Zinc Released from Injured Cells Is Acting via the Zn2+-sensing Receptor, ZnR, to Trigger Signaling Leading to Epithelial Repair*

A role for Zn2+ in accelerating wound healing is established, yet, the signaling pathways linking Zn2+ to tissue repair are not well known. We show that in the human HaCaT keratinocytes extracellular Zn2+ induces a metabotropic Ca2+ response that is abolished by silencing the expression of the G-protein-coupled receptor GPR39, suggesting that this Zn2+-sensing receptor, ZnR, is mediating the response. Keratinocytic-ZnR signaling is highly selective for Zn2+ and can be triggered by nanomolar concentrations of this ion. Interestingly, Zn2+ was also released following cellular injury, as monitored by a specific non-permeable fluorescent Zn2+ probe, ZnAF-2. Chelation of Zn2+ and scavenging of ATP from conditioned medium, collected from injured epithelial cultures, was sufficient to eliminate the metabotropic Ca2+ signaling. The signaling triggered by Zn2+, 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 Zn2+, which is either applied or released following injury, activates ZnR/GPR39 to promote signaling leading to epithelial repair.

Following injury, keratinocytes are exposed to diverse extracellular stimuli such as growth factors, cytokines, and matrix components, resulting in stimulation of cellular proliferation and migration. The release of these factors at the wound site leads to renewal of the epithelial layer in the damaged area (1). Zinc is found in the intracellular and extracellular matrix, in its free or protein-bound form and was shown to accumulate in skin tissues following injury (2). Furthermore, topical addition of zinc, in ointments or bandages, was known for many years and is also used in modern medicine to accelerate wound healing and the re-epithelialization process (2)(3)(4)(5). Consistent with a key role for Zn 2ϩ in promoting wound healing, the manifestation of dietary or genetic Zn 2ϩ deficiency are severe skin lesions and impaired wound healing that can be reversed by Zn 2ϩ supplementation (6,7). Whereas the role of Zn 2ϩ transporters in diseases linked to dyshomeostasis of this ion has been described (8 -13) cellular mechanisms linking Zn 2ϩ to keratinocytes proliferation and migration are not well understood.
Several studies have highlighted a role for Zn 2ϩ in cellular signaling (12,14). Of particular importance is the activation of mitogen-activated protein kinase (MAPK) 2 and PI3 kinase pathways, leading to enhanced cell proliferation and survival (15)(16)(17)(18)(19)(20). In epidermal tissues, higher levels of zinc have been correlated with higher mitotic activity (21). It was further shown that application of Zn 2ϩ accelerates the migration of keratinocytes via modulation of integrin receptors expression or interaction with metalloproteases (22)(23)(24)(25). The effect of Zn 2ϩ in enhancing cell proliferation is synergistic with intracellular Ca 2ϩ rise, indicating that their effects are mediated through a common pathway (26,27). Importantly, Zn 2ϩ affects cell proliferation rate prior to changes in its intracellular levels, suggesting that the extracellular Zn 2ϩ pool mediates signaling leading to epithelial repair (28).
We have previously identified a Zn 2ϩ -sensing receptor (ZnR) active in keratinocytes and other epithelial cells (16,29). ZnR activity was manifested by robust increase in intracellular Ca 2ϩ concentrations triggered by extracellular Zn 2ϩ (29). Subsequent studies have found that in neurons ZnR activity is mediated by the G-protein-coupled receptor, GPR39 (30). We demonstrate here that a ZnR/GPR39 is activated in keratinocytes, by exogenous Zn 2ϩ or by conditioned medium collected from injured cells, and up-regulates signaling and ion transport pathways necessary for wound healing.
Fluorescent Imaging of Ca 2ϩ , Zn 2ϩ , or pH-Fluorescent imaging measurements were acquired and analyzed using AIW 4 (INDEC BioSystems) and analyzed in Microsoft Excel. Graphs presented are representative graphs of at least three independent experiments. The fluorescent signal is shown as the percentage of the averaged baseline signal (R 0 ) acquired during the first 20 s of each measurement (R/R 0 ϫ 100), as previously described (29,30). The rate of fluorescent change was determined using Kaleidagraph and averaged over at least three independent experiments.
