Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a newly discovered second messenger that gates two pore channels 1 (TPC1) and 2 (TPC2) to elicit endo-lysosomal (EL) Ca²⁺ release. NAADP-induced lysosomal Ca²⁺ release may be amplified by the endoplasmic reticulum (ER) through the Ca²⁺-induced Ca²⁺ release (CICR) mechanism. NAADP-induced intracellular Ca²⁺ signals were shown to modulate a growing number of functions in the cardiovascular system, but their occurrence and role in cardiac mesenchymal stromal cells (C-MSCs) is still unknown. Herein, we found that exogenous delivery of NAADP-AM induced a robust Ca²⁺ signal that was abolished by disrupting the lysosomal Ca²⁺ store with Gly-Phe β-naphthylamide, nigericin, and bafilomycin A1, and blocking TPC1 and TPC2, that are both expressed at protein level in C-MSCs. Furthermore, NAADP-induced EL Ca²⁺ release resulted in the Ca²⁺-dependent recruitment of ER-embedded InsP₃Rs and SOCE activation. Transmission electron microscopy revealed clearly visible membrane contact sites between lysosome and ER membranes, which are predicted to provide the sub-cellular framework for lysosomal Ca²⁺ to recruit ER-embedded InsP₃Rs through CICR. NAADP-induced EL Ca²⁺ mobilization via EL TPC was found to trigger the intracellular Ca²⁺ signals whereby Fetal Bovine Serum (FBS) induces C-MSC proliferation. Furthermore, NAADP-evoked Ca²⁺ release was required to mediate FBS-induced extracellular signal-regulated kinase (ERK), but not Akt, phosphorylation in C-MSCs. These finding support the notion that NAADP-induced TPC activation could be targeted to boost proliferation in C-MSCs and pave the way for future studies assessing whether aberrant NAADP signaling in C-MSCs could be involved in cardiac disorders.

Keywords: cardiac mesenchymal stem cells; membrane contact sites; nicotinic acid adenine dinucleotide phosphate (NAADP); proliferation; store operated Ca2+ entry; two-pore channels (TPCs).

FIGURE 1

NAADP induces intracellular Ca²⁺ signals by mobilizing lysosomal Ca²⁺ in C-MSCs. (A) Exogenous administration of NAADP-AM (1 µM) indues either intracellular Ca²⁺ oscillations or a transient increase in [Ca²⁺]_i. (B) In absence of external Ca²⁺ (0Ca²⁺), NAADP-AM (1 µM) induced only a transient increase in [Ca²⁺]_i, whereas subsequent restitution of extracellular Ca²⁺ after the full recovery of [Ca²⁺]_i to the baseline resulted in a second Ca²⁺ signal that was due to extracellular Ca²⁺ entry. (C) Mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP in the presence and absence of extracellular Ca²⁺. Student’s t-test: ***p < 0.001. (D) Mean ± SE of the amplitude of NAADP-induced intracellular Ca²⁺ release and SOCE. (E) Disrupting the lysosomal Ca²⁺ store with GPN (200 μM, 30 min), nigericin (50 μM, 30 min) or bafilomycin A1 (1 μM, 30 min) severely affected the intracellular Ca²⁺ response to NAADP-AM. (F) Mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP-AM in the absence and in the presence of GPN, nigericin (Nig), or bafilomycin A1 (Baf). One-Way Anova followed by the post-hoc Dunnett’s test: ***p < 0.001.

FIGURE 2

Two-pore channels (TPCs) mediate NAADP-induced lysosomal Ca²⁺ release in C-MSCs. (A) TPC1 and TPC2 gene expression in total RNA extracts of C-MSCs. qRT-PCR data are shown as transcript abundance (genes threshold cycles [Ct] with respect to the house-keeping gene GAPDH). n = 4/group. Student’s t-test: ***p < 0.001. (B) Western Blot analysis of TPC1 and TPC2 proteins in total cellular extracts. n = 3/group. (C) The Ca²⁺ response to NAADP-AM was suppressed by incubating the cells with the following TPC inhibitors: NED-19 (100 μM, 30 min), NED-K (10 μM, 30 min), and tetrandrine (10 μM, 30 min). (D) Mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP in the absence (Ctrl) and in the presence of NED-19, NED-K and tetrandrine (Tetra). Student’s t-test: ***p < 0.001.

FIGURE 3

InsP₃Rs support the Ca²⁺ response to NAADP. (A) Administration of NAADP-AM (1 µM) after the pharmacological depletion of the ER Ca²⁺ pool with CPA (30 μm, 30 min) failed to induce intracellular Ca²⁺ release. CPA induced a transient increase in [Ca²⁺]_i that reflects passive ER Ca²⁺ leakage from the ER. (B) The intracellular Ca²⁺ transient evoked by NAADP-AM (1 µM) in the absence of extracellular Ca²⁺ (0Ca²⁺) under control conditions. (C) The intracellular Ca²⁺ release evoked by NAADP-AM (1 µM) under control (Ctrl) conditions was severely affected by blocking InsP₃Rs with 2-APB (50 μm, 30 min) or inhibiting PLC activity with U73122 (10 μm, 10 min). (D) Mean ± SE of the amplitude of the peak intracellular Ca²⁺ response to NAADP-AM under the designated treatments. Student’s t-test: ***p < 0.001.

