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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2010 August 20; 285(34): 26097–26106.
Published online 2010 June 3. doi:  10.1074/jbc.M110.107490
PMCID: PMC2924011

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

Abstract

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.

Keywords: Calcium Imaging, Calcium Intracellular Release, Cell pH, Epithelial Cell, Zinc, Na/H Exchanger, Zinc Homeostasis, Zinc Transport

Introduction

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 (2,5). Consistent with a key role for Zn2+ in promoting wound healing, the manifestation of dietary or genetic Zn2+ deficiency are severe skin lesions and impaired wound healing that can be reversed by Zn2+ supplementation (6, 7). Whereas the role of Zn2+ transporters in diseases linked to dyshomeostasis of this ion has been described (8,13) cellular mechanisms linking Zn2+ to keratinocytes proliferation and migration are not well understood.

Several studies have highlighted a role for Zn2+ 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 (15,20). In epidermal tissues, higher levels of zinc have been correlated with higher mitotic activity (21). It was further shown that application of Zn2+ accelerates the migration of keratinocytes via modulation of integrin receptors expression or interaction with metalloproteases (22,25). The effect of Zn2+ in enhancing cell proliferation is synergistic with intracellular Ca2+ rise, indicating that their effects are mediated through a common pathway (26, 27). Importantly, Zn2+ affects cell proliferation rate prior to changes in its intracellular levels, suggesting that the extracellular Zn2+ pool mediates signaling leading to epithelial repair (28).

We have previously identified a Zn2+-sensing receptor (ZnR) active in keratinocytes and other epithelial cells (16, 29). ZnR activity was manifested by robust increase in intracellular Ca2+ concentrations triggered by extracellular Zn2+ (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 Zn2+ or by conditioned medium collected from injured cells, and up-regulates signaling and ion transport pathways necessary for wound healing.

EXPERIMENTAL PROCEDURES

Cell Culture

The HaCaT cell line, a spontaneously transformed, non-tumorigenic human keratinocyte cell line (31) was cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 4 mm l-glutamine, and 0.5 μg/ml antibiotic penicillin streptomycin solution (Biological Industries, Israel).

Fluorescent Imaging of Ca2+, Zn2+, 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 (R0) acquired during the first 20 s of each measurement (R/R0 × 100), as previously described (29, 30). The rate of fluorescent change was determined using Kaleidagraph and averaged over at least three independent experiments.

For Ca2+i imaging, cells were incubated for 30 min, with 5 μm Fura-2 acetomethyl ester (TEF-Lab) in 0.1% BSA in Ringer's solution containing: 120 mm NaCl, 2.7 mm KCl, 0.8 mm MgCl2, 2 mm Hepes, 0.75 mm glucose, 1.8 mm CaCl2. For pHi measurements, cells were loaded with the pH-sensitive dye 2′, 7′-bis-(2-carboxyethyl)-5,6 carboxyfluorescein acetoxy methyl-ester (1.25 μm BCECF-AM, TEF-Lab) for 12 min. Following dye loading, the cells were washed in Ringer's solution, and the cover slides were mounted into an imaging chamber. To buffer the low Zn2+ concentrations, Ringer's solutions contained 100 μm of CaEGTA, which has affinity to Zn2+ that is six orders of magnitude higher than to Ca2+ or Mg2+ (32). Concentration of free-Zn2+ were determined using Webmaxcstandard software. Intracellular pH was calibrated by perfusing the cells with high potassium Ringer's solution (120 mm KCl replacing 120 mm NaCl) containing 5 μm nigericin, at extracellular pH levels: 6.5, 7.0, 7.5, and 8.0 (20).

To monitor extracellular Zn2+ release, the cell impermeant fluorescent dye ZnAF-2 (2 μm, Kd = 2.7 nm for Zn2+, Sigma-Aldrich) (33), was employed. ZnAF-2 was excited at 480 nm and imaged with 535-nm long pass filter.

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 ± S.E. *, p < 0.05; **, p < 0.01.

GPR39 Silencing using siRNA

For gene-silencing experiments, cells were cotransfected with either of the silencing plasmids, 3 μg 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 μm Zn2+, concentrations that induced the Ca2+ response, concomitant with the Fura-2-loading procedure, at room temperature. Because the BSA may chelate the Zn2+ 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 Zn2+-dependent Ca2+ response.

