Abstract
Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the epidermal growth factor receptor (EGFR) signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of methyltransferase like 3 (METTL3), the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3' untranslated regions (3'UTR) accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, thrombospondin-1 (TSP-1), an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.
Highlights
46 N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNAs (Roundtree et al, 2017)
En face immunofluorescence 128 staining of left common carotid artery (LCA) revealed reduced protein levels of Methyltransferase Like 3 (METTL3) and enhanced levels of VCAM129 1 in EC-Mettl3KO mice compared to right common carotid artery 117 (RCA) 2 weeks after ligation (Figure 2D and E). Overexpression of METTL3 in ECs by adeno-associated virus (AAV9-METTL3 OE) inhibited vascular adhesion molecule 1 (VCAM-1) expression induced by partial ligation in LCA endothelium (Figure 2F and G; Figure 2-figure supplement 1C and D)
These results indicate that METTL3 depletion is associated with EC activation in response to Oscillatory stress (OS). 134 OS abolished m6A prevents epidermal growth factor receptor (EGFR) mRNA degradation
Summary
46 N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNAs (Roundtree et al, 2017). This modification is reversible and is catalyzed by a multicomponent methyltransferase complex consisting of various methyltransferases including Methyltransferase Like 3 (METTL3), METTL14, Wilms Tumor 1 Associated. Protein (WTAP) and KIAA1429 (Virilizer), and is erased by demethylases such as fat mass and obesity-associated protein (FTO) or α-ketoglutarate-dependent dioxygenase alk B 52 homolog 5 (ALKBH5) (Shi et al, 2019; Huang et al, 2021). METTL14 functions as the target recognition subunit by binding 57 to RNA and recruits METTL3 to catalyze m6A formation. As the core methyltransferase subunit, METTL3 has been demonstrated to modulate key physiological processes, including spermatogenesis (Xu et al, 2017), cell reprogramming (Chen et al, 2015)
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