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

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

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

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

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

Clinical Outcomes of Critically Ill Patients Using Inhaled Nitric Oxide (iNO) during Intrahospital Transport

Critically ill patients with severe hypoxemia are often treated in the intensive care unit (ICU) with inhaled nitric oxide (iNO). These patients are at higher risk when they require intrahospital transportation. In this study, we collected clinical and laboratory data from 221 patients who were hospitalized in the general ICU and treated with iNO at Soroka Medical Center, Israel, between January 2010 and December 2019. We retrospectively compared the 65 patients who received iNO during intrahospital transportation to the 156 patients who received iNO without transportation. Among critically ill patients who were transported while being administered iNO, only one patient had an adverse event (atrial fibrillation) on transport. We found that maximal iNO dosage during ICU stay, duration of mechanical ventilation, and percent of vasopressor support were the only independent risk factors for ICU mortality in both study groups. No difference in primary outcome of ICU mortality rate was found between the critically ill patients treated with iNO during intrahospital transportation and those who were treated with iNO but not transported during the ICU stay. We anticipate that this study will advise clinical decision-making in the ICU, especially when treating patients who are administered iNO