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. 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. IntroductionFollowing 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 (1Jacinto A. Martinez-Arias A. Martin P. Nat. Cell Biol. 2001; 3: E117-123Crossref PubMed Scopus (289) Google Scholar). 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 (2Lansdown A.B. Mirastschijski U. Stubbs N. Scanlon E. Agren M.S. Wound Repair Regen. 2007; 15: 2-16Crossref PubMed Scopus (406) Google Scholar). 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 (2Lansdown A.B. Mirastschijski U. Stubbs N. Scanlon E. Agren M.S. Wound Repair Regen. 2007; 15: 2-16Crossref PubMed Scopus (406) Google Scholar, 3Barceloux D.G. J. Toxicol. Clin. Toxicol. 1999; 37: 279-292Crossref PubMed Scopus (190) Google Scholar, 4Lansdown A.B. Lancet. 1996; 347: 706-707Abstract PubMed Scopus (87) Google Scholar, 5Schwartz J.R. Marsh R.G. Draelos Z.D. Dermatol. Surg. 2005; 31 (discussion 847): 837-847Crossref PubMed Scopus (97) Google Scholar). 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 (6Jensen S.L. McCuaig C. Zembowicz A. Hurt M.A. J. Cutan. Pathol. 2008; 35: 1-13Crossref PubMed Scopus (30) Google Scholar, 7Takahashi H. Nakazawa M. Takahashi K. Aihara M. Minami M. Hirasawa T. Ikezawa Z. J. Dermatol. Sci. 2008; 50: 31-39Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Whereas the role of Zn2+ transporters in diseases linked to dyshomeostasis of this ion has been described (8Andrews G.K. Biochem. Soc. Trans. 2008; 36: 1242-1246Crossref PubMed Scopus (85) Google Scholar, 9Kambe T. Andrews G.K. Mol. Cell Biol. 2009; 29: 129-139Crossref PubMed Scopus (95) Google Scholar, 10Kambe T. Weaver B.P. Andrews G.K. Genesis. 2008; 46: 214-228Crossref PubMed Scopus (101) Google Scholar, 11Besecker B. Bao S. Bohacova B. Papp A. Sadee W. Knoell D.L. Am. J. Physiol. Lung Cell Mol. Physiol. 2008; 294: L1127-L1136Crossref PubMed Scopus (116) Google Scholar, 12Hogstrand C. Kille P. Nicholson R.I. Taylor K.M. Trends Mol. Med. 2009; 15: 101-111Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 13Taylor K.M. Biochem. Soc. Trans. 2008; 36: 1247-1251Crossref PubMed Scopus (39) Google Scholar) cellular mechanisms linking Zn2+ to keratinocytes proliferation and migration are not well understood.Several studies have highlighted a role for Zn2+ in cellular signaling (12Hogstrand C. Kille P. Nicholson R.I. Taylor K.M. Trends Mol. Med. 2009; 15: 101-111Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 14Zhao L. Chen W. Taylor K.M. Cai B. Li X. Biochem. Biophys. Res. Commun. 2007; 363: 82-88Crossref PubMed Scopus (55) Google Scholar). Of particular importance is the activation of mitogen-activated protein kinase (MAPK) 2The abbreviations used are: MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseNHENa+/H+ exchangerZnRZn2+-sensing receptorCa2+iintracellular Ca2+pHiintracellular pHDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumBSAbovine serum albumin. and PI3 kinase pathways, leading to enhanced cell proliferation and survival (15Hershfinkel M. Tamas M. Martinoia E. Molecular Biology of Metal Homeostasis and Detoxification. springer-verlag, Berlin2006Google Scholar, 16Hershfinkel M. Silverman W.F. Sekler I. Mol. Med. 2007; 13: 331-336Crossref PubMed Scopus (75) Google Scholar, 17Murakami M. Hirano T. Cancer Sci. 2008; 99: 1515-1522Crossref PubMed Scopus (251) Google Scholar, 18Kaisman-Elbaz T. Sekler I. Fishman D. Karol N. Forberg M. Kahn N. Hershfinkel M. Silverman W.F. J. Cell Physiol. 2009; 220: 222-229Crossref PubMed Scopus (26) Google Scholar, 19Dubi N. Gheber L. Fishman D. Sekler I. Hershfinkel M. Carcinogenesis. 