Ion channels in the regulation of autophagy

<p>Autophagy is a cellular process in which the cell degrades and recycles its own constituents. Given the crucial role of autophagy in physiology, deregulation of autophagic machinery is associated with various diseases. Hence, a thorough understanding of autophagy regulatory mechanisms is crucially important for the elaboration of efficient treatments for different diseases. Recently, ion channels, mediating ion fluxes across cellular membranes, have emerged as important regulators of both basal and induced autophagy. However, the mechanisms by which specific ion channels regulate autophagy are still poorly understood, thus underscoring the need for further research in this field. Here we discuss the involvement of major types of ion channels in autophagy regulation.</p

Adrenomedullin increases PC-3 and T24/83 cell adhesion, migration and invasion.

<p>(A) RT-PCR experiment showing RAMP2, RAMP3 and CLR expression in PC-3 and T24/83 cells. (B) PC-3 (left panel) and T24/83 (right panel) cell adhesion was examined by seeding 3*10⁴ and 1.5*10⁴ cells respectively per well in 96-well plates pre-coated with fibronectin, and incubated for 45 min with or without AM (200 nM) (N = 3, *P<0.05 compared with control cells). β1 integrin phosphorylation was studied by western-blotting on total proteins extracted from PC-3 and T24/83 cells seeded on fibronectin coated plates and treated with or without AM. (C) PC-3 and T24/83 cell migration was studied by Transwell assay after 8 h of treatment (N = 3, *P<0.05 compared with control cells). FAK phosphorylation was studied by western-blotting on total proteins extracted from PC-3 and T24/83 cells treated with or without AM. (D) For invasion assay, transwell membrane was pre-coated with 50 µg Matrigel, and PC-3 and T24/83 cells were let to invade for 24 h (N = 3, *P<0.05 compared with control cells).</p

The expression of Orai1, STIM1 and TRPC1 does not change during CytD and CalA treatment in both LNCaP and NED-LNCaP.

<p>(<b>A</b>) Real-time quantitative PCR for the Orai1 transcripts in both LNCaP and NED-LNCaP cells treated with 50 nM CalA for 10 min or 5 µM Cyt D for 10 min. (<b>B</b>) Real-time quantitative PCR for the STIM1 transcripts in both LNCaP and NED-LNCaP cells treated with 50 nM CalA for 10 min or 5 µM Cyt D for 10 min. (<b>C</b>) Real-time quantitative PCR for the TRPC1 transcripts in both LNCaP and NED-LNCaP cells treated with 50 nM CalA for 10 min or 5 µM Cyt D for 10 min. There was no statistical significance observed in each condition (LNCaP and NED-LNCaP). n = 3, done in triplicates.</p

Adrenomedullin induces TRPV2 translocation to plasma membrane.

<p>(A) The effect of AM (200 nM, 45 min) and TRPV2 silencing (siTRPV2, 50 nM, 48 h) on basal cytosolic calcium of PC-3 and T24-83 cells was studied by calcium imaging. (n = 120 cells, N = 4, *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM). (B) TRPV2 presence at the plasma membrane was examined by biotinylation on T24/83 cells control or either treated with AM (200 nM, 45 min) or AM and PI3K inhibitor LY294.002 (10 µM, added 5 min before AM). (C) Effect of LY294.002 on PC-3 and T24/83 cell migration examined by transwell assay after 8 h incubation with or without AM (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM). (D) Effect of LY294.002 on AM-induced migration of PC-3 and T24/83 TRPV2-silenced cells examined by transwell assay after 8 h incubation with or without AM (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM).</p

Adrenomedullin effect is mediated by TRPV2.

<p>(A) Western-blotting analysis of TRPV2 protein level in PC-3 and T24/83 cells treated with either siCTL or siTRPV2 (50 nM, 48 h). Effect of TRPV2 silencing (siTRPV2, 50 nM, 48 h) (B) on PC-3 and T24/83 cell adhesion to fibroncectin incubated or not with AM (200 nM, 45 min) (N = 3, *P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM); (C) on PC-3 and T24/83 cell migration examined by transwell assay after 8 h incubation with or without AM (N = 3 *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM); (D) on PC-3 and T24/83 cell invasion through matrigel (AM 200 nM, 24 h) (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM).</p

F-actin polymerization by calyculin A (CalA) in both control and NED-LNCaP cells.

<p>(<b>A</b>) Representative images of immunofluorescence staining of F-actin with phalloidin-FITC in NED- and NED-CalA (50 nM for 10 min)-LNCaP cells. n = 3. (<b>B</b>) Histograms representing Tg-induced SOCE quantification (Tg 1 µM, 2 mM extracellular Ca²⁺) in CT-, CT-CalA-, NED- and NED-CalA-LNCaP cells. n = 3, N = 30–40 cells, in triplicates. Asterisks denote statistical significance: * p<0.05.</p

Cytochalasine D (CytD) partially restores the store-operated current induced by TG.

<p>(<b>A</b>) The whole-cell electrophysiological recordings of SOCE induced by 1 µM TG in LNCaP cells control (CT), NED (NED), and NED LNCaP cells treated with 3 µM CytD (NED-CytD). (<b>B</b>) Histograms showing the current density quantifications in the above conditions, n = 3, N = 23; in triplicates, ** - p<0.01.</p

Regulation of Ca²⁺ homeostasis and F-actin polymerization in NED-LNCaP cells.

<p>After 4 (<b>A</b>) or one (<b>B</b>) days of culture in a charcoal-stripped culture medium used to induce NED, the capacitative Ca²⁺ entry quantified by Ca²⁺ imaging is induced by the application of 1 µM Tg in the presence of 2 mM extracellulaire CaCl_2. Asterisks denote statistical significance * - p<0.05; ** - p<0.01, n = 3, N = 30–40 cells, in triplicates. The presence of F-actin fibres was observed in both CT-LNCaP (<b>C</b>) and NED-LNCaP (<b>D</b>) using phalloidin-FITC. Scale bar equals 10 µM. n = 3. <b>E</b>, Expression of Orai1, STIM1, Orai2, and Orai3 as compared to β-Actin in LNCaP cells and NED-LNCaP cells using semiquantitative PCR. n = 2. <b>F</b>, Quantitative real-time PCR for Orai1 and STIM1 in LNCaP (CT) versus LNCaP-NED (NED), n = 3, * - P<0.05.</p

Primers and siRNA.

<p>Primers and siRNA.</p

The effects of 1,25-dihydroxyvitamin D3 on androgen-independent cell lines.

<p><b>A</b>, <b>B</b>, The effects of 1,25-dihydroxyvitamin D3 on androgen receptor-deficient DU-145 cell line in both 2 and 10% FCS-containing RPMI medium (<b>A</b> and <b>B</b>, respectively), * - P<0.05 (as compared to control), n = 3. <b>C, D</b>, The effects of 1,25-dihydroxyvitamin D3 on androgen-insensitive LNCaP C4-2 cell line in both 2 and 10% FCS-containing RPMI medium (<b>C</b> and <b>D</b>, respectively), * - P<0.05 (as compared to control), n = 3. <b>E</b>, the relative expression levels of TRPV6 channel in DU-145 cells treated with 100 µM 1,25-dihydroxyvitamin D3 for 3 days in 2 and 10% FCS-containing RPMI medium, * - P<0.05 (as compared to control), n = 3. <b>F</b>, the expression of TRPV6 channel induced by 100 nM 1,25-dihydroxyvitamin D3 for 3 days in LNCaP cells in steroid-deprived RPMI medium (LNCaP-ST), n = 3.</p