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

Kirsten Jander,Sabrina Farrokh,Niloofar Ale-Agha,Christine Goy,Corina Marziano,Swapnil K Sonkusare,Philipp Jakobs,Jan Greulich,Stefanie Gonnissen,Judith Haendeler,Joachim Altschmied

doi:10.3390/antiox10030428

Abstract

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.

Highlights

The endothelium—the innermost layer of the vessel wall—is a single layer of cells that line the inside of blood vessels
Grainyhead-like 3 (GRHL3) in cooperation with Merck Millipore, which is commercially available, we performed en face staining of the murine aorta, which is a conduit artery, and resistancesized mesenteric arteries to investigate whether GRHL3 is expressed in endothelial cells in vivo
The majority of the extra-nuclear GRHL3 was found in myoendothelial projections (MEPs) (Figure 1 and Supplementary Videos)

Summary

Introduction

The endothelium—the innermost layer of the vessel wall—is a single layer of cells that line the inside of blood vessels. They form a selective barrier between vessels and tissues and control the flow of substances into and out of a tissue. NO is produced constitutively by the endothelial NO Synthase (eNOS). Impaired endothelial cell functionality is observed in most cardiovascular diseases and is typically referred to as endothelial dysfunction. It is characterized by reduced NO bioavailability and migratory capacity as well as increased sensitivity towards apoptotic stimuli [1]

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