Abstract 446: Ampk Regulates Endothelial Mirna Biogenesis Through Nucleolin

Background: A characteristic of atherosclerosis is the focal distribution of lesions in the arterial tree despite the systemic nature of cardiovascular impairments such as hyperlipidemia. While pulsatile shear stress (PS, atheroprotective) maintains endothelial homeostasis, oscillatory shear stress (OS, atherogenic) causes endothelial dysfunction. Previously, we and others have identified AMP activated protein kinase (AMPK) is a master regulator in vascular endothelial cells (ECs) in response to PS. To further decipher the atheroprotective effect of AMPK, bioinformatic approaches identified a broad array of AMPK-regulated pathways that regulate cellular functions beyond its canonical catabolic regulation. Among other newly identified substrates, nucleolin (NCL) is a multifunctional protein and the newly identified AMPK phosphorylation of NCL S328 may be a major mechanotransduction event hindering miRNA biogenesis in ECs. Methods and Results: Using AMPK kinase assays and phospho-S328 NCL antibody, we identify that the PS-activated AMPK increased phospho-S328 NCL. Cellular fractionation and immunofluorescence indicated NCL localization throughout the nucleus and cytoplasm which was sequestered to the nucleus following AMPK activation. This indicated bipartite NCL functions, acting as a chaperone under dephospho-S328 conditions and maintaining a transcriptional function upon phosphorylation. Bioinformatics analysis validated by RNA immunoprecipitation showed that the OS-induced miR-93 and miR-484 were novel NCL-regulated miRNAs. Putative miR-93 and miR-484 targets, including eNOS and BMPR2 were cross referenced to RNA-seq data from ECs exposed to PS and OS. ECs transfected with anti-miR-93 and anti-miR-484 exhibited ratified NO bioavailability and attenuated inflammation. In response to PS stimulation, however, AMPK activation increased NCL phosphorylation resulting in its binding to the KLF2 promoter to induce KLF2 that increased NO bioavailability and inhibited miR-93 and miR-484 induction. Conclusions: These results demonstrate that AMPK can inhibit the expression of atherogenic miR-93 and miR-484 in ECs through AMPK phosphorylation of NCL S328.