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DNA methyltransferase inhibitor suppresses fibrogenetic changes in human conjunctival fibroblasts.

ABSTRACT: Purpose:This study aimed to clarify the effects of a DNA methyltransferase inhibitor on fibrogenetic changes in human conjunctival fibroblasts (HConF). Methods:HConF were pretreated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-dC) for 48 h. After one passage, the cells were treated with 5 ng/ml of transforming growth factor (TGF)-?2 for 48 h, and the expression levels of ?-smooth muscle actin (?-SMA), extracellular matrix proteins, and phosphorylated Smad3 were evaluated with western blotting. A fusion construct between the COL1A2 promoter and the luciferase gene was introduced into the HConF after the first passage, and the construct's activity was detected via a luciferase reporter gene assay. Results:TGF-?2-induced upregulation of ?-SMA was suppressed by pretreatment with 5-Aza-dC (0.1, 1.0, and 10 ?M) in a dose-dependent manner. Upregulation of type I collagen was also suppressed by 10 ?M 5-Aza-dC pretreatment. In contrast, 5-Aza-dC had no inhibitory effect on the expression of fibronectin or phosphorylated Smad3. However, COL1A2 promoter activity was suppressed with 5-Aza-dC pretreatment. Conclusions:In HConF, fibrogenetic changes were partly suppressed with a DNA methyltransferase inhibitor, suggesting an indirect inhibitory effect of the inhibitor on the COL1A2 promoter in HConF.

SUBMITTER: Yonemura H

PROVIDER: S-EPMC6707755 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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DNA methyltransferase inhibitor suppresses fibrogenetic changes in human conjunctival fibroblasts.

Yonemura Hitomi H Futakuchi Akiko A Inoue-Mochita Miyuki M Fujimoto Tomokazu T Takahashi Eri E Tanihara Hidenobu H Inoue Toshihiro T

Molecular vision 20190721

<h4>Purpose</h4>This study aimed to clarify the effects of a DNA methyltransferase inhibitor on fibrogenetic changes in human conjunctival fibroblasts (HConF).<h4>Methods</h4>HConF were pretreated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-dC) for 48 h. After one passage, the cells were treated with 5 ng/ml of transforming growth factor (TGF)-β2 for 48 h, and the expression levels of α-smooth muscle actin (α-SMA), extracellular matrix proteins, and phosphorylated Smad ...[more]

PMID: 31523116

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Project description:The role of epigenetic modifications such as DNA methylation during vertebrate sexual development is far from being clear. Using the zebrafish model, we tested the effects of one of the most common DNA methyltransferase (dnmt) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), which is approved for the treatment of acute myeloid leukaemia and is under active investigation for the treatment of solid tumours. Several dose-response experiments were carried out during two periods, including not only the very first days of development (0-6 days post-fertilization, dpf), as done in previous studies, but also, and as a novelty, the period of gonadal development (10-30 dpf).Early treatment with 5-aza-dC altered embryonic development, delayed hatching and increased teratology and mortality, as expected. The most striking result, however, was an increase in the number of females, suggesting that alterations induced by 5-aza-dC treatment can affect sexual development as well. Results were confirmed when treatment coincided with gonadal development. In addition, we also found that the adult gonadal transcriptome of 5-aza-dC-exposed females included significant changes in the expression of key reproduction-related genes (e.g. cyp11a1, esr2b and figla), and that several pro-female-related pathways such as the Fanconi anaemia or the Wnt signalling pathways were downregulated. Furthermore, an overall inhibition of genes implicated in epigenetic regulatory mechanisms (e.g. dnmt1, dicer, cbx4) was also observed.Taken together, our results indicate that treatment with a DNA methylation inhibitor can also alter the sexual development in zebrafish, with permanent alterations of the adult gonadal transcriptome, at least in females. Our results show the importance of DNA methylation for proper control of sexual development, open new avenues for the potential control of sex ratios in fish (aquaculture, population control) and call attention to possibly hidden long-term effects of dnmt therapy when used, for example, in the treatment of prepuberal children affected by some types of cancer.

Dataset Information

DNA methyltransferase inhibitor suppresses fibrogenetic changes in human conjunctival fibroblasts.

Publications

DNA methyltransferase inhibitor suppresses fibrogenetic changes in human conjunctival fibroblasts.

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