Histone demethylase KDM7A reciprocally regulates adipogenic and osteogenic differentiation via regulation of C/EBPα and canonical Wnt signalling

Abstract Recent emerging evidences revealed that epigenetic methylation of histone and DNA regulates the lineage commitment of mesenchymal progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 7A (KDM7A) on osteogenic and adipogenic differentiation. Kdm7a expression was up‐regulated in primary marrow stromal cells and established stromal ST2 line after adipogenic and osteogenic treatment. Silencing of endogenous Kdm7a in the cells blocked adipogenic differentiation whereas promoted osteogenic differentiation. Conversely, overexpression of wild‐type Kdm7a in the progenitor cells enhanced adipogenic differentiation whereas inhibited osteogenic differentiation. However, the effect of KDM7A on cell differentiation was largely attenuated when the point mutation was made that abolishes enzymatic activity of KDM7A. Mechanism investigations revealed that silencing of Kdm7a down‐regulated the expression of the CCAAT/enhancer binding protein α (C/EBPα) and secreted frizzled‐related protein 1 (Sfrp1). Chromatin immunoprecipitation (ChIP) assay revealed that KDM7A directly binds to the promoters of C/EBPα and Sfrp1 and removes the histone methylation marks H3K9me2 and H3K27me2. Furthermore, silencing of Kdm7a activated canonical Wnt signalling. Thereafter, activation of canonical Wnt signalling through silencing of Sfrp1 in ST2 attenuated the stimulation of adipogenic differentiation and inhibition of osteogenic differentiation by KDM7A. Our study suggests that KDM7A balances adipogenic and osteogenic differentiation from progenitor cells through epigenetic control of C/EBPα and canonical Wnt signalling and implicates that control of KDM7A action has an epigenetic perspective of curtailing metabolic disorders like osteoporosis.


| INTRODUCTION
Marrow stromal cells (MSCs), also known as bone marrow-derived mesenchymal progenitor cells, have the potential to differentiate into various kinds of cell types, eg, osteoblasts, chondrocytes, adipocytes and myoblasts. 1 Normally, there exists a reciprocal balance between adipogenesis and osteogenesis, 2,3 which is sometimes impaired in various human disorders including ageing, osteoporosis and obesity, etc. 4,5 It is crucial to elucidate the mechanisms that fine tune the balance between osteogenic and adipogenic differentiation.
A number of critical signalling pathways are involved in regulating the osteogenic and adipogenic commitment of MSCs, including transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signalling, canonical Wnt signalling, Notch and Hedgehogs, etc. [6][7][8][9] Besides, multiple transcription factors are critical for the differentiation of MSCs into adipocytes or osteoblasts. While runt-related transcription factor 2 (Runx2) and Osterix are required for osteogenic differentiation, 10,11 CCAAT element binding protein (C/ EBP) members and peroxisome proliferator-activated receptor γ (PPARγ) play a role in adipogenic differentiation of MSCs. 12,13 Epigenetic regulation of gene expression through histone modifications is attracting more attention as the player in lineage-specific commitment of progenitor cells. [14][15][16] Histone modifications occur usually at lysine and arginine residues, and may alter histone-DNA binding affinities and the interactions of specific transcription factors with the promoters. 17 Histone demethylases are the classes of enzymes that remove methyl groups in modified histone proteins.
Although largely unknown, recent emerging evidences have shown that histone demethylases may exert essential regulatory functions in cell fate decision of MSCs. 18 Ye et al have demonstrated that the histone demethylases KDM4B and KDM6B play a positive role in osteogenic commitment of MSCs at the expense of adipogenic differentiation. 19 The mechanism investigations showed that KDM6B increased the levels of homeobox (HOX) genes by removing H3K27me3, whereas KDM4B promoted the expression of distal-less homeobox (DLX) genes by removing H3K9me3. 19 Of interest, H3K27me3-and H3K9me3-positive MSCs of bone marrow were significantly increased in ovariectomized and ageing mice in parallel with the highly active adipogenesis. 19 Furthermore, KDM5A and KDM2A have also been recognized as osteogenic regulators, both of which negatively regulated osteogenic differentiation of MSCs. 20,21 The mechanism exploration revealed that KDM5A decreased the expression level of Runx2 through removing H3K4me3 levels from the promoter of Runx2, 20 whereas KDM2A enhanced secreted frizzled-related protein 2 (Sfrp2) transcription by decreasing histone H3K4 and H3K36 methylation at the Sfrp2 promoter. 21 The histone lysine(K)-specific demethylase 7 (KDM7) subfamily is an emerging class of transcriptional coactivators that consists of three members, KDM7A, KDM7B and KDM7C. While KDM7B plays an essential role in neuronal differentiation, craniofacial development and tumour growth, 22,23 KDM7C is involved in various biological processes including osteogenesis and adipogenesis. In osteoblasts, KDM7C promotes DNA binding of Runx2 by directly demethylating mouse Runx2 at Lys245 or human Runx2 at Lys238, rather than by demethylating histones on Runx2 target genes. 24 During adipogenesis, KDM7C physically interacts with C/EBPα and C/EBPδ and epigenetically boosts the C/EBP-driven expression of adipogenic factors. 25 Histone lysine demethylase 7A (KDM7A), also known as JHDM1D, is a member of the plant homeodomain (PHD) finger protein (PHF) family of PHD-and JmjC domain-containing histone demethylases. It is able to catalyse the removal of di-methylation marks H3K9m2 and H3K27m2 on the promoters of target genes, 26 and through this, regulates fibroblast growth factor-4 (FGF-4) expression and neural differentiation. 27 KDM7A also functions as a potential tumour suppressor through blocking tumour growth and angiogenesis. 28 Up to now, it remains unknown if KDM7A regulates adipogenic and osteogenic commitment of mesenchymal stem cells.
In this study, we identified KDM7A as a player in adipogenic and osteogenic differentiation from progenitor cells. Mechanism studies revealed that this is based upon the stimulation of C/EBPα and Sfrp1 transcription as a result of the removal of the repressive H3K9me2 and H3K27me2 marks by KDM7A from the promoter regions of C/ EBPα and Sfrp1.