2008; 29: 1692-1700Crossref PubMed Scopus (52) Google Scholar, 20Azriel-Tamir H. Sharir H. Schwartz B. Hershfinkel M. J. Biol. Chem. 2004; 279: 51804-51816Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In epidermal tissues, higher levels of zinc have been correlated with higher mitotic activity (21Rothstein J.D. Martin L. Levey A.I. Dykes-Hoberg M. Jin L. Wu D. Nash N. Kuncl R.W. Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1449) Google Scholar). It was further shown that application of Zn2+ accelerates the migration of keratinocytes via modulation of integrin receptors expression or interaction with metalloproteases (22Ravanti L. Kahari V.M. Int. J. Mol. Med. 2000; 6: 391-407PubMed Google Scholar, 23Pirila E. Korpi J.T. Korkiamaki T. Jahkola T. Gutierrez-Fernandez A. Lopez-Otin C. Saarialho-Kere U. Salo T. Sorsa T. Wound Repair Regen. 2007; 15: 47-57Crossref PubMed Scopus (61) Google Scholar, 24Tenaud I. Leroy S. Chebassier N. Dreno B. Exp. Dermatol. 2000; 9: 407-416Crossref PubMed Scopus (58) Google Scholar, 25Tenaud I. Saiagh I. Dreno B. J. Dermatolog. Treat. 2009; 20: 90-93Crossref PubMed Scopus (6) Google Scholar). The effect of Zn2+ in enhancing cell proliferation is synergistic with intracellular Ca2+ rise, indicating that their effects are mediated through a common pathway (26Huang J.S. Mukherjee J.J. Chung T. Crilly K.S. Kiss Z. Eur. J. Biochem. 1999; 266: 943-951Crossref PubMed Scopus (46) Google Scholar, 27Kiss Z. Crilly K.S. Tomono M. FEBS Lett. 1997; 415: 71-74Crossref PubMed Scopus (24) Google Scholar). 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 (28MacDonald R.S. Wollard-Biddle L.C. Browning J.D. Thornton Jr., W.H. O'Dell B.L. J. Nutr. 1998; 128: 1600-1605Crossref PubMed Scopus (39) Google Scholar).We have previously identified a Zn2+-sensing receptor (ZnR) active in keratinocytes and other epithelial cells (16Hershfinkel M. Silverman W.F. Sekler I. Mol. Med. 2007; 13: 331-336Crossref PubMed Scopus (75) Google Scholar, 29Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 11749-11754Crossref PubMed Scopus (199) Google Scholar). ZnR activity was manifested by robust increase in intracellular Ca2+ concentrations triggered by extracellular Zn2+ (29Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 11749-11754Crossref PubMed Scopus (199) Google Scholar). Subsequent studies have found that in neurons ZnR activity is mediated by the G-protein-coupled receptor, GPR39 (30Besser L. Chorin E. Sekler I. Silverman W.F. Atkin S. Russell J.T. Hershfinkel M. J. Neurosci. 2009; 29: 2890-2901Crossref PubMed Scopus (171) Google Scholar). 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.DISCUSSIONWe 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 (20Azriel-Tamir H. Sharir H. Schwartz B. Hershfinkel M. J. Biol. Chem. 2004; 279: 51804-51816Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 30Besser L. Chorin E. Sekler I. Silverman W.F. Atkin S. Russell J.T. Hershfinkel M. J. Neurosci. 2009; 29: 2890-2901Crossref PubMed Scopus (171) Google Scholar, 41Yasuda S. Miyazaki T. Munechika K. Yamashita M. Ikeda Y. Kamizono A. J. Recept. Signal Transduct. Res. 2007; 27: 235-246Crossref PubMed Scopus (78) Google Scholar). 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 (51Fitsialos G. Chassot A.A. Turchi L. Dayem M.A. LeBrigand K. Moreilhon C. Meneguzzi G. Busca R. Mari B. Barbry P. Ponzio G. J. Biol. Chem. 2007; 282: 15090-15102Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 52Yang L. Cranson D. Trinkaus-Randall V. J. Cell Biochem. 2004; 91: 938-950Crossref PubMed Scopus (57) Google Scholar, 53Gazel A. Nijhawan R.I. Walsh R. Blumenberg M. J. Cell Physiol. 2008; 215: 292-308Crossref PubMed Scopus (47) Google Scholar). 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 (54Ho Y. Samarasinghe R. Knoch M.E. Lewis M. Aizenman E. DeFranco D.B. Mol. Pharmacol. 