Lysosomal TRPML1 triggers global Ca2+ signals and nitric oxide release in human cerebrovascular endothelial cells

Abstract

Lysosomal Ca²⁺ signaling is emerging as a crucial regulator of endothelial Ca²⁺ dynamics. Ca²⁺ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP₃) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca²⁺ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca²⁺ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) induced a significant increase in [Ca²⁺]_i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca²⁺ release was sustained both by lysosomal Ca²⁺ release and ER Ca²⁺- release through inositol-1,4,5-trisphophate receptors and store-operated Ca²⁺ entry. Notably, interfering with TRPML1-mediated lysosomal Ca²⁺ mobilization led to a decrease in the free ER Ca²⁺ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca²⁺-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca²⁺ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca²⁺ store can shape endothelial Ca²⁺ signaling and Ca²⁺-dependent functions in vascular endothelial cells.

Keywords: Ca2+ signaling; InsP3 receptors; TRPML1; endothelial cells; lysosomes; nitric oxide; store-operated Ca2+ entry.

FIGURE 1

ML-SA1 evokes a dose-dependent increase in [Ca²⁺]_i in hCMEC/D3 cells. (A) Intracellular Ca²⁺ signals evoked by increasing concentration of ML-SA1 in hCMEC/D3 cells. (B) Mean ± SEM of the peak amplitude of ML-SA1-induced Ca²⁺ responses in hCMEC/D3 cells at different agonist concentrations. **** indicates p < 0.0001 (Student’s t-test and Mann-Whitney test). (C) Confocal fluorescence images of hCMEC/D3 cells loaded with LysoTracker-Red DND-99 (red) to mark acidic organelles and stained with an antibody against TRPML1 antibody (green). Nuclei were stained using DAPI (blue). Scale bar: 10 µm. (D) Representative western blotting analysis of TRPML1 on hCMEC/D3 cells lysates. Major bands of the expected molecular weights (MW) for TRPML1 (75 kDa) and the loading control protein β-actin (50 kDa) is indicated.

FIGURE 2

Lysosomal Ca²⁺ release and extracellular Ca²⁺ entry sustain TRPML-mediated Ca²⁺ signals hCMEC/D3 cells. (A) ML-SA1 induced a small transient increase in [Ca²⁺]_i in the absence of extracellular Ca²⁺. (B) Mean ± SEM of the peak amplitude of the TRPML1-induced Ca²⁺ responses in presence and absence of extracellular Ca²⁺. **** indicates p < 0.0001 (Student’s t-test). (C) Restoration of extracellular Ca²⁺ upon removal of the agonist resulted in a second bump in [Ca²⁺]_i, which was indicative of SOCE. (D) Pre-incubation with nigericin (50 μM, 20 min) under 0Ca²⁺ conditions, induced a transient increase in [Ca²⁺]_i, reflecting the depletion of the Ca²⁺ store. The subsequent application of ML-SA1 failed to induce a significant Ca²⁺ response in hCMEC/D3 cells. (E) Pharmacological (ML-SI3; 10 μM, 20 min) and genetic (siTRPML1) inhibition of TRPML1, totally abolished the Ca²⁺ response under 0Ca²⁺ condition. (F) Mean ± SEM of the amplitude of Ca²⁺ responses in cells under the designated treatments. **** indicates p < 0.0001 (Kruskal–Wallis one-way Anova test followed by Dunn’s post hoc test). (G) Representative western blotting analysis of the TRPML1 protein expressed in hCMEC/D3 (Ctrl) and hCMEC/D3 transfected with the specific siTRPML1 (siTRPML1). Major bands of the expected molecular weights (MW) for TRPML1 (75 kDa) and the loading control protein β-actin (50 kDa) is indicated. (H) Mean ± SEM of the TRPML1/β-actin ratio expression of four independent western blotting experiments. ** indicates p < 0.005 (Student’s t-test).

