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. 2020 Jun;77(12):2407-2421.
doi: 10.1007/s00018-019-03289-w. Epub 2019 Sep 12.

Histone demethylase KDM4A regulates adipogenic and osteogenic differentiation via epigenetic regulation of C/EBPα and canonical Wnt signaling

Qi Qi  1 , Yi Wang  1 , Xiaochen Wang  1 , Junying Yang  2 , Yan Xie  1 , Jie Zhou  1 , Xiaoxia Li  2 , Baoli Wang  3
Affiliations

Histone demethylase KDM4A regulates adipogenic and osteogenic differentiation via epigenetic regulation of C/EBPα and canonical Wnt signaling

Qi Qi et al. Cell Mol Life Sci. 2020 Jun.

Abstract

Epigenetic modifications play a central role in cell differentiation and development. In the current study, we have recognized lysine demethylase 4A (KDM4A) as a novel epigenetic regulator of osteoblast and adipocyte differentiation. Kdm4a expression was upregulated during osteogenesis and adipogenesis of primary marrow stromal cells and established stromal ST2 line. Overexpression of wild-type Kdm4a promoted adipogenic differentiation and blocked osteogenic differentiation of the progenitor cells. This effect was largely alleviated when the catalytically dead mutation was made. Conversely, depletion or inactivation of Kdm4a in undifferentiated progenitor cells inhibited the formation of adipocytes and promoted the differentiation of osteoblasts. Mechanism explorations showed that overexpression of Kdm4a upregulated the expression of secreted frizzled-related protein 4 (Sfrp4) and CCAAT/enhancer-binding protein α (C/ebpα). Chromatin immunoprecipitation assay demonstrated that KDM4A directly bound the promoters of Sfrp4 and C/ebpα, removed the histone methylation mark H3K9me3, and reduced DNA methylation levels of CpG in promoter regions of C/ebpα and Sfrp4. Furthermore, overexpression of Kdm4a inactivated canonical Wnt signaling. Moreover, activation of canonical Wnt signaling through silencing of Sfrp4 in ST2 attenuated the inhibition of osteogenic differentiation and the enhancement of adipogenic differentiation by KDM4A. These data have identified KDM4A as a novel regulator of osteoblast and adipocyte differentiation and suggest KDM4A inhibition as a potential therapeutic target for treating metabolic disorders such as osteoporosis.

Keywords: Adipocyte; CCAAT/enhancer-binding protein α; Differentiation; Lysine demethylase 4A; Osteoblast; Secreted frizzled-related protein 4; Wnt/β-catenin.

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Conflict of interest statement

The authors have declared no conflict of interest.

