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TET2 is involved in DNA hydroxymethylation, cell proliferation and inflammatory response in keratinocytes.

ABSTRACT: DNA methylation and hydroxymethylation are the most common epigenetic modifications associated with the cell cycle and the inflammatory response. The present study aimed to investigate the role of 5?hydroxymethyl?cytosine (5?hmC) and ten?eleven translocation?2 (TET2) in keratinocytes. Following TET2 knockdown, dot blot analysis was performed to assess the levels of 5?hmC in keratinocytes, using HaCaT cells. Subsequently, the viability and cell cycle of HaCaT cells were assessed by MTT, Cell Counting Kit?8 assay and flow cytometric assays. Cyclin?dependent kinase inhibitor 2A and proinflammatory cytokine protein and mRNA expression levels were also detected. The present results suggested that TET2 may play an important role in regulating cellular proliferation by mediating DNA hydroxymethylation in HaCaT cells. In addition, TET2 knockdown decreased the production of proinflammatory cytokines, including lipocalin 2, S100 calcium binding protein A7, matrix metallopeptidase 9, C?X?C motif chemokine ligand 1, interferon regulatory factor 7 and interleukin?7 receptor. The present study suggested that TET2 regulated cell viability, apoptosis and the expression of inflammatory mediators in keratinocytes. Collectively, the results indicated that TET2 knockdown may relieve inflammatory responses in the skin.

SUBMITTER: Liu X

PROVIDER: S-EPMC7057829 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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TET2 is involved in DNA hydroxymethylation, cell proliferation and inflammatory response in keratinocytes.

Liu Xinxin X Wang Xin X Liu Nian N Zhu Ke K Zhang Song S Duan Xiaoru X Huang Yuqiong Y Jin Zilin Z Jaypaul Himanshu H Wu Yan Y Chen Hongxiang H

Molecular medicine reports 20200218 4

DNA methylation and hydroxymethylation are the most common epigenetic modifications associated with the cell cycle and the inflammatory response. The present study aimed to investigate the role of 5‑hydroxymethyl‑cytosine (5‑hmC) and ten‑eleven translocation‑2 (TET2) in keratinocytes. Following TET2 knockdown, dot blot analysis was performed to assess the levels of 5‑hmC in keratinocytes, using HaCaT cells. Subsequently, the viability and cell cycle of HaCaT cells were assessed by MTT, Cell Coun ...[more]

PMID: 32319620

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Project description:The pathophysiology of periodontal disease involves a perturbed immune system to a dysbiotic microflora leading to unrestrained inflammation, collateral tissue damage, and various systemic complications. Gingival epithelial cells function as an important part of immunity to restrict microbial invasion and orchestrate the subsequent innate responses. A20 (TNFAIP3), an ubiquitin-editing enzyme, is one of the key regulators of inflammation and cell death in numerous tissues including gastrointestinal tract, skin, and lungs. Emerging evidence indicates A20 as an essential molecule in the oral mucosa as well. In this study, we characterized the role of A20 in human telomerase immortalized gingival keratinocytes (TIGKs) through loss and gain of function assays in preclinical models of periodontitis. Depletion of A20 through gene editing in TIGKs significantly increased IL-6 and IL-8 secretion in response to Porphyromonas gingivalis infection while A20 over-expression dampened the cytokine production compared to A20 competent cells through modulating NF-κB signaling pathway. In the subsequent experiments which assessed apoptosis, A20 depleted TIGKs displayed increased levels of cleaved caspase 3 and DNA fragmentation following P. gingivalis infection and TNF/CHX challenge compared to A20 competent cells. Consistently, there was reduced apoptosis in the cells overexpressing A20 compared to the control cells expressing GFP further substantiating the role of A20 in regulating gingival epithelial cell fate in response to exogenous insult. Collectively, our findings reveal first systematic evidence and demonstrate that A20 acts as a regulator of inflammatory response in gingival keratinocytes through its effect on NF-κB signaling and desensitizes cells to bacteria and cytokine induced apoptosis in the oral mucosa. As altered A20 levels can have profound effect on different cellular responses, future studies will determine whether A20-targeted therapies can be exploited to restrain periodontal inflammation and maintain oral mucosa tissue homeostasis.

Project description:BACKGROUND:Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor targeting estrogen receptors (ERs), has been implicated in the promotion of breast cancer. Perinatal exposure of BPA could induce longitudinal alteration of DNA hydroxymethylation in imprinted loci of mouse blood cells. To date, no data has been reported on the effects of BPA on DNA hydroxymethylation in breast cells. Therefore, we asked whether BPA can induce DNA hydroxymethylation change in human breast cells. Given that dysregulated epigenetic DNA hydroxymethylation is observed in various cancers, we wondered how DNA hydroxymethylation modulates cancer development, and specifically, whether and how BPA and its analogs promote breast cancer development via DNA hydroxymethylation. OBJECTIVES:We aimed to explore the interplay of the estrogenic activity of bisphenols at environmental exposure dose levels with TET dioxygenase-catalyzed DNA hydroxymethylation and to elucidate their roles in the proliferation of ER+ breast cancer cells as stimulated by environmentally relevant bisphenols. METHODS:Human MCF-7 and T47D cell lines were used as ER-dependent breast cell proliferation models, and the human MDA-MB-231 cell line was used as an ER-independent breast cell model. These cells were treated with BPA or bisphenol S (BPS) to examine BPA/BPS-related proliferation. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and enzyme-linked immunosorbent assays (ELISAs) were used to detect DNA hydroxymethylation. Crispr/Cas9 and RNA interference technologies, quantitative polymerase chain reaction (qPCR), and Western blot analyses were used to evaluate the expression and function of genes. Co-immunoprecipitation (Co-IP), bisulfite sequencing-PCR (BSP), and chromatin immunoprecipitation-qPCR (ChIP-qPCR) were used to identify the interactions of target proteins. RESULTS:We measured higher proliferation in ER+ breast cancer cells treated with BPA or its replacement, BPS, accompanied by an ER?-dependent decrease in genomic DNA hydroxymethylation. The results of our overexpression, knockout, knockdown, and inhibition experiments suggested that TET2-catalyzed DNA hydroxymethylation played a suppressive role in BPA/BPS-stimulated cell proliferation. On the other hand, we observed that TET2 was negatively regulated by the activation of ER? (dimerized and phosphorylated), which was also induced by BPA/BPS binding. Instead of a direct interaction between TET2 and ER?, data of our Co-IP, BSP, and ChIP-qPCR experiments indicated that the activated ER? increased the DNA methyltransferase (DNMT)-mediated promoter methylation of TET2, leading to an inhibition of the TET2 expression and DNA hydroxymethylation. CONCLUSIONS:We identified a new feedback circuit of ER? activation-DNMT-TET2-DNA hydroxymethylation in ER+ breast cancer cells and uncovered a pivotal role of TET2-mediated DNA hydroxymethylation in modulating BPA/BPS-stimulated proliferation. Moreover, to our knowledge, we for the first time established a linkage among chemical exposure, DNA hydroxymethylation, and tumor-associated proliferation. These findings further clarify the estrogenic activity of BPA/BPS and its profound implications for the regulation of epigenetic DNA hydroxymethylation and cell proliferation. https://doi.org/10.1289/EHP5862.

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TET2 is involved in DNA hydroxymethylation, cell proliferation and inflammatory response in keratinocytes.

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TET2 is involved in DNA hydroxymethylation, cell proliferation and inflammatory response in keratinocytes.

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