KDM4B modulates ERα signaling pathway to participate in vascular smooth muscle cell calcification

KDM4B modulates ERα signaling pathway to participate in vascular smooth muscle cell calcification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article KDM4B modulates ERα signaling pathway to participate in vascular smooth muscle cell calcification Yue Zhao, Fei Liu, Yang LV, Yanxia Lin, Chunyu Wang, Shengli Wang, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6522511/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Cell Death Discovery → Version 1 posted You are reading this latest preprint version Abstract Vascular calcification (VC) is recognized as an independent predictor of cardiovascular events. Although estrogen replacement is a controversial treatment due to its potential carcinogenic effects, it was considered a protective treatment against VC in postmenopausal women. Estrogen receptor α (ERα) co-regulators were considered as potential therapeutic targets for ERα-related cancers. However, ERα activity and biological function modulation of ERα co-regulators in VC remain elusive. Histone lysine demethylases 4B (KDM4B) was identified to be highly expressed in human and mouse aortic smooth muscle (ASMC) cells treated with β-phosphoglycerol and in mice overloaded with VitD3 during calcification, as evidenced by western blotting and immunofluorescence staining. Co-immunoprecipitation (Co-IP) was performed to show the association between KDM4B and ERα. Our data demonstrated that KDM4B down-regulated ERα-induced transactivation and that KDM4B depletion increased mRNA expression of endogenous ERα target gene. Furthermore, we provided the evidence to show that KDM4B associated with Polycomb repressive complex 2 (PRC2) and ERα. In addition, KDM4B depletion decreased the recruitment of PRC2 complex to estrogen response element (ERE) regions of ERα target gene, thereby down-regulating the H3K27me3 levels. Finally, KDM4B-mediated enhancement of ASMCs calcification was partially attenuated by the estrogen treatment. KDM4B inhibits ERα-induced transactivation independent of its Jumanji-C enzyme active region. Taken together, our study suggest that KDM4B acting as ERα co-repressor is involved in regulation of VC, indicating that KDM4B may be a new potential therapeutic target for VC treatment. Biological sciences/Molecular biology/Epigenetics Biological sciences/Cell biology KDM4B ERα PRC2 epigenetic regulation vascular calcification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Vascular calcification (VC) is a common degenerative phenomenon associated with aging, which is common in the end-stage of most chronic diseases and is characterized by the abnormal deposition of calcium and phosphorus onto the vascular wall [ 1 – 3 ]. VC has been recognized as an independent predictor of cardiovascular events [ 4 ]. The relationship between vascular calcification and osteoporosis is well-established [ 5 , 6 ]. Osteoporosis, characterized by excessive bone resorption over formation, leads to the release of calcium into the bloodstream, which can then ectopically deposit in blood vessels, thus promoting vascular calcification. Estrogen is recognized for its role in the prevention of osteoporosis[ 7 ] and the inhibition of vascular calcification[ 8 ]. Nevertheless, there are clear sex-specific patterns of VC, being present in more than 90% of men and 67% of women over 70 years of age. Coronary artery calcification is a component of VC, and its calcification evaluation is an independent predictor of cardiovascular adverse events in postmenopausal women. In addition to the differential lifestyle habits associated with the sexes, the cardiovascular benefits of women diminish after menopause, with the risk of cardiovascular disease increasing significantly in older women (approximately four times as much as before menopause) [ 9 ]. The incidence of coronary artery calcification is lower in premenopausal women than in men, which may be related to the protective effect of estrogen [ 10 ]. Estrogen is a vital drug component in preventing and treating postmenopausal osteoporosis, which induces bone formation by activating the estrogen receptor signaling pathway. Mechanically, estrogen has been reported to inhibit the VC process in several ways, including classically promoting E2-related gene transcription, promoting autophagy, and inhibiting the HIF-1α signaling pathway [ 8 , 11 , 12 ]. Although the biological function of the estrogen signaling pathway in VC has been investigated in recent years, the specific molecular mechanism remains unclear. Estrogen receptor α (ERα) belongs to a nuclear receptor superfamily that undergoes conformational changes and translocates from the cytoplasmic lysate to the nucleus in the presence of estrogen, thereby binding to specific estrogen response element (ERE) to regulate its downstream gene transcription [ 13 ]. Studies have shown that ERα/E2 exerts a protective effect against VC by promoting the transcriptional activity of the growth arrest-specific gene 6 (GAS6) [ 14 ]. Similarly, receptor activator of nuclear factor-Kappa B ligand (RANKL) promotes VC by regulating the expression of bone morphogenetic protein-2 (BMP2), MGP, and bone-associated protein, which is counteracted by estrogen in a receptor-dependent manner. In addition, estrogen acts mainly through the ERα to counteract these effects of RANKL stimulation [ 8 ]. Upon estrogen treatment, activated ERα recruits co-regulators that participate in ERα-mediated transcriptional activation, thereby altering chromatin structure, post-transcriptional modifications, and modulating ERα protein stability [ 15 – 17 ]. A variety of ERα co-regulators have been demonstrated to be essential for the development of breast cancer (BC) [ 18 , 19 ]. The histone-modifying enzymes and chromatin remodeling factors enhance or inhibit ERα-mediated gene transcription, referred to as ERα co-activators or co-repressors. For example, KDM3A, a histone demethylase, plays a crucial role in ERα signaling by regulating the transcription of receptor-target genes through the control of demethylation at the cis-regulatory element H3K9me1/me2. Furthermore, KDM3A has been shown to be necessary for the growth of ERα-positive BC cells [ 20 ]. In ERα-positive or ERα-negative BC cells, the deletion of KDM4A reduces the expression of ERα target genes c-Jun and cyclin D1, leading to abnormal cell proliferation [ 21 ]. The expression of KDM5A is also up-regulated in BC [ 22 – 26 ]. Mechanistically, the accumulation of p16 and p27 is promoted by blocking KDM5A-mediated H3K4me3 demethylation, leading to cell cycle arrest and aging [ 25 ]. JMJD6 regulated trans-activation of ERα-binding enhancers and their downstream target genes. Furthermore, it is a critical factor in the growth and tumorigenesis of ERα-positive BC cells [ 27 ]. These findings indicate that ERα co-regulators are essential for the development of ERα-related diseases, including BC, endometrial carcinomas, and VC. However, the modulation of ERα-induced transactivation and the biological function of ERα co-regulators in VC remain elusive. JMJD2 family members take active roles in multiple physiological processes, including cell proliferation, migration [ 28 ], gene transcription [ 29 ], and genome stability [ 30 ]. The KDM4 family, a class of demethylases, precisely removes methylation at H3K9 or H3K36 sites, playing a pivotal role in the histone code [ 28 , 31 – 33 ]. Lysine demethylase is a class of proteins that removes lysine/arginine methylation at specific histone H3/H4 [ 34 ]. Histone lysine demethylases 4B (KDM4B), consisting of 1096 amino acids, exhibits catalytic activity on histone residues H3K9me3, H3K9me2, and H3K36me3, with a preference for the H3K9Me2/3 substrate [ 32 , 35 , 36 ]. KDM4B contains a Jumanji N (JmjN) structural domain, a JmjC structural domain, two plant homology domains, and two Tudor structural domains, among which the active region for demethylase is the JmjC domain [ 34 , 35 , 37 , 38 ]. The demethylases enzyme KDM4B, a component of the demethylation module, is involved in gene transcriptional regulation. We have previously demonstrated that KDM4B is recruited to ERα target genes, where it demethylates H3K9me3 and up-regulated ERα transcription. Moreover, KDM4B plays an essential role in regulating estrogen signaling cascade, and its absence inhibits the growth of BC both in vitro and in vivo [ 39 , 40 ]. Notably, KDM4B itself is also an ERα response gene [ 41 ]. Thus, these findings suggest a positive feedback mechanism between KDM4B and ERα, where estrogen-induced KDM4B expression in turn co-regulates and up-regulates ERα target genes, thereby promoting BC growth. In addition, KDM4B can induce osteogenic differentiation of mesenchymal stem cells (MSCs). TGF-β induces the expression of KDM4B, whereas KDM4B recruits the transcription factor SMAD3 to the SOX9 promoter to inhibit the H3K9me3 modification level and promote chondrogenesis as well as differentiation of MSCs [ 42 ]. In vascular smooth muscle cells (VSMCs), KDM4B can be recruited to the STAT3 binding site of the Runx2 promoter region and inhibit the level of H3K9me3 modification, ultimately promoting Runx2 transcription [ 43 ]. Nevertheless, the mechanism underlying the effect of KDM4B on ERα-mediated gene transcription during VC remains poorly understood. In this study, we explored the potential mechanisms by which KDM4B influences the VC process in vivo and in vitro . KDM4B was highly expressed in the β-phosphoglycerol-induced VC model and a VitD3-induced VC mouse model. Furthermore, KDM4B interacted with endogenous ERα and co-repressed ERα-mediated transactivation. Unexpectedly, luciferase assay results suggest that KDM4B inhibits ERα-mediated gene transcription independent of its JmjC enzyme active region. In addition, KDM4B was required for the recruitment of PRC2 subunits to the ERE on ERα target genes, which was accompanied by the enrichment of H3K27me3 modification levels. Furthermore, KDM4B-mediated enhancement of β-phosphoglycerol (β-GP) induced calcification was attenuated by an estrogen treatment. Overall, our study identified KDM4B as a new ERα co-factor that down-regulates ERα-mediated transactivation in its demethylase-independent manner in human aortic smooth muscle cells (HASMCs) and mouse aortic vascular smooth (MOVAS) cells. Our findings demonstrated that KDM4B promotes VC via the E2/ERα signaling pathway, providing a potential therapeutic strategy for VC in females. Results KDM4B is highly expressed in β-GP-induced HASMCs and MOVAS cells calcification models Epigenetic factors that play an important role in the pathogenic regulation of cancer and other diseases’ gene transcription were selected. The expression levels of KDM family genes (KDM2A, KDM3A, KDM4A, KDM5A, KDM6A, and KDM4B) are involved in the epigenetic regulation within HASMCs with different calcification status induced following treatment with 5 mM β-GP for 0, 1, 2, 3, and 5 days. We showed that β-GP induces HASMCs calcification in a time-dependent manner. Simultaneously, the notable expression of KDM4B increased progressively as the duration of calcification extended (as shown in Supplementary Figure S1 -A). Alizarin red staining and calcium deposition quantification were performed to the measure calcification levels. We further corroborated the finding that β-GP induced HASMCs calcification in a time-dependent manner (Fig. 1 A, 1 B). The expression of calcification-related proteins was measured, with the results suggesting that runt-associated transcription factor 2 (Runx2), BMP2, and RANKL were gradually increasing. We also observed that the expression of KDM4B increased gradually with calcification stimulation time (Fig. 1 C). The mRNA levels of calcification-related factors were also analyzed, and the results indicate that Runx2 and BMP2 gradually increased, whereas alpha-smooth muscle actin (α-SMA) gradually decreased. Concurrently, an incremental increase in the mRNA expression of KDM4B was observed across various levels of calcification (Fig. 1 D). The same results were obtained for MOVAS cells (Fig. 1 E- 1 H). These results suggest that KDM4B is highly expressed in β-GP-induced calcification models and may be involved in the calcification process. KDM4B is associated with ERα in HASMCs and MOVAS cells Clinical findings showed that women have a higher risk of VC and cardiovascular disease after menopause[ 9 ]. Furthermore, the ERα signaling pathway has been suggested to be involved in inhibiting the calcification process associated with VC[ 8 ]. KDM4B is also a critical epigenetic demethylase involved in regulating transcription factors and it up-regulates ERα-target genes; hence, sustaining BC cell growth. In our effort to understand the regulatory relationship between KDM4B and ERα in VC, we investigated the association between these two proteins. Initially, immunofluorescence assays were performed to examine the subcellular distribution of KDM4B and ERα in HASMCs. Our data showed that KDM4B and ERα are predominantly distributed in the nucleus in the absence or presence of E2 treatment in HASMCs (Fig. 2 A). GST labeled ERαAF1 or ERαAF2 expression vectors, were further used for the GST pull-down experiments, revealing that KDM4B directly binds to GST-ERαAF1 and AF2 in vitro . (Fig. 2 B). In addition, HASMCs were transfected with KDM4B expression plasmid for co-IP assay. Co-IP results showed that HA-labeled KDM4B precipitated with ERα (Fig. 2 C). Further, endogenous interactions between KDM4B and ERα in HASMCs were detected using endogenous-cellular co-IP assays (Fig. 2 D, 2 E). Consistent with the previous results, endogenous interactions between KDM4B and ERα were also observed in MOVAS cells (Fig. 2 F, 2 G). HEK-293 cells were co-transfected with ERα and HA-labeled KDM4B expression plasmid for co-IP assay. Our results