NH4Cl Prepulse Paradigm for NHE1 Activity

BCECF-loaded cells were exposed to 30 mm NH4Cl in Ringer's solution, resulting in alkalinization of the cytoplasm, then Na+-free (NMDG replacing Na+) and NH4Cl- free Ringer's solution was applied, resulting in cellular acidification. Recovery of pHi, representing NHE activity, was apparent when cells were perfused with nominally Ca2+-free Ringer's solution, containing 120 mm Na+ (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 Zn2+ and inhibitors, cells were disrupted in lysis buffer and protein concentrations in the supernatant were determined using the Bio-Rad assay. Cytosolic fractions (20 μg) were separated on 10% SDS-PAGE followed by immunoblotting. Antibodies against doubly phosphorylated ERK1/2 (1:5000, Sigma), β-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 β-actin or total ERK. Each graph represents an average of at least three independent experiments.

In Vitro Scratch Assay

Cells were seeded at 2 × 104 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 (~2 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 μm CaEGTA). Agonists (Zn2+ or ATP) were applied following scratching for 15 min in Ringer's solution, the PLC inhibitor U73122 (1 μm, 30 min) or NHE1 inhibitor cariporide (0.5 μm, 60 min) were applied prior to scratching. The Ringer's solution was then replaced back to DMEM (1% FBS, 100 μm 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 μl of Ringer's solution, and the supernatant was collected. Cellular debris was removed by centrifugation (10 min, 1500 rpm). The supernatant was frozen (−80 °C) until used.

RESULTS

ZnR Signaling in HaCaT Keratinocytes

We first sought to determine if Zn2+ activates the ZnR pathway in HaCaT cells. The intracellular Ca2+ (Ca2+i) response in HaCaT cells loaded with the Ca2+-sensitive dye Fura-2 was determined following application of 50 μm free-Zn2+ (150 μm Zn2+ in the presence of 100 μm CaEGTA) in Ringer's solution. A rapid Zn2+-dependent increase of Fura-2 fluorescence was observed in the presence or absence of extracellular Ca2+ (Fig. 1A), consistent with our previous findings in primary cultures of normal human keratinocytes (29). The Zn2+-dependent [Ca2+i] rise was similar to the response triggered by ATP (100 μm), a purinergic agonist (see inset). Because Fura-2 is sensitive to Zn2+ (Kd = 2 nm), permeation of this ion can also trigger a fluorescent signal rise. To test this hypothesis Ca2+ stores were first depleted using the Ca2+ pump inhibitor thapsigargin (200 nm) and ATP (100 μm) in nominally Ca2+- free Ringer's solution. Application of Zn2+ (100 μm) following Ca2+ stores depletion, failed to elicit an increase in Fura-2 fluorescence (Fig. 1B), indicating that the response is mediated by Zn2+-dependent release of Ca2+i. The PLC inhibitor U73122 (1 μm) blocked the Zn2+-dependent [Ca2+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 Gαq or pretreatment with the Gαq inhibitor YM-254890 (1 μm) (35, 36) also abolished the Zn2+-dependent [Ca2+i] rise (Fig. 1C).

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 ...

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 Zn2+-dependent [Ca2+i] response, yet a scrambled siRNA construct (siControl) did not attenuate this response (Fig. 1D). These results suggest that extracellular Zn2+ activates a Gαq-protein coupled zinc-sensing receptor in HaCaT cells that is mediated by GPR39 (16, 29, 36).

We then studied if ZnR-dependent metabotropic Ca2+ rise will activate the MAP kinase pathway that is critical in promoting wound healing (37). Phosphorylation of extracellular-regulated kinase (ERK) 1/2 was determined in HaCaT cells treated with 50 μm Zn2+ (applied for 0 to 120 min, as indicated). Zn2+-dependent ERK1/2 phosphorylation was already evident after 10 min and peaked at 60 min (Fig. 2A). Application of ATP resulted in a similar temporal pattern of ERK1/2 phosphorylation (Fig. 2B). Zn2+-induced ERK1/2 phosphorylation was blocked by the MEK inhibitor, U0126 (1 μm) (Fig. 2C). The Gαq inhibitor YM-254890 (1 μm, 30 min) attenuated Zn2+-induced ERK1/2 phosphorylation by about 40% (Fig. 2C), suggesting that activation of the IP3 pathway by ZnR is partially mediating the Zn2+-dependent ERK1/2 activation. Intracellular Ca2+ rise activates PKC and PI3K pathways and may thereby enhance cellular proliferation (38,40). Application of the PKC inhibitor, bisindolylmaleimide I (BI, 10 nm), the PI3K inhibitor, wortmannin (100 nm), or both inhibitors, attenuated the Zn2+-dependent ERK1/2 phosphorylation by about 50% (Fig. 2D), suggesting that the two pathways converge to mediate the Zn2+-dependent activation of MAPK.

