Enhancer of zeste homolog 2 (EZH2) as a new therapeutic target to prevent indoxyl-sulfate-induced aortic valve calcification

Aortic Stenosis (AS) is highly prevalent in chronic kidney disease (CKD) patients and is associated with a poor prognosis. We recently reported that indoxyl-sulfate (IS), a toxin that accumulates in the serum as kidney function declines, promotes macrophage's inflammation and subsequent mineralization of human valvular interstitial cells (hVICs). In this context, the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2), a key modulator of macrophage inflammation, may represent an interesting therapeutic target. This work aimed to determine whether EZH2 is involved in IS-induced macrophage's polarization and subsequent hVICs mineralization. THP1-derived macrophages were exposed to increasing concentrations of IS in the presence or absence of an EZH2 inhibitor called GSK-343 (5μM). EZH2 expression and activity (evidenced by the trimethylation of the lysine 27 of the histones H3 (H3K27me3)) were assessed by western blot. Macrophages’ inflammatory potential was checked by RT-qPCR and Elisa. The impact of macrophages exposed or not to IS and/or GSK-343 on hVICs osteogenic transition was evaluated using conditioned media. HVICs osteogenic transition was assessed following runx2 expression by qRT-PCR and western blot. Mineralization was quantified by the o-cresolphthalein method. Exposure to IS upregulated macrophage's expression EZH2, an effect blocked by GSK-343. The treatment with GSK-343 decreased macrophage's secretion of IL-6 (but not that of TNF-α and IL1-β), thereby preventing the induction of runx2 and subsequent calcification of hVICs. Neither IS, nor GSK-343 modulated macrophages’ H3K27me3, suggesting that EZH2 may act as a transcriptional factor for IL-6 through its non-canonical pathway. In this latter, EZH2 is thought to promote inflammation by binding to p65, rather than through its trimethylation activity. In line with this hypothesis, exposure to IS promoted EZH2 and p65 nuclear translocation as well as their coimmunoprecipitation, two phenomena blocked by GSK343. This work demonstrates that IS induces macrophage's secretion of IL-6 through the EZH2-p65 pathway, thereby promoting hVICs osteogenic transition and mineralization. The fact that GSK-343 can block this mechanism sheds light on EZH2 as a new therapeutic target to prevent AS in CKD patients.