Fig. 8: EZH2 modulates MMT by inhibiting the expression of DUSP23 through epigenetic modification and DUSP23-meditated dephosphorylation of SMAD3 at Ser423/425, which attenuates MMT.

A Top 10 differentially expressed genes between Ezh2.iKO mice and WT mice in response to CaOx stimulation (n = 3). B Two-color immunofluorescence identifies DUSP23 (red) was decreased in F4/80⁺(green) macrophages during nephrocalcinosis and upregulated after Ezh2 deletion. C The H3K27me3 binding site is predicted on the promoter region of the Dusp23 gene by the ChIP-Seq database. ChIP assay confirmed that COM treatment significantly enhanced the binding of EZH2 and H3K27me3 on the predicted promoter region in RAW264.7 cells (n = 3). D DUSP23, H3K27me3, and total H3 were expressed by immunoblotting in the RAW264.7 cell line (n = 3). E The proportion of MMT cells in the Dusp23 overexpression macrophages was detected by flow cytometry (n = 3). F The protein sequences of human and mouse DUSP23 or SMAD3 were analyzed by BLAST. G The molecular docking analysis in humans was conducted to predict the potential amino acid binding sites between DUSP23 and SMAD3. H Co-IP assay in COM-treated RAW264.7 macrophages confirmed the interaction of SMAD3 and DUSP23. I Expression of pSMAD3 (Ser423/425), pSMAD3 (T179), and total SAMD3 was detected by immunoblotting in macrophages (n = 3). J Expression of DUSP23, pSMAD3 (Ser423/425), SMAD3, Collagen I, and α-SMA was detected by immunoblotting in macrophages (n = 3). Data are presented as the mean ± SEM. *p < 0.05 versus control group; ^#p < 0.05 versus model group. P values were determined by the two-tailed Student’s t test.