Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4

Abstract

VEGF signaling via VEGF receptor-2 (VEGFR2) is a major regulator of endothelial cell (EC) functions, including angiogenesis. Although most studies of angiogenesis focus on soluble VEGF signaling, mechanical signaling also plays a critical role. Here, we examined the consequence of disruption of mechanical signaling on soluble signaling pathways. Specifically, we observed that small interfering RNA (siRNA) knockdown of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), significantly reduced perinuclear (Golgi) VEGFR2 in human ECs with a concomitant increase in phosphorylation at Y1175 and membrane translocation. TRPV4 knockout (KO) ECs exhibited increased plasma membrane localization of phospho-VEGFR2 compared with normal ECs. The knockdown also increased phospho-VEGFR2 in whole cell lysates and membrane fractions compared with control siRNA-treated cells. siRNA knockdown of TRPV4 enhanced nuclear localization of mechanosensitive transcription factors, yes-associated protein/transcriptional coactivator with PDZ-binding motif via rho kinase, which were shown to increase VEGFR2 trafficking to the plasma membrane. Furthermore, TRPV4 deletion/knockdown enhanced VEGF-mediated migration in vitro and increased expression of VEGFR2 in vivo in the vasculature of TRPV4 KO tumors compared with wild-type tumors. Our results thus show that TRPV4 channels regulate VEGFR2 trafficking and activation to identify novel cross-talk between mechanical (TRPV4) and soluble (VEGF) signaling that controls EC migration and angiogenesis.-Kanugula, A. K., Adapala, R. K., Midha, P., Cappelli, H. C., Meszaros, J. G., Paruchuri, S., Chilian, W. M., Thodeti, C. K., Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

Keywords: angiogenesis; endothelial cell; phosphorylation.

Figure 1

TRPV4 is functionally expressed in HMECs-1. A) RT-PCR analysis of TRPV4 gene expression in HMECs-1. B) Representative traces display relative changes in cytosolic calcium in response to a selective TRPV4 agonist, GSK1016790A (GSK101; 1 nM), and antagonist, GSK2193874 (GSK2; 50 nM), in Fluo-4–loaded cells (n = 60). Arrow denotes the time when the cells were stimulated with the TRPV4 agonist. C) Quantitative analysis of cytosolic calcium influx induced by GSK101 and GSK101 + GSK2 in HMECs-1 (F/F0 = ratio of normalized Fluo-4 fluorescence intensity relative to time 0). D–F) Quantitative RT-PCR and Western blot analysis showing relative TRPV4 expression in control (control siEC) and TRPV4 siRNA transfected (TRPV4 siEC) siECs after 48 h. G) Representative traces display relative changes in cytosolic calcium in response to a selective GSK101 in Fluo-4– loaded control siECs and TRPV4 siECs (n = 60). Arrow denotes the time when the cells were stimulated with the TRPV4 agonist. H) Quantitative analysis of cytosolic calcium influx induced by GSK101 in control siECs and TRPV4 siECs (F/F0 = ratio of normalized Fluo-4 fluorescence intensity relative to time 0). Data presented are the means ± sem from at least 3 independent experiments. The significance was calculated by using Student’s t test. *P ≤ 0.05.

Figure 2

TRPV4 knockdown/deletion reduces perinuclear (Golgi) VEGFR2 expression. A) Immunofluorescence images of control siECs and TRPV4 siECs of HMECs-1 showing localization of total VEGFR2 (red). Nuclei were stained with DAPI (blue). The graph represents the quantitative analysis of the percentage of cells with perinuclear localization of VEGFR2. B) Immunofluorescence images of total VEGFR2 (red) in NECs and TRPV4KOECs. Nuclei were stained with DAPI (blue). The graph depicts the quantitative analysis of the percentage of cells with perinuclear localization of VEGFR2. Data presented are means ± sem of 3 independent experiments. *P ≤ 0.05. Scale bars, 10 μm.

Figure 3

TRPV4 knockdown/deletion increases phosphorylation (Y1175) and membrane translocation of VEGFR2. A) Immunofluorescence images of p-VEGFR2 (red) in control siECs and TRPV4 siECs. Arrows denote membrane localization of p-VEGFR2. Nuclei were stained with DAPI (blue). The graph depicts the quantitative analysis of percent cells with membrane localization of p-VEGFR2 from control and TRPV4 siECs. Data presented are means ± sem of 3 independent experiments. B) Western blot analysis represents the expression levels of total and p-VEGFR2 in whole cell lysates from control siEC and TRPV4 siEC. Relative p-VEGFR2 expression was quantified by normalizing with total VEGFR2. The graph represents the quantitative analysis of relative p-VEGFR2 expression from the whole cell lysates of control and TRPV4 siEC. C) Western blots demonstrating the expression levels of total and p-VEGFR2 in membrane fractions from control and TRPV4 siEC. β1-integrin expression was used as a loading control for plasma membrane fractions. The graph depicts the quantitative analysis of relative p-VEGFR2 expression from the membrane fractions of control and TRPV4 siEC. The given results are means ± sem from 3 independent experiments. *P ≤ 0.05. Scale bar, 10 μm.

Figure 4

TRPV4 knockdown increases YAP nuclear translocation in HMECs-1 via the rho kinase pathway. A) Immunofluorescence images of YAP (green) in control siECs and TRPV4 siECs in the presence or absence of Y-27632 (Y27; 10 μM). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. B) The graph represents the quantitative analysis of the percentage of cells with nuclear localization of YAP from control siEC, TRPV4 siEC, control siEC + Y27, and TRPV4 siEC + Y27. Data presented are means ± sem of 3 independent experiments. *P ≤ 0.05.

Figure 5

Deletion of TRPV4 increases VEGF-induced migration in vitro and VEGFR2-positive vessels in tumors in vivo. A) Migration of serum-starved NECs and TRPV4KOECs was measured by using a scratch-wound migration assay. The graph represents the percent migration of NECs and TRPV4KOECs. B) Immunofluorescence images of p-VEGFR2 (red) in migrating NECs and TRPV4KOECs. Arrows depict membrane localization of p-VEGFR2 in ECs at the wound edge. Nuclei were stained with DAPI (blue). C) Tumors were implanted into WT and TRPV4KO mice by subcutaneously injecting Lewis lung carcinoma cells, as previously described (13), and isolated on day 21. Representative images (20×) of the tumor tissue stained with CD31 (red), VEGFR2 (green), and DAPI (nuclei) were used to quantify the VEGFR2-positive vessels. D) Quantitative analysis demonstrating a significant increase in the percentage of VEGFR2-positive vessels in tumors from TRPV4KO mice compared with WT mice. Data presented are means ± sem from 3 independent experiments. *P ≤ 0.05. Scale bars, 10 μm.