GPR39/ZnR mediates Zn²⁺-dependent activation of the pro-survival protein, CLU, and rescues cells from butyrate induced cell death.

<p><b>A.</b> HT29 cells transfected with an siGPR39 construct, were treated with butyrate or without it (Ringer's solution alone), in the presence or absence of Zn²⁺ (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035482#pone-0035482-g001" target="_blank">Fig. 1</a>). Cell lysates were subjected to immunoblotting with an anti-α-CLU, actin levels are presented as control. Densitometry analysis of the results is shown in the <i>right panel</i>, normalized to anti-α-CLU expression level in control (non-transfected) cells treated with Ringer's solution (100%). n = 3. <b>B.</b> The siGPR39 cells or controls were treated with butyrate with or without Zn²⁺ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035482#pone-0035482-g001" target="_blank">Figure 1</a>. Cells were then fixed and number of cells was monitored using the SRB colorimetric assay, gray line indicates cell numbers in butyrate only treated siGPR39-transfected cells. n = 3; *p<0.05.</p

Zn²⁺ and butyrate upregulate the expression of the pro-survival protein clusterin (CLU).

<p><b>A.. </b><i>left panel</i>: Immunoblot analysis using anti α-CLU antibodies was done on lysates from HT29 cells treated with either Zn²⁺, butyrate or both, in the presence or absence of the cell impermeable Zn²⁺ chelator CaEDTA, or the kinase inhibitors as indicated. <i>right panel</i>: Densitometeric analysis of α-CLU expression. n = 3; *p<0.05. <b>B.. </b><i>left panel:</i> Immunoblot of cell lysates from HT29 cells treated as in A in the presence or absence of cariporide (0.5 µM). <i>right panel</i>: Densitometeric analysis of α-CLU expression. n = 3 *p<0.05. <b>C.</b> CLU expression was monitored using westernblot analysis in HT29 cells transfected with an siCLU or scrambled siRNA (Control) constructs. Using prolonged exposure time (180 s) CLU expression could be monitored in these cells. Actin was used as a loading control. <i>Right panel</i>: Densitometry analysis of CLU expression level in control and siCLU transfected cells. <b>D.</b> Cells were transfected with an siCLU construct and treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035482#pone-0035482-g001" target="_blank">Figure 1</a>, cell numbers were monitored using the SRB colorimetric assay and compared to control cells. n = 3, *p<0.05.</p

Extracellular Zn²⁺ reduces butyrate induced cell death and requires a functional ZnR.

<p><b>A.</b> Cell numbers in cultures treated with butyrate (30 mM, 24 h) were compared to control cultures (without butyrate, hatched bars) using the SRB colorimetric assay. Cells were treated daily with Ringer's solution (10 min) without (control) or with Zn²⁺ (80 µM Zn²⁺) n = 6, *p<0.05. <b>B.</b> The effect of Zn²⁺ desensitization of ZnR (100 µM Zn²⁺, 15 min) on cell growth was determined. 30 min following ZnR desensitization, Zn²⁺ was re-applied to activate the ZnR as in A, or desensitized-cells were treated with Ringer's solution, and subsequently cell numbers were determined using the SRB assay. Following ZnR desensitization Zn²⁺ did not enhance cell numbers significantly. Similarly, Ca²⁺ release, monitored using Fura-2 fluorescence, in response to the re-application of Zn²⁺ was almost absent following desensitization of the ZnR (right panel). <b>C.</b> The effect of Zn²⁺ on cell survival was determined following desensitization of ZnR (100 µM Zn²⁺, 15 min) or in controls (Ringer's solution, 15 min), using the SRB assay. Cells were treated with butyrate and Zn²⁺ was re-applied to control cultures or cultures previously treated for ZnR desensitization (as indicated, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035482#s2" target="_blank">Methods</a>). n = 5, *p<0.05.</p

GPR39 mediates ZnR signaling and Zn²⁺- dependent cell growth.

<p><b>A.</b> Cells were transfected with siRNA sequences compatible to GPR39 (siGPR39) or a scrambled sequence (siControl), and the mRNA and protein expression levels of GPR39 were monitored using Real-Time PCR (<i>top panel</i>) and western-blot analysis (<i>bottom panel</i>). <b>B.</b> The Zn²⁺ -dependent Ca²⁺_i responses were monitored in cells transfected with siGPR39 or siControl constructs using Fura-2 AM. ATP (50 µM) was subsequently applied to determine the integrity of the IP3 pathway. <b>C.</b> Cell numbers were determined using the SRB method in cultures transfected with siGPR39 or controls, which were treated with or without Zn²⁺ as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035482#pone-0035482-g001" target="_blank">Figure 1A</a>. n = 3 *p<0.05.</p

Zn²⁺-dependent pHi recovery following butyrate induced acid load involves PI3K signaling, and activation of NHE1 isoform, but does not contribute to cell survival.

