RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells

Ahmad F Alghanem,Beverley A Rothermel,Michael J Cross,Victoria A Simms,Emma L Wilkinson,Victoria L Heath,Maxine S Emmett,Mohammad A Aljasir,Katherine Holmes

doi:10.1007/s10456-017-9542-0

Abstract

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

Highlights

Angiogenesis is the process via which new blood vessels are formed from pre-existing vessels
We subsequently showed that Regulator of calcineurin 1 (RCAN1) was required for Vascular endothelial growth factor A (VEGF)/VEGF receptor 2 (VEGFR-2) mediated migration of endothelial cells
The duplexes utilised target both isoforms of RCAN1 (RCAN1.1 and RCAN1.4) as it was not possible to design isoform specific duplexes. short-interfering RNA (siRNA)-mediated silencing of RCAN1 resulted in a delayed VEGFR-2 downregulation following VEGF stimulation with a concomitant increase in receptor phosphorylation (Fig. 1a, b)

Summary

Introduction

Angiogenesis is the process via which new blood vessels are formed from pre-existing vessels. VEGF signalling in vascular endothelial cells is primarily mediated by VEGF receptor 2 (VEGFR-2) [6]. The binding of VEGF to VEGFR-2 results in receptor activation, characterised by receptor autophosphorylation and initiation of a series of signalling cascades within the cell resulting in migration, cell survival and proliferation [6,7,8]. Phosphorylation of the tyrosine 1175 residue within the intracellular C-terminal domain of the receptor induces activation of phospholipase C-c (PLCc) [9], which hydrolyses phosphatidylinositol (4,5)-bisphosphate (PIP2), to generate inositol (1,4,5)-trisphosphate (IP3) and Angiogenesis (2017) 20:341–358 diacylglycerol (DAG). Dephosphorylation of nuclear factor of activated T-cells (NFAT) by calcineurin results in its translocation to the nucleus, where, in cooperation with other transcription factors, it induces the transcription of a number of VEGFresponsive genes important for regulating endothelial cell physiology [11, 12]

Methods

Results

Conclusion