Identifying the role of nuclear pore components in vascular function. - PROJECT SUMMARY Endothelial cells (ECs) form the innermost lining of blood vessels to actively regulate vascular health. Fully receptive to environmental cues, ECs sense alterations in blood flow patterns and transmit hemodynamic information to the underlying vessel wall. Unidirectional laminar blood flow promotes EC quiescence and vascular health via activation of endothelial Akt signaling and corresponding changes to the transcriptome. It remains, however, unclear how mechanical forces are conveyed to the nucleus to affect transcriptional output. During the resubmission period, my laboratory identified nucleoporin93 (Nup93), a major component of the nuclear pore complex (NPC), as an indispensable player for endothelial protection. We reported aberrant nuclear Yap accumulation, a transcriptional cofactor known to trigger atherosclerosis, as the major mechanism driving endothelial inflammation in Nup93-deficient ECs. Demonstrating the importance of Yap subcellular localization, laminar flow conditions are well-known to prevent nuclear Yap localization to promote an anti-inflammatory and atheroprotective genetic profile. Intriguingly, we have identified and validated a highly conserved Akt phosphorylation motif on Nup93. Endothelial Nup93 phosphorylation may therefore occur to prevent nuclear Yap accumulation, thereby uncovering a novel mechanism of vascular protection. To understand the impact of Nup93 phosphorylation on Yap signaling and endothelial inflammation, we introduced Nup93 point-mutant constructs (phospho-impaired [Nup93-AA]; phospho-mimetic [Nup93-DD]) into primary ECs. Indeed, we find that impaired Nup93 phosphorylation (Nup93-AA) leads to nuclear Yap accumulation, EC inflammation, and endothelial permeability. Mechanistically, Nup93-AA ECs exhibit increased binding to Nup62, a nucleoporin anchored at the NPC involved in cargo transport. More importantly, endogenous mutation abolishing Nup93 phosphorylation (Nup93-AA) enhances atherosclerotic lesion formation, revealing Nup93 phosphorylation as an atheroprotective event. Based on these observations, we hypothesize that impaired Nup93 phosphorylation, coupled with increased Nup62 interaction, promotes nuclear Yap accumulation to prime the vasculature for inflammation and atherosclerosis. To test this, AIM1 will investigate Yap subcellular localization, downstream activity, and Nup93- Nup62 interaction in Nup93 mutant expressing ECs under conditions of endothelial laminar flow. AIM2 will elucidate the role of Nup93 phosphorylation in Yap-mediated endothelial inflammation and barrier permeability using classic in vitro functional models. We will also integrate our novel Nup93 phospho-deficient point-mutant (Nup93AA/AA) mouse model to define the in vivo importance of Nup93 phosphorylation in vessel homeostasis. Lastly, AIM3 will detail the consequences of impaired Nup93 phosphorylation on atherosclerotic lesion formation. These studies will include bone marrow transplant experiments and use of our newly generated Nup93 floxed conditional model to investigate the vascular and endothelial-specific importance of Nup93 phosphorylation, respectively. Overall, this proposal aims to elucidate how Akt-directed phosphorylation of Nup93, a critical nuclear pore protein, promotes endothelial and cardiovascular health, thus opening a new area of research in vascular biology.
