Jianqi Wang,Shuainan Wu,Lu Liu,Ying Pang,Zhaobao Li,Hong Mu
HDAC5-Mediated Acetylation of p100 Suppresses Its Processing
HDAC5,Inflammation,Inhibition,Periodontitis
Introduction Periodontitis is a condition involving chronic inflammation in the gums, periodontal ligaments, cementum, and alveolar bone. Nuclear factor-κB (NF-κB) activation is the prominent mediator of inflammation and osteoclast differentiation. The role of histone deacetylase 5 (HDAC5) in periodontitis development and NF-κB regulation is not fully understood. Methods We used primary mouse bone marrow–derived osteoclast cultures in vitro and a mouse model of chronic periodontists (CPD) treated with the HDAC4/5 inhibitor LMK-235. Real-time polymerase chain reaction, micro computed tomography, flow cytometry, western blot, and immunoprecipitation were used to study proinflammatory cytokines, NF-κB activation, HDAC5 activity, and the interaction of HDAC5 with NF-κB p100. Results LMK-235, a selective inhibitor of HDAC4 and HDAC5, reduced osteoclast marker gene expression (Cstk, Acp5, and Calcr) and tartrate-resistant acid phosphatase activity in primary osteoclast cultures. LMK-235 reduced the increase in cementoenamel junction–alveolar bone crest distance, inflammatory cell infiltration of gingival tissues, and expression levels of interleukin (IL)-1β, tumor necrosis factor alpha, IL-6, and IL-23a, indicating an ameliorative effect on CPD. Immunoprecipitation experiments have further confirmed p100–HDAC5 interaction, acetylation levels of p100, and NF-κB activation. Conclusions These results indicate that HDAC5 binds and deacetylates p100, leading to its activation, increased proinflammatory cytokine production, gingival infiltration, and osteoclast differentiation, thus promoting alveolar bone resorption. HDAC5 inhibition is therefore a potentially promising therapeutic strategy for the treatment of periodontitis.
HDAC5-Mediated Acetylation of p100 Suppresses Its Processing

Periodontitis is a condition involving chronic inflammation in the gums, periodontal ligaments, cementum, and alveolar bone. Nuclear factor-κB (NF-κB) activation is the prominent mediator of inflammation and osteoclast differentiation. The role of histone deacetylase 5 (HDAC5) in periodontitis development and NF-κB regulation is not fully understood.

We used primary mouse bone marrow–derived osteoclast cultures in vitro and a mouse model of chronic periodontists (CPD) treated with the HDAC4/5 inhibitor LMK-235. Real-time polymerase chain reaction, micro computed tomography, flow cytometry, western blot, and immunoprecipitation were used to study proinflammatory cytokines, NF-κB activation, HDAC5 activity, and the interaction of HDAC5 with NF-κB p100.

LMK-235, a selective inhibitor of HDAC4 and HDAC5, reduced osteoclast marker gene expression (Cstk, Acp5, and Calcr) and tartrate-resistant acid phosphatase activity in primary osteoclast cultures. LMK-235 reduced the increase in cementoenamel junction–alveolar bone crest distance, inflammatory cell infiltration of gingival tissues, and expression levels of interleukin (IL)-1β, tumor necrosis factor alpha, IL-6, and IL-23a, indicating an ameliorative effect on CPD. Immunoprecipitation experiments have further confirmed p100–HDAC5 interaction, acetylation levels of p100, and NF-κB activation.

These results indicate that HDAC5 binds and deacetylates p100, leading to its activation, increased proinflammatory cytokine production, gingival infiltration, and osteoclast differentiation, thus promoting alveolar bone resorption. HDAC5 inhibition is therefore a potentially promising therapeutic strategy for the treatment of periodontitis.