showed that ERα precipitated with the HA-labeled KDM4B (Fig. 2 H). These data demonstrated that KDM4B associates with ERα in HASMCs and MOVAS cells. KDM4B co-represses ERα-induced transactivation in HASMCs and MOVAS cells To detect the regulatory function of KDM4B on the ERα activity, luciferase assays were performed in HASMCs. Our results showed that KDM4B significantly down-regulates ERα-mediated transactivation (Fig. 3 A). To further identify the ERα domain regulated by KDM4B, we performed luciferase experiment that examined the effect of KDM4B on ERα-induced transcriptional activity containing a nonligand-dependent AF-1 domain (ERα AF-1) or a ligand-dependent AF-2 domain (ERα AF-2). We observed that KDM4B down-regulated ERα AF-2-mediated transcriptional activation in the presence of E2, whereas the ERα AF-1 activation was not regulated (Fig. 3 B). To further detect whether the demethylase activity of KDM4B is required for its regulation function on ERα-mediated transactivation, according to the previous paper [ 44 ], we constructed a KDM4B mutant expression plasmid (KDM4B HTE/ATA) carrying a demethylase loss-of-function mutation, as shown in diagram of plasmid sequencing (Fig. 3 C). Western blotting was used to verify the transfection efficiency of KDM4B full-length and its mutant counterpart (Fig. 3 D). Alternatively, the luciferase assay was performed to detect whether KDM4B down-regulates ERα-mediated transactivation depending on its demethylase activity. We observed that both KDM4B full-length or its mutant significantly down-regulated ERα-mediated transactivation in both HASMCs and MOVAS cells. This suggests that KDM4B may down-regulate ERα-mediated transactivation independent of its demethylase activity (Fig. 3 E-F). To strengthen the argument, a luciferase assay also was performed with KDM4B inhibitor, the results showed that KDM4B inhibitor has the similar effects as that of KDM4B or KDM4B mutant on ERα-mediated transactivation (Supplementary Figure S2A). Meanwhile, the results from Alizarin red staining showed that KDM4Bwt, KDM4Bmutant, or KDM4B inhibitor aggravated β-GP-induced calcification in MOVAS cells, respectively (Supplementary Figure S2B). These data suggest that the co-repression function of KDM4B on ERα-mediated transactivation may not be dependent on its demethylase activity. Furthermore, qPCR was used to examine the modulation functions KDM4B on the expression of the endogenous estrogen-responsive gene mRNA in HASMCs. Our data showed that ectopic expression of KDM4B decreased the mRNA expression of Gas6 , but not ESR1 in HASMCs and MOVAS cells (Fig. 3 G and Supplementary Figure S3A). Furthermore, western blotting showed that KDM4B overexpression decreased Gas6 protein expression in HASMCs and MOVAS cells (Fig. 3 H and Supplementary Figure S3C). Similar experiments were performed in HASMCs and MOVAS cells transfected with siKDM4B; we observed that KDM4B depletion exceedingly increased the mRNA and protein expression of Gas6 (Fig. 3 I and 3 J and Supplementary Figure S3B and S3D). Furthermore, both overexpression of the KDM4B full-length and the mutant version decreased Gas6 protein expression in HASMCs (Supplementary Figure S3E). In summary, our results suggest that KDM4B co-represses ERα-induced transactivation independent of its demethylase activity in HASMCs and MOVAS cells. KDM4B facilitates the recruitment of PRC2 core proteins to the cis -regulatory elements of ERα target genes According to a previous report, KDM4B up-regulates ERα-mediated gene transcription mainly in BC [ 40 ]. Interestingly our results demonstrate that KDM4B down-regulates ERα-mediated gene transcription. Thus, we would like to further study the mechanism through which KDM4B is involved in ERα-mediated gene transcription in VC. We sought to identify the proteins interacting with KDM4B as potential protein complexes. The STRING protein network analysis suggests that KDM4B may be associated with ERα and the subunits of the PRC2 complex (PRC2 core protein), including Enhancer of Zeste omologue 2 (EZH2) and Suppressor of zeste 12 (SUZ12) (Fig. 4 A). To determine the correlation of ERα and PRC2 core proteins and KDM4B in HASMCs and MOVAS cells, we performed co-IP experiments. Our data demonstrated that ERα and KDM4B associated with the core protein subunits of the PRC2 complex (Fig. 4 B-E). Additionally, the interactions between ERα and the PRC2 subunits were significantly reduced when KDM4B was depleted (Fig. 4 F). These results suggest that KDM4B could mediate the association of ERα and the PRC2 core protein. To further analyze the molecular mechanism underlying the influence of KDM4B on estrogen-induced gene transcription, specifically for genes such as Gas6 , we utilized the JASPAR software to predict the Estrogen response element (ERE) on Gas6 . An ERE spanning between − 72 and − 89 bp was observed from the translation start site of Gas6 (Fig. 4 G). ChIP assay was performed in HASMCs treated with or without estrogen. During estrogen induction, KDM4B or ERα was recruited into the promoter region of Gas6-ERE. The overexpression of KDM4B promoted the recruitment of PRC2 core protein, not affecting the recruitment of ERα; thus, enhancing the levels of H3K27me3 proximal to the Gas6-ERE region (Fig. 4 H). To confirm these results, ChIP analysis was further performed in cells transfected with siKDM4B. KDM4B deletion can reduce the recruitment of the PRC2 complex to the same region. In addition, in the presence of E2, KDM4B deletion decreased the levels of H3K27me3 adjacent to the Gas6-ERE promoter region, whereas no significant effects of KDM4B on H3K9me3 levels were observed (Fig. 4 I). These results indicated that KDM4B could facilitate PRC2 subunit recruitment to the promoter region of ERα target genes; thus, altering the level of H3K27me3 and enhancing gene transcription. In summary, our results suggest that KDM4B is associated with ERα and PRC2 core proteins and down-regulates ERα-mediated gene transcription in HASMCs and MOVAS cells. KDM4B aggravates β-GP-induced calcification in HASMCs and MOVAS cells We showed that KDM4B act as a co-repressor of ERα and mediates the ERα transactivation in HASMCs and MOVAS cells. Further, we examine the biological function of KDM4B in β-GP-induced calcification in HASMCs and MOVAS cells. KDM4B was knockdown following transfection with siKDM4B. To mitigate potential off-target effects of siRNA, we analyzed the impact of two distinct siRNA sequences (siKDM4B#1 and siKDM4B#3) on KDM4B, as verified by western blotting and qPCR in HASMCs (Fig. 5 A, 5 B) and MOVAS cells (Fig. 5 G). Concurrently, Alizarin red staining and quantitative calcium analyses revealed that KDM4B depletion alleviated the calcification induced by the 5 mM β-GP treatment in HASMCs and MOVAS cells (Fig. 5 C, 5 D, 5 H, 5 I). Aligning with these findings, western blotting analysis and subsequent quantification of protein expression demonstrated that KDM4B depletion led to a decrease in the expression of calcification markers such as Runx2, BMP, and RANKL over different treatment periods with 5 mM β-GP in HASMCs (Fig. 5 E, 5 F). To further reveal the role of KDM4B in calcification, western blotting analyses were performed in cells with ectopic expression of KDM4B (Fig. 6 A, 6 H). Alizarin red staining and calcium deposition indicate that KDM4B overexpression significantly aggravated β-GP-induced calcification in HASMCs (Fig. 6 B, 6 C). Furthermore, we observed enhancing calcification effects for both KDM4B full-length and KDM4B mutant overexpression (Fig. 6 E, 6 F). Accordingly, the western blot analysis of calcification markers, including Runx2, BMP2, and RANKL, indicates that both KDM4Bwt and KDM4Bmut exert comparable effects on β-GP induced calcification in HASMCs, as illustrated in Figs. 6 D and 6 G. This increase in marker expression was also observed over different days of the 5 mM β-GP treatment in HASMCs, as confirmed by western blotting analysis and subsequent quantification of protein expression (Supplementary Figure S4A, 4B). These effects were also observed in MOVAS cells (Fig. 6 I, 6 J, 6 K, 6 L). Taken together, our results suggest that KDM4B may play an essential role in calcification, and this function may not be influenced by its deubiquitinase activity. KDM4B-mediated enhancement of β-GP-induced calcification is partially attenuated by estrogen treatment The ERα signaling pathway is essential for suppression of VC. Having established in this study that KDM4B acting as a co-repressor of ERα inhibits ERα-mediated transactivation. Next, we thus turned to examine whether KDM4B-mediated enhancement of β-GP induced calcification is related with ERα signaling. Alizarin red staining and quantitative calcium analysis showed that KDM4B overexpression increased calcification following the β-GP treatment, which was partially attenuated by the estrogen treatment (Fig. 7 A, 7 B). In addition, the pro-calcific effect of KDM4B overexpression on β-GP-induced calcification, as detected by the expression of calcification markers Runx2, BMP2, and RANKL, was attenuated by estrogen treatment in HASMCs (Figs. 7 C, 7 D, 7 E). Additionally, Alizarin red staining and quantitative calcium analysis revealed that KDM4B depletion reduced calcification in the presence of the β-GP treatment, the estrogen treatment at least partially attenuates KDM4B-mediated enhancement of β-GP-induced calcification (Fig. 7 F, 7 G). Western blotting results showed that KDM4B depletion could decrease β-GP-induced calcification indicated by the expression of calcification markers Runx2, BMP2, and RANKL was also attenuated by the estrogen treatment in HASMCs or MOVAS (Fig. 7 H, Supplementary Fig. 5A, 5B). These results suggest that the function of KDM4B on β-GP-induced calcification is, if not all, but partially related to estrogen/ERα signaling pathway. KDM4B is highly expressed in a VitD3-induced calcification mouse model Ultimately, an in vivo calcification model was generated to confirm the in vitro findings. We generated a mouse calcification model using VitaminD3. After the calcification protocol, we isolated the aortas and conducted western blotting and immunohistochemistry. The protein expression of KDM4B in the calcified aged mouse was higher than that in the control group (Fig. 8 A). Immunohistochemical results showed that the KDM4B expression was enhanced in the ovary removal group (OVX) and further increased in the OVX with VitD3-induced calcification (OVX + VC) group. The expression levels of calcification marker BMP2 were increased and the expression levels of α-SMA were decreased in OVX + VC group (Fig. 8 B). Our results suggest that KDM4B is highly expressed in VitD3-induced calcification mice. Discussion In recent studies, the estrogen/ERα signaling pathway has been shown to play a significant role in VC. Estrogen inhibits the VC process in several ways, including the promotion of E2-induced gene transcription, promoting autophagy, and inhibiting the HIF-1α signaling pathway [ 8 , 11 ]. ERα as a typical transcription factor mediates E2-induced gene transcription and inhibits VC. Co-activators and co-repressors of ERα are epigenetic enzymes and chromatin remodelers that activate or repress ERα-mediated gene transcription. KDM4B as a histone demethylase could regulate demethylation at histone H3K9 and H3K27. However, the epigenetic events responsible for the calcification process associated with KDM4B are mainly unknown. We, therefore, explored the mechanism underlying the function of KDM4B on modulation of ERα-mediated transactivation in VC. Our study has shown that KDM4B suppresses ERα-mediated gene transcription independent of its demethylase activity. KDM4B interacts with the PRC2 complex to be recruited to the promoter region of ERα target gene. In addition, KDM4B-mediated enhancement of ASMCs calcification was partially attenuated by the estrogen treatment (Fig. 8 C). Therefore, KDM4B may be a new potential therapeutic target for VC treatment. It has been reported that the KDM4 family is a class of demethylases that can precisely remove H3K9 or H3K36, thereby playing a central role in the histone code. A previous study demonstrated the relationship between KDM4B and ERα[ 45 ]. For example, KDM4B acts as an ERα cofactor that promotes mammary gland development and maturation. H3K9, KDM3A, and KDM4B synergistically regulate ERα activity through an autoregulatory loop that facilitates the recruitment of each co-activating enzyme to chromatin [ 45 ]. KDM4B recruitment within the upstream regulatory region of the ERα gene and demethylation of inhibitory H3K9me3 markers allow GATA-3 binding to drive receptor expression. This indicates the importance of KDM4B in the ERα signaling cascade and suggest a potential therapeutic target for BC therapy [ 40 ]. Deletion of KDM4B reduced WEE1, CCND1, and CCNA1 transcription and disrupted the destrier-induced cell cycle G1-S phase transition in BC cells, resulting in BC cell arrest in the G2-M or G1-S phase inhibition of BC occurrence [ 40 , 41 , 46 ]. We discovered that KDM4B overexpression promotes AR recruitment to the c-Myc gene enhancers and induces H3K9 demethylation; hence, increasing AR-dependent c-Myc mRNA transcription, which regulates sensitivity to next-generation AR-targeted therapies [ 47 ]. Although KDM4B enzymatic activity is required to enhance AR transcriptional activity, we have previously shown that KDM4B is an androgen-regulated demethylase that affects the transcriptional activity of AR not only through demethylation activity, but also through the regulation of ubiquitinylation [ 48 ]. KDM4B-regulated alternative splicing promotes AR-V7 expression and KDM4B promotes PCa cell growth via AR-V7 under androgen-deprivation conditions [ 49 ]. In our study, we used immunofluorescence to assess whether KDM4B and ERα co-localized in the nucleus in the presence or absence of