| Quantitative RT-PCR
RNA was extracted using a total RNA isolation kit (Gmbiolab, Taiwan). After reverse transcription with 1 μg of the total RNA and random primers, the cDNA was PCR-amplified on a real-time PCR cycler using a SYBR Green fluorescence PCR kit (Sangon Biotech, Shanghai, China) with gene specific primers. RT-PCR amplifications were carried out for one cycle of 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds, 57°C for 10 seconds and 72°C for 10 seconds. β-actin was used as internal control. The expression levels of target genes were measured by the comparative Ct (ΔΔCt) method. The sequences of the primers are listed in Table S1.

| Lentiviral packaging and infection
To

| Oil-red O staining
Differentiated adipocytes were fixed in 4% paraformaldehyde, then washed with deionized water. After incubating with 60% isopropanol for 2 minutes, the cells were stained with oil-red O solution (0.5% oil red O in isopropanol/water = 3:2) for 5 minutes. For oil-red O quantification, isopropanol was added to dissolve the stain. Light absorbance was measured at 520 nm.  Park, CA, USA) was finally used to visualize the results.

| Statistical analysis
Data are expressed as means ± SD. For the relative mRNA expression analysis, the means of the control groups were set to 1. Statistical analysis was performed with the independent t test or one-way ANOVA. If the one-way ANOVA result was significant, a post hoc comparison was performed with the least significant difference (LSD) test. A value of P < 0.05 indicated statistical significance.

| Kdm7a was expressed in bone and adipose tissue and increased during osteoblast and adipocyte differentiation
We examined the expression levels of Kdm7a in various tissues in 1month-old mice. Kdm7a mRNA was highly expressed in bone and skeletal muscle, and moderately expressed in spleen and heart.
Kdm7a was expressed in relatively low level in other indicated tissues ( Figure 1A). qRT-PCR analysis revealed that Kdm7a expression during osteogenic differentiation ( Figure 1D,E). These results suggest that KDM7A has a regulatory role in adipogenic and/or osteogenic differentiation.

| Silencing of endogenous Kdm7a in ST2 cells inhibited adipocyte formation and promoted osteoblast differentiation
Two independent siRNAs targeting different coding regions of By contrast, the Kdm7a siRNAs positively affected the differentiation of ST2 cells into osteoblasts, revealed by enhanced ALP staining ( Figure 2F). Consistently, the mRNA levels of Runx2, Osterix, Alp, Osteopontin (Opn) and Osteocalcin (Oc) were increased 72 hours after osteogenic treatment ( Figure 2G). Moreover, the protein levels of Runx2, Osterix, ALP and Osteopontin were also substantially increased 72 hours after osteogenic treatment ( Figure 2H).

| Kdm7a overexpression in ST2 cells induced adipocyte formation and inhibited osteoblast differentiation and the effect is dependent on its demethylase activity
In order to further demonstrate whether KDM7A regulates adipogenic and osteogenic differentiation and whether this is dependent on its demethylase activity, we generated a wild-type construct and a catalytically dead Kdm7a mutant in which histidine 282 was changed to alanine, disrupting the non-haem metal binding site required for its demethylase activity. 26

| KDM7A targets C/EBPα and canonical Wnt signalling to regulate ST2 cells differentiation through removing H3K9me2 and H3K27me2
The commitment of MSCs is regulated by a complex and highly  The effect of methylation status on transcription depends on the exact residue targeted and the degree of methylation. Trimethylation of H3K4, H3K36 and H3K79 is a representative marker for euchromatin, which is loosely packed, so accessible for transcription factors. In contrast, mono-methylation of H4K20, and di-/tri-methylation of H3K9 or H3K27 mark heterochromatin, which is too tightly packed to be accessed by transcription factors. 24 In summary, our work has identified novel KDM7A functions as a reciprocal regulator of osteogenesis and adipogenesis. The function is based upon its epigenetic regulation of SFRP1 and C/ EBPα. Our study provides insights into the epigenetic mechanisms underlying the lineage commitment of MSCs. These findings may contribute to new therapeutic clues for metabolic disorders such as osteoporosis.