Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells

Abstract

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2–4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1–4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

Effects of transforming growth factor‐β1 on plasminogen activation in stem cells from the apical papilla: role of activating receptor‐like kinase 5/Smad2 and mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) signalling

“…Previous studies have found the stimulation of Smad2/3, ERK and p38 signalling pathways by TGF‐β1 in dental pulp cells (Wang et al ). TGF‐β1 may activate Smad2, MEK/ERK and TAK1 phosphorylation in dental pulp cells and SCAP, suggesting its role in regulation of SCAP bioactivity (Chang et al , ). Thus, the role of ALK5/Smad2 and MEK/ERK signallings in regulation of plasminogen activation systems of SCAP was investigated.…”

“…Culture medium was collected for measurement of PAI‐1, uPA and suPAR concentrations by ELISA kits (R & D Systems, Minneapolis, MN, USA) according to the manufacture’s instruction (Chang et al ). Cell number was estimated by 3‐(4,5‐dimethyldiazol‐2‐yl)‐2,5 diphenyl tetrazolium bromide (MTT, Sigma‐Aldrich) assay as described before (Chang et al , , ). In short, MTT was added to SCAP cell culture at a final concentration of 0.5 mg mL −1 and incubated for 2 h. Cultured medium was decanted and then insoluble formazan generated by mitochondrial dehydrogenase of viable SCAP was eluted by 300 μL of DMSO.…”

“…After overnight of cell attachment, cells were exposed to different concentrations of TGF‐β1 for 24 h. Culture medium was aspirated. Cells were rinsed with PBS and then lysis buffer containing 10 mmol L − 1 Tris‐HCl, pH 7; 140 mmol L − 1 sodium chloride; 3 mmol L − 1 magnesium chloride; 0.5% NP‐40; 2 mmol L − 1 phenylmethylsulfonyl fluoride; 1% aprotinin; and 5 mmol L − 1 dithiothreitol (Chang et al , ) was used for dissolving of cells and harvesting of proteins. Equal amounts of protein (30–50 μg/lane) in cell lysates were applied for 12.5% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE).…”

Exogenous and endogenous HDAC inhibitor effects in Rubinstein-Taybi syndrome models

“…We tested three different concentrations for each HDACi (Supplementary file 2) and selected the maximum dose and timing of exposure ensuring acceptable LCLs survival (data not shown). Cells were incubated with vehicles (H2O or DMSO) at maximum time (24h), TSA 2µM for 2h, SAHA 2µM for 24h, VPA 2mM for 24h or NaB 5mM for 24h as suggested from the literature (Supplementary file 2) (Chang et al, 2018;Chriett et al, 2019;Freese et al, 2019;Gottlicher M et al, 2001;Schölz et al, 2015;Tarasenko et al, 2018).…”

Gut microbiota orchestrates PTH action in bone: role of butyrate and T cells

“…2,3 Butyrate, a well-known histone deacetylase inhibitor, has been shown to be a regulator of osteoblast and osteoclast metabolism. 4,5 In vivo, butyrate increased bone mass in mice by inhibiting osteoclast differentiation and bone resorption through metabolic reprogramming of osteoclasts. 5 Because butyrate stimulates Tregs and Tregs are needed for the bone anabolic activity of PTH, the group of Roberto Pacifici decided to examine the roles of the microbiota and butyrate in the regulation of bone responses to intermittent PTH administration.…”