Statistical analysis was performed using Student's t test, applied following Levene's test for homogeneity of variances, or ANOVA analysis as relevant. Multiple comparisons were performed by student-Newman-Keuls or Dunnet method as appropriate. Non-parametric test included Mann Whitney and Kruskal Wallis test. Multiple comparisons followed by Fisher's protected level of significance method, were adjusted for the non-parametric setting. The data are presented as mean Ϯ S.E. *, p Ͻ 0.05; **, p Ͻ 0.01.
GPR39 Silencing using siRNA-For gene-silencing experiments, cells were cotransfected with either of the silencing plasmids, 3 g of siGPR39 or siT1R3 or a scrambled (siControl) siRNA construct (Sigma-Aldrich) in 35-mm plates, using the transfection reagent DreamfectGold (OZBiosciences) as described by the manufacturer. Cells were imaged 48 h after transfection. Analysis of cells co-transfection with YFP indicated that more than 90% of the cells were transfected using this reagent. The target sequence of the GPR39 for siRNA was CCATGGAGTTCTACAGC ATtt and that of T1R3 was CUUAGGA UGAAGGGGGACUtt.
Desensitization Protocol-Cells seeded on glass coverslips were incubated for 30 min with 50 M Zn 2ϩ , concentrations that induced the Ca 2ϩ response, concomitant with the Fura-2loading procedure, at room temperature. Because the BSA may chelate the Zn 2ϩ and change the effective amount of this ion in the solution, the Ringer's solution during loading did not contain BSA. In some of the experiments the cells were then allowed to recover in BSA containing Ringer's solution. For the prolonged recovery periods, cells were loaded immediately prior to imaging. Fluorescence imaging was performed as above to study the Zn 2ϩ -dependent Ca 2ϩ response. NH 4 Cl Prepulse Paradigm for NHE1 Activity-BCECFloaded cells were exposed to 30 mM NH 4 Cl in Ringer's solution, resulting in alkalinization of the cytoplasm, then Na ϩ -free (NMDG replacing Na ϩ ) and NH 4 Cl-free Ringer's solution was applied, resulting in cellular acidification. Recovery of pH i , representing NHE activity, was apparent when cells were perfused with nominally Ca 2ϩ -free Ringer's solution, containing 120 mM Na ϩ (20). ERK1/2 Phosphorylation by Western Blotting-HaCaT cells were grown to confluency in 60-mm dishes and then starved overnight for serum. Following treatment, with Zn 2ϩ and inhibitors, cells were disrupted in lysis buffer and protein concentrations in the supernatant were determined using the Bio-Rad assay. Cytosolic fractions (20 g) were separated on 10% SDS-PAGE followed by immunoblotting. Antibodies against doubly phosphorylated ERK1/2 (1:5000, Sigma), ␤-actin (1:40,000) or total ERK (1:5000) were detected and quantified digitally using Chemimager 5 (Alpha-Innotech, Labtrade) (20). Densitometry analysis was performed using ImageJ software. The values presented are normalized to ␤-actin or total ERK. Each graph represents an average of at least three independent experiments.
In Vitro Scratch Assay-Cells were seeded at 2 ϫ 10 4 cells/ well in 96-well plates and grown to confluency and deprived of serum for 24 h. A scratch was performed using 1 ml plastic pipette tip (ϳ2 mm in width), as previously described (34), in 37°C Ringer's solution and subsequently washed extensively with DMEM (supplemented with 1% FBS and 100 M CaEGTA). Agonists (Zn 2ϩ or ATP) were applied following scratching for 15 min in Ringer's solution, the PLC inhibitor U73122 (1 M, 30 min) or NHE1 inhibitor cariporide (0.5 M, 60 min) were applied prior to scratching. The Ringer's solution was then replaced back to DMEM (1% FBS, 100 M CaEGTA). The rate of scratch closure was determined by acquiring bright field images immediately after performing the scratch, and after 24 h. Area measurements of the cell-free region were performed using ImageJ software, and the ratio of denuded areas at these times are presented.