FIGURE 3

ER Ca²⁺ release and SOCE contribute to the TRPML1-mediated Ca²⁺ signals in hCMEC/D3 cells. (A, B) pre-incubation with CPA (10 μM, 30 min) or 2-APB (50 μM, 30 min) significantly reduced the ML-SA1-induced Ca²⁺ response under 0Ca²⁺ conditions. (C) Mean ± SEM of the amplitude of the Ca²⁺ responses in cells under the designated treatments. **** indicates p < 0.0001 (Kruskal–Wallis one-way Anova test followed by Dunn’s post hoc test). (D) The Orai1 blockers, BTP-2 (20 μM, 20 min) and Pyr6 (20 μM, 20 min), reduced the ML-SA1-evoked Ca²⁺ response. (E) Mean ± SEM of the amplitude of Ca²⁺ responses in cells under the designated treatments. Each drug totally inhibited the Ca²⁺ response. **** indicates p < 0.0001 (Kruskal–Wallis one-way Anova test followed by the Dunn’s post hoc test).

FIGURE 4

The inhibition of TRPML1 reduces ER Ca²⁺ release. (A) The transient Ca²⁺ response induced by CPA (10 µM) under 0 Ca²⁺ conditions is strongly reduced in the presence of ML-SI3 (10 μM, 20 min) and in hCMEC/D3 cells transfected with the selective siTRPML1. (B) Mean ± SEM of the amplitude of the Ca²⁺ responses in cells under the designated treatments. **** indicates p < 0.0001 (Kruskal–Wallis one-way Anova test followed by the Dunn’s post hoc test).

FIGURE 5

The endogenous ligand PI(3,5)P₂ mimics the Ca²⁺ response to ML-SA1. (A) 10 µM PI(3,5)P₂ induced a long-lasting increase in [Ca²⁺]_i, mimicking the ML-SA1-evoked Ca²⁺ response. (B) Restoration of extracellular Ca²⁺ upon removal of the agonist PI(3,5)P₂ resulted in a second bump in [Ca²⁺]_i, which was indicative of SOCE. (C) Depletion of lysosomal Ca²⁺ stores with 50 µM nigericin abolished the PI(3,5)P₂ –induced Ca²⁺ response. (D). Pharmacological (ML-SI3; 10 μM, 20 min) and genetic (siTRPML1) inhibition of TRPML1 totally abolished the PI(3,5)P₂ –induced Ca²⁺ response, in absence and presence of extracellular Ca²⁺. (E) Mean ± SEM of the amplitude of Ca²⁺ responses in the absence of extracellular Ca²⁺ (0Ca²⁺) under the designated treatments. Each drug totally inhibited the Ca²⁺ response. **** indicates p < 0.0001 (Kruskal–Wallis one-way Anova test followed by the Dunn’s post hoc test).

FIGURE 6

TRPML1 induces Ca²⁺-dependent NO release in hCMEC/D3 cells. (A) 100 μM ML-SA1 evoked a slow, but sustained increase in DAF-FM fluorescence, which reflected NO release and was inhibited by L-NIO (10 μM, 1 h) and BAPTA-AM (20 μM, 2 h). (B) Pharmacological (ML-SI3; 10 μM, 20 min) and genetic (siTRPML1) inhibition of TRPML1, significantly abolished the ML-SA1-induced NO response. (C) Mean ± SEM of the amplitude of NO responses in presence of extracellular Ca²⁺. **** indicates p < 0.0001 (Kruskal–Wallis one-way Anova test followed by the Dunn’s post hoc test).

FIGURE 7

TRPML1 mediates ATP-induced intracellular Ca²⁺ release and NO production. (A) The Ca²⁺ response to ATP (100 μM) under 0 Ca²⁺ conditions was strongly reduced in the presence of ML-SI3 (10 μM, 20 min) or apilimod (100 nM, 20 min) and in hCMEC/D3 cells transfected with the selective siTRPML1. (B) Mean ± SEM of the amplitude of the Ca²⁺ responses in cells under the designated treatments. **** indicates p < 0.0001 (Kruskal–Wallis one-way Anova test followed by Dunn’s post hoc test). (C). ATP (100 μM) induced a sustained increase in DAF-DM fluorescence, which reflected NO release and was inhibited in the presence of ML-SI3 (10 μM, 20 min) and in hCMEC/D3 cells transfected with the selective siTRPML1. (D) Mean ± SEM of the amplitude of the Ca²⁺ responses in cells under the designated treatments. **** indicates p < 0.0001 (Kruskal–Wallis one-way Anova test followed by the Dunn’s post hoc test).