Inactivation of the Calcium Sensing Receptor Inhibits E-cadherin-mediated Cell-Cell Adhesion and Calcium-induced Differentiation in Human Epidermal Keratinocytes

Extracellular Ca2+ (Ca2+o) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca2+ signaling during Ca2+o-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca2+o-induced increase in intracellular free calcium (Ca2+i) and expression of terminal differentiation genes, while promoting apoptosis. Ca2+o also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca2+o concentration ([Ca2+]o) from 0.03 to 2 mm rapidly induced the co-localization of α-, β-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca2+o-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca2+o-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of the E-cadherin-catenin-PI3K complex was sensitive to the pharmacologic inhibition of Src family tyrosine kinases but was not affected by inhibition of Ca2+o-induced rise in Ca2+i. Inhibition of CaR expression blocked the Ca2+o-induced tyrosine phosphorylation of β-, γ-, and p120-catenin, PI3K, and the tyrosine kinase Fyn and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca2+o-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling. Extracellular Ca2+ (Ca2+o) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca2+ signaling during Ca2+o-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca2+o-induced increase in intracellular free calcium (Ca2+i) and expression of terminal differentiation genes, while promoting apoptosis. Ca2+o also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca2+o concentration ([Ca2+]o) from 0.03 to 2 mm rapidly induced the co-localization of α-, β-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca2+o-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca2+o-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of the E-cadherin-catenin-PI3K complex was sensitive to the pharmacologic inhibition of Src family tyrosine kinases but was not affected by inhibition of Ca2+o-induced rise in Ca2+i. Inhibition of CaR expression blocked the Ca2+o-induced tyrosine phosphorylation of β-, γ-, and p120-catenin, PI3K, and the tyrosine kinase Fyn and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca2+o-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling. Keratinocytes proliferate and differentiate in cultures in a manner recapitulating the process of epidermal differentiation in vivo (1Eckert R.L. Crish J.F. Robinson N.A. Physiol. Rev. 1997; 77: 397-424Crossref PubMed Scopus (345) Google Scholar). Raising the [Ca2+] 2The abbreviations used are: Ca2+oextracellular Ca2+;Ca2+i, intracellular Ca2+CaRcalcium sensing receptorPLCphospholipase CAJadherens junctionPI3Kphosphatidylinositol 3-kinaseBAPTAbis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-AM2APB2-aminoethoxydiphenyl boratemAbmonoclonal antibodyPBSphosphate-buffered salineNHKnormal human keratinocytes. o above 0.07 mm triggers an acute and then a sustained increase in Ca2+i, and the onset of terminal differentiation (2Pillai S. Bikle D.D. J. Cell Physiol. 1991; 146: 94-100Crossref PubMed Scopus (126) Google Scholar, 3Sharpe G.R. Fisher C. Gillespie J.I. Greenwell J.R. Arch. Dermatol. Res. 1993; 284: 445-450Crossref PubMed Scopus (36) Google Scholar). Blocking the increase in Ca2+i with BAPTA, a Ca2+i chelator, inhibits the ability of Ca2+o to induce differentiation (4Li L. Tucker R.W. Hennings H. Yuspa S.H. Cell Growth & Differ. 1995; 6: 1171-1184PubMed Google Scholar, 5Li L. Tucker R.W. Hennings H. Yuspa S.H. J. Cell Physiol. 1995; 163: 105-114Crossref PubMed Scopus (84) Google Scholar). Previous studies demonstrated an obligatory role for the CaR, a G-protein-coupled receptor, in mediating Ca2+ signaling and Ca2+o-induced keratinocyte differentiation (6Tu C.L. Chang W. Bikle D.D. J. Biol. Chem. 2001; 276: 41079-41085Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 7Komuves L. Oda Y. Tu C.L. Chang W.H. Ho-Pao C.L. Mauro T. Bikle D.D. J. Cell Physiol. 2002; 192: 45-54Crossref PubMed Scopus (67) Google Scholar, 8Tu C.L. Chang W. Bikle D.D. J. Investig. Dermatol. 2007; 127: 1074-1083Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Activation of the CaR by Ca2+o stimulates phospholipase C (PLC) to convert phosphatidylinositol 4,5-bisphosphate into diacylglylcerol and inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate in turn induces Ca2+ release from internal stores and increases Ca2+i (9Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). Inhibition of CaR expression led to a decline of Ca2+i pools and altered cellular Ca2+ handling, hence reducing the Ca2+i response to Ca2+o and impairing differentiation in keratinocytes (8Tu C.L. Chang W. Bikle D.D. J. Investig. Dermatol. 