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 ...

High Affinity and Selectivity of the Keratinocytic-ZnR

Because the concentration of free Zn2+ 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 μm (free-Zn2+ buffered with EGTA, see “Experimental Procedures”) was applied to HaCaT cells loaded with Fura-2. Amplitude of the [Ca2+i] responses triggered by Zn2+ were fitted using the Michaelis-Menten equation and exhibited a K0.5 of 450 ± 50 pm (Fig. 3A). We then asked whether MAPK signaling could also be triggered by similar low Zn2+ concentrations. Cells were treated with the same Zn2+ concentrations for 30 min. The stimulatory effect of Zn2+ on ERK1/2 phosphorylation was already apparent upon addition of 1 nm and was 3-fold higher at 50 μm Zn2+ (Fig. 3B). Thus, the affinity of the keratinocytic-ZnR for Zn2+ is well within the physiological range of Zn2+ levels in the epidermis.

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 Zn ...

Other trace elements, such as copper, are also used in vivo to enhance wound healing (24). To determine the metal selectivity of the ZnR, Ca2+i responses, following the administration of Ni2+, Fe2+, Cu2+, and Pb2+ in the presence of CaEGTA allowing free-ion concentration of 5, 30, 10, and 6 μm, respectively, were compared with the response triggered by 10 μm Zn2+ (Fig. 3C). All the cations tested, but Zn2+, failed to evoke a significant change in [Ca2+i], indicating that the keratinocytic ZnR is selectively activated by this ion, in agreement with ZnR/GPR39 selectivity in other cell types (29, 41).

Zn2+ Released from Injured Cells Is Sufficient to Activate ZnR Signaling

The high affinity and the specificity of the keratinocytic-ZnR suggest that endogenous Zn2+ which may be released upon cellular injury can act as a paracrine agonist via the ZnR. To determine if Zn2+ 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 Zn2+-selective fluorescent dye ZnAF-2 (2 μm, (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 Zn2+ chelator, CaEDTA (100 μm, Fig. 4B). Whereas CaEDTA can also chelate other ions the selectivity of Zn-AF-2 to Zn2+ (43) indicates that this ion is indeed released following injury. Thus, our findings suggest that Zn2+ is released or secreted from cells upon injury.

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 ...

We next assessed if the release of endogenous Zn2+ following injury of epithelial cultures triggers ZnR activation. We therefore monitored the Ca2+ response following application of conditioned medium obtained from mechanically injured cells (see “Experimental Procedures”). Application of conditioned medium was followed by a massive [Ca2+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 Ca2+ 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 Ca2+i response (about 50 ± 3% of the control, Fig. 4, C and D), suggesting that while ATP is activating metabotropic Ca2+i signaling, a significant residual response is maintained in its absence. To determine if Zn2+ mediates this residual response, we applied conditioned medium containing both the extracellular Zn2+ chelator CaEDTA (100 μm) and apyrase. This resulted in complete elimination of the Ca2+ response (about 10 ± 2% compared with control, Fig. 4D), suggesting that the residual activity was triggered by Zn2+. Indeed, the residual response to application apyrase-treated conditioned medium (ΔR/R0 of 31 ± 4%) is similar to the response triggered by application of 50 μm Zn2+ (ΔR/R0 of 33 ± 2%). Although CaEDTA can also chelate other heavy metals, it is unlikely that these ions triggered the Ca2+i rise because of the metal selectivity of ZnR response (Fig. 3C).