calcification and E2. Co-IP results confirmed that they associated with each other in HASMCs and MOVAS cells. Interestingly, luciferase assay showed that KDM4B inhibited ERα-mediated gene transcription, whereas a mutant version of KDM4B with loss of demethylase activity (KDM4B HTE/ATA) had no synergistic effect on ERα in HASMCs and MOVAS cells. These results suggest that the down-regulation of ERα by KDM4B is independent of the enzymatic activity and is independent of the JmjC domain. Alternatively, KDM4B may bind to the original reader but not to another reader. The intricate chromatin environment may dictate the diverse functions exhibited by different tissues and various cell types. Histone methyltransferase complexes consist primarily of histone methyltransferases and histone interacting proteins that play a critical role in chromatin remodeling, histone modification, cell differentiation, or tumorigenesis in mammalian cells[ 50 ]. The PRC2 core subcomplex has H3K27 methyltransferase activity, which contains EZH2, SUZ12, and other potentially combinatorial proteins [ 50 – 54 ]. The STRING database was used to predict the proteins that may be related to KDM4B, and we discovered that KDM4B may interact with EZH2 and SUZ12 in the presence of ESR1 (ERα). In HASMCs and MOVAS cells, we provide evidence that KDM4B could interact with the PRC2 subcomplex alongside ERα, thereby influencing the spatial interaction between PRC2 proteins and ERα. On chromatin, KDM4B can affect the recruitment of PRC2 complexes to specific DNA regions. In addition, KDM4B promoted the recruitment of PRC2 complex core proteins and further altered the histone modification level in the promoter regions of ERα target genes. These results suggest that KDM4B, an ERα co-regulator and a PRC2 complex bridge protein, might be involved in complex chromatin events essential for epigenetic modulation of ERα-mediated gene transcription in VC. It has been reported that KDM4B plays crucial roles during bone formation. KDM4B further enhances the expression of DLX2 and DLX5 by removing H3K9me3 marks; hence, facilitating osteogenic commitment [ 55 , 56 ]. In the process of osteoclast formation, KDM4B physically binds to CCAR1-MED1 to form a complex, which localizes to the promoter of multiple osteoclast-related genes and alters the chromatin structure through the action of histone demethylase, thereby promoting the expression of related genes [ 57 ]. Moreover, although it has also been shown that KDM4B knockdown weakened the osteogenic differentiation in MSCS, the audiogenic differentiation was enhanced. Loss of KDM4B increases H3K9me3, which reduces bone formation and increases bone marrow fat, thereby exacerbating bone aging and osteoporosis [ 58 ]. In our study, KDM4B was highly expressed in the calcification model and we showed it played a role modulating ERα-mediated gene transcription regulation. Ectopic expression of KDM4B aggravated mineralization and increased calcium content as well as the expression levels of calcification markers (BMP2, Runx2, and RANKL); thus, indicating that calcification is exacerbated by KDM4B. Additionally, KDM4B-mediated enhancement of β-GP-induced calcification was attenuated by the estrogen treatment. This suggests that E2/ERα underlies the function of KDM4B in VC. Our data have revealed that KDM4B acts as a ERα-mediated transcriptional regulator independent of its demethylase activity. Currently, VC is considered an independent predictor of cardiovascular events. For example, simple supplementation with estrogen might alleviate VC; however, such treatment might be associated with numerous estrogen-specific adverse and might not be conducive to treating diseases [ 10 , 59 , 60 ]. Our findings uncover an epigenetic factor that acts as a novel co-regulator of ERα, participating in the VC pathway. This discovery offers vital insights for postponing the onset of cardiovascular disease in older adults. Furthermore, our findings indicate that KDM4B could be utilized as a marker for predicting VC and might even emerge as a viable therapeutic target for treating this condition. Our study has demonstrated that KDM4B is highly expressed in β-GP-induced HASMCs and MOVAS cell calcification models. KDM4B co-represses ERα-induced transactivation independent of its demethylase activity in HASMCs and MOVAS cells. Furthermore, it facilitates the recruitment of PRC2 core proteins to the cis-regulatory elements of ERα target genes. Therefore, we believe that KDM4B is a potential therapeutic target for calcification, providing a novel insight into clinical treatment. Experimental procedures Cell line culture and in vitro calcification model HASMCs and MOVAS cells were cultured in Dulbecco's modified Eagle medium (DMEM, SERAXPRO). The HEK293 cell was cultured in DMEM (SERAXPRO). Cells were cultured with 10% fetal bovine serum (FBS, SERAXPRO), 100 U/mL penicillin and 100 µg/mL streptomycin (Glenview, Florida, USA) in a 37°C incubator with a humidified, under 5% CO 2 atmosphere. Next, 17-estradiol (E2, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethanol (Aladdin Scientific, Riverside, CA, USA). Cells were cultured in phenol red-free DMEM or RMPI1640 when needed to add an E2 stimulus. Culture dishes were purchased from Guangzhou Jet Bio-Filtration Co. Ltd., China. When VSMC reaches 50 ~ 70% confluency, VSMCs are treated with calcification medium containing 1% FBS and 5 mM β-GP. The calcification medium was changed every 2 days to induce calcification in VSMC. Antibodies The following antibodies were used in this study: anti-KDM4B (Cell Signaling Technology, Danvers, MA, USA, #8639), anti-KDM4B (ABclonal, Woburn, MA, USA, #A6670), anti-ERα (Cell Signaling Technology, #D8H8), anti-Gas6 (Proteintech, Sankt-Leon Rot, Germany, #13795-1-AP), anti-β-actin (Proteintech, #66009-1-AP), anti-EZH2 (Proteintech, #21800-1-AP), anti-SUZ12 (Cell Signaling Technology, #3737), anti-H3K9me3 (Cell Signaling Technology, #13969) and anti-H3K27me3 (Cell Signaling Technology, #9733), anti-Rabbit/Mouse (ABclonal), anti-IgG (Proteintech, #10238-1-AP), anti-HA (ABclonal M20003M), BMP2 (Proteintech, #18933-1-AP), anti-Runx2 (Sigma, #AV36678), and anti-RANKL (Proteintech, #23408-1-AP). Transfections, siRNA, and luciferase dual reporter assay The HA-KDM4B plasmid was purchased from Addgene (Plasmid #24181). According to a previous study, the HA-KDM4B mutant was cloned into the PCMV-HA vector [ 44 ]. Expression plasmids for ERα, ERα-AF1, and ERα-AF2 were kindly provided by Dr Shigeaki Kato [ 61 ]. Final constructs were verified using DNA sequencing. siRNA against KDM4B were purchased from JTS Scientific (London, UK). All sequences are listed in Supplementary Table S1 . Cell lines were co-transfected with the listed constructs according to the manufacturer's instructions using jetPRIME™ DNA Transfection Reagent (Polyplus transfection). KDM4B and its truncated mutants, ERα, and ERE-tk-Luc were co-transfected with a plasmid containing Renilla luciferase (pRL). After co-transfection, cells are cultured in medium containing 3% charcoal-stripped fetal bovine serum (CS-FBS) for 6 h with or without E2 stimulation. After another day, cells were collected for dual-luciferase reporter assay (Promega, Madison, WI, USA). Western blotting analysis, Co-Immunoprecipitation, and glutathione sepharose (GST) pull-down assays Western blotting analysis was performed using a standard procedure introduced in previous studies [ 62 , 63 ]. In brief, cells or vascular tissues were lysed on ice for 30 min using lysis buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% NP‑40, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA and protease inhibitor cocktail (B14001, Houston, TX, USA)]. Lysates were vortexed every 10 min and then centrifuged at 13000 × g for 20 min at 4°C. Next, the supernatant protein contents were measured using G250, and 30–50 µg lysates were prepared for blotting. Samples were loaded into 8% or 12% polyacrylamide gels, separated by SDS-PAGE, and transferred onto PVDF membranes (ISEQ00010, Millipore, Burlington, MA, USA) under 80 v for 120 min. Next, membranes were blocked with 5% nonfat milk in TBST solution [20 mM Tris (pH 7.4), 137 mM NaCl, and 0.05% Tween‑20] for 1 h at room temperature and probed with primary antibodies overnight at 4°C. The next day, after washing the membranes with TBST solution three times, the membranes were incubated with secondary antibodies for 1 h at room temperature. Finally, the membranes were detected by chemiluminescence. Co-immunoprecipitation begins with whole cell lysis after 2 h of purification with anti-IgG. Protein G beads were purchased from GE Healthcare (Chicago, Il, USA) and concentrated into sepharose for overnight rotation of antibody-protein interactions. The GST pull-down assay was performed using a standard protocol introduced in a previous study [ 62 ]. GST alone proteins and GST fusion proteins, including GST-ERα 29-180aa and GST-ERα 282-595aa, were expressed in BL21 and bound to GST-sepharose beads according to the manufacturer's instructions (GE Healthcare). HA-KDM4B expression plasmid was constructed to synthesize the HA-KDM4B protein. Equal amounts of GST alone or conjugated GST-sepharose beads of GST fusion protein with in vitro translated HA-KDM4B protein were incubated overnight at 4°C. The precipitated proteins were washed 4 times with binding buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 10% glycerol, and 1% Nonidet P-40). Western blotting and Coomassie brilliant blue staining were used to detect binding proteins. Confocal immunofluorescence imaging Cells are fixed in 4% paraformaldehyde for 15 min at room temperature and then blocked in 1% donkey serum albumin. Cells were incubated with primary antibodies overnight at 4°C and then conjugated with secondary antibody to FITC, Cy 3, or Cy 5 (Jackson Immunoresearch Laboratories Inc., West Gove, PA, USA). DAPI (Roche, Zurich, Switzerland) staining was used to observe nuclear morphology. RNA and quantitative real-time PCR (qPCR) After transfection of control and KDM4B siRNA, RNA was extracted. At 4–6 h after transfection, estrogen and the same amount of EtOH were added to a final concentration of 100 nM for 16–18 h. Total RNA was extracted with RNA Trizol (TAKARA Bio, Tokyo, Japan), and the PimeScript RT-PCR Kit (TAKARA Bio) was used to reverse transcribe the cDNA. The SYBR premeraseTaq Kit (TAKARA Bio) was used for real-time PCR detection of Roche's LightCycler96. All primers for qPCR are listed in Supplementary Table S2. Statistical analysis was performed using PRISM GraphPad 8. Each experiment was performed at least in triplicate and significance was tested using Student's t -tests. Chromatin immunoprecipitation (ChIP) ChIP assays were performed using a standard protocol [ 64 ]. HASMCs were transfected with HA-tagged plasmids to overexpress KDM4B, or KDM4B siRNA to knockdown its expression. After transfection, cells were cultured in phenol-red DMEM medium containing 10% CS-FBS for 2 days. When the cell confluency reached 80%, cells were stimulated with 100 nM E2 or an equivalent amount of EtOH for 12 h. After the experiment, qPCR was performed using DNA as the template (primers are listed in Supplement Table S3). Alizarin Red staining Cells were washed thrice with PBS solution, fixed with 4% paraformaldehyde (#AR-0211, Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) for 30 min at room temperature, then washed with PBS solution and stained with 1% Alizarin red (pH 4.2, #G1452, Solarbio, Beijing, China) for 30 min at room temperature. Finally, stained cells were rinsed 3 times with distilled water at room temperature and imaged. Quantification of calcium deposition Cells or the vascular tissues were washed with PBS solution and then decalcified with 0.6 M HCL at 37°C for 24 h. The supernatant was collected and used for calcium quantification (Biosino Bio-Technology And Science Inc., Beijing, China). Next, cells or tissues were lysed (0.1 M NaOH, 0.1% SDS) for 30 min, then centrifuge at 12,000 × g for 20 min at 4°C, and the protein concentration was measured using a BCA kit (Cat NO: KGP903, Keygen Biotech, Nanjing, China) to normalize the calcium levels. Animal calcification model Mice were brought from Beijing Vitonglihua Experimental Animal Technology Limited Company (China). All experiments were approved by the Institutional Animal Protection and Use Committee (IACUC) of China Medical University and performed in accordance with the IACUC guidelines. To induce VC phenotypes in vivo , we used classical and recognized mouse calcification models. Mice were injected subcutaneously with VitD3 solution [5 × 10 5 UI/kg Vit D3 mixed in corn oil], weighed once every 3 days, and sacrificed on the 7th day. Statistical analysis All results are presented as the mean ± standard deviation (SD) and analyses were performed using the PRISM GraphPad 8 software program. A two-sided Student's t -test was used to determine the significant differences in calcium deposition, the real-time PCR data, and luciferase assay. Significance was measured using p-values (* p < .05, ** p < .01, and *** p < .001). Abbreviations VC vascular calcification ASMCs aortic smooth muscle cells HASMCs human aortic smooth muscle cells MOVAS mouse aortic vascular smooth KDM4B Histone lysine demethylases 4B PRC2 Polycomb repressive complex 2 BMP2 bone morphogenic protein 2 OVX ovary removal group β-GP β-phosphoglycerol ERα estrogen receptorα ERE estrogen response element GAS6 Growth arrest-specific gene 6 BC breast cancer JmjN Jumanji N MSC mesenchymal stem cells VSMCs vascular smooth muscle cells CS-FBS charcoal-stripped fetal bovine serum ChIP chromatin immunoprecipitation Co-IP co-immunoprecipitation qPCR quantitative polymerase chain reaction EZH2 Zeste omologue 2 SUZ12 suppressor of zeste 12 GST glutathione sepharose. Declarations Acknowledgements We appreciate Dr. Xiaoxu Zhou and Dr. Tao Sun for their excellent technical assistance. Author Contributions Conceptualisation: Fei Liu, Wen Tian and Yue Zhao; Methodology: Fei Liu; Software: Baosheng Zhou, Hao Li, and Mengsu Cao; Validation: Chunyu Wang, and Yue Zhao; Formal analysis: Fei Liu, and Yue Zhao; Investigation: Fei Liu, Baosheng Zhou, Yanxia Lin; Resources: Yang Lv, Shengli Wang, Kai Zeng, Xiaocen Chang, and Lin Lin; Writing: Fei Liu, Kato Shigeaki, Wen Tian and Yue Zhao; Supervision: Wen Tian, and Yue Zhao. