ABSTRACT
OBJECTIVE: Sister-of-Mammalian Grainyhead (SOM) is a member of the Grainyhead family of transcription factors. In humans, 3 isoforms are derived from differential first exon usage and alternative splicing and differ only in their N terminal domain. SOM2, the only variant also present in mouse, induces endothelial cell migration and protects against apoptosis. The functions of the human specific isoforms SOM1 and SOM3 have not yet been investigated. Therefore we wanted to elucidate their functions in endothelial cells. APPROACH AND RESULTS: Overexpression of SOM1 in primary human endothelial cells induced migration, phosphorylation of Akt1 and endothelial nitric oxide synthase, and protected against apoptosis, whereas SOM3 had opposite effects; isoform-specific knockdowns confirmed the disparate effects on apoptosis. After reporter assays demonstrated that both are active transcription factors, microarray analyses revealed that they induce different target genes, which could explain the different cellular effects. Overexpression of SOM3 in zebrafish embryos resulted in increased lethality and severe deformations, whereas SOM1 had no deleterious effect. CONCLUSIONS: Our data demonstrate that the splice variant-derived isoforms SOM1 and SOM3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.
Subject(s)
DNA-Binding Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Transcription Factors/metabolism , Animals , Apoptosis , Cell Movement , DNA-Binding Proteins/genetics , Enzyme Activation , Gene Expression Profiling/methods , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Humans , MCF-7 Cells , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Oligonucleotide Array Sequence Analysis , Phosphorylation , Protein Isoforms , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Transcription Factors/genetics , Transcription, Genetic , Transfection , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Migratory capacity and resistance to apoptosis are crucial for proper endothelial function. In a screen for anti-apoptotic genes in a breast cancer cell line, we identified Grainyhead like 3 (GRHL3). Therefore, the aim of our study was to investigate whether GRHL3 is expressed in endothelial cells and moreover, to determine its role in migration, apoptosis and senescence. GRHL3 is expressed in human endothelial cells. GRHL3 is required for endothelial cell migration. The underlying mechanism is independent of vascular endothelial growth factor. GRHL3 induces Akt and endothelial nitric oxide synthase phosphorylation and its expression is increased by physiological concentrations of nitric oxide. Nitric oxide dependent migration is completely dependent on GRHL3 expression. Moreover, GRHL3 inhibits apoptosis of endothelial cells in an eNOS-dependent manner. Thus, loss of GRHL3 may result in endothelial dysfunction in vivo. One may consider new therapeutic strategies with the aim to conserve GRHL3 expression in the vasculature.