Conditioned Medium-Conditioned medium was produced from confluent monolayers of HaCaT cells seeded on 60-mm dishes. HaCaT cells were grown to confluency and were then starved for serum for 24 h, to remove exogenous growth factors. The monolayers were washed with Ringer's solution, scraped with a rubber policeman into 100 l of Ringer's solution, and the supernatant was collected. Cellular debris was removed by centrifugation (10 min, 1500 rpm). The supernatant was frozen (Ϫ80°C) until used.

RESULTS
ZnR Signaling in HaCaT Keratinocytes-We first sought to determine if Zn 2ϩ activates the ZnR pathway in HaCaT cells. The intracellular Ca 2ϩ (Ca 2ϩ i ) response in HaCaT cells loaded with the Ca 2ϩ -sensitive dye Fura-2 was determined following application of 50 M free-Zn 2ϩ (150 M Zn 2ϩ in the presence of 100 M CaEGTA) in Ringer's solution. A rapid Zn 2ϩ -dependent increase of Fura-2 fluorescence was observed in the presence or absence of extracellular Ca 2ϩ (Fig. 1A), consistent with our previous findings in primary cultures of normal human keratinocytes (29). The Zn 2ϩ -dependent [Ca 2ϩ i ] rise was similar to the response triggered by ATP (100 M), a purinergic agonist (see inset). Because Fura-2 is sensitive to Zn 2ϩ (K d ϭ 2 nM), permeation of this ion can also trigger a fluorescent signal rise. To test this hypothesis Ca 2ϩ stores were first depleted using the Ca 2ϩ pump inhibitor thapsigargin (200 nM) and ATP (100 M) in nominally Ca 2ϩ -free Ringer's solution. Application of Zn 2ϩ (100 M) following Ca 2ϩ stores depletion, failed to elicit an increase in Fura-2 fluorescence (Fig. 1B), indicating that the response is mediated by Zn 2ϩ -dependent release of Ca 2ϩ i . The PLC inhibitor U73122 (1 M) blocked the Zn 2ϩ -dependent [Ca 2ϩ i ] rise (Fig. 1C), whereas its inactive analogue, U73343, did not attenuate this response (not shown). Similarly, pretreatment of HaCaT cells with Pasteurella multocida toxin, which induces uncoupling of PLC from G␣ q or pretreatment with the G␣ q inhibitor YM-254890 (1 M) (35,36) also abolished the Zn 2ϩ -dependent [Ca 2ϩ i ] rise (Fig. 1C).
The orphan GPCR, GPR39, was recently linked to ZnR signaling in neurons (30), we therefore asked if GPR39 also mediates keratinocytic-ZnR activity. Transfection of HaCaT cells with an siRNA (siGPR39) construct targeted to silence GPR39 expression (see inset in Fig. 1D) was followed by elimination of the Zn 2ϩ -dependent [Ca 2ϩ i ] response, yet a scrambled siRNA construct (siControl) did not attenuate this response (Fig. 1D). These results suggest that extracellular Zn 2ϩ activates a G␣ q -protein coupled zinc-sensing receptor in HaCaT cells that is mediated by GPR39 (16,29,36).
High Affinity and Selectivity of the Keratinocytic-ZnR-Because the concentration of free Zn 2ϩ in the epidermis is estimated to be in the nanomolar range (2) we performed a dose-response analysis to determine if the affinity of the keratinocytic-ZnR is physiologically relevant. Zinc, at concentrations between 0.5 nM and 100 M (free-Zn 2ϩ buffered with EGTA, see "Experimental Procedures") was applied to HaCaT cells loaded with Fura-2. Amplitude of the [Ca 2ϩ i ] responses  ). B, application of Zn 2ϩ following depletion of Ca 2ϩ i stores using thapsigargin (200 nM) and ATP (100 M) in nominally Ca 2ϩ -free Ringer's solution, notice that following application of Zn 2ϩ no rise in fluorescence is seen for the same time period shown in A. C, Zn 2ϩ -dependent Ca 2ϩ i response was monitored in cells pretreated with the PLC inhibitor, U73122 (3 M), the G␣ q inhibitor, YM-254890 (1 M), and following treatment with Pasteurella multocida toxin (12 h). Inhibitors of the IP 3 pathway or depletion of Ca 2ϩ stores blocked the Zn 2ϩ -dependent Ca 2ϩ rise. D, Zn 2ϩ (100 M) -dependent Ca 2ϩ 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 g/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 Zn 2ϩ -dependent response. (n ϭ 5 for each treatment). triggered by Zn 2ϩ were fitted using the Michaelis-Menten equation and exhibited a K 0.5 of 450 Ϯ 50 pM (Fig. 3A). We then asked whether MAPK signaling could also be triggered by similar low Zn 2ϩ concentrations. Cells were treated with the same Zn 2ϩ concentrations for 30 min. The stimulatory effect of Zn 2ϩ on ERK1/2 phosphorylation was already apparent upon addition of 1 nM and was 3-fold higher at 50 M Zn 2ϩ (Fig. 3B). Thus, the affinity of the keratinocytic-ZnR for Zn 2ϩ is well within the physiological range of Zn 2ϩ levels in the epidermis.
