TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation

Abstract

Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells (epithelial) themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation in vivo. Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation.

Figure 1. TEC exhibit increased proliferation which is inhibited by pharmacological activation of TRPV4.

(A) XTT assay showing increased proliferation of tumor endothelial cells (TEC), compared to normal endothelial cells (NEC), which was significantly (p ≤ 0.05) reduced by GSK (100 nM). NS = non-significant. The data shown is mean ± SEM from five independent experiments. Significance was determined using Student’s paired t test and significance was set at p ≤ 0.05. (B) Concentration-dependent reduction in TEC proliferation by GSK measured using Calcein-AM (p ≤ 0.05). Note that GSK did not affect NEC proliferation. Significance was determined using Student’s paired t test and ANOVA with Tukey’s post-hoc analysis and significance was set at p ≤ 0.05. (C) Western blot analysis of PCNA expression in NEC and TEC. Quantitative analysis of the Western blots showing a significant (p ≤ 0.05) increase in PCNA expression in TEC. PCNA levels were normalized to tubulin and expressed as a fold change compared to NEC. (D) Quantitative analysis of BrdU positive cells in NEC and TEC (percentage of BrdU positive cells from the total number of cells (1000 cells for condition)) and expressed as fold change relative to NEC (p ≤ 0.05). (E) Western blot analysis of PCNA expression in TEC untreated or treated with GSK. Quantitative analysis of the Western blots showing a significant (p ≤ 0.05) decrease in PCNA expression in TEC treated with GSK. PCNA levels were normalized to tubulin and expressed as a fold change relative to TEC). (F) Quantitative analysis of BrdU positive cells in TEC untreated or treated with GSK (percentage of BrdU positive cells from the total number of cells (1000 cells for condition)) and expressed as fold change compared to TEC (p ≤ 0.05). All the data shown is mean ± SEM from at least three independent experiments.

Figure 2. TRPV4 pharmacological activation did not inhibit LLC cell proliferation.

(A) Western blots depicting TRPV4 expression in Lewis Lung Carcinoma (LLC) cells. NEC were used as a positive control for TRPV4 expression. (B) XTT assay showing no change in LLC proliferation by GSK (100 nM). NS = non-significant. Data shown are ± SEM from four independent experiments.

Figure 3. Pharmacological activation of TRPV4 decreases TEC proliferation via modulation of ERK1/2 but not AKT pathway.

(A) Representative Western blots showing ERK1/2 phosphorylation in control and GSK treated NEC and TEC. (B) Densitometric analysis of the Western blots showing a decrease in ERK1/2 phosphorylation in TEC treated with GSK (100 nM). ERK1/2 phosphorylation was measured by normalizing phospho-ERK1/2 to total-ERK1/2 and was expressed as a fold change relative to NEC. (C) Representative Western blots depicting ERK1/2 phosphorylation TEC transfected with TRPV4-EGFP. (D) Densitometric analysis of the Western blots for ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured by normalizing phospho-ERK1/2 to total-ERK1/2 and was expressed as a fold change relative to TEC. All the data shown is mean ± SEM from at least three independent experiments.

Figure 4. TEC express increased levels of proliferation-associated genes.

Relative gene expression of cell-cycle associated genes in NEC and TEC. EC were treated with GSK (100 nM) for 24 h and subsequently lysed for RNA isolation. cDNA was prepared and qPCR analysis was performed using Fast SYBR green master mix (Applied Biosystems). Gene expression was first normalized to GAPDH and presented as relative expression to NEC. All the data shown is mean ± SEM from at least three independent experiments.

Figure 5. Pharmacological activation of TRPV4 inhibits EC proliferation in vivo.

(A) Tumors were implanted into C57BL/6 mice by subcutaneously injecting LLC cells. Mice were treated with GSK (+GSK) or saline (−GSK), as previously described, and isolated on day 21. Representative images (20X) of the tumor tissue stained with CD31 (red), ki-67 (green), and DAPI (nuclei) were used to quantify the proliferation of TEC. Scale bar = 10 μm (B) Quantitative analysis demonstrating a significant (p ≤ 0.05) decrease in the percentage of proliferating vessels (CD31+ki-67 positive vessels divided by CD31 positive vessels) in tumors treated with GSK (+GSK) compared to untreated tumors (−GSK).