The inhibition of DNMT using AZA
The inhibition of DNMT1 using 5-AZA-dC or DNMT1 siRNA increased TIMP2 protein and mRNA expression, suggesting that TIMP2 expression is down-regulated by DNA methylation in the HDFs. In addition, 5-AZA-dC treatment led to a dose-dependent decrease of DNMT1 protein expression. 5-AZA-dC is a cytosine analog with the capacity to inhibit either DNMT1 activity by irreversibly binding to the enzyme or DNMT1 protein expression by triggering ubiquitin-E3 ligase activity for DNMT1 degradation [32,33]. This process may occur even in the absence of DNA replication, when DNMT1 is in its free, unbound state . An overexpression of DNMT1, on the other, significantly decreased TIMP2 expression. Our data indicate a strong correlation between DNMT1 and TIMP2 and suggest that DNMT1 induces methylation in TIMP2 promoter, resulting in decreased TIMP2 expressions. Analysis by MSP on TIMP2 promoter in UV-irradiated dermal fibroblasts revealed increased methylation, in contrast to the partial methylation seen for the non-UV-irradiated (Fig. 5). This finding is in accordance with a recent study that elucidated increased recruitment of DNMTs at the TIMP2 promoter by performing ChIP assays in ovarian cancer SKF38393 HCl . For our future studies, we plan to investigate in depth the DNA methylation process by performing pyrosequencing in the TIMP2 promoter region.
We cannot rule out the possibility of other epigenetic regulators that may influence this outcome. Aside from DNMT1, in vivo and in vitro results showed increased expressions of MBD1 in UV-irradiated human skin. MBDs have been reported to contribute to the DNA methylation machinery by recruiting DNMT1 and other histone methylation enzymes and ensuring transcriptional inactivation . Further inspection into the role of MBD1 and histone methylation enzymes such as H3K27me3 and chromatin remodeling, which have been reported to be associated with the gene silencing of TIMPs, would be favorable [35,37].
Our study presents previously unreported evidences on the changes in DNA methylation-associated TIMP2 expressions as a response to UV exposure of the human skin. It also reveals that UV irradiation of the human skin decreases TIMP2 expressions through an epigenetic regulation that may involve DNMT1-mediated DNA hypermethylation. Epigenetic modification by DNMTs can lead to various biological consequences related to matrix remodeling, inflammation, and immune regulation in addition to skin aging and cancer . For example, inhibition of DNMTs by 5-AZA-dC increased COL1A1 and COL1A2 in cardiac fibroblasts and HDFs, respectively [26,38]. Silencing of DNMT1 prevented the activation of JAK2/STAT3 pathway via SOCS1 promoter in lipopolysaccharide-treated macrophages . On other hand, overexpression of DNMT1 in macrophages led to proinflammatory cytokine production and atherosclerosis in a mouse model . Moreover, since TIMPs and MMPs have been associated not only with photoaging and cancer , but also with inflammation, cell migration, cell proliferation, and apoptosis [6,7,27], our findings raise possibilities for therapeutic modalities in such fields using epigenetic modulations. In addition, future studies are needed to elucidate the crosstalk among epigenetic modulators in the regulation of TIMP2 in photoaged human skin in vivo.
Conflict of interest
Acknowledgements This research was supported by the Basic Science Research program of the National Research Foundation (NRF) of Korea funded by the Ministry of Education (grant number: 2017R1D1A1B03031122), and by the Korea Health Technology R&D Project of the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C0089).