2008; 74: 1141-1151Crossref PubMed Scopus (78) Google Scholar, 55Sarfaraz S. Afaq F. Adhami V.M. Malik A. Mukhtar H. J. Biol. Chem. 2006; 281: 39480-39491Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). However, in HaCaT cells upon exposure to UVA, sustained ERK1/2 phosphorylation via activation of metalloproteinases may serve as a survival signal suppressing apoptosis (56Han W. He Y.Y. Photochem. Photobiol. 2009; 85: 997-1003Crossref PubMed Scopus (24) Google Scholar). 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 (57Campbell M. Trimble E.R. Circ. Res. 2005; 96: 197-206Crossref PubMed Scopus (67) Google Scholar, 58Greco S. Storelli C. Marsigliante S. J. Endocrinol. 2006; 188: 79-89Crossref PubMed Scopus (49) Google Scholar) and in melanoma cell growth (59Sinnberg T. Lasithiotakis K. Niessner H. Schittek B. Flaherty K.T. Kulms D. Maczey E. Campos M. Gogel J. Garbe C. Meier F. J. Invest. Dermatol. 2009; 129: 1500-1515Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 60Lasithiotakis K.G. Sinnberg T.W. Schittek B. Flaherty K.T. Kulms D. Maczey E. Garbe C. Meier F.E. J. Invest. Dermatol. 2008; 128: 2013-2023Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar).The NHE1, once thought to was also shown to cell proliferation and migration L. Int. J. Biochem. Cell Biol. 2005; 37: PubMed Scopus Google Scholar, J. S. 2004; PubMed Scopus Google Scholar, D.L. Pharmacol. Toxicol. PubMed Scopus Google Scholar, M. H. D.L. Am. J. Physiol. Cell Physiol. 2004; PubMed Scopus Google Scholar, L. P. P. A. J. Cell Biol. 2009; PubMed Scopus (75) Google for by promoting the D.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and cell F. M. K. P. M. A. I. E. 2007; PubMed Scopus Google Scholar) or via its interaction with D.L. J. Cell Biol. 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Dermatol. 2005; Full Text Full Text PDF PubMed Scopus Google we show that the exchanger NHE1 is activated in skin cells following cellular The robust up-regulation of NHE1 activity by Zn2+ in keratinocytes suggests an role for this ion in The of found in the layer of the skin is essential for of the M. I. M. J. Invest. Dermatol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, R. S. N. Eur. J. 2009; PubMed Scopus Google Scholar). Zn2+ deficiency the response D.L. Bao S. B. Med. 2009; 37: PubMed Scopus Google Scholar) and a role for this ion in wound healing was to be an A effect of Zn2+ may of the and the epithelial that is by NHE1 activity or expression M. I. M. J. Invest. Dermatol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C. G. H. A. Eur. J. Cell Biol. 2008; PubMed Scopus Google Scholar, L. M. A. J. S. J. A. C. J. Physiol. 2007; PubMed Scopus Google Scholar). results that following skin injury, the release of Zn2+ or its topical application may of the and an molecule, from Zn2+, that is released from epithelial is ATP J. Cell Sci. 1997; PubMed Google which can a metabotropic Ca2+ response mediated via the receptors L. Cranson D. Trinkaus-Randall V. J. Cell Biochem. 2004; 91: 938-950Crossref PubMed Scopus (57) Google Scholar, M. S. A. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. B. Invest. Sci. 2005; 46: PubMed Scopus Google Scholar). results show that Zn2+ and ATP are released following injury and can Ca2+ signaling and activation of the MAPK pathway leading to up-regulation of NHE1 activity. Interestingly, activation of the ZnR pathway in cells induces the release of ATP H. Hershfinkel M. Biochem. Biophys. Res. Commun. 2005; PubMed Scopus (30) Google this may a for the release of this that further the metabotropic Ca2+ signaling. The role of ATP in wound healing is Activation of the receptor and Ca2+ release shown to and to enhanced wound closure J. K. J. A. J. Cell Sci. 2007; PubMed Scopus Google Scholar, I. L. C. V. Trinkaus-Randall V. Exp. Res. 2007; 85: PubMed Scopus (49) Google Scholar). that ATP ERK1/2 and PI3K activation and cell migration S. J. E. F. F. G. J. H. M. J. 2007; PubMed Scopus Google Scholar). as well as L. Cranson D. Trinkaus-Randall V. J. Cell Biochem. 