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Declaration of interests The authors have no relevant competing interests to declare in relation to this manuscript. Funding disclosure This study was supported by the Liaoning Provincial Project of Applied Basic Research (2022JH2/101300061 for Wen Tian); National Natural Science Foundation of China (32170603, 31871286 for Yue Zhao, 82273123 for Chunyu Wang, 32100440 for Ge Sun); China Postdoctoral Science Foundation (276066) for Ge Sun; Foundation of Liaoning Province of China (LJKZ0756 for Shengli Wang); Local projects supported by the central government (2022JH6/100100035 for Yue Zhao); Foreign expert project of Ministry of Science and Technology (G2022006007L for Yue Zhao). Ethics approval and consent to participate Mice were brought from Beijing Vitonglihua Experimental Animal Technology Limited Company (China). All experiments were approved by the Institutional Animal Protection and Use Committee (IACUC) of China Medical University and performed in accordance with the IACUC guidelines. Consent for publication Not applicable. Supporting information This article contains supporting information. 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Int J Biol Sci,2015; 11(9): 992-1005. https://dx.doi.org/10.7150/ijbs.10918. Jia P, Wu N, Yang H, Guo Y, Guo X,Sun Y. Different roles of BAG3 in cardiac physiological hypertrophy and pathological remodeling. Transl Res,2021; 23347-61. https://dx.doi.org/10.1016/j.trsl.2021.02.004. Nelson JD, Denisenko O,Bomsztyk K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc,2006; 1(1): 179-185. https://dx.doi.org/10.1038/nprot.2006.27. Additional Declarations There is a duality of interest Supplementary Files SupportingInformation0422.docx Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Cell Death Discovery → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6522511","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":450980118,"identity":"e5e8e251-3f34-4261-a373-d39bc508eeb6","order_by":0,"name":"Yue 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University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Li","suffix":""},{"id":450980132,"identity":"f1a4777f-06d3-446b-a28b-7368cbbf34b9","order_by":14,"name":"Xihong Hu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xihong","middleName":"","lastName":"Hu","suffix":""},{"id":450980133,"identity":"4a83d855-9a7d-41fa-8713-69a75ef93c31","order_by":15,"name":"Shigeaki Kato","email":"","orcid":"https://orcid.org/0000-0002-5564-1695","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shigeaki","middleName":"","lastName":"Kato","suffix":""},{"id":450980134,"identity":"cd74f423-2231-4e4c-bc93-aeb842946206","order_by":16,"name":"Wen Tian","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Tian","suffix":""}],"badges":[],"createdAt":"2025-04-24 16:15:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6522511/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6522511/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-025-02765-6","type":"published","date":"2025-10-07T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82815192,"identity":"62e9eb9f-5c82-4d8a-b251-59216119efca","added_by":"auto","created_at":"2025-05-15 14:10:20","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1164259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B is highly expressed in β-GP-induced calcification models in HASMCs and MOVAS cells. \u003c/strong\u003e(A) Alizarin red staining on different days in Human aortic vascular smooth muscle-derived cells (HASMCs) following 5 mM β-GP treatment. (B) Quantification of calcium deposition on different days in HASMCs treated with 5 mM β-GP. (C) Western blotting analysis for KDM4B, Runx2, BMP, and RANKL expression in HASMCs treated with 5 mM β-GP (β-actin was used as a loading control and the day 0 was set as the control in each parameter). (D) qRT-PCR for KDM4B, Runx2, BMP, and α-SMA expression in HASMCs treated with 5 mM β-GP. Statistical significance was determined using the Student \u003cem\u003et\u003c/em\u003e-tests. Error bars represent mean ± SD. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, and ns stand for non-significant. (E) Alizarin red staining on different days in MOVAS cells following 5 mM β-GP treatment. (F) Quantification of calcium deposition in MOVAS cells treated with 5 mM β-GP. (G) Western blotting analysis for KDM4B, Runx2, BMP, and RANKL expression in MOVAS cells treated with 5 mM β-GP (β-actin was used as a loading control and the day 0 was set as the control in each parameter). (H) qRT-PCR for KDM4B, Runx2, BMP, and α-SMA expression in MOVAS cells treated with 5 mM β-GP. Statistical significance was determined using the Student \u003cem\u003et\u003c/em\u003e-tests. Error bars represent mean ± SD. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, and ns stand for non-significant. Data were presented as the mean ± SD of triplicate experiments.\u003c/p\u003e","description":"","filename":"1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/f8ed7030393ffacf661e7176.jpeg"},{"id":82813987,"identity":"7415ce02-e3e6-4cb3-a70b-19e88a42d2b4","added_by":"auto","created_at":"2025-05-15 14:02:20","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":435320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B associates with ERα in HASMCs and MOVAS cells.\u003c/strong\u003e (A) The distribution of KDM4B and ERα in HASMCs treated with or without E2 (E2, 100 nM) and β-GP. Cells were stained with anti-DAPI (Blue), anti-ERα (Green), and anti-KDM4B (Red). Scale bars were 50 μm. (B) KDM4B directly binds GST-ERαAF1 and GST-ERαAF2 \u003cem\u003ein vitro\u003c/em\u003e. The GST-ERαAF1 and GST-ERαAF2 proteins were expressed in prokaryotic cells and purified using GST beads and then incubated with lysate of HEK-293 T cells expressing HA-KDM4B. After being washed with cold PBS, the eluted complexes were subjected to western blotting and detected with specific antibodies. (C) HASMC cells were co-transfected with PcDNA3.1/HA-KDM4B and ERα plasmids and treated with or without estrogen. Whole-cell extracts were immunoprecipitated with the anti-HA antibody or IgG after transfection for two days. Input means 5% of the entire extract for each column. (D, E) Co-IP experiments show endogenous KDM4B and ERα associated with each other in HASMC cells. Reciprocal Co-IP and immunoblotting were performed with antibodies as indicated. A 5% fraction of the input cell lysate before immunoprecipitation was loaded as a control. (F, G) Co-IP experiments show the interaction between endogenous KDM4B and ERα in MOVAS cells. (H) HEK293 cells were co-transfected with PcDNA3.1/HA-KDM4B and ERα plasmids and treated with or without estrogen (E2, 100 nM). Whole-cell extracts were immunoprecipitated with the anti-HA antibody or IgG after transfection for two days. Input means 5% of the whole extract for each column.\u003c/p\u003e","description":"","filename":"2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/cbef00c6ff265c77440d3b0f.jpeg"},{"id":82813990,"identity":"c2aed46f-6fa0-43f5-909f-ecc049a2299c","added_by":"auto","created_at":"2025-05-15 14:02:20","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":990980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B co-represses ERα-induced transactivation independent of its demethylase activity in HASMCs and MOVAS cells.\u003c/strong\u003e (A) KDM4B co-represses ERα-induced transactivation. HASMCs were co-transfected with ERE-tk-luc and pRL-tk expression plasmids together with HA-KDM4B or PcDNA3.1 plasmid (vector) as indicated and treated with or without E2. The expression levels of HA-KDM4B were detected using western blotting with anti-HA. (B) KDM4B co-represses the transcriptional activity induced by ERα or its two truncated mutants (ERαAF1 and ERαAF2) in HASMCs. (C) Plasmid sequencing diagram of KDM4B and KDM4Bmut (HTE/ATA) (loss of function of KDM4B demethylase activity mutation). (D) HASMCs were transfected with HA-KDM4Bwt (overexpress KDM4B full length) and the HA-KDM4Bmutant (HTE/ATA) and cultured in growth medium for three days. (E) KDM4B (HTE/ATA) carrying a loss of function in demethylase activity exhibits reduced co-activation function on ERα action in HASMCs. (F) KDM4B (HTE/ATA) carrying a loss of function in demethylase activity exhibits reduced coactivation function on ERα action in MOVAS cells. Relative luciferase activity represents the mean value at least three replicates. Student \u003cem\u003et\u003c/em\u003e-tests were used, and error bars represent mean ± SD. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, and ns stand for non-significant. (G, I) qPCR analysis was examined for the effects of KDM4B on mRNA expression of endogenous ERα target genes in HASMCs. Statistical significance was determined using the Student \u003cem\u003et\u003c/em\u003e-tests. Error bars represent mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, and ns stands for non-significance. (H, J) The effects of KDM4B on protein expression of endogenous ERα target genes in HASMCs. β-actin was used as a control.\u003c/p\u003e","description":"","filename":"3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/fd278056f3d87e31d7167027.jpeg"},{"id":82813995,"identity":"03f2b568-c279-41f9-855b-570ab2285993","added_by":"auto","created_at":"2025-05-15 14:02:20","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1003310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B facilitates the recruitment of PRC2 core proteins to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-regulatory elements of ERα target genes.\u003c/strong\u003e (A) The STRING protein network analysis suggests that KDM4B might associate with ERα and the subunits of PRC2 complex (PRC2 core proteins), including EZH2 and SUZ12. (B-C) The Co-IP assays show an association between ERα and PRC2 core proteins andKDM4B in HASMCs. (D-E) The Co-IP assays show an association between ERα and PRC2 core proteins andKDM4B in MOVAS cells. (F) The Co-IP experiment in HASMCs harboring control siRNA or siKDM4B were performed using anti-ERα and IgG. Precipitated proteins were determined by western blotting with antibodies against PRC2 complex core proteins or KDM4B/ERα as indicated. The location of the star indicates where the protein was expressed. (G) A diagram indicating the KDM4B/ERα enrichments and modification of H3K9me3 at an independent ERE upstream of the TSS region of the Gas6 gene. The location of the EREs is indicated in the diagram above (Green rectangle). (H) ChIP assays were conducted using the specified antibodies to demonstrate protein recruitment, the effect of KDM4B on the recruitment of these proteins, and the levels of histone modification on Gas6-ERE. HASMCs were transfected with an overexpression construct for KDM4B, either with or without treatment with estrogen (100 nM). (I) ChIP assays for HASMCs transfected with either control siRNA or siKDM4B following treatment with or without estrogen (100 nM).\u003c/p\u003e","description":"","filename":"4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/274265f3a21855d196b8891d.jpeg"},{"id":82816881,"identity":"c2a7e07c-1b4f-4796-9306-775e1b8da916","added_by":"auto","created_at":"2025-05-15 14:26:20","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":504648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B depletion alleviates β-GP-induced calcification in HASMCs and MOVAS cells. All groups were treated with or without 5 mM β-GP for five days before quantifying the calcification levels.\u003c/strong\u003e(A-B) The depletion of KDM4B was achieved using three different siRNAs (siKDM4B#1, siKDM4B#2, and siKDM4B#3) and was detected through western blotting and quantitative real-time PCR (qPCR) in HASMCs. (C) Alizarin red staining of HASMCs transfected with siKDM4B#1 and siKDM4B#3. (D) Quantifying calcium deposition in HASMCs transfected with siKDM4B#1 and siKDM4B#3. (E-F) Western blotting analysis and quantification of protein expression for KDM4B, Runx2, BMP, and RANKL expression in HASMCs (β-actin was used as a loading control). The KDM4B knockdown was used as the control for each parameter. (G) The depletion of KDM4B in MOVAS cells was achieved siKDM4B#3 and was detected through western blotting. (H) Alizarin red staining in MOVAS cells transfected with siKDM4B#3. (I) Quantification of calcium deposition in MOVAS cells transfected with siKDM4B#3. Data were collected from 3 replicates and expressed as the mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, and ns stands for non-significant.\u003c/p\u003e","description":"","filename":"5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/aabfaeec683822d3f8e9a3e6.jpeg"},{"id":82814019,"identity":"a8993c24-7bfb-49a9-9ff6-88571e5878e9","added_by":"auto","created_at":"2025-05-15 14:02:22","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1297144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEctopic expression of KDM4B aggravates β-GP induced calcification in HASMCs and MOVAS cells.