Impaired autophagy triggered by HDAC9 in mesenchymal stem cells accelerates bone mass loss

Background Bone mass loss in aging is linked with imbalanced lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs). Recent studies have proved that histone deacetylases (HDACs) are regarded as key regulators of bone remodeling. However, HDACs involve in regulating BMMSC bio-behaviors remain elusive. Here, we investigated the ability of HDAC9 on modulation of autophagy and its significance in lineage differentiation of BMMSCs. Methods The effects of HDAC9 on lineage differentiation of BMMSCs and autophagic signaling were assessed by various biochemical (western blot and ChIP assay), morphological (TEM and confocal microscopy), and micro-CT assays. Results Sixteen-month mice manifested obvious bone mass loss and marrow fat increase, accompanied with decreased osteogenic differentiation and increased adipogenic differentiation of BMMSCs. Further, the expression of HDAC9 elevated in bone and BMMSCs. Importantly, HDAC9 inhibitors recovered the lineage differentiation abnormality of 16-month BMMSCs and reduced p53 expression. Mechanistically, we revealed that HDAC9 regulated the autophagy of BMMSCs by controlling H3K9 acetylation in the promoters of the autophagic genes, ATG7, BECN1, and LC3a/b, which subsequently affected their lineage differentiation. Finally, HDAC9 inhibition improved endogenous BMMSC properties and promoted the bone mass recovery of 16-month mice. Conclusions Our data demonstrate that HDAC9 is a key regulator in a variety of bone mass by regulating autophagic activity in BMMSCs and thus a potential target of age-related bone loss treatment.

myoblasts a, d p53, p-p53, in skeletal muscle (a) and myoblasts (d) from young and aged mice were examined by western blotting. b, e The expression of HDAC9 in skeletal muscle (b) and myoblasts (e) from young and aged mice were examined by RT-PCR. c, f The expressions of HDAC9 and the levels of acetylation of H3K9 in skeletal muscle (c) and myoblasts (f) from young and aged mice were examined by western blotting. Data are the mean ± s.d. of triplicate samples. *P < 0.05, **P < 0.01, a, c unpaired two-tailed Student's t-test.
sFig. 3 HDACs inhibitors treatment promoted osteogenic differentiation and inhibited adipogenic differentiation in BMMSCs a, b BMMSCs were treated with different dose of HDAC9 inhibitors (TSA (a) or NaB (b)), and the expression of HDAC9 and the levels of acetylation of H3K9 were detected by western blotting.
c Alizarin Red staining was performed and osteogenesis-related proteins were detected with western blotting in aged BMMSCs treated with HDACs inhibitors, TSA or NaB. d Oil Red O staining was performed and adipogenesis-related proteins were detected with western blotting in aged BMMSCs treated with HDACs inhibitors, TSA or NaB. Scale bars = 100 μm. *P < 0.05, **P < 0.01, One-way analysis of variance (ANOVA).

sFig. 4 The silence efficiency of HDAC9
a The expression of HDAC9 mRNA were examined by qRT-PCR in aged BMMSCs after transfected with an HDAC9 siRNA for 48 hours. b, c Protein level of HDAC9 (b) and acetylation of H3K9 (c) were examined by western blotting in aged BMMSCs after transfection with an HDAC9 siRNA for 48 hours. d HDAC9 mRNA expression was measured by qRT-PCR 3 days and 7 days after siHDAC9 transfection in aged BMMSCs. e HDAC9 protein expression was measured by western blotting 3 days and 7 days after siHDAC9 transfection in aged BMMSCs. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. *P < 0.05, **P < 0.01. a unpaired two-tailed Student's t-test. d One-way analysis of variance (ANOVA).
sFig. 5 HDAC9 siRNA restored the number of autophagosomes in aged BMMSCs a Transmission electron microscopy (TEM) was used to detect autophagosomes in young and aged BMMSCs and cells treated with chloroquine (CQ). Scale bars = 1 μm. b Transmission electron microscopy (TEM) was used to detect autophagosomes in aged BMMSCs respectively transfected with Nc siRNA, HDAC9 siRNA and cells treated with CQ, respectively. Scale bars = 1 μm. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. *P < 0.05, ** P < 0.01, *** P < 0.001, One-way analysis of variance (ANOVA).

sFig. 6 HDAC9 siRNA treatment decreased the levels of acetylation of H3K9 in aged BMMSCs cultured in vitro
To evaluate the effect of HDAC9 removing acetyl groups from H3K9, the expression of HDAC9 and the levels of acetylation of H3K9 were examined by western blotting in aged BMMSCs transfected with an HDAC9 siRNA and those cells treated with CQ. The data are presented as the means ± s.d. of each independent experiment performed in triplicate.

sFig. 7 The silence efficiency of BECN1 in aged BMMSCs cultured in vitro
Protein expression of Beclin1 was examined by western blotting in aged BMMSCs transfected with a BECN1 siRNA 48 hours later. The data are presented as the means ± s.d. of each independent experiment performed in triplicate.

sFig. 8
The thickness of growth plate in femora were no changes in aged mice treatment with HDAC9 shRNA lentivirus The chondrocytes in growth plate of femur were recognized with Alcian blue staining and thickness of growth plate was quantitative analyzed. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. One-way analysis of variance (ANOVA).