Decitabine effect on COL1, COL3, MMP2 and MMP9 in TGF-β1 treated mare endometrial fibroblasts

Abstract

Endometrial chronic degenerative fibrosis in mares, known as endometrosis is detrimental to fertility. Endometrium type I (COL1) and III (COL3) collagen accumulation and periglandular fibrosis characterize endometrosis. Transforming growth factor β1 (TGFβ1), is a main pro-fibrotic signal for myofibroblast differentiation, promoting collagen synthesis by epigenetic mechanisms in humans. Collagen turnover is controlled by metalloproteinase-2 (MMP-2), MMP-9 and their inhibitors. Because epigenetic alterations may change fibroblast phenotype and control fibroproliferative diseases, we hypothesized that endometrial fibroblasts might be under epigenetic regulation and modulate endometrosis. Epigenetic changes can be reversed and are promising for therapy. Nevertheless, this has not been demonstrated in mares. DNA methylation, a stable epigenetic marker, can be assessed through DNA methyltransferases (DNMTs) action. This study aimed to evaluate if transcription of some genes involved in endometrosis (COL1A1, COL3A1, MMP2 and MMP9) was altered with endometrial fibrosis, and if they were epigenetically modulated, through DNA methylation. Therefore, in vitro epigenetic modulation of equine endometrial fibroblasts treated with TGF-β1 was assessed, and the epigenetic inhibitor, 5-aza-2′-deoxycytidine (5-aza-dc or decitabine), was used to impair fibrogenesis. Fibroblasts from mare endometria (n=5) were treated with TGF-β1 (10 ng/mL) for 48h, followed by decitabine (1 µM) for 48h. As controls, fibroblasts were incubated in culture medium, or with decitabine or TGF-β1, for 96h. TGF-β1 and decitabine effects on DNA methyltransferases(DNMT1, DNMT3A and DNMT3B), COL1A1, COL3A1, MMP2 and MMP9 mRNA levels (qPCR) were evaluated in fibroblasts, and collagen (EIA) and metalloproteinases MMP-2 and -9 (zymography) in conditioned medium. TGF-β1 increased DNMT3A mRNA levels (P<0.05), COL1 and COL3 mRNA (P<0.01 and P<0.001, respectively) and protein (P<0.05). It also decreased MMP2 mRNA levels and enzymatic activity (P<0.0001 and P<0.05, respectively). Decitabine diminished DNMT3A mRNA levels (P<0.001), COL1 (P<0.001) and COL3 (P<0.01) mRNA and protein (P<0.01 and P<0.05, respectively). It increased MMP2 (P<0.01) and MMP9 mRNA (P<0.001), and enzymatic activity (P<0.05 and P<0.01, respectively), in TGF-β1 challenged fibroblasts. Data show an epigenetic involvement in the pathogenesis of endometrosis, as depicted by downregulation of TGF-β1 profibrotic effect in endometrial fibroblasts treated with decitabine. Decitabine influences MMPs reflected by increasing MMP2 and 9 mRNA and enzymatic activity in TGF-β1 treated endometrial fibroblasts. Furthermore, the hypermethylation (indicated by increased DNMT3A mRNA levels) that occurred with TGF-β1 treatment, seems to inhibit the expression and activity of MMP2. Data are in agreement with previous results and suggest that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation. Further studies will allow to better understand endometrosis epigenetics modulation. UIDB/00276/2020; LA/P/0059/2020; 2022.09161.PTDC