\u003c/strong\u003e (A) Western blotting of HASMCs overexpressing KDM4B following transfection with the HA-KDM4B. (B, E) Alizarin red staining of HASMCs. (C, F) Quantification of calcium deposition in HASMCs. (D, G) Western blotting analysis for HA-KDM4B, Runx2, BMP2, RANKL, and β-actin (used as a loading control). The KDM4Bwt or KDM4Bmut were overexpress in HASMCs treated with β-GP and used as the control condition for each parameter. (H) Western blotting of MOVAS cells overexpressing KDM4B following transfection with the HA-KDM4B. (I, L) Alizarin red staining of MOVAS cells. (J) Quantification of calcium deposition in MOVAS. (K) Western blotting analysis and quantification of protein expression for HA-KDM4B, Runx2, RANKL, and β-actin (used as a loading control). The KDM4Bwt or KDM4Bmut were overexpress in MOVAS cells treated with β-GP and used as the control condition for each parameter. Data were collected from 3 replicates and expressed as the mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, and ns stands for non-significant.\u003c/p\u003e","description":"","filename":"6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/f67f3a6f9772c0a1f9598674.jpeg"},{"id":82815716,"identity":"9533fc28-40bb-4a8b-9048-049d6091b1af","added_by":"auto","created_at":"2025-05-15 14:18:20","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":522271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B-mediated enhancement of β-GP-induced calcification is attenuated by the estrogen treatment.\u003c/strong\u003e (A) Alizarin red staining of HASMCs. (B) Quantification of calcium deposition in HASMCs. Data were collected from 3 replicates and expressed as the mean ± SD. (C-E) Western blotting analysis and quantification of protein expression for KDM4B, Runx2, BMP2, RANKL, and β-actin (used as a loading control). HASMCs with KDM4B knockdown followed by treatment with or without β-GP were used as the control for each parameter. (F) Alizarin red staining of HASMCs. (G) Quantification of calcium deposition in HASMCs. (H) Western blotting analysis and quantification of protein expression for KDM4B, Runx2, BMP 2, RANKL, and β-actin (β-actin was used as a loading control). HASMCs with KDM4B knockdown followed by β-GP treatment was used as the control for each parameter. Data are expressed as the mean ± SD of triplicate experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, and ns stands for non-significant.\u003c/p\u003e","description":"","filename":"7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/84940bd74d51b820af32f9da.jpeg"},{"id":82815194,"identity":"e9bcff92-0727-4ab4-b956-2da0c7cd5039","added_by":"auto","created_at":"2025-05-15 14:10:20","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":332063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B is highly expressed in a VitD3-induced calcification mouse model. To construct a calcification model, WT and OVX mice were treated with or without 5 × 10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e UI/kg VitD3, respectively. \u003c/strong\u003e(A) Mice were sacrificed and aortas were isolated for western blotting analysis of Runx2, RANKL, and β-actin (used as a loading control). (B) KDM4B, BMP2, and α-SMA immunostaining expression in the VC model. (C) Schematic diagram illustrating the modulatory function of KDM4B on ERα action and the role of KDM4B in promoting VC.\u003c/p\u003e","description":"","filename":"8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/999d30c75966e99658100659.jpeg"},{"id":93009022,"identity":"b3c8af84-2f13-47ae-a65c-f781f74395a2","added_by":"auto","created_at":"2025-10-08 07:06:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7778941,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/b4d97e2a-9aa0-4d6e-971b-80277f5f699d.pdf"},{"id":82815713,"identity":"5a017dcb-3ec5-43bf-af23-137fb56eabb2","added_by":"auto","created_at":"2025-05-15 14:18:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1745870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupportingInformation0422.docx","url":"https://assets-eu.researchsquare.com/files/rs-6522511/v1/88fee73fbb45898e4ecb10c5.docx"}],"financialInterests":"There is a duality of interest","formattedTitle":"KDM4B modulates ERα signaling pathway to participate \nin vascular smooth muscle cell calcification","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVascular calcification (VC) is a common degenerative phenomenon associated with aging, which is common in the end-stage of most chronic diseases and is characterized by the abnormal deposition of calcium and phosphorus onto the vascular wall [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. VC has been recognized as an independent predictor of cardiovascular events [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The relationship between vascular calcification and osteoporosis is well-established [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Osteoporosis, characterized by excessive bone resorption over formation, leads to the release of calcium into the bloodstream, which can then ectopically deposit in blood vessels, thus promoting vascular calcification. Estrogen is recognized for its role in the prevention of osteoporosis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and the inhibition of vascular calcification[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nevertheless, there are clear sex-specific patterns of VC, being present in more than 90% of men and 67% of women over 70 years of age. Coronary artery calcification is a component of VC, and its calcification evaluation is an independent predictor of cardiovascular adverse events in postmenopausal women. In addition to the differential lifestyle habits associated with the sexes, the cardiovascular benefits of women diminish after menopause, with the risk of cardiovascular disease increasing significantly in older women (approximately four times as much as before menopause) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The incidence of coronary artery calcification is lower in premenopausal women than in men, which may be related to the protective effect of estrogen [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Estrogen is a vital drug component in preventing and treating postmenopausal osteoporosis, which induces bone formation by activating the estrogen receptor signaling pathway. Mechanically, estrogen has been reported to inhibit the VC process in several ways, including classically promoting E2-related gene transcription, promoting autophagy, and inhibiting the HIF-1α signaling pathway [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although the biological function of the estrogen signaling pathway in VC has been investigated in recent years, the specific molecular mechanism remains unclear.\u003c/p\u003e \u003cp\u003eEstrogen receptor α (ERα) belongs to a nuclear receptor superfamily that undergoes conformational changes and translocates from the cytoplasmic lysate to the nucleus in the presence of estrogen, thereby binding to specific estrogen response element (ERE) to regulate its downstream gene transcription [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Studies have shown that ERα/E2 exerts a protective effect against VC by promoting the transcriptional activity of the growth arrest-specific gene 6 (GAS6) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, receptor activator of nuclear factor-Kappa B ligand (RANKL) promotes VC by regulating the expression of bone morphogenetic protein-2 (BMP2), MGP, and bone-associated protein, which is counteracted by estrogen in a receptor-dependent manner. In addition, estrogen acts mainly through the ERα to counteract these effects of RANKL stimulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Upon estrogen treatment, activated ERα recruits co-regulators that participate in ERα-mediated transcriptional activation, thereby altering chromatin structure, post-transcriptional modifications, and modulating ERα protein stability [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA variety of ERα co-regulators have been demonstrated to be essential for the development of breast cancer (BC) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The histone-modifying enzymes and chromatin remodeling factors enhance or inhibit ERα-mediated gene transcription, referred to as ERα co-activators or co-repressors. For example, KDM3A, a histone demethylase, plays a crucial role in ERα signaling by regulating the transcription of receptor-target genes through the control of demethylation at the cis-regulatory element H3K9me1/me2. Furthermore, KDM3A has been shown to be necessary for the growth of ERα-positive BC cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In ERα-positive or ERα-negative BC cells, the deletion of KDM4A reduces the expression of ERα target genes c-Jun and cyclin D1, leading to abnormal cell proliferation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The expression of KDM5A is also up-regulated in BC [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Mechanistically, the accumulation of p16 and p27 is promoted by blocking KDM5A-mediated H3K4me3 demethylation, leading to cell cycle arrest and aging [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. JMJD6 regulated trans-activation of ERα-binding enhancers and their downstream target genes. Furthermore, it is a critical factor in the growth and tumorigenesis of ERα-positive BC cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These findings indicate that ERα co-regulators are essential for the development of ERα-related diseases, including BC, endometrial carcinomas, and VC. However, the modulation of ERα-induced transactivation and the biological function of ERα co-regulators in VC remain elusive.\u003c/p\u003e \u003cp\u003eJMJD2 family members take active roles in multiple physiological processes, including cell proliferation, migration [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], gene transcription [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and genome stability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The KDM4 family, a class of demethylases, precisely removes methylation at H3K9 or H3K36 sites, playing a pivotal role in the histone code [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Lysine demethylase is a class of proteins that removes lysine/arginine methylation at specific histone H3/H4 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Histone lysine demethylases 4B (KDM4B), consisting of 1096 amino acids, exhibits catalytic activity on histone residues H3K9me3, H3K9me2, and H3K36me3, with a preference for the H3K9Me2/3 substrate [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. KDM4B contains a Jumanji N (JmjN) structural domain, a JmjC structural domain, two plant homology domains, and two Tudor structural domains, among which the active region for demethylase is the JmjC domain [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The demethylases enzyme KDM4B, a component of the demethylation module, is involved in gene transcriptional regulation. We have previously demonstrated that KDM4B is recruited to ERα target genes, where it demethylates H3K9me3 and up-regulated ERα transcription. Moreover, KDM4B plays an essential role in regulating estrogen signaling cascade, and its absence inhibits the growth of BC both in \u003cem\u003evitro\u003c/em\u003e and in \u003cem\u003evivo\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Notably, KDM4B itself is also an ERα response gene [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Thus, these findings suggest a positive feedback mechanism between KDM4B and ERα, where estrogen-induced KDM4B expression in turn co-regulates and up-regulates ERα target genes, thereby promoting BC growth. In addition, KDM4B can induce osteogenic differentiation of mesenchymal stem cells (MSCs). TGF-β induces the expression of KDM4B, whereas KDM4B recruits the transcription factor SMAD3 to the SOX9 promoter to inhibit the H3K9me3 modification level and promote chondrogenesis as well as differentiation of MSCs [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In vascular smooth muscle cells (VSMCs), KDM4B can be recruited to the STAT3 binding site of the Runx2 promoter region and inhibit the level of H3K9me3 modification, ultimately promoting Runx2 transcription [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Nevertheless, the mechanism underlying the effect of KDM4B on ERα-mediated gene transcription during VC remains poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, we explored the potential mechanisms by which KDM4B influences the VC process \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. KDM4B was highly expressed in the β-phosphoglycerol-induced VC model and a VitD3-induced VC mouse model. Furthermore, KDM4B interacted with endogenous ERα and co-repressed ERα-mediated transactivation. Unexpectedly, luciferase assay results suggest that KDM4B inhibits ERα-mediated gene transcription independent of its JmjC enzyme active region. In addition, KDM4B was required for the recruitment of PRC2 subunits to the ERE on ERα target genes, which was accompanied by the enrichment of H3K27me3 modification levels. Furthermore, KDM4B-mediated enhancement of β-phosphoglycerol (β-GP) induced calcification was attenuated by an estrogen treatment. Overall, our study identified KDM4B as a new ERα co-factor that down-regulates ERα-mediated transactivation in its demethylase-independent manner in human aortic smooth muscle cells (HASMCs) and mouse aortic vascular smooth (MOVAS) cells. Our findings demonstrated that KDM4B promotes VC via the E2/ERα signaling pathway, providing a potential therapeutic strategy for VC in females.