Abstract 5557: Sister Of Mammalian Grainyhead 3 - A Newly Identified Transcription Factor - Is Required For Endothelial Cell Migration And Sprouting
Sister of mammalian grainyhead 3 (GRHL3) is a member of the grainyhead transcription factors that are highly conserved from fly to human. The three known human isoforms are derived from alternative splicing and show different expression patterns. GRHL3 deficient mice show pronounced spina bifida and impaired wound healing indicating defects in cell migration. Therefore the aim of this study was to investigate whether GRHL3 is indeed a new player for endothelial cell (EC) migration and sprout formation and what are the underlying mechanisms. Overexpression of GRHL3 isoforms 1 and 2 induced migration of ECs comparable to the promigratory stimulus vascular endothelial growth factor (VEGF), whereas isoform 3, which lacks the transactivation domain present in the other isoforms, suppressed migration of ECs (empty vector: 9 +/− 3 migrated cells, empty vector + VEGF: 131 +/− 21; GRHL3 isoform 1: 172 +/− 49; isoform 2: 149 +/− 52; isoform 3: 3 +/− 1, p<0.05 vs empty vector). Similar results were obtained in a spheroid assay, where isoforms 1 and 2 increased cumulative sprout length, whereas isoform 3 did not show any effect. To get insights into the underlying mechanisms, we next investigated the known VEGF-induced activators of EC migration -phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Indeed, overexpression of GRHL3 isoforms 1 and 2 increased Akt (Ser473) and eNOS(Ser1179) phosphorylation, while isoform 3 reduced eNOS phosphorylation significantly. Next, we investigated the Src kinase family, the role of which in cell migration and sprout formation has been controversly discussed. Blockade of the Src kinase family by incubation with PP2 inhibited migration and increased GRHL3 expression as measured by real time PCR. Moreover, mouse embryonic fibroblasts (MEFs), deficient for Src, Fyn and Yes showed enhanced migratory and proliferative capacity and increased endogenous GRHL3 expression as determined by immunoblot. In conclusion, we identified GRHL3 as a new transcription factor which importantly contributes to EC migration and sprout formation via induction of Akt and eNOS activation. Inhibition of Src expression increased GRHL3 expression and EC and fibroblasts migration.