAG–PCAF, as indicated. (B) HEK-293T cells were co-transfected with HA–p300 and/or FLAG–PCAF, as indicated. (A and B) Lysates were immunoprecipitated with an anti-(acetyl lysine) antibody. Immunoprecipitated samples (top panel) and whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of NFATc1, PCAF and actin. (C and D) HEK-293T cells were co-transfected with an OSCAR luciferase reporter (C) or a TRAP luciferase reporter (D) together with empty vector, NFATc1, PCAF and/or p300, as indicated. Relative intensities of the bands on each gel, measured by densitometry, are shown below each lane. Results represent the means±S.D. for three independent replicate experiments. #P<0.05, *P<0.01 and **P<0.001. Ac-Lys, acetyl lysine; IP, immunoprecipitation; Luc, luciferase; WCE, whole-cell extract.

Next, we examined the effect of p300 on NFATc1 transactivation activity using a luciferase reporter assay. The NFATc1-mediated transactivation of the promoters of its target genes TRAP and OSCAR was strongly increased by PCAF or p300 (Figures 6C and 6D). Together, PCAF and p300 enhanced NFATc1 transactivation activity more than either one alone. Taken together, our results suggest that p300 increases the stability of PCAF protein via acetylation, thereby increasing PCAF-mediated NFATc1 acetylation and stability.

Down-regulation of PCAF reduces NFATc1 acetylation and stability

Since PCAF acts as a major HAT in RANKL-induced NFATc1 acetylation, we investigated its physiological role in NFATc1 acetylation and stability using PCAF siRNAs. Down-regulation of PCAF expression by siRNA strongly attenuated PCAF- or RANKL-induced NFATc1 acetylation and stability (Figures 7A and 7B). In addition, silencing of PCAF in BMMs significantly inhibited RANKL-induced osteoclastogenesis (Figures 7C and 7D), and NFATc1 acetylation and stability (Figure 7E). These results suggest that PCAF plays an important role in RANKL-induced NFATc1 acetylation, as well as osteoclastogenesis.

Figure 7 Down-regulation of PCAF reduces NFATc1 acetylation and stability

(A and B) HEK-293T cells (A) or RANK-expressing HEK-293 cells (B) were co-transfected with HA–NFATc1, FLAG–PCAF and/or a PCAF siRNA-expressing vector (PCAF-si) in the absence or presence of RANKL, as indicated. Lysates were immunoprecipitated with an anti-(acetyl lysine) antibody. Immunoprecipitated samples (top panel) and whole-cell extracts (bottom panels) were subjected to Western blot analysis for detection of NFATc1, PCAF and actin. (C and D) BMMs were transduced with pSuper-retro (control) or PCAF siRNA-expressing (PCAF-si) retrovirus and cultured for 3 days with M-CSF and RANKL. (C) Cultured cells were fixed and stained for TRAP. (D) Numbers of TRAP-positive MNCs were counted. (E) BMMs were transduced with pSuper-retro (control) or PCAF siRNA-expressing (PCAF-si) retrovirus and cultured for 3 days with M-CSF and RANKL. Lysates were immunoprecipitated with an anti-(acetyl lysine) antibody. Immunoprecipitated samples (top panel) and whole-cell extracts (bottom panels) were subjected to Western blotting for detection of NFATc1, PCAF and actin. Relative intensities of the bands on each gel, measured by densitometry, are shown below each lane. Results represent the means±S.D. for three independent replicate experiments. #P<0.05 and *P<0.01. Ac-Lys, acetyl lysine; IP, immunoprecipitation; WCE, whole-cell extracts.

DISCUSSION

Recently, we discovered that M-CSF induces NFATc1 degradation through Cbl-mediated NFATc1 ubiquitination during late-stage osteoclastogenesis [23]. In the present study, we investigated the post-translational modifications of NFATc1 regulated by RANKL, and elucidated the factors involved in determining the state of post-translational modification of NFATc1. The present study provides the first evidence for the post-translational modification of NFATc1 mediated by RANKL.

