Abstract P314: Grainyhead-like 3 Plays an Essential Role in Endothelial Cell Function in a NO-Dependent Manner

Abstract

Aging of human endothelial cells (EC) is associated with reduced nitric oxide (NO) bioavailability, decreased migratory capacity and an increase in Src kinase activation and apoptosis sensitivity. We recently identified the transcription factor grainyhead-like 3 (GRHL3) as a pro-migratory transcription factor in EC. A role for GRHL3 in aging processes was suggested by reduced expression in brains from old mice. Therefore, we wanted to investigate the regulation of GRHL3 by NO and Src kinase and GRHL3 effects on NO-bioavailabilty, apoptosis and migration. We treated EC either with physiological concentrations of NO or the Src kinase inhibitor PP2. In both cases GRHL3 expression was increased (3.75 fold and 4.50 fold, respectively). In addition, both treatments induced migration and inhibited apoptosis. Interestingly, overexpression of GRHL3 activated endothelial nitric oxide synthase (eNOS), its upstream regulator Akt and subsequently increased the S-NO content of EC. This demonstrates that GRHL3 enhances NO-bioavailability in EC, which is inseparably tied to apoptosis protection and migration. Along this line, GRHL3 overexpression reduced apoptosis of EC (1.89 fold reduction of basal apoptosis vs. empty vector transfected cells). Interestingly, this anti-apoptotic effect was dependent on NO synthesis by eNOS, since the eNOS inhibitor L-NMMA completely abrogated the protective effect of GRHL3. Having demonstrated a pro-migratory effect of GRHL3, we wanted to know whether this effect is mediated by induction of vascular endothelial growth factor (VEGF) expression. Surprisingly, GRHL3 overexpression did not change VEGF protein levels. To exclude a bystander effect of GRHL3 in EC migration, we knocked down expression with shRNA. Reduction of GRHL3 mRNA levels decreased basal and NO-induced EC migration (scr: 73 +/− 16 migrated cells; shGRHL3: 26 +/− 12 migrated cells; scr+NO: 149 +/− 19 migrated cells; shGRHL3+NO: 36 +/− 10 migrated cells) demonstrating an essential role in this process. Taken together, these data suggest that GRHL3 is essential for EC functions compromised during aging.