FIGURE 4

Ultrastructural analysis of C-MSCs. Several examples of membrane contacts between lysosomes (Ly) and ER are shown. (A) Note the closeness of the lysosomes with the cell nucleus and ER. Scale bar: 1 µm. (B) The lysosome-ER contact site is indicated (arrow and inset). Scale bar: 1 μm; inset scale bar: 100 nm. (C) The arrow indicates the membrane contact site. Scale bar: 500 nm. (D) Extensive contact (arrows) between the lysosomal and the ER membranes. Scale bar: 500 nm. (E) Note the contact (arrows) between the ribosomes-rich ER and the lysosome. Scale bar: 200 nm. (F) Two close lysosomes; the one on the right is in tight contact with the ER (arrows and inset). Scale bar: 1 μm; inset scale bar: 100 nm.

FIGURE 5

NAADP-AM-induced lysosomal Ca²⁺ mobilization is functionally coupled to SOCE in C-MSCs. (A) The influx of extracellular Ca²⁺ evoked by NAADP-AM (1 µM) upon depletion of intracellular Ca²⁺ stores (Ctrl) was severely affected by inhibiting SOCE with Pyr6 (10 μM, 10 min) or BTP-2 (20 μm, 20 min). The previous NAADP-AM-evoked endogenous Ca²⁺ release has not been shown. (B) Mean ± SE of the amplitude of NAADP-AM-evoked Ca²⁺ entry evoked by nigericin in the absence (Ctrl) and presence of Pyr-6 and BTP-2. Student’s t-test: ***p < 0.001. (C) Nigericin-evoked extracellular Ca²⁺ entry was attenuated or inhibited by, respectively, blocking SOCE with BTP-2 (20 μM, 20 min) or Pyr6 (10 μM, 10 min). (D) Mean ± SE of the amplitude of Ca²⁺ entry evoked by nigericin in the absence (Ctrl) and presence of Pyr-6 and BTP-2. Student’s t-test: ***p < 0.001.

FIGURE 6

Fetal bovine serum (FBS)-induces intracellular Ca²⁺ release from endogenous stores and enhance SOCE. (A) 20% FBS induced oscillatory or transient increases in [Ca²⁺]_i. (B) Under 0Ca²⁺ conditions, 20% FBS induced a transient elevation in [Ca²⁺]_i. Subsequent re-addition of extracellular Ca²⁺, 100 s after agonist removal from the bath, resulted in a second bump in [Ca²⁺]_i that was indicative of SOCE. (C) The intracellular Ca²⁺ release evoked by 20% FBS (Ctrl) was inhibited by depleting the ER Ca²⁺ pool with CPA (30 μM, 30 min), by blocking InsP₃Rs with 2-APB (50 μM, 30 min), or inhibiting PLC with U73122 (10 μM, 10 min). (D) Mean ± SE of the amplitude of the peak intracellular Ca²⁺ response to 20% FBS under the designated treatments. (E) Subsequent to store depletion by 20% of FBS application (data are not shown her(E), FBS were washed out from bath, then extracellular Ca²⁺ added to the bath to bath in the presence and absence of SOCE inhibitors, Pyr6 (10 μm, 10 min) or BTP-2 (20 μm, 20 min). (F) Mean ± SE of the amplitude of Ca²⁺ entry evoked by 20% FBS in the absence (Ctrl) and presence of Pyr6 and BTP-2. The asterisk indicates ***p < 0.001.

FIGURE 7

TPCs mediate 20% FBS-induced intracellular Ca²⁺ release. (A) Intracellular Ca²⁺ release induced by 20% FBS was abolished upon depletion of the lysosomal Ca²⁺ pool with either GPN (200 μM, 30 min) or nigericin (50 μM, 30 min). (B) Mean ± SE of the amplitude of the intracellular Ca²⁺ peak evoked by 20% FBS under the designated treatments. (C) 20% FBS induced an intracellular Ca²⁺ transient that was significantly reduced or inhibited by blocking TPCs with, respectively, NED-K (10 μM, 30 min), tetrandrine (10 μM, 30 min) or NED-19 (100 μM, 30 min). (D) Mean ± SE of the amplitude of the intracellular Ca²⁺ peak evoked by 20% FBS in the absence (Ctrl) or presence of NED-19, NED-K or tetrandrine (Tetra). Student’s t-test: ***p < 0.001.

FIGURE 8

TPCs mediate 20% FBS-induced proliferation and ERK phosphorylation in C-MSCs. Following 4 h of growth without serum, cells were treated with NED-19 (100 μM, 30 min) and subsequently stimulated with 20% FBS. (A) Following 24 and 48 h of FBS stimulation, the medium was removed, cells detached from the plates, and counted by hemocytometer (n = 3/group). Student’s t-test: *p < 0.05. (B) The cells were lysate after 60 min of FBS stimulation. Total protein extract from treated cells was subjected to Western blot analysis to visualize active phosphorylated form and total of ERK and AKT using specific antibodies. Phospho-ERK1/2 and Phospho-AKT levels were corrected by total ERK1/2 and AKT densitometry respectively. (C) Western blot data are presented as the fold change of target protein expression. The results are expressed as mean ± SEM (n = 3/group). Student’s t-test: *p < 0.05.