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.
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.
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.
Maintenance of apoptosis protection and migratory capacity are absolutely required for proper endothelial function. Several pathways are described, which are necessary for these processes including activation of the protein kinase B (Akt) and the endothelial nitric oxide synthase (eNOS). Recently, we identified a new player, the transcription factor sister-of-mammalian grainyhead (SOM)/Grainyhead-like 3, which demonstrated anti-apoptotic and pro-migratory properties in endothelial cells (EC). In humans, three isoforms of SOM are expressed, two of which, SOM1 and SOM3, are derived from an alternatively spliced transcript. SOM3 lacks exon 2, described to contain the putative transactivation domain, and thus, has been suggested to be a repressor. SOM1 and SOM3 are co-expressed in numerous tissues, however, their exact cellular functions have not been investigated so far. Here we demonstrate that SOM1 and SOM3 are expressed in primary human EC. In contrast to published data, which were based on a yeast two hybrid analysis with isolated domains, we could show that both full length proteins are transcriptional activators. Moreover, they have opposing functions in EC and in a whole animal model. Overexpression of SOM1 inhibited apoptosis and induced migration of EC. The latter effect could be due to the observed activation of eNOS and its upstream regulator Akt. On the contrary, SOM3 has no effect on apoptosis and significantly inhibits migration and eNOS activation. To assess the in vivo relevance of the opposing effects of SOM1 and SOM3, we made use of zebrafish as a whole animal model. SOM3, but not SOM1, induced severe malformations in embryos and reduced the number of normally developed embryos significantly. Microarray analyses showed that SOM1 and SOM3 have some overlapping, but an even larger number of distinct target genes. Our data suggest that the opposing effects of the two SOM isoforms are mediated by transcriptome alterations and that a switch in isoform expression could change the cellular fate with dramatic consequences for a whole organism.
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