Zinc released from injured cells is acting via the Zn2+-sensing receptor, ZnR, to trigger signaling leading to epithelial repair

Abstract

A role for Zn(2+) in accelerating wound healing is established, yet, the signaling pathways linking Zn(2+) to tissue repair are not well known. We show that in the human HaCaT keratinocytes extracellular Zn(2+) induces a metabotropic Ca(2+) response that is abolished by silencing the expression of the G-protein-coupled receptor GPR39, suggesting that this Zn(2+)-sensing receptor, ZnR, is mediating the response. Keratinocytic-ZnR signaling is highly selective for Zn(2+) and can be triggered by nanomolar concentrations of this ion. Interestingly, Zn(2+) was also released following cellular injury, as monitored by a specific non-permeable fluorescent Zn(2+) probe, ZnAF-2. Chelation of Zn(2+) and scavenging of ATP from conditioned medium, collected from injured epithelial cultures, was sufficient to eliminate the metabotropic Ca(2+) signaling. The signaling triggered by Zn(2+), via ZnR, or by ATP further activated MAP kinase and induced up-regulation of the sodium/proton exchanger NHE1 activity. Finally, activation of ZnR/GPR39 signaling or application of ATP enhanced keratinocytes scratch closure in an in vitro model. Thus our results indicate that extracellular Zn(2+), which is either applied or released following injury, activates ZnR/GPR39 to promote signaling leading to epithelial repair.

Figures

FIGURE 1.

Extracellular Zn2+ triggers a ZnR-dependent Ca2+i response mediated by the Gq protein-coupled receptor GPR39 in HaCaT keratinocytes. A, application of extracellular Zn2+ (50 μm) to Fura-2 loaded HaCaT cells. A robust rise in Fura-2 fluorescence following application of Zn2+ is shown both in the absence or presence of extracellular Ca2+. To the right, fluorescent images of the culture before and after application of Zn2+. The Ca2+i rise triggered by ATP (100 μm) is shown (top right). B, application of Zn2+ following depletion of Ca2+i stores using thapsigargin (200 nm) and ATP (100 μm) in nominally Ca2+-free Ringer's solution, notice that following application of Zn2+ no rise in fluorescence is seen for the same time period shown in A. C, Zn2+-dependent Ca2+i response was monitored in cells pretreated with the PLC inhibitor, U73122 (3 μm), the Gαq inhibitor, YM-254890 (1 μm), and following treatment with Pasteurella multocida toxin (12 h). Inhibitors of the IP3 pathway or depletion of Ca2+ stores blocked the Zn2+-dependent Ca2+ rise. D, Zn2+ (100 μm) -dependent Ca2+ response in HaCaT cells transfected with siGPR39, or a scrambled control siRNA (siControl). Inset shows immunoblot of the expression level of GPR39 in HEK293 cells transfected with GPR39 plasmid (0.33 μg/60 mm plate) together with the indicated siRNA constructs: either siGPR39, siControl, or siT1R3 aimed to silence a non-related GPCR. Silencing of GPR39 eliminated the Zn2+-dependent response. (n = 5 for each treatment).

FIGURE 2.

ZnR activates the MAPK pathway in HaCaT keratinocytes. Immunoblot of ERK1/2 phosphorylation following application of either 50 μm Zn2+ or the extracellular Zn2+ chelator, CaEGTA to chelate residual Zn2+ (A), or 100 μm ATP (B) for the indicated times. Quantification of the phosphorylation level normalized to β-actin expression is shown in lower panels. C, Zn2+-dependent ERK1/2 phosphorylation was determined in the presence of the MEK inhibitor U0126 (1 μm); the Gαq inhibitor, YM-254890 (1 μm); the PI3K inhibitor, wortmannin (100 nm), or the PKC inhibitor, BI (10 nm) as indicated. Quantification of ERK1/2 phosphorylation normalized to β-actin expression, shown to the right, indicating that inhibition of either the IP3, MAPK, or PI3K pathways reduced the Zn2+-dependent ERK1/2 phosphorylation. (n = 5 for each treatment; *, p < 0.05; **, p < 0.001 compared with control; #, p < 0.05 compared with Zn2+).

FIGURE 3.

Affinity and selectivity of the ZnR to Zn2+ in HaCaT keratinocytes. A, dose-response of the intracellular Ca2+ rise triggered by extracellular Zn2+ in HaCaT cells. The maximal Cai2+ response (monitored as in 1) following application of the indicated Zn2+ concentration was averaged and plotted versus the applied free-Zn2+ concentration (see “Experimental Procedures”). B, cells were treated with varying concentrations of Zn2+ and then ERK1/2 phosphorylation was monitored. Quantification of ERK1/2 phosphorylation normalized to total ERK1/2 levels is shown in bottom panel (n = 4 for each concentration; *, p < 0.05; **, p < 0.001 compared with EGTA). Zn2+-dependent ERK1/2 phosphorylation was already evident in the presence of 1 nm Zn2+ indicating that the keratinocytic ZnR has a high affinity to Zn2+. C, cells were exposed to the indicated divalent ions, while monitoring the Ca2+i response. None of the other heavy metals applied triggered a significant rise in Ca2+i, indicating that the ZnR response is Zn2+ selective.

FIGURE 4.

