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.

Keywords: HDAC inhibitor; bone resorption; butyric acid; osteoblasts; osteoprotegerin/RANKL; periodontal/root canal pathogens.

Figure 1

The stimulation of the histone H3 acetylation of MG63 cells as analyzed by immunofluorescent staining (IF) and Western blotting. (A) IF pictures of Ac-H3 expression: Control (120 min) and butyrate (8 mM)-treated MG-63 cells for 120 min. (B) IF pictures of Ac-H3 expression: Control (24 h) and butyrate (8 mM)-treated MG63 cells for 24 h, 400×, original magnification, (C) Western blotting of control and 8 mM butyrate-treated MG-63 cells for 24 h. One representative IF study result was shown. GADPH: Glyceroaldehyde-3-dehydrogenase. MW: Molecular weight (KD).

Figure 2

Morphologic changes of MG-63 cells (10⁴ cells/well) after exposure to different concentrations of butyrate for three days. (A) Control, (B) 4 mM butyrate, (C) 8 mM butyrate, (D) 16 mM butyrate. 100× original magnification (bar = 100 μm). One representative result was shown.

Figure 3

Effect of butyrate on the cell viability of MG63 cells: (A) MG63 cells (1 × 10,000 cells/24-well) were exposed to butyrate for 3 days, (B) roughly confluent MG63 cells (1 × 100,000 cells/24-well) were exposed to butyrate for three days. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results were expressed as a percentage of control (Mean ± SE). Statistically significant difference when compared with the control (p < 0.05) denoted by *.

Figure 4

Effect of butyrate on the induction of the apoptosis and necrosis of MG63 cells as analyzed by propidium iodide (PI) + annexin V flow cytometry. UL (upper left): Necrosis, UR (upper right) and LR (lower right): Apoptosis. One representative PI and annexin V flow cytometry histogram was shown. (A) Control and (B) 16 mM butyrate-treated cells.

Figure 5

Effect of 24-h exposure to butyrate on the osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression of MG63 osteoblastic cells. (A) RT-PCR analysis of mRNA expression, (B) Western blot analysis of OPG and RANKL protein expression, (C) ELISA analysis of OPG level in culture medium. Statistically significant differences are denoted by * and ** (p < 0.05 and p < 0.01) when compared with the control, respectively.

Figure 6

OPG secretion of MG-63 cells after three days of exposure to (A) butyrate, (B) phenylbutyrate, (C) valproic acid, (D) trichostatin for three days. The OPG level in the culture medium was determined by ELISA and expressed as mean ± SE (pg/mL). Statistically significant differences are denoted by * and ** (p < 0.05 and p < 0.01) when compared with the control, respectively.

Figure 7

Effect of three days of exposure to butyrate on (A) 8-isoprostane, (B) pro-collagen I, (C) MMP-2, (D) osteonectin, (E) osteocalcin, and (F) osteopontin (OPN) secretion of MG-63 cells as analyzed by ELISA. Results were expressed as mean ± SE. Statistically significant differences are denoted by * when compared with the control (p < 0.05). (G) Effect of butyrate on the alkaline phosphatase (ALP) activity of MG-63 cells. One representative ALP staining picture was shown.