KDM4B down-regulates ERα signaling independent on its demethylase activity in vascular calcification

Abstract

Aims: Vascular Calcification (VC) is recognized as an independent predictor of cardiovascular events. Estrogen replacement was reported as protective treatment against vascular calcification in postmenopausal women, while it is controversial because of its potential carcinogenicity. ERα co-regulators have been putatively considered as potential therapeutic targets for ERα-related cancers. However, the modulation of ERα action and biological function of ERα co-regulators in vascular calcification are still elusive. Methods and Results: KDM4B (Histone lysine demethylases 4B) was identified to be highly expressed in β-phosphoglycerol treated human and mouse aortic smooth muscle cells (ASMCs) and VitD3-overloaded mice during calcification by Western blot and immunofluorescence staining. Co-immunoprecipitation (Co-IP) and confocal immunofluorescence imaging were performed to show the association between KDM4B and ERa. Luciferase reporter assay demonstrated that KDM4B downregulated ERa-induced transactivation, and qPCR results showed that KDM4B depletion increased mRNA expression of endogenous ERa target gene; the results by co-IP showed that KDM4B associated with PRC2 complex and ERa. ChIP assay (Chromatin immunoprecipitation) demonstrated that KDM4B depletion decreased the recruitment of PRC2 (Polycomb repressive complex 2) complex to estrogen response element (ERE) regions, thereby down-regulating the level of H3K27me3. Finally, KDM4B-mediated enhancement of ASMCs calcification was attenuated by the estrogen treatment. Conclusion: KDM4B inhibits ERα-induced transactivation independent of its JmjC enzyme active region. KDM4B is involved in vascular calcification via down-regulation of ERα action. KDM4B associates with PRC2 complex to be recruited to ERE element of ERα downstream target gene, thereby modulating histone H3K27me3 modification on the ERE region, suggesting KDM4B acts as a new potential therapeutic target for VC.