Hypermethylation of Klotho and Peroxisome Proliferator-Activated Receptor γ Concomitant with Overexpression of DNA Methyltransferase 1 in Adenomyosis

Abstract

Cellular senescence is known to be involved in tissue repair, but its role in adenomyosis remains unclear. This study was tasked to evaluate the expression of Klotho, a well-known aging-suppressing protein, as well as PPARγ and DNMT1 in adenomyotic lesions (AD) in comparison with that of control endometrium (CT). We performed immunohistochemistry analysis of markers of cellular senescence p16 and p21, along with Klotho, PPARγ and DNMT1 in CT and AD samples, followed by the quantification of gene expression of Klotho, PPARγ and DNMT1 in epithelial organoids derived from AD and CT samples and methylation-specific PCR to evaluate promoter methylation status. The effect of forced expression and knockdown of DNMT1 on Klotho and PPARγ expression in ectopic endometrial epithelial cells was evaluated. We found that both p16 and p21 immunoreactivity in AD was significantly higher while that of Klotho and PPARγ was significantly lower than CT samples, which was concomitant with elevated immunoexpression of DNMT1. The results were confirmed by transcriptional analysis using epithelial organoids derived from AD and CT samples. In addition, the promoter regions of both Klotho and PPARγ genes were hypermethylated in AD as compared with CT, and treatment with HDAC and DNMT inhibitors reactivated the expression of both Klotho and PPARγ. Forced expression of DNMT1 resulted in downregulation of both Klotho and PPARγ but its knockdown increased their expression. Thus, overexpression of DNMT1 seems to facilitate the promoter hypermethylation of both Klotho and PPARγ in AD, resulting in their reduced expression that is suggestive of the role of senescence in adenomyosis.

Keywords: Adenomyosis; DNMT1; Fibrosis; Klotho; Methylation; PPARγ; Senescence.