Periodontitis (PD) is a chronic inflammatory condition mediated by the bacterial flora of the oral cavity. It may affect all parts of the tooth, including gums, periodontal ligaments, cementum, and alveolar bone., Bone destruction resulting from osteoclast activation in advanced PD eventually results in tooth loss., Inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) facilitate the gingival tissue infiltration with the CD45-/CD11b-positive myeloid cells., Osteoclast differentiation from monocytes/macrophage precursors is positively regulated by macrophages, colony-stimulating factor (CSF)-1, IL-23a, and the secretion of receptor activator for nuclear factor-κB (NF-κB) ligand (RANKL) that interact with osteoclast receptor activator for NF-κB (RANK). Distinct from predominantly proinflammatory canonical NF-κB activation that is based on IκBα, RelA, and NF-κB p50 complex formation and p50 subunit nuclear translocation, transcription factors activated in the noncanonical NF-κB pathway p52/RelB NF-κB complex depend on their precursor nuclear factor NF-κB p100 subunit (p100), the precursor and feedback inhibitor of p52 and RelB. RANKL-RNAK ligation activated NF-κB–inducing kinase (NIK), which phosphorylates IκB kinase-α, triggering p100 phosphorylation and processing., RANKL-RANK–activated noncanonical NF-κB p100/p52 is the essential pathway in osteoclastogenic bone disease. Regulators of this pathway have therapeutic potential for inflammation-related bone disease, including periodontic diseases. Histone deacetylases (HDACs) play a crucial role in gene expression regulation by modifying chromosome structural modification through histone deacetylation. HDAC has also been shown to regulate protein function through direct binding and interaction. LMK-235 is a potent, selective inhibitor of HDAC4/5. Inhibition of HDACs was demonstrated to reduce NF-κB p65 activation in vitro. In this study, we explore the effect of LMK-235 on p100 deacetylation and its effect on PD and osteoclast differentiation.

All animal experiments were approved by the ethics committee of Cangzhou Central Hospital and performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals. Wild-type (WT) mice on C57BL/6 background at 8 weeks old were purchased from Cyagen Biosciences Inc. B6N.129-Map3k14 tm1Rds /J (NIK−/−) mice (025557) on B6/129 background at 8 weeks old were purchased from the Jackson Laboratory.

LADMAC (ATCC) was used as a source of CSF-1 for bone marrow macrophage differentiation. LADMAC cell–conditioned media (LCM) was produced as reported previously.

The tibial bones were carefully dissected from the WT and NIK-/- mice. Bone marrow cells were flushed from femurs and tibia of age-matched mice (6–8 weeks) by perfusion with 2 mL of phosphate buffer saline (PBS, Gibco). The resulting cell suspension was further dissociated by pipetting and centrifuged at 300 g. Bone marrow–derived macrophages were differentiated by culturing bone marrow cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum and 30% LCM for 4 to 6 days. Osteoclast differentiation was induced by culturing bone marrow–derived macrophages in LCM supplemented with mouse RANKL (Thermo Fisher Scientific; 50 ng/mL) for 4 days.

Primary osteoclast culture cells were treated with LMK-235 (5, 10, and 20 nM; SelectChem) by adding LMK-235 to RANKL differentiation media. Dimethyl sulfoxide (DMSO) was used as a control. Primary osteoclast cultures were pretreated with LMK-235 (20 nM) for 2 hours and then stimulated with lipopolysaccharide (LPS; Escherichia coli 055:B5; Sigma-Aldrich) 1 µg/mL for 6 and 24 hours or Pam3csk4 (tlrl-pms; InvivoGen) for 6 hours.

The total RNA was extracted using an RNeasy Plus Mini Kit (Qiagen) according to the kit protocol. The RNA was reverse-transcribed to complementary DNA (cDNA) using Maxima H Minus cDNA Synthesis Master Mix with double-strand–specific DNase (dsDNase; Thermo Fisher Scientific). Gene expression was detected using Power SYBR Green Master Mix (Thermo Fisher Scientific) with the mouse-specific primers using 10 ng of the initial cDNA template. Amplicons were detected by QuantStudio 3 real-time polymerase chain reaction (RT-PCR; Applied Biosystems). Cycle-threshold values of the target genes were normalised to GAPDH, and the fold change was calculated using the (2−ΔΔCT) method.

An Acid Phosphatase Assay Kit (Abcam) detected TRAP activity. Briefly, cells were washed with PBS and lysed in citrate buffer + 0.1% Triton X-100 for 5 minutes of incubation at room temperature. The lysates were transferred to 96 well plates. Then 100 µL p-nitrophenyl phosphate and disodium salt were added to the wells. After another 5 minutes of incubation, the reaction was stopped by adding 50 µL 0.5 M NaOH. The optical density (OD) was detected at a 405-nm absorption peak using an 800 TS Absorbance Reader (BioTek Instruments). The OD in DMSO treated control was considered as 100% osteoclastogenesis.

The mice were anaesthetised with ketamine hydrochloride (100 mg/kg body weight) and xylazine (4 mg/kg body weight). Subgingival tissues of first and second molars (M1 and M2) were ligated with silk sutures (3/0; Johnson & Johnson Medical Ltd) soaked in propylene glycol as the continuous loop on one side of the maxilla bone. Water in cages was supplemented with 5% high-sucrose drinking water for 4 weeks. Four weeks later, the ligation was removed and LMK-235 (S7569; Selleckchem) 20 mg/kg was administered via oral gavage for 4 weeks.

