HDAC1 overexpression inhibits steroid-induced apoptosis of mouse osteocyte-like MLO-Y4 cells by inducing SP1 deacetylation

doi:10.12122/j.issn.1673-4254.2025.01.02

Abstract

Abstract:

Objective To explore the mechanism by which histone deacetylase 1 (HDAC1) regulates steroid-induced apoptosis of mouse osteocyte-like MLO-Y4 cells. Methods MLY-O4 cells were treated with 400 nmol/L trichostatin A (TSA) or 1 mmol/L dexamethasone for 24 h or transfected with a HDAC1-overexpressing vector prior to TSA or dexamethasone treatment. The changes in the expressions of HDAC1, SP1, cleaved caspase-3 and Bax, SP1 acetylation level, cell proliferation, and cell apoptosis were examined. The interaction between HDAC1 and SP1 was determined with immunoprecipitation assay and Western blotting. Results Treatment with dexamethasone significantly increased cell apoptosis, enhanced the expressions of cleaved caspase-3 and Bax, reduced HDAC1 expression, and suppressed proliferation of MLO-Y4 cells. Both TSA and dexamethasone obviously increased SP1 acetylation level and the expression of SP1 in MLO-Y4 cells. HDAC1 overexpression in the cells significantly attenuated the effect of TSA and dexamethasone, promoted cell proliferation, lowered the expressions of SP1, cleaved caspase-3 and Bax, and inhibited dexamethasone-induced cell apoptosis. Immunoprecipitation assay and Western blotting demonstrated the interaction between HDAC1 and SP1 in the cells. Conclusion HDAC1 inhibits dexamethasone-induced apoptosis and promotes proliferation of cultured mouse osteocytes by suppressing SP1 expression via promoting its deacetylation.

Key words: steroid-induced osteonecrosis femoral head, SP1, HDAC1, deacetylation

Shenyao ZHANG, Min LU, Gaoyan KUANG, Xiaotong XU, Jun FU, Churan ZENG. HDAC1 overexpression inhibits steroid-induced apoptosis of mouse osteocyte-like MLO-Y4 cells by inducing SP1 deacetylation[J]. Journal of Southern Medical University, 2025, 45(1): 10-17.

Figures/Tables 7

Fig.1 Cell apoptosis and HDAC1 expression in control and dexamethasone-treated mouse osteocyte-like MLO-Y4 cells. A: Cell apoptosis detected by flow cytometry. B: Expression levels of cleaved caspase-3, Bax and HDAC1 in the cells detected by Western blotting. **P<0.01, ***P<0.001.

Tab.1 A450 nm in MLO-Y4 cells in CCK-8 assay 24, 48 and 72 h after dexamethasone treatment (n=3, Mean±SD)

Group	Treatment time (h)			F P
Group	24	48	72	F P
Control	0.59±0.07	1.12±0.09	1.25±0.09	52.89	0.009
Steroid	0.55±0.09	0.76±0.08	0.95±0.09	27.32	0.028
t	0.501	4.299	3.308
P	0.643	0.013	0.03

Fig.2 Identification of transfection efficiency of HDAC1 overexpression vector. A: qPCR for HDAC1 expression; B: western blot for HDAC1 protein. *P < 0.05, ***P<0.001.

Fig.3 Up-regulation of HDAC1 inhibits apoptosis of dexamethasone-treated MLO-Y4 cells. A: Cell apoptosis detected by flow cytometry. B: Expressions of cleaved caspase-3 and Bax detected by Western blotting. *P<0.05, ***P<0.001.

Tab.2 A450 nm at 24, 48 and 72 h after the transfection of HDAC1 overexpressing vector

Group	Treatment time (h)			F	P
Group	24	48	72	F	P
Steroid	0.56±0.07	0.79±0.09	0.96±0.11	47.72	0.016
Steroid+oe-NC	0.45±0.04	0.80±0.09	0.97±0.12	15.12	0.048
Steroid+oe-HDAC1	0.59±0.08*	1.15±0.11*	1.32±0.10*	30.63	0.031
F	2.177	8.92	7.279
P	0.195	0.016	0.025

Fig.4 Identification of the interaction between HDAC1 and SP1 by immunoprecipitation.

Fig.5 Regulation of protein acetylation levels by the HDAC1 axis. A: Cellular acetylation levels detected by immunoprecipitation assay and Western blotting. B: SP1 protein detected by Western blotting. *P<0.05, **P<0.01.

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