Zinc Sensing Receptor Signaling, Mediated by GPR39, Reduces Butyrate-Induced Cell Death in HT29 Colonocytes via Upregulation of Clusterin

Zinc enhances epithelial proliferation, protects the digestive epithelial layer and has profound antiulcerative and antidiarrheal roles in the colon. Despite the clinical significance of this ion, the mechanisms linking zinc to these cellular processes are poorly understood. We have previously identified an extracellular Zn2+ sensing G-protein coupled receptor (ZnR) that activates Ca2+ signaling in colonocytes, but its molecular identity as well as its effects on colonocytes' survival remained elusive. Here, we show that Zn2+, by activation of the ZnR, protects HT29 colonocytes from butyrate induced cell death. Silencing of the G-protein coupled receptor GPR39 expression abolished ZnR-dependent Ca2+ release and Zn2+-dependent survival of butyrate-treated colonocytes. Importantly, GPR39 also mediated ZnR-dependent upregulation of Na+/H+ exchange activity as this activity was found in native colon tissue but not in tissue obtained from GPR39 knock-out mice. Although ZnR-dependent upregulation of Na+/H+ exchange reduced the cellular acid load induced by butyrate, it did not rescue HT29 cells from butyrate induced cell death. ZnR/GPR39 activation however, increased the expression of the anti-apoptotic protein clusterin in butyrate-treated cells. Furthermore, silencing of clusterin abolished the Zn2+-dependent survival of HT29 cells. Altogether, our results demonstrate that extracellular Zn2+, acting through ZnR, regulates intracellular pH and clusterin expression thereby enhancing survival of HT29 colonocytes. Moreover, we identify GPR39 as the molecular moiety of ZnR in HT29 and native colonocytes

Inhibition of ZnR signaling reverses the pro-survival effects of Zn²⁺.

<p><b>A–B.</b> HT29 cells were treated with Zn²⁺ for 10 min in Ringer's solution following 30–60 min incubation with the indicated concentrations of the MEK1/2 inhibitor U0126 or the PI3 kinase inhibitors wortmanin and LY294002. Cell lysates were immunoblotted with an anti ERK1/2 or p-ERK1/2 (A), and with an anti-AKT or p-AKT (B). Desnitometry analyses are presented, <i>bottom panels</i>, the results are normalized to the maximal activation obtained by the Zn²⁺ treatment. n = 3 *p<0.05 compared to control and #p<0.05 compared to Zn²⁺ treated cells. <b>C.</b> Cell numbers were determined in cells treated with butyrate and Zn²⁺ in the presence or absence of U0126 (1 µM), wortmannin (30 nM) or both. n = 3 * p<0.05 compared to control and #p<0.05 compared to Zn²⁺ treated cells.</p

Zn²⁺-dependent activation of NHE in the colon is mediated by GPR39.

<p><b>A–B.</b> Colon epithelium was obtained from WT and GPR39 KO mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035482#s2" target="_blank">Materials and Methods</a>. Colonocytes were loaded with the intracellular pH-sensitive dye (BCECF, 5 µM) inset was imaged at 440 nm using a 10× objective, showing the cells surrounding crypts are loaded with the dye. The NH₄Cl prepulse paradigm was employed to determine NHE activity. Representative traces of the experiments from WT (A) and KO (B) tissues are shown in the <i>left panels</i>. In the <i>right panels</i>, the bar graph represents the average rate of Na⁺-dependent H⁺ efflux following acidification as calculated from the traces, and the effect of Zn²⁺ is presented as a fold increase of the basal NHE activity (control). n = 5, *p<0.05. <b>C.</b> The rate of Na⁺-dependent H⁺ efflux following acidification was monitored in GPR39 KO tissues exposed to short acidification period (control) or a prolonged acidification (by maintaining a Na⁺-free Ringer's solution for 10 min following initial acidification) and is presented as a fold increase of the Na/H exchange rate obtained in the short acidification. Representative traces are shown in the <i>left panel</i>. Na⁺/H⁺ exchange activity was determined as the rate of pHi recovery, averaged rates are presented in <i>right panel</i>. 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

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⁺ 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

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