Other trace elements, such as copper, are also used in vivo to enhance wound healing (24). To determine the metal selectivity of the ZnR, Ca 2ϩ i responses, following the administration of Ni 2ϩ , Fe 2ϩ , Cu 2ϩ , and Pb 2ϩ in the presence of CaEGTA allowing free-ion concentration of 5, 30, 10, and 6 M, respectively, were compared with the response triggered by 10 M Zn 2ϩ (Fig.  3C). All the cations tested, but Zn 2ϩ , failed to evoke a significant change in [Ca 2ϩ i ], indicating that the keratinocytic ZnR is selectively activated by this ion, in agreement with ZnR/GPR39 selectivity in other cell types (29,41).
Zn 2ϩ Released from Injured Cells Is Sufficient to Activate ZnR Signaling-The high affinity and the specificity of the keratinocytic-ZnR suggest that endogenous Zn 2ϩ which may be released upon cellular injury can act as a paracrine agonist via the ZnR. To determine if Zn 2ϩ is released upon injury in the keratinocyte model, a scratch was made across a monolayer of HaCaT cells while monitoring changes in the fluorescence of the non-permeant Zn 2ϩ -selective fluorescent dye ZnAF-2 (2 M, (42)). A sharp rise in the extracellular fluorescence was observed immediately following formation of the scratch (Fig.  4A). This signal was largely reduced in the presence of the extracellular Zn 2ϩ chelator, CaEDTA (100 M, Fig. 4B). Whereas CaEDTA can also chelate other ions the selectivity of Zn-AF-2 to Zn 2ϩ (43) indicates that this ion is indeed released following injury. Thus, our findings suggest that Zn 2ϩ is released or secreted from cells upon injury.
We next assessed if the release of endogenous Zn 2ϩ following injury of epithelial cultures triggers ZnR activation. We therefore monitored the Ca 2ϩ response following application of conditioned medium obtained from mechanically injured cells (see "Experimental Procedures"). Application of conditioned medium was followed by a massive [Ca 2ϩ i ] rise (Fig. 4, C and D) and ERK1/2 phosphorylation (Fig. 4E) in HaCaT cultures. Because conditioned medium may also contain ATP that can trigger a Ca 2ϩ rise (44), the same experiment was conducted in the presence of the ATP scavenger apyrase (0.66 units/ml). In the presence of apyrase, application of conditioned medium triggered an attenuated Ca 2ϩ i response (about 50 Ϯ 3% of the control, Fig. 4, C and D), suggesting that while ATP is activating metabotropic Ca 2ϩ i signaling, a significant  The maximal Ca i 2ϩ response (monitored as in 1) following application of the indicated Zn 2ϩ concentration was averaged and plotted versus the applied free-Zn 2ϩ concentration (see "Experimental Procedures"). B, cells were treated with varying concentrations of Zn 2ϩ and then ERK1/2 phosphorylation was monitored. Quantification of ERK1/2 phosphorylation normalized to total ERK1/2 levels is shown in bottom panel (n ϭ 4 for each concentration; *, p Ͻ 0.05; **, p Ͻ 0.001 compared with EGTA). Zn 2ϩdependent ERK1/2 phosphorylation was already evident in the presence of 1 nM Zn 2ϩ indicating that the keratinocytic ZnR has a high affinity to Zn 2ϩ . C, cells were exposed to the indicated divalent ions, while monitoring the Ca 2ϩ i response. None of the other heavy metals applied triggered a significant rise in Ca 2ϩ i , indicating that the ZnR response is Zn 2ϩ selective.