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eKDM4B is highly expressed in β-GP-induced HASMCs and MOVAS cells calcification models\u003c/h2\u003e \u003cp\u003eEpigenetic factors that play an important role in the pathogenic regulation of cancer and other diseases\u0026rsquo; gene transcription were selected. The expression levels of KDM family genes (KDM2A, KDM3A, KDM4A, KDM5A, KDM6A, and KDM4B) are involved in the epigenetic regulation within HASMCs with different calcification status induced following treatment with 5 mM β-GP for 0, 1, 2, 3, and 5 days. We showed that β-GP induces HASMCs calcification in a time-dependent manner. Simultaneously, the notable expression of KDM4B increased progressively as the duration of calcification extended (as shown in Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-A). Alizarin red staining and calcium deposition quantification were performed to the measure calcification levels. We further corroborated the finding that β-GP induced HASMCs calcification in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The expression of calcification-related proteins was measured, with the results suggesting that runt-associated transcription factor 2 (Runx2), BMP2, and RANKL were gradually increasing. We also observed that the expression of KDM4B increased gradually with calcification stimulation time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The mRNA levels of calcification-related factors were also analyzed, and the results indicate that Runx2 and BMP2 gradually increased, whereas alpha-smooth muscle actin (α-SMA) gradually decreased. Concurrently, an incremental increase in the mRNA expression of KDM4B was observed across various levels of calcification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The same results were obtained for MOVAS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These results suggest that KDM4B is highly expressed in β-GP-induced calcification models and may be involved in the calcification process.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKDM4B is associated with ERα in HASMCs and MOVAS cells\u003c/h3\u003e\n\u003cp\u003eClinical findings showed that women have a higher risk of VC and cardiovascular disease after menopause[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, the ERα signaling pathway has been suggested to be involved in inhibiting the calcification process associated with VC[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. KDM4B is also a critical epigenetic demethylase involved in regulating transcription factors and it up-regulates ERα-target genes; hence, sustaining BC cell growth. In our effort to understand the regulatory relationship between KDM4B and ERα in VC, we investigated the association between these two proteins. Initially, immunofluorescence assays were performed to examine the subcellular distribution of KDM4B and ERα in HASMCs. Our data showed that KDM4B and ERα are predominantly distributed in the nucleus in the absence or presence of E2 treatment in HASMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). GST labeled ERαAF1 or ERαAF2 expression vectors, were further used for the GST pull-down experiments, revealing that KDM4B directly binds to GST-ERαAF1 and AF2 \u003cem\u003ein vitro\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In addition, HASMCs were transfected with KDM4B expression plasmid for co-IP assay. Co-IP results showed that HA-labeled KDM4B precipitated with ERα (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFurther, endogenous interactions between KDM4B and ERα in HASMCs were detected using endogenous-cellular co-IP assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Consistent with the previous results, endogenous interactions between KDM4B and ERα were also observed in MOVAS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). HEK-293 cells were co-transfected with ERα and HA-labeled KDM4B expression plasmid for co-IP assay. Our results showed that ERα precipitated with the HA-labeled KDM4B (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These data demonstrated that KDM4B associates with ERα in HASMCs and MOVAS cells.\u003c/p\u003e\n\u003ch3\u003eKDM4B co-represses ERα-induced transactivation in HASMCs and MOVAS cells\u003c/h3\u003e\n\u003cp\u003eTo detect the regulatory function of KDM4B on the ERα activity, luciferase assays were performed in HASMCs. Our results showed that KDM4B significantly down-regulates ERα-mediated transactivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To further identify the ERα domain regulated by KDM4B, we performed luciferase experiment that examined the effect of KDM4B on ERα-induced transcriptional activity containing a nonligand-dependent AF-1 domain (ERα AF-1) or a ligand-dependent AF-2 domain (ERα AF-2). We observed that KDM4B down-regulated ERα AF-2-mediated transcriptional activation in the presence of E2, whereas the ERα AF-1 activation was not regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To further detect whether the demethylase activity of KDM4B is required for its regulation function on ERα-mediated transactivation, according to the previous paper [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], we constructed a KDM4B mutant expression plasmid (KDM4B HTE/ATA) carrying a demethylase loss-of-function mutation, as shown in diagram of plasmid sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Western blotting was used to verify the transfection efficiency of KDM4B full-length and its mutant counterpart (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Alternatively, the luciferase assay was performed to detect whether KDM4B down-regulates ERα-mediated transactivation depending on its demethylase activity. We observed that both KDM4B full-length or its mutant significantly down-regulated ERα-mediated transactivation in both HASMCs and MOVAS cells. This suggests that KDM4B may down-regulate ERα-mediated transactivation independent of its demethylase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). To strengthen the argument, a luciferase assay also was performed with KDM4B inhibitor, the results showed that KDM4B inhibitor has the similar effects as that of KDM4B or KDM4B mutant on ERα-mediated transactivation (Supplementary Figure S2A). Meanwhile, the results from Alizarin red staining showed that KDM4Bwt, KDM4Bmutant, or KDM4B inhibitor aggravated β-GP-induced calcification in MOVAS cells, respectively (Supplementary Figure S2B). These data suggest that the co-repression function of KDM4B on ERα-mediated transactivation may not be dependent on its demethylase activity. Furthermore, qPCR was used to examine the modulation functions KDM4B on the expression of the endogenous estrogen-responsive gene mRNA in HASMCs. Our data showed that ectopic expression of KDM4B decreased the mRNA expression of \u003cem\u003eGas6\u003c/em\u003e, but not \u003cem\u003eESR1\u003c/em\u003e in HASMCs and MOVAS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and Supplementary Figure S3A). Furthermore, western blotting showed that KDM4B overexpression decreased Gas6 protein expression in HASMCs and MOVAS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and Supplementary Figure S3C). Similar experiments were performed in HASMCs and MOVAS cells transfected with siKDM4B; we observed that KDM4B depletion exceedingly increased the mRNA and protein expression of Gas6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ and Supplementary Figure S3B and S3D). Furthermore, both overexpression of the KDM4B full-length and the mutant version decreased Gas6 protein expression in HASMCs (Supplementary Figure S3E). In summary, our results suggest that KDM4B co-represses ERα-induced transactivation independent of its demethylase activity in HASMCs and MOVAS cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKDM4B facilitates the recruitment of PRC2 core proteins to the\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-regulatory elements of ERα target genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to a previous report, KDM4B up-regulates ERα-mediated gene transcription mainly in BC [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Interestingly our results demonstrate that KDM4B down-regulates ERα-mediated gene transcription. Thus, we would like to further study the mechanism through which KDM4B is involved in ERα-mediated gene transcription in VC. We sought to identify the proteins interacting with KDM4B as potential protein complexes. The STRING protein network analysis suggests that KDM4B may be associated with ERα and the subunits of the PRC2 complex (PRC2 core protein), including Enhancer of Zeste omologue 2 (EZH2) and Suppressor of zeste 12 (SUZ12) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To determine the correlation of ERα and PRC2 core proteins and KDM4B in HASMCs and MOVAS cells, we performed co-IP experiments. Our data demonstrated that ERα and KDM4B associated with the core protein subunits of the PRC2 complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E). Additionally, the interactions between ERα and the PRC2 subunits were significantly reduced when KDM4B was depleted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results suggest that KDM4B could mediate the association of ERα and the PRC2 core protein.\u003c/p\u003e \u003cp\u003eTo further analyze the molecular mechanism underlying the influence of KDM4B on estrogen-induced gene transcription, specifically for genes such as \u003cem\u003eGas6\u003c/em\u003e, we utilized the JASPAR software to predict the Estrogen response element (ERE) on \u003cem\u003eGas6\u003c/em\u003e. An ERE spanning between \u0026minus;\u0026thinsp;72 and \u0026minus;\u0026thinsp;89 bp was observed from the translation start site of \u003cem\u003eGas6\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). ChIP assay was performed in HASMCs treated with or without estrogen. During estrogen induction, KDM4B or ERα was recruited into the promoter region of Gas6-ERE. The overexpression of KDM4B promoted the recruitment of PRC2 core protein, not affecting the recruitment of ERα; thus, enhancing the levels of H3K27me3 proximal to the Gas6-ERE region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). To confirm these results, ChIP analysis was further performed in cells transfected with siKDM4B. KDM4B deletion can reduce the recruitment of the PRC2 complex to the same region. In addition, in the presence of E2, KDM4B deletion decreased the levels of H3K27me3 adjacent to the Gas6-ERE promoter region, whereas no significant effects of KDM4B on H3K9me3 levels were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). These results indicated that KDM4B could facilitate PRC2 subunit recruitment to the promoter region of ERα target genes; thus, altering the level of H3K27me3 and enhancing gene transcription. In summary, our results suggest that KDM4B is associated with ERα and PRC2 core proteins and down-regulates ERα-mediated gene transcription in HASMCs and MOVAS cells.\u003c/p\u003e\n\u003ch3\u003eKDM4B aggravates β-GP-induced calcification in HASMCs and MOVAS cells\u003c/h3\u003e\n\u003cp\u003eWe showed that KDM4B act as a co-repressor of ERα and mediates the ERα transactivation in HASMCs and MOVAS cells. Further, we examine the biological function of KDM4B in β-GP-induced calcification in HASMCs and MOVAS cells. KDM4B was knockdown following transfection with siKDM4B. To mitigate potential off-target effects of siRNA, we analyzed the impact of two distinct siRNA sequences (siKDM4B#1 and siKDM4B#3) on KDM4B, as verified by western blotting and qPCR in HASMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and MOVAS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Concurrently, Alizarin red staining and quantitative calcium analyses revealed that KDM4B depletion alleviated the calcification induced by the 5 mM β-GP treatment in HASMCs and MOVAS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Aligning with these findings, western blotting analysis and subsequent quantification of protein expression demonstrated that KDM4B depletion led to a decrease in the expression of calcification markers such as Runx2, BMP, and RANKL over different treatment periods with 5 mM β-GP in HASMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eTo further reveal the role of KDM4B in calcification, western blotting analyses were performed in cells with ectopic expression of KDM4B (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Alizarin red staining and calcium deposition indicate that KDM4B overexpression significantly aggravated β-GP-induced calcification in HASMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Furthermore, we observed enhancing calcification effects for both KDM4B full-length and KDM4B mutant overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Accordingly, the western blot analysis of calcification markers, including Runx2, BMP2, and RANKL, indicates that both KDM4Bwt and KDM4Bmut exert comparable effects on β-GP induced calcification in HASMCs, as illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG. This increase in marker expression was also observed over different days of the 5 mM β-GP treatment in HASMCs, as confirmed by western blotting analysis and subsequent quantification of protein expression (Supplementary Figure S4A, 4B). These effects were also observed in MOVAS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Taken together, our results suggest that KDM4B may play an essential role in calcification, and this function may not be influenced by its deubiquitinase activity.\u003c/p\u003e\n\u003ch3\u003eKDM4B-mediated enhancement of β-GP-induced calcification is partially attenuated by estrogen treatment\u003c/h3\u003e\n\u003cp\u003eThe ERα signaling pathway is essential for suppression of VC. Having established in this study that KDM4B acting as a co-repressor of ERα inhibits ERα-mediated transactivation. Next, we thus turned to examine whether KDM4B-mediated enhancement of β-GP induced calcification is related with ERα signaling. Alizarin red staining and quantitative calcium analysis showed that KDM4B overexpression increased calcification following the β-GP treatment, which was partially attenuated by the estrogen treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In addition, the pro-calcific effect of KDM4B overexpression on β-GP-induced calcification, as detected by the expression of calcification markers Runx2, BMP2, and RANKL, was attenuated by estrogen treatment in HASMCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Additionally, Alizarin red staining and quantitative calcium analysis revealed that KDM4B depletion reduced calcification in the presence of the β-GP treatment, the estrogen treatment at least partially attenuates KDM4B-mediated enhancement of β-GP-induced calcification (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Western blotting results showed that KDM4B depletion could decrease β-GP-induced calcification indicated by the expression of calcification markers Runx2, BMP2, and RANKL was also attenuated by the estrogen treatment in HASMCs or MOVAS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, Supplementary Fig.