CEJ-ABC was analysed by rodent µCT (Rigaku Corporation). The imaging was performed at 90 kV and 200 mA for 3 minutes with a slice thickness of 50 µm. Two-dimensional (2D) images were acquired and analysed using TRI/3D-BON (Ratoc System Engineering Co., Ltd.).

At the experimental end point, mice were sacrificed by CO2 inhalation, and gingival tissues were collected (10 mice per group). Gingival tissues were dissected and dissociated by maceration and incubation with Trypsin-EDTA 0.25% (Thermo Fisher Scientific) and DNase and collagenase II for 40 minutes; the resulting cell suspension was washed with PBS and incubated with 2.5% bovine serum albumin (BSA) in PBS for 15 minutes and then with rat CD45 Alexa Flour 488 and CD11b APC for 30 minutes and washed with PBS. The negative population gate was set against cell suspension and incubated for 30 minutes with rat IgG2b kappa Alexa Flour 488 and Mouse IgG1 APC Isotype Control. The stained cell populations were recorded using the CyanADP flow cytometer (Beckman Coulter), and the resulting 2D plots were analysed using FlowJo (FlowJo LLC).

IL-1b assay kit (R&D Systems), IL-6 assay kit (R&D Systems), TNF-α assay kit (R&D Systems), and IL-23a assay kit (Abcam) were used according to the kit instructions. OD was detected using Synergy H1 (Biotek) recorded at 450 nm.

Western blot was performed as previously described., Osteoclast cultures were washed with PBS and lysed by radioimmunoprecipitation assay buffer containing FAST protease inhibitors (Sigma-Aldrich). The protein concentration was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), and 30 µg of total protein per lane was separated by 4%–20% gradient sodium dodecyl sulfate–polyacrylamide gel. Separated proteins were transferred to 0.45-μm pore-size nitrocellulose membranes, with 30 to 50 μg total protein per lane. The membranes were blocked with 5% BSA (Sigma-Aldrich) at room temperature for 1 hour and then incubated overnight at 4 °C with the primary antibodies. After being washed with a PBS-Tween 20 buffer (Sigma-Aldrich), the blots were incubated with a horseradish peroxidase–conjugated secondary antibody (1:1000; Biorad) for 1 hour at room temperature. The signals were developed with enhanced chemiluminescence with a SuperSignal West Pico Kit (Thermo Fisher Scientific).

HEK293T (CRL-3216) cells were purchased from ATCC and grown in complete DMEM. HDAC5-flag plasmid (Addgene) and human influenza hemagglutinin (HA)-tagged p100 plasmid (Addgene) were transfected to mouse HEK293 cells using a lipofectamine 3000 transfection kit (Thermo Fisher Scientific). Briefly, 3 µL lipofectamine 3000 (100022052, Thermo Fisher Scientific) was diluted in 250 µL Opti-MEM Reduced Serum Medium (31985062; Thermo Fisher Scientific). The reagent P3000 (100022058; Thermo Fisher Scientific) was diluted in 250 µL Opti-MEM, and 1 µg of HDAC5-flag DNA was combined with reagent P3000. The solutions were gently mixed by inverting the tubes. After 10 minutes of incubation at room temperature, the mixture was added to HEK293T cells and incubated overnight. HEK cells were harvested for pull-down assays into Pierce TM IP Lysis buffer (87787; Thermo Fisher Scientific) supplemented containing FAST protease inhibitors (Cat#: S8830, Sigma-Aldrich). The 500 μg total protein was incubated with 2.5 µg of FLAG antibody or rabbit immunoglobulin G control at 4 °C overnight. Then immune complexes were conjugated with a 25-µL Protein A/G bead (Santa Cruz Biotechnology) for 2 hours. Proteins were eluted with RIPA buffer supplemented with β-Mercaptoethanol. The β-actin from total lysate was used as the loading control. For the whole osteoclast acetylation experiment, native p100 was pulled down by the p100 antibody from 500 μg of osteoclast cultures lysates by overnight incubation at 4 °C, and the acetylation level was determined by the acetylated-lysine antibody.

The statistical analysis and bar graph design were performed using GraphPad Prism software. The results are presented as mean ± SD. One-way analysis of variance (ANOVA) analysed multiple groups with Tukey multiple comparisons, and the 2 groups were compared with an unpaired t test (2-tailed). A P value <.05 was considered significant.

