WNK and TGF-beta in Endothelial Migration

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Abstract The long term goal of this research is to determine the molecular mechanisms underlying the interactions of WNK1 with TGFβ signaling on endothelial plasticity and homeostasis. Failed angiogenesis in endothelial-specific WNK1 null mice led to embryonic death. We have found two processes important for endothelial plasticity that are coordinately regulated by TGFβ and WNK1: conversion of cells to a migratory phenotype and tight junction breakdown that occurs with complete endothelial-mesenchymal transition. These processes contribute to normal vascular physiology and to pathophysiology. We will investigate how signaling pathways regulated by TGFβ and WNK1 intersect in controlling endothelial cell behavior and characteristics. WNK-selective kinase inhibitors, protein depletion, gene editing, proximity ligation, gene expression and other biochemical means will be used in cell- and tissue-based assays to discover the mechanisms through which WNK1 mediates dynamic reorganization of endothelial cell structures underlying normal and pathophysiological angiogenesis in tandem with TGFβ. In the first specific aim, we will determine interactions between TGFβ and WNK1 signaling pathways that regulate expression of the mesenchymal transcription factor Slug (Snai2) and subsequent induction of endothelial cell motility. We hypothesize that WNK1 activates Slug expression and migration through actions on TGFβ-regulated SMADs. Slug promotes endothelial cell migration and remodeling by inducing proteins that repress cell-cell adhesion and enhance a migratory mesenchymal phenotype. In the second specific aim, we will determine how WNK1 promotes TGFβ-induced tight junction disassembly. In response to TGFβ, the WNK1 substrate kinase OSR1 binds to tight junction proteins along with other signaling proteins and TGFβ receptors to break down tight junctions. Inhibiting WNK1 kinase activity prevents co-immunoprecipitation of OSR1 with occludin and prevents TGFβ-induced tight junction disassembly in endothelial cells. We hypothesize that WNK1 is required for TGFβ-induced tight junction disassembly through actions of its substrate kinase OSR1. We will analyze how WNK1/OSR1 participate in TGFβ-initiated tight junction break down to discover the steps requiring their cooperation. We will identify components in OSR1-occludin complexes and the effects of interfering with OSR1 function on tight junction breakdown. Essential events will be established using rescue strategies. Our results will define the extent of cooperation between TGFβ and WNK1 signaling mechanisms and uncover opportunities for therapeutic targeting of the WNK1 pathway in disease. Our findings will lead to a better understanding of normal and pathological angiogenesis.