Figure 1.
Extracellular Zn2+ reduces butyrate induced cell death and requires a functional ZnR.
A. 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. 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). C. 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 Methods). n = 5, *p<0.05.
Figure 2.
Inhibition of ZnR signaling reverses the pro-survival effects of Zn2+.
A–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, bottom panels, 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. C. 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.
Figure 3.
GPR39 mediates ZnR signaling and Zn2+- dependent cell growth.
A. 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 (top panel) and western-blot analysis (bottom panel). 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. C. Cell numbers were determined using the SRB method in cultures transfected with siGPR39 or controls, which were treated with or without Zn2+ as in Figure 1A. n = 3 *p<0.05.
Figure 4.
Zn2+-dependent activation of NHE in the colon is mediated by GPR39.
A–B. Colon epithelium was obtained from WT and GPR39 KO mice as described in Materials and Methods. 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 left panels. In the right panels, 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. C. 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 left panel. Na+/H+ exchange activity was determined as the rate of pHi recovery, averaged rates are presented in right panel. n = 3, *p<0.05.
Figure 5.
Zn2+-dependent pHi recovery following butyrate induced acid load involves PI3K signaling, and activation of NHE1 isoform, but does not contribute to cell survival.
A. The pH sensitive dye BCECF was used to monitor pHi 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. pHi was monitored following application of butyrate and Zn2+, as in A, in the presence of the PI3 kinase inhibitor (wortmannin). C. Averaged rates of pHi recovery following addition of Na+ as determined from the traces. n = 3; *p<0.05. D. Cell survival of HT29 colonocytes was measured using the SRB assay. Cells were treated with butyrate and Zn2+, as in Figure 1, in the presence or absence of 0.5 µM cariporide. n = 3; *p<0.05.
Figure 6.
Zn2+ and butyrate upregulate the expression of the pro-survival protein clusterin (CLU).
A. left panel: 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. right panel: Densitometeric analysis of α-CLU expression. n = 3; *p<0.05. B. left panel: Immunoblot of cell lysates from HT29 cells treated as in A in the presence or absence of cariporide (0.5 µM). right panel: Densitometeric analysis of α-CLU expression. n = 3 *p<0.05. C. 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. Right panel: Densitometry analysis of CLU expression level in control and siCLU transfected cells. D. Cells were transfected with an siCLU construct and treated as in Figure 1, cell numbers were monitored using the SRB colorimetric assay and compared to control cells. n = 3, *p<0.05.
Figure 7.
GPR39/ZnR mediates Zn2+-dependent activation of the pro-survival protein, CLU, and rescues cells from butyrate induced cell death.
A. 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 Fig. 1). 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 right panel, normalized to anti-α-CLU expression level in control (non-transfected) cells treated with Ringer's solution (100%). n = 3. B. The siGPR39 cells or controls were treated with butyrate with or without Zn2+ as described in Figure 1. 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.