Activation of ZnR signaling by endogenous Zn2+, released during cellular injury. A, changes in fluorescence of the cell impermeable Zn2+-sensitive dye ZnAF-2 (2 μm), at the indicated time a scratch was performed with a pipette tip (see “Experimental Procedures”). The scratching was followed by robust rise in ZnAF-2 fluorescence. Images of cell culture before and after the scratch are shown below, and the circles show regions of interest used for averaging ZnAF-2 fluorescence. B, averaged ZnAF-2 response following injury in the absence or presence of the non-permeable Zn2+ chelator CaEDTA (n = 4, *, p < 0.05). C, supernatant obtained from injured cultures (conditioned medium, see “Experimental Procedures”) was applied alone or in the presence of the ATP scavenger, apyrase (0.66 units/ml), or apyrase with the extracellular Zn2+ chelator, CaEDTA (100 μm) to intact HaCaT cells loaded with Fura-2-AM. Fluorescent images of the Ca2+ response in these cultures following application of the conditioned medium as indicated are shown above the graph. D, averaged intracellular Ca2+ signals from C are shown (n = 5; *, p < 0.05; **, p < 0.01). Application of both CaEDTA and apyrase eliminated the Ca2+ rise triggered by application of conditioned medium. E, conditioned medium was applied (10 min) to HaCaT cell cultures and ERK1/2 phosphorylation was monitored. ERK1/2 phosphorylation is significantly increased upon application of the conditioned medium (n = 3; *, p < 0.05).

FIGURE 5.

ZnR desensitization by Zn2+. A, HaCaT cells were pretreated with 50 μm Zn2+ for 30 min and allowed to recover (see “Experimental Procedures”). At 1 or 5 h later Zn2+ (50 μm) or ATP (100 μm, see inset) was re-applied while monitoring the Ca2+i response. Cells that were pretreated with Zn2+ did not respond to re-application of Zn2+, while a robust Ca2+ signal was monitored following application of ATP. B, averaged Ca2+ response to Zn2+ re-applied at the indicated times following desensitization (n = 3, *, p < 0.05). C, phosphorylation of ERK1/2 in response to re-application of Zn2+ at 5 h following pre-application of Zn2+, quantification normalized to β-actin expression is shown below (n = 5, *, p < 0.05). Similar to the effect on Ca2+ signaling, desensitization by Zn2+ attenuated the subsequent Zn2+-dependent phosphorylation of ERK1/2.

FIGURE 6.

Stimulation of Na+/H+ exchange by Zn2+ via the ZnR, in HaCaT keratinocytes. HaCaT cells loaded with the pH-sensitive dye, BCECF-AM (1 μm), were acidified by NH4Cl pre-pulse protocol (see “Experimental Procedures”) upon addition of extracellular Na+ (at time indicated by the bar) alkalinization is observed and the rate of pHi recovery during this phase represents NHE activity. The pHi recovery rate was measured in either: (A) control cells (B) in the presence of the NHE1 inhibitor, cariporide (1 μm), (C) following application of Zn2+ (50 μm, 15 min), (D) application of Zn2+ in the presence of cariporide (0.5 μm), or (E) application of Zn2+ in the presence of the ERK1/2 inhibitor, U0126 (1 μm). F, averaged rates of pHi recovery, associated with NHE1 activity, of A–E. G, NH4Cl prepulse protocol was performed following application of 100 μm ATP (10 min). H, averaged rate of pHi recovery following application of ATP in the presence or absence of U0126. (n = 7 for each treatment, **, p < 0.01 compared with control, ##, p < 0.01 compared with Zn2+).

FIGURE 7.

Extracellular Zn2+ via the ZnR promotes in vitro scratch closure. A, scratch closure assay was performed on confluent monolayers of HaCaT cells and the scratch closure rate was determined by comparing the denuded area immediately and 24 h later (see “Experimental Procedures”). This assay was repeated in cells treated with CaEGTA or Zn2+ (50 μm) in the presence of: low, 0.05 mm, or high, 1.8 mm, Ca2+ concentration in the extracellular Ringer's solution; the PLC inhibitor, U73122 (1 μm); the NHE1 inhibitor, cariporide (0.5 μm), or ATP (100 μm). B, averaged rates of scratch closure during 24 h following injury, normalized to the rate monitored in Zn2+-treated cultures, is shown. (n = 8 for Zn2+ and 4 for all other treatments, **, p < 0.01 compared with CaEGTA; #, p < 0.01 compared with Zn2+).

FIGURE 8.

ZnR/GPR39 mediates the Zn2+-dependent up-regulation of NHE1 activity and scratch closure. A, HaCaT cells were transfected with siGPR39 or siControl constructs and the NH4Cl prepulse paradigm was performed as in Fig. 6, recovery rate was measured in cells treated with Zn2+ or in control cells. Averaged rates of pHi recovery, representing NHE1 activity are shown to the right (n = 5 for each treatment, **, p < 0.01 compared with siControl cells without Zn2+). B, in vitro scratch assay was performed as in Fig. 7 in cultures transfected with siControl or siGPR39. Cultures were treated with Zn2+ or CaEGTA, and injured areas were monitored immediately and 24 h following scratching. Averaged rates of scratch closure recovery normalized to the recovery rate of control cells treated with Zn2+ are shown below (n = 7 for each treatment, **, p < 0.01 compared with siControl cells without Zn2+).