residual response is maintained in its absence. To determine if Zn 2ϩ mediates this residual response, we applied conditioned medium containing both the extracellular Zn 2ϩ chelator CaEDTA (100 M) and apyrase. This resulted in complete elimination of the Ca 2ϩ response (about 10 Ϯ 2% compared with control, Fig. 4D), suggesting that the residual activity was triggered by Zn 2ϩ . Indeed, the residual response to application apyrase-treated conditioned medium (⌬R/R 0 of 31 Ϯ 4%) is similar to the response triggered by application of 50 M Zn 2ϩ (⌬R/R 0 of 33 Ϯ 2%). Although CaEDTA can also chelate other heavy metals, it is unlikely that these ions triggered the Ca 2ϩ i rise because of the metal selectivity of ZnR response (Fig. 3C). ZnR Undergoes Profound Desensitization by Zn 2ϩ -An important hallmark of GPCRs is functional desensitization by their ligands, a process designed to regulate intracellular signaling and to prevent excessive stimulation (19,45). Because unlike many other ligands Zn 2ϩ is not degraded nor quickly removed from the extracellular region (46), effective desensitization of ZnR is of particular relevance. Pre-exposure of HaCaT cells to Zn 2ϩ (50 M, 30 min) resulted in complete inhibition of the Zn 2ϩ -dependent [Ca 2ϩ i ] response, monitored immediately after or up to 6 h following the desensitization process (Fig. 5, A  and B). The pretreatment with Zn 2ϩ did not reduce the ATPdependent response indicating that the IP 3 pathway remained intact (see inset in Fig. 5A). The Zn 2ϩ -dependent Ca 2ϩ response recovered to 75 Ϯ 2% of the control only 16 h following pre-exposure to Zn 2ϩ (Fig. 5B). Thus, following the preapplication of Zn 2ϩ the ZnR undergoes complete and prolonged functional desensitization. We then used the desensitization paradigm (50 M Zn 2ϩ for 30 min) and monitored Zn 2ϩ -dependent ERK1/2 phosphorylation at 5 h following desensitization. The pre-exposure to Zn 2ϩ largely inhibited the Zn 2ϩ -induced ERK1/2 phosphorylation (Fig. 5C). This suggests that a functional ZnR is required for mediating Zn 2ϩ -dependent signaling in HaCaT keratinocytes.
The Keratinocytic-ZnR Regulates the Sodium-Proton Exchanger, NHE1-As the NHE exchangers are regulated by MAPK activation (47) and enhance cell proliferation and . Activation of ZnR signaling by endogenous Zn 2؉ , released during cellular injury. A, changes in fluorescence of the cell impermeable Zn 2ϩsensitive dye ZnAF-2 (2 M), 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 Zn 2ϩ chelator CaEDTA (n ϭ 4, *, p Ͻ 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 Zn 2ϩ chelator, CaEDTA (100 M) to intact HaCaT cells loaded with Fura-2-AM. Fluorescent images of the Ca 2ϩ response in these cultures following application of the conditioned medium as indicated are shown above the graph. D, averaged intracellular Ca 2ϩ signals from C are shown (n ϭ 5; *, p Ͻ 0.05; **, p Ͻ 0.01). Application of both CaEDTA and apyrase eliminated the Ca 2ϩ 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 ϭ 3; *, p Ͻ 0.05). i response. Cells that were pretreated with Zn 2ϩ did not respond to re-application of Zn 2ϩ , while a robust Ca 2ϩ signal was monitored following application of ATP. B, averaged Ca 2ϩ response to Zn 2ϩ re-applied at the indicated times following desensitization (n ϭ 3, *, p Ͻ 0.05). C, phosphorylation of ERK1/2 in response to re-application of Zn 2ϩ at 5 h following pre-application of Zn 2ϩ , quantification normalized to ␤-actin expression is shown below (n ϭ 5, *, p Ͻ 0.05). Similar to the effect on Ca 2ϩ signaling, desensitization by Zn 2ϩ attenuated the subsequent Zn 2ϩ -dependent phosphorylation