<p><b>A.</b> The pH sensitive dye BCECF was used to monitor pH_i in HT29 control cells, cells pretreated with Zn²⁺ (80 µM, 2 min) or cells treated with Zn²⁺ and the NHE1 inhibitor cariporide (0.5 µM). Cells were superfused with 30 mM butyrate (pH 7.4) in Na⁺-free Ringer's solution, and then Na⁺ was added to the Ringer's solution. Representative traces are shown. <b>B.</b> pHi was monitored following application of butyrate and Zn²⁺, as in A, in the presence of the PI3 kinase inhibitor (wortmannin). <b>C.</b> Averaged rates of pH_i recovery following addition of Na⁺ as determined from the traces. n = 3; *<i>p</i><0.05. <b>D.</b> Cell survival of HT29 colonocytes was measured using the SRB assay. Cells were treated with butyrate and Zn²⁺, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035482#pone-0035482-g001" target="_blank">Figure 1</a>, in the presence or absence of 0.5 µM cariporide. n = 3; *<i>p</i><0.05.</p

Effect of NCLX expression or activity on glucose dependent cytosolic calcium responses.

<p><b>A.</b> Real time PCR analysis of mRNA NCLX expression normalized to GAPDH in pancreatic primary β cells transfected with siNCLX vs. siControl, n = 3 (*P<0.05). <b>B.</b> Silencing NCLX expression inhibits glucose-induced Ca²⁺ entry in primary β cells. Representative fluorescent traces of cytosolic Ca²⁺ in pancreatic primary β cells transfected with either siNCLX or siControl loaded with Fura 2 AM and stimulated with high glucose following the same experimental paradigm described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g002" target="_blank">Fig. 2A</a>. <b>Insert.</b> Shows representative images of MIN6 cells co-transfected with the Dharmacon siGLO Red transfection reagent. The scale bar is 10 µm. <b>C.</b> NCLX dominant negative construct inhibits glucose dependent cytosolic Ca²⁺ changes in primary β cells. Representative fluorescent traces of primary β cells transfected with dnNCLX or control vector (pcDNA) loaded with Fura 2 AM and treated with high glucose when indicated. <b>D.</b> Averaged rates of cytosolic Ca²⁺ responses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g004" target="_blank">Fig. 4B</a>, n = 10 (*P<0.05). <b>E.</b> Averaged rates of cytosolic Ca²⁺ responses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g004" target="_blank">Fig. 4C</a>, n = 10 (*P<0.05). <b>F.</b> Averaged cytosolic Ca²⁺ amplitudes of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g004" target="_blank">Fig. 4B</a>, n = 10 (*P<0.05). <b>G.</b> Averaged cytosolic Ca²⁺ amplitudes of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g004" target="_blank">Fig. 4C</a>, n = 10 (*P<0.05).</p

Effect of NCLX on mitochondrial Ca²⁺ transport, metabolic rate in resting and high glucose dependent manner.

<p><b>A.</b> Knocked down of NCLX modulates mitochondrial calcium transport. Pancreatic primary β cells were infected with lenti-pericam viral particles and transfected with either siNCLX or siControl and superfused with the indicated high glucose Ringer solution. <b>Insert.</b> Representative image of pancreatic primary β cell infected with lenti-pericam. The scale bar is 10 µm. <b>B.</b> Averaged mitochondrial Ca²⁺ influx rates of pancreatic primary β cells of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g005" target="_blank">Fig. 5A</a>, n = 3 (*P<0.05). <b>C.</b> Averaged mitochondrial Ca²⁺ efflux rates of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g005" target="_blank">Fig. 5A</a>, n = 3 (*P<0.05). <b>D.</b> Effect of NCLX on respiratory chain activity determined by monitoring NAD(P)H intrinsic fluorescence in pancreatic primary β cells, transfected with either siNCLX or siControl before and after application of high glucose Ringer solution. FCCP or high glucose Ringer's solution was added where indicated.</p

NCLX is expressed in mitochondria of pancreatic β cells and mediates mitochondrial Ca²⁺ transport.

