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      METTL14 aggravates endothelial inflammation and atherosclerosis by increasing FOXO1 N6-methyladeosine modifications

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          Abstract

          Aims: The N6-methyladenosine (m 6A) modification plays an important role in various biological processes, but its role in atherosclerosis remains unknown. The aim of this study was to investigate the role and mechanism of m 6A modification in endothelial cell inflammation and its influence on atherosclerosis development.

          Methods: We constructed a stable TNF-α-induced endothelial cell inflammation model and assessed the changes in the expression of m 6A modification-related proteins to identify the major factors involved in this process. The m 6A-modified mRNAs were identified by methylated RNA immunoprecipitation (RIP) sequencing and forkhead box O1 (FOXO1) was selected as a potential target. Through cytological experiments, we verified whether methyltransferase-like 14 (METTL14) regulates FOXO1 expression by regulating m 6A-dependent mRNA and protein interaction. The effect of METTL14 on atherosclerosis development in vivo was verified using METTL14 knockout mice.

          Results: These findings confirmed that METTL14 plays major roles in TNF-α-induced endothelial cell inflammation. During endothelial inflammation, m 6A modification of FOXO1, an important transcription factor, was remarkably increased. Moreover, METTL14 knockdown significantly decreased TNF-α-induced FOXO1 expression. RIP assay confirmed that METTL14 directly binds to FOXO1 mRNA, increases its m 6A modification, and enhances its translation through subsequent YTH N6-methyladenosine RNA binding protein 1 recognition. Furthermore, METTL14 was shown to interact with FOXO1 and act directly on the promoter regions of VCAM-1 and ICAM-1 to promote their transcription, thus mediating endothelial cell inflammatory response. In vivo experiments showed that METTL14 gene knockout significantly reduced the development of atherosclerotic plaques.

          Conclusion: METTL14 promotes FOXO1 expression by enhancing its m 6A modification and inducing endothelial cell inflammatory response as well as atherosclerotic plaque formation. Decreased expression of METTL14 can inhibit endothelial inflammation and atherosclerosis development. Therefore, METTL14 may serve as a potential target for the clinical treatment of atherosclerosis.

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          Most cited references28

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          ALKBH5-dependent m6A demethylation controls splicing and stability of long 3'-UTR mRNAs in male germ cells.

          N6-methyladenosine (m6A) represents one of the most common RNA modifications in eukaryotes. Specific m6A writer, eraser, and reader proteins have been identified. As an m6A eraser, ALKBH5 specifically removes m6A from target mRNAs and inactivation ofAlkbh5leads to male infertility in mice. However, the underlying molecular mechanism remains unknown. Here, we report that ALKBH5-mediated m6A erasure in the nuclei of spermatocytes and round spermatids is essential for correct splicing and the production of longer 3'-UTR mRNAs, and failure to do so leads to aberrant splicing and production of shorter transcripts with elevated levels of m6A that are rapidly degraded. Our study identified reversible m6A modification as a critical mechanism of posttranscriptional control of mRNA fate in late meiotic and haploid spermatogenic cells.
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            FTO-Dependent N 6 -Methyladenosine Regulates Cardiac Function During Remodeling and Repair

            Despite its functional importance in various fundamental bioprocesses, the studies of N6-methyladenosine (m6A) in the heart are lacking. Here we show that, fat mass and obesity-associated (FTO), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling and regeneration. We used clinical human samples, preclinical pig and mouse models and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO using AAV9 (in vivo), adenovirus (both in vivo and in vitro) and siRNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility and cardiac function post-ischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing (MeRIP-seq) to map transcriptome-wide m6A, and MeRIP qPCR assays to map and validate m6A in individual transcripts, in healthy and failing hearts and myocytes. We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is carried out by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. Additionally, we demonstrate that FTO overexpression in mouse models of MI decreased fibrosis and enhanced angiogenesis. Collectively, our study demonstrates the functional importance of FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.
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              Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter.

              Transcriptional activation of a gene involves an orchestrated recruitment of components of the basal transcription machinery and intermediate factors, concomitant with an alteration in local chromatin structure generated by posttranslational modifications of histone tails and nucleosome remodeling. We provide here a comprehensive picture of events resulting in transcriptional activation of a gene, through evaluating the estrogen receptor-alpha (NR3A1) target pS2 gene promoter in MCF-7 cells. This description integrates chromatin remodeling with a kinetic evaluation of cyclical networks of association of 46 transcription factors with the promoter, as determined by chromatin immunoprecipitation assays. We define the concept of a "transcriptional clock" that directs and achieves the sequential and combinatorial assembly of a transcriptionally productive complex on a promoter. Furthermore, the unanticipated findings of key roles for histone deacetylases and nucleosome-remodeling complexes in limiting transcription implies that transcriptional activation is a cyclical process that requires both activating and repressive epigenetic processes.
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                Author and article information

                Journal
                Theranostics
                Theranostics
                thno
                Theranostics
                Ivyspring International Publisher (Sydney )
                1838-7640
                2020
                11 July 2020
                : 10
                : 20
                : 8939-8956
                Affiliations
                [1 ]Department of Cardiology, Henan Provincial People's Hospital, Department of Cardiology of Central China Fuwai Hospital, Henan Key Laboratory for Coronary Heart Disease Prevention and Control, Central China Fuwai Hospital of Zhengzhou University, Zhengzhou, Henan, 450003, China.
                [2 ]Department of Cardiology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450003, China.
                [3 ]Heart Center of Henan Provincial People's Hospital, Central China Fuwai Hospital, Key Laboratory for Cardiac Regenerative Medicine, National Health Commission & Central China Fuwai Hospital of Zhengzhou University, Zhengzhou, Henan, 450003, China.
                [4 ]Department of Cardio-Thoracic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310003, China.
                [5 ]Department of Hematology, Henan Provincial People's Hospital, Zhengzhou, Henan, 450003, China.
                [6 ]Department of Pathology, Henan Provincial People's Hospital, Zhengzhou, Henan, 450003, China.
                [7 ]Department of Hepatobiliary and Pancreatic Surgery, Zhengzhou People's Hospital, Zhengzhou, Henan, 450003, China.
                [8 ]School of Life Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China.
                Author notes
                ✉ Corresponding author: Professor Muwei Li (E-mail: lmw0207@ 123456gs.zzu.edu.cn ) and Chuanyu Gao (E-mail: gaocy1102@ 123456163.com ), Department of Cardiology, Henan Provincial People's Hospital, Department of Cardiology of Central China Fuwai Hospital, Henan Key Laboratory for Coronary Heart Disease Prevention and Control, Central China Fuwai Hospital of Zhengzhou University, Zhengzhou, Henan, 450003,China. Assistant professor Shuai Wang (E-mail: wangshuai@ 123456westlake.edu.cn ), School of Life Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China. Phone: +86 (0371) 58681135; Fax: +86 (0571) 85271986.

                *These authors contributed equally to this work.

                Competing Interests: The authors have declared that no competing interest exists.

                Article
                thnov10p8939
                10.7150/thno.45178
                7415798
                32802173
                ff9cd711-a705-4dfb-a8c0-a2fb2c197d4c
                © The author(s)

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

                History
                : 21 February 2020
                : 26 June 2020
                Categories
                Research Paper

                Molecular medicine
                mettl14,m6a modification,endothelial inflammation,foxo1,atherosclerosis
                Molecular medicine
                mettl14, m6a modification, endothelial inflammation, foxo1, atherosclerosis

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