Abstract
Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.
Highlights
There are 58 human receptor tyrosine kinases (RTKs) which are sub-classified into 20 families
Upon stimulation with saturating levels of Vascular endothelial growth factor A (VEGF-A) (1.25 nM), the peak level of VEGFR2 activation achieved in response to VEGFA165 (Fig. 1C) was comparable to that induced by vascular endothelial growth factor (VEGF)-A121 (Fig. 1D)
At this VEGF-A concentration, activated VEGFR2 appeared to be dephosphorylated at an increased rate upon treatment with VEGF-A121 (Fig. 1D) compared to cells treated with VEGF-A165 (Fig. 1C)
Summary
There are 58 human receptor tyrosine kinases (RTKs) which are sub-classified into 20 families. These Type I membrane proteins regulate animal development, health and disease states (Lemmon and Schlessinger, 2010). Upon ligand-binding, RTK monomers undergo dimerisation followed by trans-autophosphorylation of cytoplasmic tyrosine residues, enabling the recruitment and phosphorylation of a vast array of signal transduction enzymes and adaptor proteins. RTKs are key targets for new therapeutics but successful drug design is complicated by the increasing. The physiological relevance for the expression of numerous ligands is unclear; many studies have based their conclusions on studying the effects of a single ligand for a specific RTK
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