2004; 91: 938-950Crossref PubMed Scopus (57) Google show that ATP activates the MAPK pathway leading for to epithelial cell migration M. S. A. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). Importantly, our results indicate that the signaling triggered by ATP induced up-regulation of NHE1 activity and enhanced in vitro scratch results further a role for ATP in promoting wound healing, as of Zn2+ and scavenging of ATP the Ca2+ response triggered by conditioned that following injury, Zn2+ and ATP are of the Ca2+ signaling that is essential for wound of the keratinocytic-ZnR activity mediated by GPR39 is by the of its signaling pathway in epithelial proliferation and migration. the highly selective we further show release of Zn2+ to the extracellular medium following injury, indicating that changes in the of extracellular Zn2+ this The of Zn2+ released following a scratch or in the conditioned medium was sufficient to the high affinity ZnR-dependent Ca2+ signaling in keratinocytic the effects of Zn2+ on wound healing previously our results the between Zn2+ and this process via the specific of that not the ZnR may in the an to accelerate wound IntroductionFollowing 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 (1Jacinto A. Martinez-Arias A. Martin P. Nat. Cell Biol. 2001; 3: E117-123Crossref PubMed Scopus (289) Google Scholar). 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 (2Lansdown A.B. Mirastschijski U. Stubbs N. Scanlon E. Agren M.S. Wound Repair Regen. 2007; 15: 2-16Crossref PubMed Scopus (406) Google Scholar). 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 (2Lansdown A.B. Mirastschijski U. Stubbs N. Scanlon E. Agren M.S. Wound Repair Regen. 2007; 15: 2-16Crossref PubMed Scopus (406) Google Scholar, 3Barceloux D.G. J. Toxicol. Clin. Toxicol. 1999; 37: 279-292Crossref PubMed Scopus (190) Google Scholar, 4Lansdown A.B. Lancet. 1996; 347: 706-707Abstract PubMed Scopus (87) Google Scholar, 5Schwartz J.R. Marsh R.G. Draelos Z.D. Dermatol. Surg. 2005; 31 (discussion 847): 837-847Crossref PubMed Scopus (97) Google Scholar). 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 (6Jensen S.L. McCuaig C. Zembowicz A. Hurt M.A. J. Cutan. Pathol. 2008; 35: 1-13Crossref PubMed Scopus (30) Google Scholar, 7Takahashi H. Nakazawa M. Takahashi K. Aihara M. Minami M. Hirasawa T. Ikezawa Z. J. Dermatol. Sci. 2008; 50: 31-39Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Whereas the role of Zn2+ transporters in diseases linked to dyshomeostasis of this ion has been described (8Andrews G.K. Biochem. Soc. Trans. 2008; 36: 1242-1246Crossref PubMed Scopus (85) Google Scholar, 9Kambe T. Andrews G.K. Mol. Cell Biol. 2009; 29: 129-139Crossref PubMed Scopus (95) Google Scholar, 10Kambe T. Weaver B.P. Andrews G.K. Genesis. 2008; 46: 214-228Crossref PubMed Scopus (101) Google Scholar, 11Besecker B. Bao S. Bohacova B. Papp A. Sadee W. Knoell D.L. Am. J. Physiol. Lung Cell Mol. Physiol. 2008; 294: L1127-L1136Crossref PubMed Scopus (116) Google Scholar, 12Hogstrand C. Kille P. Nicholson R.I. Taylor K.M. Trends Mol. Med. 2009; 15: 101-111Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 13Taylor K.M. Biochem. Soc. Trans. 2008; 36: 1247-1251Crossref PubMed Scopus (39) Google Scholar) cellular mechanisms linking Zn2+ to keratinocytes proliferation and migration are not well understood.Several studies have highlighted a role for Zn2+ in cellular signaling (12Hogstrand C. Kille P. Nicholson R.I. Taylor K.M. Trends Mol. Med. 2009; 15: 101-111Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 14Zhao L. Chen W. Taylor K.M. Cai B. Li X. Biochem. Biophys. Res. Commun. 