\u0026nbsp;5A, 5B). These results suggest that the function of KDM4B on β-GP-induced calcification is, if not all, but partially related to estrogen/ERα signaling pathway.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKDM4B is highly expressed in a VitD3-induced calcification mouse model\u003c/h2\u003e \u003cp\u003eUltimately, an \u003cem\u003ein vivo\u003c/em\u003e calcification model was generated to confirm the \u003cem\u003ein vitro\u003c/em\u003e findings. We generated a mouse calcification model using VitaminD3. After the calcification protocol, we isolated the aortas and conducted western blotting and immunohistochemistry. The protein expression of KDM4B in the calcified aged mouse was higher than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Immunohistochemical results showed that the KDM4B expression was enhanced in the ovary removal group (OVX) and further increased in the OVX with VitD3-induced calcification (OVX\u0026thinsp;+\u0026thinsp;VC) group. The expression levels of calcification marker BMP2 were increased and the expression levels of α-SMA were decreased in OVX\u0026thinsp;+\u0026thinsp;VC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Our results suggest that KDM4B is highly expressed in VitD3-induced calcification mice.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent studies, the estrogen/ERα signaling pathway has been shown to play a significant role in VC. Estrogen inhibits the VC process in several ways, including the promotion of E2-induced gene transcription, promoting autophagy, and inhibiting the HIF-1α signaling pathway [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. ERα as a typical transcription factor mediates E2-induced gene transcription and inhibits VC. Co-activators and co-repressors of ERα are epigenetic enzymes and chromatin remodelers that activate or repress ERα-mediated gene transcription. KDM4B as a histone demethylase could regulate demethylation at histone H3K9 and H3K27. However, the epigenetic events responsible for the calcification process associated with KDM4B are mainly unknown. We, therefore, explored the mechanism underlying the function of KDM4B on modulation of ERα-mediated transactivation in VC. Our study has shown that KDM4B suppresses ERα-mediated gene transcription independent of its demethylase activity. KDM4B interacts with the PRC2 complex to be recruited to the promoter region of ERα target gene. In addition, KDM4B-mediated enhancement of ASMCs calcification was partially attenuated by the estrogen treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Therefore, KDM4B may be a new potential therapeutic target for VC treatment.\u003c/p\u003e \u003cp\u003eIt has been reported that the KDM4 family is a class of demethylases that can precisely remove H3K9 or H3K36, thereby playing a central role in the histone code. A previous study demonstrated the relationship between KDM4B and ERα[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. For example, KDM4B acts as an ERα cofactor that promotes mammary gland development and maturation. H3K9, KDM3A, and KDM4B synergistically regulate ERα activity through an autoregulatory loop that facilitates the recruitment of each co-activating enzyme to chromatin [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. KDM4B recruitment within the upstream regulatory region of the ERα gene and demethylation of inhibitory H3K9me3 markers allow GATA-3 binding to drive receptor expression. This indicates the importance of KDM4B in the ERα signaling cascade and suggest a potential therapeutic target for BC therapy [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Deletion of KDM4B reduced WEE1, CCND1, and CCNA1 transcription and disrupted the destrier-induced cell cycle G1-S phase transition in BC cells, resulting in BC cell arrest in the G2-M or G1-S phase inhibition of BC occurrence [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. We discovered that KDM4B overexpression promotes AR recruitment to the c-Myc gene enhancers and induces H3K9 demethylation; hence, increasing AR-dependent c-Myc mRNA transcription, which regulates sensitivity to next-generation AR-targeted therapies [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Although KDM4B enzymatic activity is required to enhance AR transcriptional activity, we have previously shown that KDM4B is an androgen-regulated demethylase that affects the transcriptional activity of AR not only through demethylation activity, but also through the regulation of ubiquitinylation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. KDM4B-regulated alternative splicing promotes AR-V7 expression and KDM4B promotes PCa cell growth via AR-V7 under androgen-deprivation conditions [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In our study, we used immunofluorescence to assess whether KDM4B and ERα co-localized in the nucleus in the presence or absence of calcification and E2. Co-IP results confirmed that they associated with each other in HASMCs and MOVAS cells. Interestingly, luciferase assay showed that KDM4B inhibited ERα-mediated gene transcription, whereas a mutant version of KDM4B with loss of demethylase activity (KDM4B HTE/ATA) had no synergistic effect on ERα in HASMCs and MOVAS cells. These results suggest that the down-regulation of ERα by KDM4B is independent of the enzymatic activity and is independent of the JmjC domain. Alternatively, KDM4B may bind to the original reader but not to another reader. The intricate chromatin environment may dictate the diverse functions exhibited by different tissues and various cell types.\u003c/p\u003e \u003cp\u003eHistone methyltransferase complexes consist primarily of histone methyltransferases and histone interacting proteins that play a critical role in chromatin remodeling, histone modification, cell differentiation, or tumorigenesis in mammalian cells[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The PRC2 core subcomplex has H3K27 methyltransferase activity, which contains EZH2, SUZ12, and other potentially combinatorial proteins [\u003cspan additionalcitationids=\"CR51 CR52 CR53\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The STRING database was used to predict the proteins that may be related to KDM4B, and we discovered that KDM4B may interact with EZH2 and SUZ12 in the presence of ESR1 (ERα). In HASMCs and MOVAS cells, we provide evidence that KDM4B could interact with the PRC2 subcomplex alongside ERα, thereby influencing the spatial interaction between PRC2 proteins and ERα. On chromatin, KDM4B can affect the recruitment of PRC2 complexes to specific DNA regions. In addition, KDM4B promoted the recruitment of PRC2 complex core proteins and further altered the histone modification level in the promoter regions of ERα target genes. These results suggest that KDM4B, an ERα co-regulator and a PRC2 complex bridge protein, might be involved in complex chromatin events essential for epigenetic modulation of ERα-mediated gene transcription in VC.\u003c/p\u003e \u003cp\u003eIt has been reported that KDM4B plays crucial roles during bone formation. KDM4B further enhances the expression of DLX2 and DLX5 by removing H3K9me3 marks; hence, facilitating osteogenic commitment [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In the process of osteoclast formation, KDM4B physically binds to CCAR1-MED1 to form a complex, which localizes to the promoter of multiple osteoclast-related genes and alters the chromatin structure through the action of histone demethylase, thereby promoting the expression of related genes [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Moreover, although it has also been shown that KDM4B knockdown weakened the osteogenic differentiation in MSCS, the audiogenic differentiation was enhanced. Loss of KDM4B increases H3K9me3, which reduces bone formation and increases bone marrow fat, thereby exacerbating bone aging and osteoporosis [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In our study, KDM4B was highly expressed in the calcification model and we showed it played a role modulating ERα-mediated gene transcription regulation. Ectopic expression of KDM4B aggravated mineralization and increased calcium content as well as the expression levels of calcification markers (BMP2, Runx2, and RANKL); thus, indicating that calcification is exacerbated by KDM4B. Additionally, KDM4B-mediated enhancement of β-GP-induced calcification was attenuated by the estrogen treatment. This suggests that E2/ERα underlies the function of KDM4B in VC. Our data have revealed that KDM4B acts as a ERα-mediated transcriptional regulator independent of its demethylase activity. Currently, VC is considered an independent predictor of cardiovascular events. For example, simple supplementation with estrogen might alleviate VC; however, such treatment might be associated with numerous estrogen-specific adverse and might not be conducive to treating diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Our findings uncover an epigenetic factor that acts as a novel co-regulator of ERα, participating in the VC pathway. This discovery offers vital insights for postponing the onset of cardiovascular disease in older adults. Furthermore, our findings indicate that KDM4B could be utilized as a marker for predicting VC and might even emerge as a viable therapeutic target for treating this condition.\u003c/p\u003e \u003cp\u003eOur study has demonstrated that KDM4B is highly expressed in β-GP-induced HASMCs and MOVAS cell calcification models. KDM4B co-represses ERα-induced transactivation independent of its demethylase activity in HASMCs and MOVAS cells. Furthermore, it facilitates the recruitment of PRC2 core proteins to the cis-regulatory elements of ERα target genes. Therefore, we believe that KDM4B is a potential therapeutic target for calcification, providing a novel insight into clinical treatment.\u003c/p\u003e"},{"header":"Experimental procedures","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell line culture and in vitro calcification model\u003c/h2\u003e \u003cp\u003eHASMCs and MOVAS cells were cultured in Dulbecco's modified Eagle medium (DMEM, SERAXPRO). The HEK293 cell was cultured in DMEM (SERAXPRO). Cells were cultured with 10% fetal bovine serum (FBS, SERAXPRO), 100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin (Glenview, Florida, USA) in a 37\u0026deg;C incubator with a humidified, under 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Next, 17-estradiol (E2, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethanol (Aladdin Scientific, Riverside, CA, USA). Cells were cultured in phenol red-free DMEM or RMPI1640 when needed to add an E2 stimulus. Culture dishes were purchased from Guangzhou Jet Bio-Filtration Co. Ltd., China. When VSMC reaches 50\u0026thinsp;~\u0026thinsp;70% confluency, VSMCs are treated with calcification medium containing 1% FBS and 5 mM β-GP. The calcification medium was changed every 2 days to induce calcification in VSMC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eThe following antibodies were used in this study: anti-KDM4B (Cell Signaling Technology, Danvers, MA, USA, #8639), anti-KDM4B (ABclonal, Woburn, MA, USA, #A6670), anti-ERα (Cell Signaling Technology, #D8H8), anti-Gas6 (Proteintech, Sankt-Leon Rot, Germany, #13795-1-AP), anti-β-actin (Proteintech, #66009-1-AP), anti-EZH2 (Proteintech, #21800-1-AP), anti-SUZ12 (Cell Signaling Technology, #3737), anti-H3K9me3 (Cell Signaling Technology, #13969) and anti-H3K27me3 (Cell Signaling Technology, #9733), anti-Rabbit/Mouse (ABclonal), anti-IgG (Proteintech, #10238-1-AP), anti-HA (ABclonal M20003M), BMP2 (Proteintech, #18933-1-AP), anti-Runx2 (Sigma, #AV36678), and anti-RANKL (Proteintech, #23408-1-AP).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTransfections, siRNA, and luciferase dual reporter assay\u003c/h2\u003e \u003cp\u003eThe HA-KDM4B plasmid was purchased from Addgene (Plasmid #24181). According to a previous study, the HA-KDM4B mutant was cloned into the PCMV-HA vector [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Expression plasmids for ERα, ERα-AF1, and ERα-AF2 were kindly provided by Dr Shigeaki Kato [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Final constructs were verified using DNA sequencing. siRNA against KDM4B were purchased from JTS Scientific (London, UK). All sequences are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eCell lines were co-transfected with the listed constructs according to the manufacturer's instructions using jetPRIME\u0026trade; DNA Transfection Reagent (Polyplus transfection). KDM4B and its truncated mutants, ERα, and ERE-tk-Luc were co-transfected with a plasmid containing Renilla luciferase (pRL). After co-transfection, cells are cultured in medium containing 3% charcoal-stripped fetal bovine serum (CS-FBS) for 6 h with or without E2 stimulation. After another day, cells were collected for dual-luciferase reporter assay (Promega, Madison, WI, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis, Co-Immunoprecipitation, and glutathione sepharose (GST) pull-down assays\u003c/h2\u003e \u003cp\u003eWestern blotting analysis was performed using a standard procedure introduced in previous studies [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In brief, cells or vascular tissues were lysed on ice for 30 min using lysis buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% NP‑40, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA and protease inhibitor cocktail (B14001, Houston, TX, USA)]. Lysates were vortexed every 10 min and then centrifuged at 13000 \u0026times; g for 20 min at 4\u0026deg;C. Next, the supernatant protein contents were measured using G250, and 30\u0026ndash;50 \u0026micro;g lysates were prepared for blotting. Samples were loaded into 8% or 12% polyacrylamide gels, separated by SDS-PAGE, and transferred onto PVDF membranes (ISEQ00010, Millipore, Burlington, MA, USA) under 80 v for 120 min. Next, membranes were blocked with 5% nonfat milk in TBST solution [20 mM Tris (pH 7.4), 137 mM NaCl, and 0.05% Tween‑20] for 1 h at room temperature and probed with primary antibodies overnight at 4\u0026deg;C. The next day, after washing the membranes with TBST solution three times, the membranes were incubated with secondary antibodies for 1 h at room temperature. Finally, the membranes were detected by chemiluminescence.