Figures

Fig. 1
Fig. 1
Kdm4a expression increased during osteogenic and adipogenic differentiation. Kdm4a expression was examined using RT-PCR in various tissues of mice. The level of Kdm4a in intestine was set to 1 (a). Kdm4a expression in ST2 after osteogenic (b) and adipogenic (c) treatment was examined using qRT-PCR. The level of Kdm4a at d 0 was set to 1. Values are mean ± SD (n = 3). *P < 0.05 vs. vehicle
Fig. 2
Fig. 2
Kdm4a overexpression in ST2 promoted adipogenic differentiation and inhibited osteogenic differentiation. Overexpression of Kdm4a in ST2 was verified using qRT-PCR (a). The protein levels of KDM4A and H3K9me3 were examined (b). Cell growth rate was examined in ST2 following overexpression of Kdm4a (c). Differentiated adipocytes were stained with oil-red O (d). Oil-red O extracted with isopropanol was measured at OD520 (e). The mRNA (f) and protein (g) levels of adipogenic factors were examined. Differentiated osteoblasts were subjected to ALP staining (h). The mRNA (i) and protein (j) levels of osteogenic factors were examined. Values are mean ± SD. a, b, e, f, g, i, jn = 3; cn = 8. *P < 0.05 vs. vector
Fig. 3
Fig. 3
ML324 treatment blocked adipocyte formation and promoted osteoblast differentiation. Effect of ML324 on H3K9me3 was examined using Western blotting (a). Cell growth rate was examined in ST2 following ML324 treatment at 2, 4 and 8 μM (b). Differentiated adipocytes were stained with oil-red O (c). Oil-red O extracted with isopropanol was measured at OD520 (d). The mRNA (e) and protein (f) levels of adipogenic factors were examined. Differentiated osteoblasts were subjected to ALP staining (g). The mRNA (h) and protein (i) levels of osteogenic factors were examined. Values are mean ± SD. a, d, e, f, h, in = 3; bn = 8. *P < 0.05 vs. vehicle treatment
Fig. 4
Fig. 4
Silencing of Kdm4a in ST2 inhibited adipogenic differentiation and promoted osteogenic differentiation. The knockdown of Kdm4a mRNA in ST2 cells was verified using qRT-PCR (a). Differentiated adipocytes were stained with oil-red O (b). Oil-red O extracted with isopropanol was measured at OD520 (c). The mRNA (d) and protein (e) levels of adipogenic factors were examined. Differentiated osteoblasts were subjected to ALP staining (f). The mRNA (g) and protein (h) levels of osteogenic factors were examined. Values are mean ± SD (n = 3). *P < 0.05 vs. control siRNA
Fig. 5
Fig. 5
Kdm4a depletion in primary marrow stromal cells stimulated osteogenic differentiation at the expense of adipogenic differentiation. The silencing of Kdm4a mRNA in MSCs was verified using qRT-PCR (a). Differentiated adipocytes were stained with oil-red O (b). Oil-red O extracted with isopropanol was measured at OD520 (c). The mRNA levels of adipogenic factors were examined (d). Differentiated osteoblasts were subjected to ALP staining (e). The mRNA levels of osteogenic factors were examined (f). Values are mean ± SD (n = 3). *Significant vs. control lentivirus, P < 0.05
Fig. 6
Fig. 6
KDM4A controlled lineage commitment by demethylating H3K9me3 on Sfrp4 and C/ebpα promoters. The mRNA (a, c) and protein (b) levels of C/EBPα and SFRP4 were examined after Kdm4a overexpression (a, b) or ML324 treatment (c). The protein levels of the major components of canonical Wnt signaling were examined following Kdm4a overexpression (d). KDM4A occupancy on the promoters of C/ebpα and Sfrp4 was examined using ChIP assay (e, f). Binding of H3K9me3 to the promoters of C/ebpα and Sfrp4 was examined using ChIP assay (g, h). Values are mean ± SD (n = 3). *P < 0.05 vs. vector
Fig. 7
Fig. 7
KDM4A decreased DNA methylation on Sfrp4 and C/ebpα promoters. The methylation state of CpG sites proximal to the transcription start sites (TSS) of C/ebpα and Sfrp4 promoters are shown (a, b). The bisulfite-converted amplicons of C/ebpα and Sfrp4 promoters in ST2 after Kdm4a overexpression were run on gel. “M” indicates methylated sequence-specific PCR; “U” indicates unmethylated sequence-specific PCR (c). The level of DNA methylation was also determined using real-time qPCR (d). The interaction among H3K9me3, DNMT3B and MeCP2 were detected using co-IP (e). *P < 0.05 vs. vector
Fig. 8
Fig. 8
Silencing of Sfrp4 attenuated KDM4A promotion of adipocyte differentiation and suppression of osteoblast differentiation. ST2 cells were co-transfected with Kdm4a-WT or the vector, and Sfrp4 siRNA or the control siRNA. Differentiated adipocytes were stained with oil-red O (a). Oil-red O extracted with isopropanol was measured at OD520 (b). The mRNA (c) and protein (d) levels of adipogenic factors were examined. Differentiated osteoblasts were subjected to ALP staining (e). The mRNA (f) and protein (g) levels of osteogenic factors were examined. Values are mean ± SD (n = 3). *P < 0.05 vs. Vector plus Ctrl siRNA, #P < 0.05 vs. Kdm4a plus Ctrl siRNA
Fig. 9
Fig. 9
β-catenin transcriptionally regulated Kdm4a expression. Putative β-catenin binding sites on the mouse Kdm4a promoter are shown (a). The effect of β-catenin on the expression level of KDM4A was studied using qRT-PCR (b) and Western blotting (c). Luciferase assay was done to measure the promoter activity of wild-type (d) or mutant (e) Kdm4a promoter. Values are mean ± SD. b, cn = 3; d, en = 4. *P < 0.05 vs. vector (b) or Ctrl siRNA (c). *P < 0.05 vs. pGL4.14 plus vector; #P < 0.05 vs. WT or mutant promoter plus vector
Fig. 10
Fig. 10
Schematic diagram depicting the mechanism for KDM4A in regulating MSCs differentiation
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