The Epigenetic Mechanism Of Histone Demethylase KDM4B Corepressive Era-Mediated Transcription And Its Role In Vascular Calcification

Objective: Vascular Calcification(VC)is the pathology of several diseases and is also valued as an independent predictor of cardiovascular events.Vascular smooth muscle cells(VSMCs)play an important role in the process of vascular calcification,which mainly changes from a contractile phenotype to an osteoblast-like phenotype.Studies have shown that vascular calcification is present in over 90% of men and over 67% of women over 70.Estrogen receptor alpha(ERα)/E2 has been reported to play a protective role against vascular calcification by promoting the transcriptional activity of the GAS6 gene.It is important to study vascular calcification pathogenesis in depth to reduce cardiovascular events.Steroid receptor superfamily members include ER,which consists of two isoforms,ERα and ERβ,which are encoded by different genes.Erα has 595 amino acids.ERαInitiating downstream target gene transcription on ERE after binding to the ligand E2 requires the participation of co-regulators,which are divided into co-inhibitors and coactivators according to their effect on transcriptional activity.It has been reported that coregulators involved in the regulation of gene transcription play a key role in the occurrence and development of diseases.Therefore,the discovery of new co-regulators is key to the exploration of vascular calcification.ERα-mediated gene transcription plays an important role in vascular calcification.It suggests that the discovery and exploration of new ERαcoregulators and their regulatory effects on transcription factor-mediated gene transcription may reveal new therapeutic targets for vascular calcification.Dynamic reversal of histone methylation and demethylation has been reported to affect many biological processes and play a key role in various diseases such as tumors,osteoporosis,cardiovascular disease,and so on.KDM4 B protein length is 1096 amino acids,which can catalyze histone residue activity.DM4 B is a member of the KDM4/JMJD2 family of histone demethylases.KDM4 B is highly expressed in estrogen receptor(ER)-positive breast cancer and is a critical regulatory gene.However,the function of KDM4 B as an(ER)co-regulator in vascular calcification needs to be further explored.In this study,our objectives were the priority:(1)Verify the role of KDM4 B in β-GP induced calcification models in HASMCs and MOVASs;(2)Explore whether KDM4 B associates with ERα and;(3)To explore whether KDM4 B is involved in ERα-mediated gene transcription;(4)To explore the further molecular mechanisms by which ERα-mediated gene transcription is regulated by KDM4B;(5)Explore the biological function of KDM4 B in vascular calcification.Methods:(1)The correlation of KDM4 B protein expression levels and MOVASs in β-GPinduced HASMCs calcification model and aortic calcification in mouse were analyzed by immunohistochemical(IHC)staining and western blotting;(2)Co-immunoprecipitation assay(co-IP)to find the possibility of KDM4 B interacting with ERα;Immunofluorescence experiments verified the localization of KDM4 B and ERα in HASMCs.(3)Luciferase reporter gene experiments verify the role of KDM4 B on the ERα-mediated gene transcription,and real-time q-PCR and western blot verified the effect of KDM4 B on ERα-E2 signaling pathway and gene expression after estrogen treatment;(4)The molecular mechanism of KDM4 B affecting ERα-E2 signaling pathway was discussed.The interaction of KDM4 B,ERα and PRC2 between core complex members was detected through the experiments of co-immunoprecipitation.In addition,the experiments of chromatin co-immunoprecipitation(ChIP)was used to verify whether knockdown or overexpression of KDM4 B affects the function of core members of the PRC2 complex on the ERα target genes transcriptional activity.(5)Calcium quantification alizarin red staining,and western blot were used to detect the role of KDM4 B in vascular calcification.Results:(1)Alizarin red staining,calcium quantification,Western blot and immunofluorescence staining showed that KDM4 B was highly expressed in the aorta of HASMCs,MOVASs and vitd3-overloaded mice during calcification.(2)In HASMCs,MOVASs and HEK-293 cells,we found that KDM4 B could interact with ERα by co-IP.Through immunofluorescence localization experiments,KDM4 B and ERα can be colocalized in the nucleus under E2 stimulation.(3)In HASMCs and MOVASs,luciferase reporter gene detection found that KDM4 B downregulated the transcriptional activity of ERα,real-time PCR found that,after estrogen treatment,KDM4 B affected ERα-E2 signaling pathway and the expression of gene;(4)With accordance to the protein immunoprecipitation experiments,it was found that KDM4 B could interact with the PRC2 complex and ERα,which affected the interaction between ERα and the PRC2 complex.Using the ChIP assays experiment,we found that si KDM4 B affected the recruitment of PRC2 complex on Gas6-ERE regions and influence the modification level of H3K27me3;(5)We have found the knockdown of KDM4 B could greatly alleviate β-GP induced calcification in HASMCs and MOVASs.Conversely,calcification was aggravated transfected with HA-KDM4 B by Alizarin red staining,calcium quantitative assay and Western blotting.Conclusion: KDM4 B is highly expressed in β-GP induced calcification model.KDM4 B interacts with ERα and PRC2 complex,and co-recruit to ERα downstream target genes estrogen response element,changing the histone H3K27me3 modification level and downregulate ERα-mediated gene transcription,which is independent of its demethylase activity.Estrogen could attenuate KDM4B-induced β-GP induced calcification.