ZnR Undergoes Profound Desensitization by Zn2+

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 Zn2+ 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 Zn2+ (50 μm, 30 min) resulted in complete inhibition of the Zn2+-dependent [Ca2+i] response, monitored immediately after or up to 6 h following the desensitization process (Fig. 5, A and B). The pretreatment with Zn2+ did not reduce the ATP-dependent response indicating that the IP3 pathway remained intact (see inset in Fig. 5A). The Zn2+-dependent Ca2+ response recovered to 75 ± 2% of the control only 16 h following pre-exposure to Zn2+ (Fig. 5B). Thus, following the pre-application of Zn2+ the ZnR undergoes complete and prolonged functional desensitization. We then used the desensitization paradigm (50 μm Zn2+ for 30 min) and monitored Zn2+-dependent ERK1/2 phosphorylation at 5 h following desensitization. The pre-exposure to Zn2+ largely inhibited the Zn2+-induced ERK1/2 phosphorylation (Fig. 5C). This suggests that a functional ZnR is required for mediating Zn2+-dependent signaling in HaCaT keratinocytes.

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 ...

The Keratinocytic-ZnR Regulates the Sodium-Proton Exchanger, NHE1

As the NHE exchangers are regulated by MAPK activation (47) and enhance cell proliferation and migration (48) we asked if ZnR stimulates NHE activity. Basal activity of the Na+/H+ exchanger in HaCaT cells, loaded with the pH-sensitive dye BCECF, was determined by monitoring the rate of pHi recovery following acid load induced by NH4Cl prepulse. Addition of Na+ following the acidification, to control cells was followed by rapid pHi recovery to resting level (Fig. 6A, 0.17 ± 0.01 pH units/min). This recovery phase was completely inhibited following application of the NHE1 blocker, cariporide (0.5 μm, Fig. 6B, 0.02 ± 0.01 pH units/min). The pHi recovery rate was then determined following pre-application of Zn2+ (50 μm, 15 min), a paradigm which does not lead to Zn2+ permeation but activates the ZnR (see Fig. 1B). ZnR activation was followed by enhanced pHi recovery rate (Fig. 6C, 0.27 ± 0.02 pH units/min), that was also inhibited by cariporide (Fig. 6D, 0.05 ± 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 μm) was added 10 min before application of Zn2+. The addition of this ERK1/2 inhibitor resulted in inhibition of the stimulatory effect of Zn2+ on NHE1 activity (Fig. 6E).

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 ...

We then studied the role of the purinergic agonist ATP in regulating NHE1 activity. Application of ATP (100 μm, 10 min) was also followed by enhanced NHE1 activity (Fig. 6G, 0.4 ± 0.02 pH units/min) that was reversed by the inhibition of the MAPK pathway using U0126 (1 μm, Fig. 6H, 0.16 ± 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 Zn2+ is released from injured cells, such transient release of extracellular Zn2+ may activate the ZnR. To study whether ZnR activity underlies Zn2+-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 μm Zn2+ for 15 min. Application of Zn2+, 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 Zn2+ in the solutions. Application of ATP, also enhanced scratch closure (by 30% compared with control). Because extracellular Ca2+ may alter the balance between differentiation and proliferation of keratinocytes (31, 50) we assessed the scratch closure rate in the presence of extracellular Ca2+ (1.8 mm). Similar Zn2+-dependent closure rates were measured in the presence or absence of Ca2+e, (Fig. 7A) indicating that at least during the first 24 h, extracellular Ca2+ 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 Zn2+. The PLC inhibitor (U73122, 1 μm), completely blocked the Zn2+-dependent enhancement of scratch closure (Fig. 7A). Application of the NHE1 inhibitor, cariporide (0.5 μm) also reduced Zn2+-mediated enhancement of the scratch closure rate, to a level similar to the control (Fig. 7A). Thus, our results (Fig. 7B) indicate that Zn2+, via ZnR signaling, activates the IP3 pathway and up-regulates NHE1 and thereby enhances scratch closure.

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 ...