NFATc1 is strongly induced by stimulation with RANKL, and its mRNA expression is up-regulated by c-Fos and NF-κB [8,29]. In addition to the role of RANKL in the transcriptional induction of NFATc1 during osteoclastogenesis, we found that RANKL can induce the accumulation of NFATc1, which is regulated by acetylation of NFATc1 protein via a transcription-independent mechanism.

We found that NFATc1 acetylation was mediated by the HAT activity of PCAF through physical interaction with NFATc1 and was enhanced by HDAC inhibitors. Also, RANKL further increased the acetylation of NFATc1 induced by PCAF. Another HAT, p300, also acetylates non-histone proteins and is known to interact with various transcription factors [30,31]. We found that p300 also induces NFATc1 acetylation, but to a much lesser extent than does PCAF. However, p300 did induce PCAF acetylation and stability to a significant degree, thereby increasing NFATc1 protein levels. These results suggest that PCAF functions as a major regulator of RANKL-induced NFATc1 acetylation, whereas p300 may be a lesser regulator of RANKL-induced NFATc1 acetylation via indirect means.

Several previous reports have shown that PCAF-mediated acetylation of various proteins, including E2F1, NF-E4 and p53, increased the function and stability of the target protein [17,32,33]. In the present study, we observed that the forced expression of PCAF increased the stability and transactivation activity of NFATc1. Based on these results, it appears that NFATc1 is a novel substrate of PCAF, which plays a critical role in NFATc1 activation in osteoclasts.

HDACs can inhibit lysine acetylation by removing acetyl groups from their substrates [34]. NFATc1 acetylation by RANKL was strongly enhanced by treatment with HDAC inhibitors during osteoclastogenesis. Conversely, HDAC5 greatly inhibited NFATc1 acetylation. Furthermore, overexpression of HDAC5 reduced the stability and transactivation activity of NFATc1, and attenuated RANKL-induced osteoclast formation. These results indicate that NFATc1 deacetylation by HDACs plays a negative regulatory role in RANKL-induced osteoclast differentiation.

It should be noted that two HDAC inhibitors, trichostatin A and sodium butyrate, have been reported to suppress rather than enhance osteoclast differentiation by blocking the RANKL/TNF-induced activation of NF-κB and MAPK (mitogen-activated protein kinase) signalling pathways [35]. Since HDAC inhibitors block RANKL-induced early signalling pathways, which are upstream of NFATc1, HDAC inhibitors might be predicted to attenuate RANKL-induced osteoclastogenesis, although HDAC inhibitors could induce NFATc1 acetylation which is accompanied by stabilization and transactivation of NFATc1.

Recently, Li et al. [36] also showed that miRNA (microRNA) designed to specifically down-regulate HDAC5 (miR-2861) induced osteoblast differentiation in vivo, whereas it did not affect osteoclast formation [36]. This lack of effect of HDAC5 suppression on osteoclasts in vivo may be due to redundant compensatory mechanisms from other HDAC(s). In the present study, we found that HDAC5 greatly inhibits NFATc1 acetylation, and that HDAC6 inhibits NFATc1 acetylation slightly. However, HDACs 1, 2 and 4 did not affect NFATc1 acetylation. These results indicate that all HDACs do not have the same inhibitory effect on NFATc1 acetylation and osteoclast differentiation. Although HDAC5 can inhibit osteoclast differentiation by inhibition of NFATc1 acetylation, other HDAC(s) may have an effect on osteoclast differentiation through different mechanisms. Such mechanisms may induce osteoclastogenesis without affecting NFATc1 acetylation, since HDAC inhibitors have been reported to suppress osteoclast differentiation, as noted above [35]. Thus further studies are necessary to better understand the effect of each HDAC family protein on NFATc1 acetylation and osteoclast differentiation.

