Regulation of shelterin proteins TERF2IP and TRF2 by the MLL2-H3K4me3-p65 axis drives hyperglycemia-dependent endothelial senescence

Endothelial senescence has been linked with several cardiovascular diseases. Dysregulation of TRF2 and TERF2IP, proteins of the shelterin complex, causes cellular senescence. However, it is still unknown whether exposure to hyperglycemia alters levels of proteins of the shelterin complex, further dictating the senescent phenotype of endothelial cells (EC). In this study, we observed elevated protein levels of p21 and p53 in EC upon exposure to Intermittent High Glucose(IHG). Furthermore, exposure to hyperglycemia increased levels of TERF2IP and TRF2 proteins. Interestingly, we observed a robust induction in the p65 protein level as well as it's phosphorylation upon intermittent hyperglycemia challenge. ChIP-qPCR analysis revealed enhanced H3K4me3 enrichment in the gene promoters of p65, TERF2IP, and TRF2. Inhibition of H3K4me3 deposition either by pharmacological inhibitor or siRNA-mediated knockdown of MLL2 reduced the expression of p65, TERF2IP and TRF2 and restored the levels of senescence markers, namely p53 and p21. Interestingly, pharmacological inhibition of NF-κB signaling reduced the expression of TERF2IP and TRF2 levels, reversing the levels of p53 and p21. Co-immunoprecipitation and co-localization analysis revealed an association between nuclear p65 and MLL2 proteins in EC exposed to hyperglycemia. Knockdown of either TERF2IP or TRF2 impaired intermittent hyperglycemia-induced p53 and p21 expression and associated endothelial senescence.