of ERK1/2. migration (48) we asked if ZnR stimulates NHE activity. Basal activity of the Na ϩ /H ϩ exchanger in HaCaT cells, loaded with the pH-sensitive dye BCECF, was determined by monitoring the rate of pH i recovery following acid load induced by NH 4 Cl prepulse. Addition of Na ϩ following the acidification, to control cells was followed by rapid pH i recovery to resting level (Fig.  6A, 0.17 Ϯ 0.01 pH units/min). This recovery phase was completely inhibited following application of the NHE1 blocker, cariporide (0.5 M, Fig. 6B, 0.02 Ϯ 0.01 pH units/min). The pH i recovery rate was then determined following pre-application of Zn 2ϩ (50 M, 15 min), a paradigm which does not lead to Zn 2ϩ permeation but activates the ZnR (see Fig. 1B). ZnR activation was followed by enhanced pH i recovery rate (Fig. 6C, 0.27 Ϯ 0.02 pH units/min), that was also inhibited by cariporide (Fig.  6D, 0.05 Ϯ 0.02 pH units/min) indicating that ZnR is up-regulating the activity of NHE1. To determine if NHE1 activation is mediated by the MAPK pathway (47,49) the ERK1/2 inhibitor U0126 (1 M) was added 10 min before application of Zn 2ϩ . The addition of this ERK1/2 inhibitor resulted in inhibition of the stimulatory effect of Zn 2ϩ on NHE1 activity (Fig. 6E).
We then studied the role of the purinergic agonist ATP in regulating NHE1 activity. Application of ATP (100 M, 10 min) was also followed by enhanced NHE1 activity (Fig. 6G, 0.4 Ϯ 0.02 pH units/min) that was reversed by the inhibition of the MAPK pathway using U0126 (1 M, Fig. 6H, 0.16 Ϯ 0.03 pH units/min). Thus, both the ZnR-and ATP-mediated signaling upregulate the rate of NHE1-dependent recovery from acid load via the MAPK pathway.

ZnR Activity Promotes in Vitro
Scratch Closure-Our results (Fig.  4) indicate that Zn 2ϩ is released from injured cells, such transient release of extracellular Zn 2ϩ may activate the ZnR. To study whether ZnR activity underlies Zn 2ϩ -dependent enhanced wound healing, an in vitro scratch assay was performed on confluent HaCaT cultures and the rate of closure was determined by comparing the cell denuded areas immediately and 24 h following formation of the scratch. To fully activate the ZnR response, as determined by the dose response analyses (Fig. 4), cultures were treated with 50 M Zn 2ϩ for 15 min. Application of Zn 2ϩ , using the ZnR activation paradigm, was followed by an increase in the closure rate, which was about 20% higher than that of CaEGTA-treated cells (Fig. 7A), that was used to chelate residual Zn 2ϩ in the solutions. Application of ATP, also enhanced scratch closure (by 30% compared with control). Because extracellular Ca 2ϩ may alter the balance between differentiation and proliferation of keratinocytes (31,50) we assessed the scratch closure rate in the presence of extracellular Ca 2ϩ (1.8 mM). Similar Zn 2ϩ -dependent closure rates were measured in the presence or absence of Ca 2ϩ e, (Fig. 7A) indicating that at least during the first 24 h, extracellular Ca 2ϩ does not affect scratch closure rates. To determine if keratinocytic-ZnR signaling is triggering the enhanced scratch closure, the PLC and NHE1 inhibitors were applied prior to addition of Zn 2ϩ . The PLC inhibitor (U73122, 1 M), completely blocked the Zn 2ϩ -dependent enhancement of scratch closure (Fig. 7A). Application of the NHE1 inhibitor, cariporide (0.5 M) also reduced Zn 2ϩ -mediated enhancement of the scratch closure rate, to a level similar to the control (Fig.  7A). Thus, our results (Fig. 7B) indicate that Zn 2ϩ , via ZnR signaling, activates the IP 3 pathway and up-regulates NHE1 and thereby enhances scratch closure.