To explore the regulatory mechanism of noncanonical NF-κB signaling in osteoclasts cells, we analysed the binding protein of p100 through STRING interaction network analysis (https://string-db.org/); we found that the p100 molecule can bind to multiple proteins, including HDAC4 and HDAC5 (Figure 1A). To verify the functions of the above molecules, we used the HDAC4/5 inhibitor LMK-235 to treat RANKL-induced primary osteoclast cultures. LMK-235 (10, 20 nM) significantly inhibited the osteoclast-specific genes Acp5, Calcr, and Cstk (Figure 1B) at mRNA levels. Furthermore, alkaline phosphatase activity, the main protein level indicator of osteoclastogenesis, was also significantly reduced by LMK-235 treatment in a dose-dependent manner (Figure 1C).

Treatment with LMK-235 (20 mg/kg) for 4 weeks significantly reduced the cementoenamel junction–alveolar bone crest (CEJ-ABC) distance increase as analysed by µCT when compared with vehicle-treated controls (Figure 2A). Interestingly, NIK−/- mice with Map3k14 ablation and reduced noncanonical NF-κB activation did not experience significantly increased CEJ-ABC distance, nor were these mice significantly affected by LMK-235 treatment (Figure 2A). Flow cytometry analysis of dissociated gingival tissues in the chronic periodontis (CPD) model demonstrated substantial reduction of myeloid (CD45/CD11b) positive immune cell infiltration in LMK-235–treated mice (Figure 2B). Consistently, RT-PCR analysis of the mRNA extracted from gingival tissue of CPD mice similarly indicated a significant reduction of IL-1b, TNF-α, IL-6, and IL-23a transcription in LMK-235–treated mice (Figure 2C).

Primary osteoclast cultures were pretreated with LMK-235 and stimulated with LPS for 6 hours, inducing both canonical and noncanonical NF-κB activation. The mRNA levels of IL-1b, IL-6, TNF-α, and IL-23a were determined by RT-PCR (Figure 3A). LMK-235 treatment did not affect the transcription of IL-1b, IL-6, or TNF-α but significantly reduced IL-23a gene levels. The mRNA-level data were further confirmed by ELISA detection of the respective protein expression following 24 hours of LPS stimulation, achieving the same results with only IL-23a being downregulated in LMK-235 (Figure 3B), but not other proinflammatory cytokines. Similar results were achieved when osteoclast cultures were pretreated with LMK-235 and then stimulated with the noncanonical NF-κB activator Pam3csk4 for 6 hours. Consistent with LPS results, only IL-23a was affected by the LMK-235 treatment (Figure 3C), but it did not affect the expression of other cytokines. These data indicate that LMK-235 predominately affects the noncanonical NF-κB activation pathway.

To study the effect of inhibiting HDAC5 on noncanonical NF-κB pathways, we pretreated primary cultured osteoclasts with LMK-235 at 20 nM before RANKL stimulation for 0, 12, and 24 hours. The NF-κB p52 subunit decreased in the nuclear fraction, indicating activation of the noncanonical NF-κB pathway with the inhibition of HDAC4/5 (Figure 4A). The interaction of HDAC5 with p100 was further confirmed by the overexpression of Flag-tagged HDAC5 and human influenza HA-tagged p100 in HEK293 cells following Flag antibody pull-down, and HA-p100 was strongly detected in the pull-down lanes HDAC5 (Figure 4B). The increase of acetylation levels of p100 was further confirmed by p100 antibody pull-down from primary osteoclast cultures and detection of the increase of acetylated-lysine in the pull-down lanes of LMK-235–treated cultures (Figure 4C).

Consistent with Figure 1C, primary bone marrow cultures isolated from WT animals demonstrated decreased osteoclast markers Acp5, Clcr, and Cstk mRNA levels when treated with LMK-235. However, bone marrow cultures isolated from NIK−/− mice indicated baseline decreases in mRNA levels of osteoclast markers, which were not significantly affected by LMK-235 treatment (Figure 4D), suggesting impaired NF-κB activation and osteoclast differentiation in NIK−/− mice.

