Mechanism of PAI-1 Polarization to Myoendothelial Junctions

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Abstract During the course of metabolic syndrome, there is a significant increase in plasminogen activator inhibitor-1 (PAI-1); however the pathological role that PAI-1 plays during the course of the disease is not clear. We have recently identified PAI-1 as being key to regulation of myoendothelial junctions (MEJs), which are critical heterocellular structures linking the endothelium and smooth muscle in the resistance vasculature, coordinating communication between the two cell types. In mice with metabolic syndrome, we have shown that PAI-1 accumulates in MEJs and increases the total number of these structures, indicating a possible role in altering heterocellular communication. In order to understand how PAI-1 may alter MEJ formation, we used TNF-a to mimic the effect of metabolic syndrome and demonstrate significant increases in PAI-1 mRNA throughout endothelium, MEJ, and smooth muscle. However, after application of TNF-a, there was only a significant increase in PAI-1 protein at the MEJ. This indicates that PAI-1 may be locally translated at the MEJ. Recently, the serpine binding protein 1 (SERBP1) has been described as a novel RNA binding protein (RBP) specifically for PAI-1 mRNA that promotes stabilization of the mRNA for translation. However, an RBP requires anchoring to the cytoskeleton to remain in a specific area of the cell (e.g., the MEJ). To that end, we discovered a microtubule binding domain in the novel protein nicotinamide phosphoribosyltransferase (NAMPT), demonstrated to increase concurrently with PAI-1 during the course of metabolic syndrome, which we found to be localized to the MEJ. Therefore, we hypothesize that SERBP1 and NAMPT act together as a PAI-1 RBP complex to localize PAI-1 mRNA to the MEJ for the rapid and specific dissemination of PAI-1 protein. To test this hypothesis, we have put forth two specific aims: 1) PAI-1 mRNA is locally translated at the MEJ and 2) NAMPT regulates localization of SERBP1 to the MEJ. This proposal will focus on how PAI-1 is capable of being polarized to the MEJ using such techniques as FlAsH/ReAsH to examine nascent PAI-1 protein production and how RBP proteins could be anchored to the cytoskeleton using peptides against the microtubule binding domain of NAMPT. The aggregate of the experiments will provide for the first time a pathological mechanism for the effect of increased PAI-1 seen in metabolic syndrome.