GPR39 Is Essential for Mediating the Effects of Zn 2ϩ in HaCaT Keratinocytes-GPR39 silencing resulted in elimination of the Zn 2ϩ -dependent Ca 2ϩ response (Fig. 1) suggesting that this receptor mediates ZnR signaling in keratinocytes. We therefore asked if ZnR/GPR39 is mediating the effects of Zn 2ϩ on NHE1 activity and scratch closure. HaCaT cells were transfected with the siGPR39 or siControl constructs and the NH 4 Cl prepulse paradigm was applied in BCECF-loaded cells. Following the transfection basal activity of NHE1 in the HaCaT cells was completely attenuated to a similar level with both siRNA constructs (Fig. 8A). Cells were then treated with Zn 2ϩ (as in Fig. 6) and pH i recovery from acid load was monitored. While in the control cells Zn 2ϩ strongly enhanced pH i recovery (0.04 Ϯ Finally, we determined the effect of Zn 2ϩ on scratch closure rate following GPR39 silencing. HaCaT cells were transfected with the siGPR39 or siControl constructs and the in vitro scratch assay was performed on confluent cultures as described in Fig. 7. Scratch closure rates were significantly enhanced following application of Zn 2ϩ treatment to cultures transfected with siControl (by about 70% compared with control cells treated with CaEGTA, Fig. 8B). In contrast, Zn 2ϩ application did not significantly enhance scratch closure rate in the siGPR39 transfected cultures. Following transfection with either the siControl or siGPR39 constructs, the rate of recovery in control cells, not treated with Zn 2ϩ , was largely attenuated compared with the naïve cultures, consistent with the low basal activity of NHE1 (Fig. 8A). Our results therefore suggest that ZnR/GPR39 is essential for mediating the effects of Zn 2ϩ on scratch closure.

DISCUSSION
We demonstrate here that extracellular Zn 2ϩ , at the concentrations found in the epidermis, acts as a signaling molecule, triggering Ca 2ϩ release from thapsigargin-sensitive stores. The response to Zn 2ϩ is mediated through a G␣ q -coupled receptor, GPR39, which activates PLC and the IP 3 pathway, similar to the ZnR-response in colonocytes and neurons (20,30,41). Analysis of the Zn 2ϩ -dependent Ca 2ϩ response, indicated that the keratinocytic-ZnR has high affinity and selectivity for Zn 2ϩ . Importantly, we show that following epithelial injury endogenous Zn 2ϩ is released and may act as a paracrine agonist triggering ZnR signaling leading to activation of intracellular signaling pathways associated with wound healing.
Our results demonstrate ZnR-dependent activation of the MAPK pathway in keratinocytic cells. Activation of this pathway triggers the majority of the early genes induced in a scratch model and is essential for wound healing (51)(52)(53). Interestingly, a long-lasting, marked increase in ERK1/2 phosphorylation was apparent following a brief exposure to Zn 2ϩ . Sustained ERK1/2 phosphorylation may be, upon certain conditions, referred to as a precursor of cell death (54,55). However, in HaCaT cells upon exposure to UVA, sustained ERK1/2 phosphorylation via activation of metalloproteinases may serve as a survival signal suppressing apoptosis (56). The enhanced migration of HaCaT cells, monitored using the in vitro scratch assay, following keratinocytic-ZnR activation suggests that ZnR-dependent ERK1/2 phosphorylation leads to cell growth. ZnR-dependent activation of ERK1/2 was partially mediated by PKC and the PI3K pathway, suggesting that cross-talk between these pathways may mediate the effects of Zn 2ϩ on epithelial repair. These signaling pathways are also involved in enhanced epithelial breast cancer cell proliferation following activation of the bradykinin receptor (57,58) and in melanoma cell growth (59,60).
The NHE1, once thought to merely regulate pH i , was also shown to enhance cell proliferation and migration (48,(61)(62)(63)(64), for example, by promoting the G2/M transition (65) and cell volume (66) or via its functional interaction with cytoskeletal elements (67). Our results show that ZnR-or ATP-dependent metabotropic Ca 2ϩ signaling induce up-regulation of NHE1 activity and thereby enhance the recovery of intracellular pH from acid load. Moreover, inhibition of the Zn 2ϩ -induced NHE1 up-regulation by cariporide resulted in attenuated migration and scratch closure. Phosphorylation of NHE1 by ERK1/2 plays a pivotal role in the activation of the exchanger by growth factors, hormone stimulation (47,68) and also by Zn 2ϩ (20). Inhibition of the MAPK pathway resulted in attenuation of the stimulatory effect of Zn 2ϩ via the ZnR on NHE1 also in keratinocytes.
