Regulation Of Histone Demethylase KDM4A In Adipocyte And Osteoblast Differentiation

Objective The adipogenesis and osteogenesis of bone marrow stromal stem cells(BMSCs)are competing and reciprocal.And the imbalance between adipogenesis and osteogenesis results in bone-related diseases.While the differentiation of BMSCs is regulated by a complex network of signaling pathways and multiple genes,little is known about how epigenetic regulation in BMSCs differentiation.In this study,we investigated the functional role of lysine-specific demethylase 4a(KDM4A)and explored its molecular mechanisms during the BMSCs differentiation in vitro and in vivo.Methods 1.The Kdm4 a expression was quantified in various tissues of mice and BMSCs,ST2 stromal cells after adipogenic and osteogenic treatment.Using over-expression,knockdown,and enzymatic inhibition,we detected the m RNA and protein levels of osteoblast-specific and adipocyte-specific transcriptional factors and mark genes.Meanwhile,the potential of ST2 cells and BMSCs to differentiate into adipocytes and osteoblasts was assessed by Oil staining and ALP staining.2.Furthermore,we screened transcriptional factors by q RT-PCR and investigated the changes of methylation in Cp G islands and H3K9me3 on the promoter of target genes by Ch IP,MSP and co-IP to find out the mechanisms of KDM4 A in regulating BMSCs lineage determination.We also performed bioinformatics prediction and dual luciferase reporter assay to confirm which gene regulate transcription of Kdm4 a.3.Obesity model was constructed and mice were tail vein injected with Kdm4 a KD-LV.The changes of biochemical index in serum were detected,and qualities and size of adipocytes were measured.Results 1.The expression level of Kdm4 a was most highly expressed in bone,and most lowly in intestine.The expression profile of Kdm4 a during adipogenic differentiation was increased after adipogenic treatment,and then maintained at a high level until day3.During osteogenic differentiation the Kmd4 a expression was remarkably decreased through day 5.Enforced expression of Kdm4 a in ST2 cells and BMSCs promoted adipogenesis and inhibited osteogenic differentiation.Whereas knockdown of Kdm4 a expression inhibited adipogenesis and promoted osteogenic differentiation.The mutant in KDM4 A active site of enzyme catalysis leaded to attenuated ability in regulating BMSCs differentiation.We demonstrated KDM4 A inhibitor ML324 could efficiently block its catalytic activity and epigenetically boost osteogenic differentiation in ST2 cells.2.By using quantitative analysis,we identified that C/EBP? and Sfrp4 upregulated by KDM4 A.KDM4A removed H3K9me3 on promoter of target genes,and therefore decreased protein levels(DNMT3B,Me CP2)recruited by H3K9me3.By decreasing Cp G dinucleotide methylation of proximal to transcription start sites within C/EBP? and Sfrp4 promoters,KDM4 A activated transcription of target gene.Furthermore we verified that KDM4 A attenuates adipogenesis via inhibiting Sfrp4 expression.Kdm4 a was also predicted to be potential target of ?-catenin.Luciferase assay also verified that ?-catenin suppress transcription of Kdm4 a.3.We also demonstrated that Kdm4 a KD-LV treatment surpressed the accumulation of body weight and fat,and decreased the serum level of total cholesterol,glucose and free fatty acid and improved glucose tolerance in HFD-fed mice.Conclusion KDM4 A promote adipogenesis and inhibit osteogenic differentiation in ST2 cells and BMSCs.Inhibition of Kdm4 a enzymatic activity resulted in impaired regulation of differentiation potential.ML324 attenuated regulation ability of KDM4 A on the differentiation of BMSCs.KDM4 A removed tri-methylation marks of H3K9,which interact with DNMT3 B and Me CP2 to suppress C/EBP? and Sfrp4 expression.And ?-catenin reduced transcript level of Kdm4 a.Therefore Kdm4a/Wnt/?-catenin forms a feedback loop to regulate the differentiation of BMSCs.Kdm4 a KD-LV protected HFD-fed mice from diet-induced obesity and improved obesity-related impaired glucose regulation.