Abstract P257: Isoform-Specific Effects of the Transcription Factor Sister-of-Mammalian Grainyhead on Endothelial Cells and in Vivo

Abstract

Apoptosis and reduced migratory capacity of human endothelial cells (EC) are hallmarks for the development of atherosclerosis. TNFalpha has been described as one apoptotic stimulus, which is increased during cardiovascular disease. However, recent findings support the hypothesis that TNFalpha can induce survival genes before committing cells to apoptosis. In a screen for anti-apoptotic genes regulated by TNFalpha we have identified the transcription factor Sister-of-Mammalian Grainyhead/Grainyhead-like 3 (SOM/GRHL3). In humans two RNAs are transcribed from the gene, one of which is alternatively spliced, yielding the protein isoforms SOM1 and SOM3, the latter being an N-terminally truncated version. We have found that both isoforms are expressed in EC. Since nothing is known about the function of these proteins in EC, we investigated their functional properties and role in migration and apoptosis. To analyze their transcription factor activity we established a SOM-dependent reporter system by inserting tandem SOM binding sites and corresponding mutants upstream of a minimal promoter driving luciferase expression. To assess transcriptional activation by SOM1 and SOM3 we cotransfected these reporters with expression vectors for both proteins. In contrast to previously published work, in which isolated SOM domains fused to a Gal4 DNA binding domain were used, we found that both full length proteins are active transcription factors. We next investigated the influence of SOM1 and SOM3 on EC functions. Surprisingly, overexpression of isoform 1 induced migration and inhibited apoptosis, whereas isoform 3 had opposite effects. Along the same lines, SOM1, but not SOM3 activated endothelial nitric oxide synthase and Akt. To investigate whether these isoforms have different functions also in vivo, we overexpressed them in zebrafish embryos. SOM3 but not SOM1 overexpression led to increased lethality, a strong reduction in normal phenotype and a 10 fold higher frequency in heavy deformations. The effects observed on EC migration and apoptosis as well as on zebrafish development suggest that these isoforms activate different sets of target genes, which we are currently identifying by microarray analysis.