Extra-Nuclear Functions of the Transcription Factor Grainyhead-Like 3 in the Endothelium-Interaction with Endothelial Nitric Oxide Synthase.

We previously demonstrated that the transcription factor Grainyhead-like 3 (GRHL3) has essential functions in endothelial cells by inhibiting apoptosis and promoting migration as well as activation of endothelial nitric oxide synthase (eNOS). We now show that a large portion of the protein is localized to myo-endothelial projections of murine arteries suggesting extra-nuclear functions. Therefore, we generated various deletion mutants to identify the nuclear localization signal (NLS) of GRHL3 and assessed potential extra-nuclear functions. Several large-scale deletion mutants were incapable of activating a GRHL3-dependent reporter construct, which could either be due to deficiencies in transcriptional activation or to impaired nuclear import. One of these mutants encompassed a predicted bipartite NLS whose deletion led to the retention of GRHL3 outside the nucleus. Interestingly, this mutant retained functions of the full-length protein as it could still inhibit pathways inducing endothelial cell apoptosis. As apoptosis protection by GRHL3 depends on NO-production, we examined whether GRHL3 could interact with eNOS and showed a direct interaction, which was enhanced with the extra-nuclear GRHL3 variant. The observation that endogenous GRHL3 also interacts with eNOS in intact murine arteries corroborated these findings and substantiated the notion that GRHL3 has important extra-nuclear functions in the endothelium.

Fetal gender and gestational age differentially affect PCSK9 levels in intrauterine growth restriction.

BACKGROUND: Maternal and fetal Low Density Lipoprotein-Cholesterol (LDL-C) concentrations are compromised in intrauterine growth restriction (IUGR). Generally, LDL-C catabolism is under control of PCSK9 by binding to the LDL-receptor leading to its degradation. Hence, we hypothesized a role for PCSK9 in the modulation of lipid metabolism and placental transport in IUGR. METHODS: 172 women, 70 IUGR and 102 controls were included in the study. Maternal and fetal serum PCSK9 levels and lipid profiles including LDL-C were measured. Placental LDL-receptor and PCSK9 expressions were estimated by tissue microarray immunohistochemistry, and analyzed by two blinded observers using an immunoreactivity score. Non-parametric tests and multivariate regression analyses were used for statistical estimations. RESULTS: PCSK9 levels in the maternal and fetal compartment independently predicted LDL-C levels (maternal compartment: adjusted R 2 = 0.2526; coefficient b i = 0.0938, standard error s bi =0.0217, rpartial = 0.4420, t-value = 4.323, p < 0.0001; fetal compartment: adjusted R 2 = 0.2929; b i = 0.1156, s bi =0.020, rpartial = 0.5494, t-value = 5.81, p < 0.0001). We did not find significant differences in maternal PCSK9 concentrations between IUGR and controls. However, we found lower fetal serum PCSK9 concentrations in IUGR than in controls (IUGR median 137.1 ng/mL (95% CI 94.8-160.0) vs. controls 176.8 (154.6-202.5), p = 0.0005). When subgrouping according to early onset, late onset IUGR, and fetal gender differences remained consistent only for male neonates born before 34 weeks of gestation. In the placenta we found no correlation between PCSK9 and LDL-receptor expression patterns. However, the LDL-receptor was significantly upregulated in IUGR when compared to controls (p = 0.0063). CONCLUSIONS: Our results suggest that PCSK9 play a role in impaired fetal growth by controlling fetal LDL-C metabolism, which seems to be dependent on gestational age and fetal gender. This underlines the need to identify subgroups of IUGR that may benefit from individualized and gender-specific pharmacotherapy in future studies.

Critical regulators of endothelial cell functions: for a change being alternative.

SIGNIFICANCE: The endothelium regulates vessel dilation and constriction, balances hemostasis, and inhibits thrombosis. In addition, pro- and anti-angiogenic molecules orchestrate proliferation, survival, and migration of endothelial cells. Regulation of all these processes requires fine-tuning of signaling pathways, which can easily be tricked into running the opposite direction when exogenous or endogenous signals get out of hand. Surprisingly, some critical regulators of physiological endothelial functions can turn malicious by mere alternative splicing, leading to the expression of protein isoforms with opposite functions. RECENT ADVANCES: While reviewing the evidence of alternative splicing on cellular physiology, it became evident that expression of splice factors and their activities are regulated by externally triggered signaling cascades. Furthermore, genome-wide identification of RNA-binding sites of splicing regulatory proteins now offer a glimpse into the splicing code responsible for alternative splicing of molecules regulating endothelial functions. CRITICAL ISSUES: Due to the constantly growing number of transcript and protein isoforms, it will become more and more important to identify and characterize all transcripts and proteins regulating endothelial cell functions. One critical issue will be a non-ambiguous nomenclature to keep consistency throughout different laboratories. FUTURE DIRECTIONS: RNA-deep sequencing focusing on exon-exon junction needs to more reliably identify alternative splicing events combined with functional analyses that will uncover more splice variants contributing to or inhibiting proper endothelial functions. In addition, understanding the signals mediating alternative splicing and its regulation might allow us to derive new strategies to preserve endothelial function by suppressing or upregulating specific protein isoforms. Antioxid. Redox Signal. 22, 1212-1229.

Two isoforms of Sister-Of-Mammalian Grainyhead have opposing functions in endothelial cells and in vivo.

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.