2007; 363: 82-88Crossref PubMed Scopus (55) Google Scholar). Of particular importance is the activation of mitogen-activated protein kinase (MAPK) 2The abbreviations used are: MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseNHENa+/H+ exchangerZnRZn2+-sensing receptorCa2+iintracellular Ca2+pHiintracellular pHDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumBSAbovine serum albumin. and PI3 kinase pathways, leading to enhanced cell proliferation and survival (15Hershfinkel M. Tamas M. Martinoia E. Molecular Biology of Metal Homeostasis and Detoxification. springer-verlag, Berlin2006Google Scholar, 16Hershfinkel M. Silverman W.F. Sekler I. Mol. Med. 2007; 13: 331-336Crossref PubMed Scopus (75) Google Scholar, 17Murakami M. Hirano T. Cancer Sci. 2008; 99: 1515-1522Crossref PubMed Scopus (251) Google Scholar, 18Kaisman-Elbaz T. Sekler I. Fishman D. Karol N. Forberg M. Kahn N. Hershfinkel M. Silverman W.F. J. Cell Physiol. 2009; 220: 222-229Crossref PubMed Scopus (26) Google Scholar, 19Dubi N. Gheber L. Fishman D. Sekler I. Hershfinkel M. Carcinogenesis. 2008; 29: 1692-1700Crossref PubMed Scopus (52) Google Scholar, 20Azriel-Tamir H. Sharir H. Schwartz B. Hershfinkel M. J. Biol. Chem. 2004; 279: 51804-51816Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In epidermal tissues, higher levels of zinc have been correlated with higher mitotic activity (21Rothstein J.D. Martin L. Levey A.I. Dykes-Hoberg M. Jin L. Wu D. Nash N. Kuncl R.W. Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1449) Google Scholar). It was further shown that application of Zn2+ accelerates the migration of keratinocytes via modulation of integrin receptors expression or interaction with metalloproteases (22Ravanti L. Kahari V.M. Int. J. Mol. Med. 2000; 6: 391-407PubMed Google Scholar, 23Pirila E. Korpi J.T. Korkiamaki T. Jahkola T. Gutierrez-Fernandez A. Lopez-Otin C. Saarialho-Kere U. Salo T. Sorsa T. Wound Repair Regen. 2007; 15: 47-57Crossref PubMed Scopus (61) Google Scholar, 24Tenaud I. Leroy S. Chebassier N. Dreno B. Exp. Dermatol. 2000; 9: 407-416Crossref PubMed Scopus (58) Google Scholar, 25Tenaud I. Saiagh I. Dreno B. J. Dermatolog. Treat. 2009; 20: 90-93Crossref PubMed Scopus (6) Google Scholar). The effect of Zn2+ in enhancing cell proliferation is synergistic with intracellular Ca2+ rise, indicating that their effects are mediated through a common pathway (26Huang J.S. Mukherjee J.J. Chung T. Crilly K.S. Kiss Z. Eur. J. Biochem. 1999; 266: 943-951Crossref PubMed Scopus (46) Google Scholar, 27Kiss Z. Crilly K.S. Tomono M. FEBS Lett. 1997; 415: 71-74Crossref PubMed Scopus (24) Google Scholar). 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 (28MacDonald R.S. Wollard-Biddle L.C. Browning J.D. Thornton Jr., W.H. O'Dell B.L. J. Nutr. 1998; 128: 1600-1605Crossref PubMed Scopus (39) Google Scholar).We have previously identified a Zn2+-sensing receptor (ZnR) active in keratinocytes and other epithelial cells (16Hershfinkel M. Silverman W.F. Sekler I. Mol. Med. 2007; 13: 331-336Crossref PubMed Scopus (75) Google Scholar, 29Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 11749-11754Crossref PubMed Scopus (199) Google Scholar). ZnR activity was manifested by robust increase in intracellular Ca2+ concentrations triggered by extracellular Zn2+ (29Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 11749-11754Crossref PubMed Scopus (199) Google Scholar). Subsequent studies have found that in neurons ZnR activity is mediated by the G-protein-coupled receptor, GPR39 (30Besser L. Chorin E. Sekler I. Silverman W.F. Atkin S. Russell J.T. Hershfinkel M. J. Neurosci. 2009; 29: 2890-2901Crossref PubMed Scopus (171) Google Scholar). 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