GPR39 Is Essential for Mediating the Effects of Zn2+ in HaCaT Keratinocytes

GPR39 silencing resulted in elimination of the Zn2+-dependent Ca2+ response (Fig. 1) suggesting that this receptor mediates ZnR signaling in keratinocytes. We therefore asked if ZnR/GPR39 is mediating the effects of Zn2+ on NHE1 activity and scratch closure. HaCaT cells were transfected with the siGPR39 or siControl constructs and the NH4Cl 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 Zn2+ (as in Fig. 6) and pHi recovery from acid load was monitored. While in the control cells Zn2+ strongly enhanced pHi recovery (0.04 ± 0.01 pH units/min versus 0.32 ± 0.04 pH units/min following Zn2+ treatment), in siGRP39-treated cells application of Zn2+ failed to upregulate the activity of NHE1 (0.04 ± 0.02 pH units/min versus 0.06 ± 0.02 pH units/min following Zn2+ treatment). These results are in agreement with the pharmacological demonstration of the major role of ZnR in Zn2+-dependent up-regulation of NHE1 (Fig. 6) and further indicate that Zn2+, via ZnR/GPR39 up-regulates NHE1 activity.

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 ...

Finally, we determined the effect of Zn2+ 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 Zn2+ treatment to cultures transfected with siControl (by about 70% compared with control cells treated with CaEGTA, Fig. 8B). In contrast, Zn2+ 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 Zn2+, 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 Zn2+ on scratch closure.

DISCUSSION

We demonstrate here that extracellular Zn2+, at the concentrations found in the epidermis, acts as a signaling molecule, triggering Ca2+ release from thapsigargin-sensitive stores. The response to Zn2+ is mediated through a Gαq-coupled receptor, GPR39, which activates PLC and the IP3 pathway, similar to the ZnR-response in colonocytes and neurons (20, 30, 41). Analysis of the Zn2+-dependent Ca2+ response, indicated that the keratinocytic-ZnR has high affinity and selectivity for Zn2+. Importantly, we show that following epithelial injury endogenous Zn2+ 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 (51,53). Interestingly, a long-lasting, marked increase in ERK1/2 phosphorylation was apparent following a brief exposure to Zn2+. 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 Zn2+ 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 pHi, was also shown to enhance cell proliferation and migration (48, 61,64), 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 Ca2+ signaling induce up-regulation of NHE1 activity and thereby enhance the recovery of intracellular pH from acid load. Moreover, inhibition of the Zn2+-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 Zn2+ (20). Inhibition of the MAPK pathway resulted in attenuation of the stimulatory effect of Zn2+ via the ZnR on NHE1 also in keratinocytes.

In agreement with other works (69, 70), we show that the Na+/H+ exchanger NHE1 is activated in skin cells following cellular acidification. The robust up-regulation of NHE1 activity by Zn2+ 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). Zn2+ 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 Zn2+ 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 Zn2+ or its topical application may enhance formation of the barrier, and thereby, exert an anti-inflammatory effect.

Another molecule, apart from Zn2+, that is released from epithelial cultures is ATP (44), which can trigger a metabotropic Ca2+ response mediated via the purinergic receptors (52, 75, 76). Our results show that both Zn2+ and ATP are released following injury and can independently induce Ca2+ 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 Ca2+ signaling. The role of ATP in wound healing however is still controversial. Activation of the purinergic receptor and subsequent Ca2+ 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 Zn2+ and scavenging of ATP eliminated the Ca2+ response triggered by conditioned medium. This indicates that following injury, Zn2+ and ATP are two major mediators of the Ca2+ 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 Zn2+ to the extracellular medium following keratinocyte injury, indicating that transient changes in the concentration of extracellular Zn2+ occur during this process. The concentration of Zn2+ released following a scratch or in the conditioned medium was sufficient to activate the high affinity ZnR-dependent Ca2+ signaling in keratinocytic cultures. Although the effects of Zn2+ on wound healing were previously described, our results provide the first mechanistic link between Zn2+ 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.

Acknowledgments

The Gαq inhibitor, YM-254890, was a kind gift from Astellas Pharma Inc. We thank Dr. Sharon Herrmann for technical assistance.

*This work was supported by the Israel Science Foundation (Grant 585/05, to M. H.) and by the German Israeli Foundation (Grant 912-90.11/2006, to M. H.).

2The abbreviations used are:

MAPK
mitogen-activated protein kinase
ERK
extracellular signal-regulated kinase
NHE
Na+/H+ exchanger
ZnR
Zn2+-sensing receptor
Ca2+i
intracellular Ca2+
pHi
intracellular pH
DMEM
Dulbecco's modified Eagle's medium
FBS
fetal bovine serum
BSA
bovine serum albumin.

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