<p><b>A</b>. Immunoblot analysis of NCLX expression in total lysate and isolated mitochondria in MIN6 cells (20 µg). <b>B.</b> Immunoblot analysis of NCLX expression in siNCLX vs. siControl (20 µg) transfected MIN6 cell lysates. VDAC and β Actin were used as mitochondrial and cytosolic markers, respectively. <b>C.</b> Knock down of NCLX expression increases Ca²⁺ influx and inhibits mitochondrial Ca²⁺ efflux. At the indicated time, cells were superfused with high K⁺ Ringer solution while monitoring mitochondrial Ca²⁺ in MIN6 cells transfected with mito-pericam and either siNCLX or siControl. <b>D.</b> Dominant negative NCLX construct increases Ca²⁺ influx and inhibits mitochondrial Ca²⁺ efflux. Representative fluorescent traces of pancreatic MIN6 cells co-transfected with mito-pericam and either dnNCLX or control vector (pcDNA), while applying the same experimental paradigm described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>. <b>Insert.</b> Representative images of MIN6 cells co-transfected with mito-pericam. The scale bar is 10 µm. <b>E.</b> Averaged rates of mitochondrial Ca²⁺ influx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>, D, n = 9 (*P<0.05). <b>F.</b> Averaged rates of mitochondrial Ca²⁺ efflux of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>, D, n = 9 (*P<0.05). <b>G.</b> Silencing of NCLX expression inhibits mitochondrial Ca²⁺ efflux following a metabotropic cytosolic Ca²⁺ response. Cells were co-transfected with mito-pericam and either siNCLX or siControl and superfused with Ca²⁺ free Ringer solution containing 50 µM ATP, while monitoring the Ca²⁺ response. <b>H.</b> Averaged rates of mitochondrial Ca²⁺ influx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1G</a>, n = 7 (*P<0.05). <b>I.</b> Averaged rates of mitochondrial Ca²⁺ efflux of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1G</a>, n = 7 (*P<0.05).</p

Role of NCLX in cytosolic Ca²⁺ responses.

<p><b>A.</b> Silencing of NCLX expression inhibits cytosolic Ca²⁺ responses. MIN6 cells either transfected with siNCLX or siControl were loaded with Fura 2 AM and depolarized with high K⁺ Ringer solution, while monitoring cytosolic Ca²⁺ responses. <b>B.</b> Dominant negative mutant NCLX, dnNCLX inhibits cytosolic Ca²⁺ responses. MIN6 cells transfected with either dnNCLX or control vector (pcDNA) were loaded with Fura 2 AM and cytosolic Ca²⁺ was monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3A</a>. <b>C.</b> Averaged rates of cytosolic Ca²⁺ responses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3A</a>, n = 12 (*P<0.05). <b>D.</b> Averaged rates of cytosolic Ca²⁺ responses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3B</a>, n = 12 (*P<0.05). <b>E.</b> Averaged cytosolic Ca²⁺ response amplitude of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3A</a>, n = 12 (*P<0.05). <b>F.</b> Averaged cytosolic Ca²⁺ response amplitude of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3B</a>, n = 12 (*P<0.05).</p

NCLX mediates glucose dependent mitochondrial Ca²⁺ transport and modulates the basal mitochondrial membrane potential and calcium resting levels.

<p><b>A.</b> Silencing of NCLX expression blocks the glucose dependent mitochondrial Ca²⁺ efflux. The mitochondrial Ca²⁺ transport was monitored in MIN6 cells co-transfected with mito-pericam and either siNCLX or siControl. Cells were first superfused with low glucose (3 mM) Ringer followed by high glucose (20 mM) Ringer solution. <b>B.</b> Averaged rates of mitochondrial Ca²⁺ influx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g002" target="_blank">Fig. 2A</a>, n = 11 (*P<0.05). <b>C.</b> Averaged rates of mitochondrial Ca²⁺ efflux of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g002" target="_blank">Fig. 2A</a>, n = 11 (*P<0.05). <b>D.</b> Silencing NCLX modulates the basal but not glucose dependent change in mitochondrial membrane potential. Changes in mitochondrial membrane potential were monitored in MIN6 cells transfected with siNCLX or siControl, superfused continuously with 0.05 µM TMRM. FCCP 5 µM was added in the indicated times to calibrate the signal. <b>E.</b> Effect of knock down of NCLX expression on mitochondrial resting Ca²⁺ in MIN6 cells transfected with siNCLX vs. siControl. Averaged mitochondrial Ca²⁺ basal signals, n = 10 (*P<0.05).</p