\u003c/p\u003e \u003cp\u003eCo-immunoprecipitation begins with whole cell lysis after 2 h of purification with anti-IgG. Protein G beads were purchased from GE Healthcare (Chicago, Il, USA) and concentrated into sepharose for overnight rotation of antibody-protein interactions.\u003c/p\u003e \u003cp\u003eThe GST pull-down assay was performed using a standard protocol introduced in a previous study [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. GST alone proteins and GST fusion proteins, including GST-ERα 29-180aa and GST-ERα 282-595aa, were expressed in BL21 and bound to GST-sepharose beads according to the manufacturer's instructions (GE Healthcare). HA-KDM4B expression plasmid was constructed to synthesize the HA-KDM4B protein. Equal amounts of GST alone or conjugated GST-sepharose beads of GST fusion protein with \u003cem\u003ein vitro\u003c/em\u003e translated HA-KDM4B protein were incubated overnight at 4\u0026deg;C. The precipitated proteins were washed 4 times with binding buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 10% glycerol, and 1% Nonidet P-40). Western blotting and Coomassie brilliant blue staining were used to detect binding proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConfocal immunofluorescence imaging\u003c/h2\u003e \u003cp\u003eCells are fixed in 4% paraformaldehyde for 15 min at room temperature and then blocked in 1% donkey serum albumin. Cells were incubated with primary antibodies overnight at 4\u0026deg;C and then conjugated with secondary antibody to FITC, Cy 3, or Cy 5 (Jackson Immunoresearch Laboratories Inc., West Gove, PA, USA). DAPI (Roche, Zurich, Switzerland) staining was used to observe nuclear morphology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNA and quantitative real-time PCR (qPCR)\u003c/h2\u003e \u003cp\u003eAfter transfection of control and KDM4B siRNA, RNA was extracted. At 4\u0026ndash;6 h after transfection, estrogen and the same amount of EtOH were added to a final concentration of 100 nM for 16\u0026ndash;18 h. Total RNA was extracted with RNA Trizol (TAKARA Bio, Tokyo, Japan), and the PimeScript RT-PCR Kit (TAKARA Bio) was used to reverse transcribe the cDNA. The SYBR premeraseTaq Kit (TAKARA Bio) was used for real-time PCR detection of Roche's LightCycler96. All primers for qPCR are listed in Supplementary Table S2. Statistical analysis was performed using PRISM GraphPad 8. Each experiment was performed at least in triplicate and significance was tested using Student's \u003cem\u003et\u003c/em\u003e-tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eChIP assays were performed using a standard protocol [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. HASMCs were transfected with HA-tagged plasmids to overexpress KDM4B, or KDM4B siRNA to knockdown its expression. After transfection, cells were cultured in phenol-red DMEM medium containing 10% CS-FBS for 2 days. When the cell confluency reached 80%, cells were stimulated with 100 nM E2 or an equivalent amount of EtOH for 12 h. After the experiment, qPCR was performed using DNA as the template (primers are listed in Supplement Table S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAlizarin Red staining\u003c/h2\u003e \u003cp\u003eCells were washed thrice with PBS solution, fixed with 4% paraformaldehyde (#AR-0211, Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) for 30 min at room temperature, then washed with PBS solution and stained with 1% Alizarin red (pH 4.2, #G1452, Solarbio, Beijing, China) for 30 min at room temperature. Finally, stained cells were rinsed 3 times with distilled water at room temperature and imaged.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of calcium deposition\u003c/h2\u003e \u003cp\u003eCells or the vascular tissues were washed with PBS solution and then decalcified with 0.6 M HCL at 37\u0026deg;C for 24 h. The supernatant was collected and used for calcium quantification (Biosino Bio-Technology And Science Inc., Beijing, China). Next, cells or tissues were lysed (0.1 M NaOH, 0.1% SDS) for 30 min, then centrifuge at 12,000 \u0026times; g for 20 min at 4\u0026deg;C, and the protein concentration was measured using a BCA kit (Cat NO: KGP903, Keygen Biotech, Nanjing, China) to normalize the calcium levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnimal calcification model\u003c/h2\u003e \u003cp\u003eMice were brought from Beijing Vitonglihua Experimental Animal Technology Limited Company (China). All experiments were approved by the Institutional Animal Protection and Use Committee (IACUC) of China Medical University and performed in accordance with the IACUC guidelines. To induce VC phenotypes \u003cem\u003ein vivo\u003c/em\u003e, we used classical and recognized mouse calcification models. Mice were injected subcutaneously with VitD3 solution [5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e UI/kg Vit D3 mixed in corn oil], weighed once every 3 days, and sacrificed on the 7th day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analyses were performed using the PRISM GraphPad 8 software program. A two-sided Student's \u003cem\u003et\u003c/em\u003e-test was used to determine the significant differences in calcium deposition, the real-time PCR data, and luciferase assay. Significance was measured using p-values (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.01, and *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evascular calcification\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASMCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eaortic smooth muscle cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHASMCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman aortic smooth muscle cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMOVAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emouse aortic vascular smooth\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKDM4B\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHistone lysine demethylases 4B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRC2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolycomb repressive complex 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBMP2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebone morphogenic protein 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOVX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eovary removal group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eβ-GP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eβ-phosphoglycerol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eERα\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eestrogen receptorα\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eERE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eestrogen response element\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAS6\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGrowth arrest-specific gene 6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebreast cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJmjN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eJumanji N\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emesenchymal stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVSMCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evascular smooth muscle cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCS-FBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echarcoal-stripped fetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echromatin immunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCo-IP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eco-immunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEZH2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eZeste omologue 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSUZ12\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esuppressor of zeste 12\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglutathione sepharose.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate Dr. Xiaoxu Zhou and Dr. Tao Sun for their excellent technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualisation: Fei Liu, Wen Tian and Yue Zhao; Methodology: Fei Liu; Software: Baosheng Zhou, Hao Li, and Mengsu Cao; Validation: Chunyu Wang, and Yue Zhao; Formal analysis: Fei Liu, and Yue Zhao; Investigation: Fei Liu, Baosheng Zhou, Yanxia Lin; Resources: Yang Lv, Shengli Wang, Kai Zeng, Xiaocen Chang, and Lin Lin; Writing: Fei Liu, Kato Shigeaki, Wen Tian and Yue Zhao; Supervision: Wen Tian, and Yue Zhao.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant competing interests to declare in relation to this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by\u0026nbsp;the Liaoning Provincial Project of Applied Basic Research (2022JH2/101300061 for Wen Tian); National Natural Science Foundation of China\u0026nbsp;(32170603, 31871286 for Yue Zhao,\u0026nbsp;82273123 for Chunyu Wang, 32100440 for Ge Sun); China Postdoctoral Science Foundation (276066) for Ge Sun;\u0026nbsp;Foundation of Liaoning Province of China (LJKZ0756 for Shengli Wang); Local projects supported by the central government (2022JH6/100100035 for Yue Zhao); Foreign expert project of Ministry of Science and Technology (G2022006007L for Yue Zhao).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were brought from Beijing Vitonglihua Experimental Animal Technology Limited Company (China). All experiments were approved by the Institutional Animal Protection and Use Committee (IACUC) of China Medical University and performed in accordance with the IACUC guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article contains supporting information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Data\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Data are available at\u0026nbsp;\u003cstrong\u003e\u003cem\u003eCell Death and Differentiation.\u003c/em\u003e\u003c/strong\u003e online.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNicoll R,Henein M. 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Although estrogen replacement is a controversial treatment due to its potential carcinogenic effects, it was considered a protective treatment against VC in postmenopausal women. Estrogen receptor α (ERα) co-regulators were considered as potential therapeutic targets for ERα-related cancers. However, ERα activity and biological function modulation of ERα co-regulators in VC remain elusive. Histone lysine demethylases 4B (KDM4B) was identified to be highly expressed in human and mouse aortic smooth muscle (ASMC) cells treated with β-phosphoglycerol and in mice overloaded with VitD3 during calcification, as evidenced by western blotting and immunofluorescence staining. Co-immunoprecipitation (Co-IP) was performed to show the association between KDM4B and ERα. Our data demonstrated that KDM4B down-regulated ERα-induced transactivation and that KDM4B depletion increased mRNA expression of endogenous ERα target gene. Furthermore, we provided the evidence to show that KDM4B associated with Polycomb repressive complex 2 (PRC2) and ERα. In addition, KDM4B depletion decreased the recruitment of PRC2 complex to estrogen response element (ERE) regions of ERα target gene, thereby down-regulating the H3K27me3 levels. Finally, KDM4B-mediated enhancement of ASMCs calcification was partially attenuated by the estrogen treatment. KDM4B inhibits ERα-induced transactivation independent of its Jumanji-C enzyme active region. Taken together, our study suggest that KDM4B acting as ERα co-repressor is involved in regulation of VC, indicating that KDM4B may be a new potential therapeutic target for VC treatment.\u003c/p\u003e","manuscriptTitle":"KDM4B modulates ERα signaling pathway to participate \nin vascular smooth muscle cell calcification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 14:02:15","doi":"10.21203/rs.3.rs-6522511/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"74328a39-7379-4e0d-a8de-e2e05bb902c3","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47980869,"name":"Biological sciences/Molecular biology/Epigenetics"},{"id":47980870,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-10-08T07:06:17+00:00","versionOfRecord":{"articleIdentity":"rs-6522511","link":"https://doi.org/10.1038/s41420-025-02765-6","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2025-10-07 04:00:00","publishedOnDateReadable":"October 7th, 2025"},"versionCreatedAt":"2025-05-15 14:02:15","video":"","vorDoi":"10.1038/s41420-025-02765-6","vorDoiUrl":"https://doi.org/10.1038/s41420-025-02765-6","workflowStages":[]},"version":"v1","identity":"rs-6522511","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6522511","identity":"rs-6522511","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}