In agreement with other works (69,70), we show that the Na ϩ /H ϩ exchanger NHE1 is activated in skin cells following cellular acidification. The robust up-regulation of NHE1 activity by Zn 2ϩ in keratinocytes suggests an important physiological role for this ion in pH regulation. The acidic surface, pH of 6.0, found in the upper layer of the skin is essential for formation of the permeability barrier (70,71). Zn 2ϩ deficiency enhances the inflammatory response (72) and a prominent role for this ion in wound healing was suggested to be an antimicrobial effect. A beneficial effect of Zn 2ϩ may therefore require strengthening of the proton gradient and the epithelial permeability barrier that is regulated by NHE1 activity or expression (70,73,74). Our results suggest that following skin injury, the release of Zn 2ϩ or its topical application may enhance formation of the barrier, and thereby, exert an anti-inflammatory effect.
Another molecule, apart from Zn 2ϩ , that is released from epithelial cultures is ATP (44), which can trigger a metabotropic Ca 2ϩ response mediated via the purinergic receptors (52,75,76). Our results show that both Zn 2ϩ and ATP are released following injury and can independently induce Ca 2ϩ signaling and activation of the MAPK pathway leading to up-regulation of NHE1 activity. Interestingly, activation of the ZnR pathway in salivary gland cells induces the release of ATP (36), this may provide a mechanism for the release of this molecule that will further enhance the metabotropic Ca 2ϩ signaling. The role of ATP in wound healing however is still controversial. Activation of the purinergic receptor and subsequent Ca 2ϩ release were shown to induce EGFR transactivation and lead to enhanced wound closure (77,78). Another study however, claimed that ATP inhibited serum-induced ERK1/2 and PI3K activation and attenuated cell migration (79). Our results, as well as others (52), show that ATP activates the MAPK pathway leading for example to enhance airway epithelial cell migration (75). Importantly, our results indicate that the downstream signaling triggered by ATP induced up-regulation of NHE1 activity and enhanced in vitro scratch closure. Our results further support a role for ATP in promoting wound healing, as chelation of Zn 2ϩ and scavenging of ATP eliminated the Ca 2ϩ response triggered by conditioned medium. This indicates that following injury, Zn 2ϩ and ATP are two major mediators of the Ca 2ϩ signaling that is essential for wound healing.
The physiological significance of the keratinocytic-ZnR activity mediated by GPR39 is demonstrated by the involvement of its downstream signaling pathway in epithelial proliferation and migration. Using the highly selective probe ZnAF-2, we further show release of Zn 2ϩ to the extracellular medium following keratinocyte injury, indicating that transient changes in the concentration of extracellular Zn 2ϩ occur dur- A, HaCaT cells were transfected with siGPR39 or siControl constructs and the NH 4 Cl prepulse paradigm was performed as in Fig. 6, recovery rate was measured in cells treated with Zn 2ϩ or in control cells. Averaged rates of pH i recovery, representing NHE1 activity are shown to the right (n ϭ 5 for each treatment, **, p Ͻ 0.01 compared with siControl cells without Zn 2ϩ ). B, in vitro scratch assay was performed as in Fig. 7 in cultures transfected with siControl or siGPR39. Cultures were treated with Zn 2ϩ 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 Zn 2ϩ are shown below (n ϭ 7 for each treatment, **, p Ͻ 0.01 compared with siControl cells without Zn 2ϩ ).
ing this process. The concentration of Zn 2ϩ released following a scratch or in the conditioned medium was sufficient to activate the high affinity ZnR-dependent Ca 2ϩ signaling in keratinocytic cultures. Although the effects of Zn 2ϩ on wound healing were previously described, our results provide the first mechanistic link between Zn 2ϩ and this process via the specific ZnR/GPR39. Generation of agonists that will activate, but will not desensitize, the ZnR may in the future provide an effective approach to accelerate wound healing.