5-Aza-2′-Deoxycytidine (5-Aza-dC, Decitabine) Inhibits Collagen Type I and III Expression in TGF-β1-Treated Equine Endometrial Fibroblasts

SIMPLE SUMMARY: Endometrosis is a major cause of infertility in mares and involves the excessive deposition of extracellular matrix in the mare’s endometrium, such as collagen and α smooth muscle actin (α-SMA). Collagen is formed by activated fibroblasts, which are mainly stimulated by transforming growth factor β1 (TGF-β1). Alterations in fibroblast phenotype are linked with epigenetic alterations. Unlike genetic alterations, epigenetic alterations are changes in gene function without DNA nucleotide sequence modification. Epigenetic changes can be reversed and are therefore extremely promising for therapeutic use. DNA methylation analysis is one of the most used methods to detect epigenetic changes. It can be assessed by measuring DNA methylating enzymes (DNMT1, DNMT3A, and DNMT3B). Thus, the aims of this study were to investigate the in vitro epigenetic regulation of mare endometrial fibrogenesis through DNMTs transcription and the effect of the epigenetic inhibitor 5-aza-2′-deoxycytidine (5-aza-dC or decitabine) on collagen expression in mare endometrial fibroblasts challenged with TGF-β1. It was observed that TGF-β1 upregulated DNMT3A, COLs, and α-SMA transcripts and COLs secretion. The increase in DNMT3A and COLs (transcripts and protein) after TGF-β1 stimulation of equine endometrial fibroblasts was reduced after treatment with the demethylating agent 5-aza-Dc, suggesting an epigenetic regulation of mare endometrial fibrosis. ABSTRACT: Endometrosis negatively affects endometrial function and fertility in mares, due to excessive deposition of type I (COL1) and type III (COL3) collagens. The pro-fibrotic transforming growth factor (TGF-β1) induces myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, and collagen synthesis. In humans, fibrosis has been linked to epigenetic mechanisms. To the best of our knowledge, this has not been described in mare endometrium. Therefore, this study aimed to investigate the in vitro epigenetic regulation in TGF-β1-treated mare endometrial fibroblasts and the use of 5-aza-2′-deoxycytidine (5-aza-dC), an epigenetic modifier, as a putative treatment option for endometrial fibrosis. Methods and Results: The in vitro effects of TGF-β1 and of 5-aza-dC on DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), COL1A1, COL3A1, and α-SMA transcripts were analyzed in endometrial fibroblasts, and COL1 and COL3 secretion in a co-culture medium. TGF-β1 upregulated DNMT3A transcripts and collagen secretion. In TGF-β1-treated endometrial fibroblasts, DNA methylation inhibitor 5-aza-dC decreased collagen transcripts and secretion, but not α-SMA transcripts. Conclusion: These findings suggest a possible role of epigenetic mechanisms during equine endometrial fibrogenesis. The in vitro effect of 5-aza-dC on collagen reduction in TGF-β1-treated fibroblasts highlights this epigenetic involvement. This may pave the way to different therapeutic approaches for endometrosis.