HDAC inhibition has been shown to reduce chronic inflammatory diseases, including inhibition of the RANKL-RANK pathway. HDACs are major gene expression regulators; some HDAC members interact with transcription factors directly. In the current study, we demonstrated that inhibition of HDAC5 by LMK-235 reduced osteoclast differentiation marker expression in vitro and ameliorated experimental CPD. Increased RANK expression sensitised precursor cells for osteoclastic differentiation. NF-κB signaling participates in the regulation of osteoclast differentiation, activity, and survival., The inhibitory protein IκB localises NF-κB in the cytoplasm until it is activated by dimerising with Rel family proteins, promoting nuclear translocation of the p52 subunit. Noncanonical NF-κB signaling, however, leads to ubiquitination of p100, but instead of proteasomal degradation, p100 is processed to p52 and the resulting RelB:p52 heterodimers translocate to the nucleus. In osteoclastogenesis expression, p52 levels increase within 2 hours after RANKL treatment and remain increased without changes in RelA or p50 mRNA levels. Our overexpression experiments in HEK293 cells definitively demonstrated the direct interaction of HDAC5 with the p100 subunit of the NF-κB pathway, and LMK-235 inhibition of HDAC5 results in an increase of p100 acetylation well as reduction of NF-κB p52 nuclear presence, presumably due to proteasomal degradation of acidulated p100, establishing deacetylation as the regulatory factor for noncanonical NF-κB activation. NIK-/- mice that lack p52 signaling indicate resistance to antigen-induced arthritis resulting from T cell responses., Indeed, in our study, NIK-deficient mice did not indicate substantial PD development and were unaffected by LMK-235 treatment in vivo and primary osteoclast cultures, indicating that the NIK is part of the RANKL/RANK axis upstream of HDAC's regulation (Figure 5). In CPD in vivo, we observed decreased expression of proinflammatory cytokines IL-1b, TNF-α, IL-6, and IL-23a in gingival tissues. However, in the primary osteoclast cultures, when stimulated by LPS which induces both canonical and noncanonical NF-κB or Pam3CSK4 which only induces noncanonical NF-κB pathway activation, only IL-23a expression was inhibited by LMK-235 treatment. Infiltrating monocytes and macrophages are the tissue's main sources of these inflammatory cytokines mediated by toll-like receptor (TLR) signaling. In the osteoclast cultures, however, IL-23a is upregulating RANK expression and mediating osteoclast-like cells, indicating an IL-23a–mediated positive feedback loop in osteoclasts that in turn facilitates activation and recruitment of macrophages. These results may also suggest the more broad effect of LMK-235 in other cell types, including infiltrating macrophages in the gingival tissues. Further studies include replicating the effects of LMK-235 in the human microflora inoculation model in rats and testing LMK-235 on the nonrodent model of PD. On the molecular level, the combination of LMK-235 with TLR inhibitors may potentiate the effects of HDAC5 inhibition, as both arcs of NF-κB activation through colony stimulating factor receptor and TLR would be disrupted. Investigation of the effects of cytokines through recombinant cytokines and macrophage condition media on LMK-235–treated osteoclasts, as well as the direct effect of LMK-235 on macrophages, would allow further understanding of the role of HDAC5 inhibition and p100 acetylation in the PD inflammatory process. Currently, the treatment of PD is limited to surgical removal of the affected tissue, topical and oral antibiotic use, anti-inflammatory treatment, and enhanced oral hygiene. None of the treatment protocols include direct modulation of bone resorption and osteoclast activity. Currently, several ongoing clinical trials already use HDAC inhibitors in human patients, predominantly in cancers such as multiple myeloma. Whilst systemic application of LMK-235 for the treatment of PD may be undesirable due to potential side effects and systemic toxicity, the topical use in combination with antibiotic treatment or a small dose intraperiodontal pocket delivery may mitigate the issue of systemic side effects, opening a new prospect for PD therapies that directly target osteoclast activity and inflammatory infiltration of the gum tissues.

The LMK-235 inhibitor used in this study inhibits HDAC4 and 5. Our in vitro experiments have ascertained the functions of HDAC5 in noncanonical NF-κB activation. However, the HDAC4 contribution to the observed in vivo and in vitro results requires further study. Primary osteoclast cultures were validated by the expression of osteoclast-specific markers Acp5, Calcr, and Cstk and TRAP activity. However, we can not fully exclude the possibility of the presence of a small population of the other cell types. The ligature-induced PD model is well established and accepted. However, it includes a substantial inflammatory component of the foreign body presence. The effect of HDAC5 inhibition on other animal models of PD, such as microflora inoculation, warrants further investigation.

This work has identified HDAC5 as a direct regulator of NF-κB p100 subunit deacetylation which facilitates p52 noncanonical NF-κB activation as part of osteoclast-mediated bone resorption and PD development.

None disclosed.