The function of WNK1/OSR1 in cell migration and angiogenesis

Abstract

Deletion of the WNK1 (with no lysine kinase-1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial-specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive-1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to be stimulated by TGF-β/Smad signaling which induces epithelial-mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF-βII receptor at tight junctions which mediates ubiquitination of GTP-RhoA and dissolution of tight junction upon activation by TGF-β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for the expression of TGF-β/Smad-induced EMT factors, suppression of RhoA activity and disruption of cell-cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 siRNA-mediated knockdown decreased the expression of TGF-β-induced EMT mediators such as Slug, the receptor tyrosine kinase Axl and the transcription factor p63 (ΔNp63α) (normalized densitometry values to control; siWNK1-Slug: 0.5 ± 0.25, Axl: 0.2 ± 0.05, p63: 0.35 ± 0.15 vs. siControl: 1, n = 2) in MCFDCIS cancer cell line. Similarly, WNK1 pharmacological inhibition decreased Axl expression (normalized densitometry values to control; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n = 3, *p<0.05) in primary HUVECs (Human Umbilical Vein Endothelial Cells). This suggests that WNK1 is involved in Slug/p63/Axl network in endothelial and cancer cells. We found that knockdown of WNK1 increased F-actin content (normalized arbitrary fluorescence units to control; siWNK1: 1.6 ± 0.3 vs. siControl: 1, n = 2, 4–5 replicates per experiment) in HeLa cells. Because RhoA activity regulates actin dynamics, RhoA activity was measured in WNK1 depleted cells and the ratio of GTP-bound RhoA to total RhoA was found to be significantly increased (siWNK1: 3.08 ± 0.75 vs. siControl: 0.34 ± 0.05, n = 3, *p<0.035). Finally, we determined that OSR1 and Occludin, a tight junction protein are co-localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co-localization during TGF-β signaling in primary HUVECs (Pearson's coefficient; Control: 0.08 ± 0.2 vs. TGF-β: 0.61 ± 0.15 vs. TGF-β+WNK1i: 0.12 ± 0.25, n = 2, 2 replicates per experiment). These data suggest WNK1-dependent function of OSR1 in cell migration and angiogenesis through effects on GTP-RhoA degradation. Combined, these data indicate the importance of WNK1 function in endothelial and cancer cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis, ischemia-reperfusion injury as well as cancer. Support or Funding Information Early part of this work was supported by NIH 5R01GM053032-16 grant. Current work is being supported by the Welch Foundation grant I-1243. Co-localization of OSR1 and Occludin in primary HUVECs This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.