Post-translational modifications, such as phosphorylation, ubiquitination and acetylation, are crucial for regulating the function of many eukaryotic proteins [37,38]. Proteasome-dependent ubiquitination can induce the degradation of target proteins, whereas acetylation can regulate a wide variety of cellular events by inducing the stability of target proteins [38,39]. Because ubiquitination and acetylation are based on the nature of the modified lysine residues, two post-translational modifications targeted to the same residue can generate a great potential for cross-regulation [12]. In previous studies [23], and the present study, we showed that M-CSF and RANKL can regulate the protein expression levels of NFATc1 during osteoclastogenesis by ubiquitination and acetylation processes. Although M-CSF and RANKL are necessary for sufficient osteoclast differentiation and function, they play distinct roles in the post-translational modification of NFATc1. M-CSF might induce ubiquitination of NFATc1 located in the cytoplasm only through activation of cytoplasmic adaptor molecules, the Cbl family proteins [23]. Therefore this raises the possibility that RANKL may protect from the M-CSF-mediated ubiquitination of NFATc1 by NFATc1 acetylation of the same lysine residues. RANKL induces the importation of NFATc1 into the nucleus and, in turn, NFATc1 can be acetylated by PCAF. Further studies will be required to elucidate the mechanisms of cross-regulation of NFATc1 by ubiquitination and acetylation during osteoclastogenesis, and whether post-translational modifications of NFATc1, such as ubiquitination, acetylation and deacetylation, are controlled by NFATc1 localization.

In summary the results of the present study show that a direct interaction between NFATc1 and PCAF in osteoclasts mediates RANKL-induced acetylation of NFATc1 and thereby enhances the stability and transcriptional activity of NFATc1. HDACs such as HDAC5 physically interact with NFATc1 and reverse HAT-mediated acetylation of NFATc1. The results of the present study represent the first direct evidence that RANKL induces the post-translational modification of NFATc1 via HAT-mediated NFATc1 acetylation. Thus our results suggest that the expression level of NFATc1 protein during osteoclastogenesis, which is regulated by the balance between acetylation and deacetylation, is important for osteoclast formation.

AUTHOR CONTRIBUTION

Jung Ha Kim, Kabsun Kim, Bang Ung Youn, Hye Mi Jin, Ji-Young Kim, Jang Bae Moon and Aeran Ko performed the research and analysed the results. Sang-Beom Seo and Kwang-Youl Lee contributed vital reagents and analytical tools. Nacksung Kim designed the research and wrote the paper.

FUNDING

This work was supported, in part, by a Korea Science and Engineering Foundation (KOSEF) National Research Laboratory (NRL) Program grant funded by the Korean government (MEST) [grant number R0A-2007-000-20025-0]; and the Korea Science and Engineering Foundation through the Medical Research Center for Gene Regulation at Chonnam National University [grant number R13-2002-013-03001-0].

Acknowledgments

We thank Dr T. Kitamura for Plat E cells.

Abbreviations: BMM, bone marrow-derived macrophage; CBP, CREB (cAMP-response-element-binding protein)-binding protein; CHX, cycloheximide; FBS, fetal bovine serum; GST, glutathione transferase; HA, haemagglutinin; HAT, histone acetyltransferase; HDAC, histone deacetylase; HEK, human embryonic kidney; HEK-293T, HEK-293 cells expressing the large T-antigen of SV40 (simian virus 40); M-CSF, macrophage colony stimulating factor; MNC, multinuclear cell; NFAT, nuclear factor of activated T-cells; NF-κB, nuclear factor κB; OSCAR, osteoclast-associated receptor; PCAF, p300/CBP-associated factor; RANK, receptor activator of NF-κB; RANKL, RANK ligand; RT, reverse transcription; siRNA, small interfering RNA; TNF, tumour necrosis factor; TRAP, tartrate-resistant acid phosphatase

References

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