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

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.

Keywords: 5-aza-dC; DNMTs; collagen; demethylating inhibitor; endometrosis; epigenetics; fibroblasts; mare.

Figure 1

Representative pictures of immunofluorescence staining of vimentin (A,B) in cultured fibroblasts. (C) DAPI staining. Scale bar = 50 μm (magnification: 20,40).

Figure 2

Relative levels of (A) collagen type I (COL1A1), (B) collagen type III (COL3A1), and (C) α-smooth muscle actin (α-SMA) mRNA, and of (D) COL1 and (E) COL3 protein concentrations in mare endometrial fibroblasts after incubation with TGF-β1 (10 ng/mL) for 48 h and 96 h; n = 5. All values are expressed as percentage of change from respective hour control (non-treated fibroblasts). Bars represent mean ± SEM. Asterisks indicate significant differences between treatment and the respective control (** p < 0.01, *** p < 0.001, **** p < 0.0001); a and b letters indicate significant differences between treatment hours (a—p < 0.05; b—p < 0.01; c—p < 0.001).

Figure 3

Relative DNA methyltransferases ((A)—DNMT1, (B)—DNMT3A, and (C)—DNMT3B) mRNA levels in mare endometrial fibroblasts after incubation with TGF-β1 (10 ng/mL) for 48 h and 96 h; n = 5. All values are expressed as percentage of change from respective hour control (non-treated fibroblasts). Bars represent mean ± SEM. Asterisks indicate significant differences from the respective control (** p < 0.01, *** p < 0.001).

Figure 4

Relative levels of (A) collagen type I (COL1A1), (B) collagen type III (COL3A1), and (C) α-smooth muscle actin (α-SMA) mRNA, and of (D) COL1 and (E) COL3 protein concentrations in non-treated (control-C) endometrial fibroblasts or treated with 5-aza-dC (1 µM), TGF-β1 (10 ng/mL) for 96 h or both combined (TGF-β1 followed by 5-aza-dC at 48 h—total cell incubation of 96 h); n = 5. Each treatment was compared to respective control (all groups with control C and TGF-β1 + 5-aza-dC with TGF-β1). Bars represent mean ± SEM. Asterisks indicate significant differences between treatments (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

Relative DNA methyltransferases ((A)—DNMT1, (B)—DNMT3A and (C)—DNMT3B) mRNA levels in non-treated (control-C) mare endometrial fibroblasts or treated with 5-aza-dC (1 µM), TGF-β1 (10 ng/mL) for 96 h or both combined (TGF-β1 followed by 5-aza-dC at 48 h—total cell incubation of 96 h); n = 5. Each treatment was compared to respective control (all groups with control C and TGF-β1 + 5-aza-dC with TGF-β1); n = 5. Bars represent mean ± SEM. Asterisks indicate significant differences between treatments (* p < 0.05, *** p < 0.001).

Figure 6

Relative levels of (A) collagen type I (COL1A1), (B) collagen type III (COL3A1), and (C) α-smooth muscle actin (α-SMA) mRNA, and of (D) COL1 and (E) COL3 protein concentrations in mare endometrial fibroblasts after incubation with TGF-β1 (10 ng/mL) for 48 h and 96 h; n = 5. All values are expressed as percentage of change from respective hour control (non-treated fibroblasts). Bars represent mean ± SEM. Asterisks indicate significant differences between treatment and the respective control (* p < 0.05; ** p < 0.01); a and b letters indicate significant differences between treatment hours (a—p < 0.05; b—p < 0.01).

Figure 7

Relative DNA methyltransferases ((A)—DNMT1, (B)—DNMT3A, and (C)—DNMT3B) mRNA levels in mare endometrial fibroblasts after incubation with 5-aza-dC (1 µM) for 48 h and 96 h; n = 5. All values are expressed as percentage of change from respective hour control (non-treated fibroblasts). Bars represent mean ± SEM. Asterisks indicate significant differences from the respective control (* p < 0.05, ** p < 0.01, *** p < 0.001). a, b, and c letters indicate significant differences between treatment hours (a—p < 0.05; b—p < 0.01; c—p < 0.001).

Figure 8

Relative levels of (A) collagen type I (COL1A1), (B) collagen type III (COL3A1), and (C) DNA methyltransferase 3A (DNMT3A) mRNA, and of (D) COL1 and (E) COL3 protein concentrations in non-treated (control-C) endometrial fibroblasts or treated with TGF-β1 (10 ng/mL) for 96 h or TGF-β1 (10 ng/mL) + 5-aza-dC (1 µM) (TGF-β1 followed by 5-aza-dC at 48 h—total cell incubation of 96 h); n = 5. Bars represent mean ± SEM. Asterisks indicate significant differences between treatments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).