2007; 127: 1074-1083Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). extracellular Ca2+;Ca2+i, intracellular Ca2+ calcium sensing receptor phospholipase C adherens junction phosphatidylinositol 3-kinase bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-AM 2-aminoethoxydiphenyl borate monoclonal antibody phosphate-buffered saline normal human keratinocytes. One of the immediate cellular responses to Ca2+o in epithelial cells is the formation of cell-cell contacts, a process mediated by the adhesion molecule E-cadherin (10Perez-Moreno M. Jamora C. Fuchs E. Cell. 2003; 112: 535-548Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). E-cadherin is a major classical cadherin in keratinocytes and is expressed throughout the epidermis (11Furukawa F. Fujii K. Horiguchi Y. Matsuyoshi N. Fujita M. Toda K. Imamura S. Wakita H. Shirahama S. Takigawa M. Microsc. Res. Tech. 1997; 38: 343-352Crossref PubMed Scopus (71) Google Scholar). Upon Ca2+o stimulation, the extracellular portion of E-cadherin interacts with E-cadherin molecules on the surface of neighboring cells, whereas its cytoplasmic tail interacts with β- (or γ-), α-, and p120-catenins to form the core adhesive structure of adherens junctions (AJ) (12Pokutta S. Weis W.I. Annu. Rev. Cell Dev. Biol. 2007; 23: 237-261Crossref PubMed Scopus (285) Google Scholar). E-cadherin-mediated cell adhesion plays key roles in remodeling of epithelial cell-cell interaction and maintaining proper epidermal differentiation (10Perez-Moreno M. Jamora C. Fuchs E. Cell. 2003; 112: 535-548Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 13Furukawa F. Takigawa M. Matsuyoshi N. Shirahama S. Wakita H. Fujita M. Horiguchi Y. Imamura S. J. Dermatol. 1994; 21: 802-813Crossref PubMed Scopus (38) Google Scholar). Loss of E-cadherin in the epidermis leads to a loss of AJ and impaired terminal differentiation (14Young P. Boussadia O. Halfter H. Grose R. Berger P. Leone D.P. Robenek H. Charnay P. Kemler R. Suter U. EMBO J. 2003; 22: 5723-5733Crossref PubMed Scopus (114) Google Scholar). The sequential binding of catenins physically links E-cadherin to the actin cytoskeleton and other signaling molecules, including phosphatidylinositol 3-kinase (PI3K) (10Perez-Moreno M. Jamora C. Fuchs E. Cell. 2003; 112: 535-548Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 15Wheelock M.J. Johnson K.R. Curr. Opin. Cell Biol. 2003; 15: 509-514Crossref PubMed Scopus (235) Google Scholar). Keratinocyte differentiation induced by Ca2+o necessitates the activation of PI3K. Pharmacological inhibition of PI3K blocks the expression of late differentiation markers and induces apoptosis in differentiating keratinocytes (16Calautti E. Li J. Saoncella S. Brissette J.L. Goetinck P.F. J. Biol. Chem. 2005; 280: 32856-32865Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar). Through interactions with the E-cadherin-catenin complex PI3K is recruited to the cell membrane, where it converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate. Phosphatidylinositol 3,4,5-triphosphate in turn binds and activates PLCγ1 (17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar), which is required for maintaining the Ca2+o-induced increase in Ca2+i crucial for keratinocyte differentiation (18Xie Z. Bikle D.D. J. Biol. Chem. 1999; 274: 20421-20424Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). E-cadherin by antibody E-cadherin expression by activation of PI3K keratinocyte differentiation (16Calautti E. Li J. Saoncella S. Brissette J.L. Goetinck P.F. J. Biol. Chem. 2005; 280: 32856-32865Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, Z. Bikle D.D. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). E-cadherin, studies demonstrated that the Ca2+o-induced of PI3K to cell membrane, activation of PI3K, and keratinocyte differentiation β- and but not Z. Bikle D.D. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). cell adhesion plays a key role not in cellular and in but also in cellular signals that keratinocyte differentiation. Keratinocyte differentiation induced by Ca2+o is by tyrosine phosphorylation (17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar, E. E. M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, E. C. Dev. 1995; PubMed Scopus Google Scholar). studies demonstrated that tyrosine kinase is for the assembly of AJ and the interaction of PI3K with the E-cadherin-catenin Pharmacological inhibition of tyrosine kinase the formation of AJ and activation of PI3K by Ca2+o (16Calautti E. Li J. Saoncella S. Brissette J.L. Goetinck P.F. J. Biol. Chem. 2005; 280: 32856-32865Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar, J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). differentiating Ca2+o-induced assembly of E-cadherin-catenin complex and the of PI3K to E-cadherin by tyrosine phosphorylation of β-, γ-, and p120-catenin E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar). The of E-cadherin/PI3K signaling by Ca2+o activation of Src family tyrosine Src family tyrosine kinases blocks formation of AJ E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar), Ca2+o activation of PI3K, and expression of differentiation markers in keratinocytes (17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar). with the that Src family kinases keratinocyte cell-cell adhesion, Fyn tyrosine kinase with E-cadherin at the cell membrane E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar). keratinocytes tyrosine phosphorylation of β-, γ-, and p120-catenin, in cell adhesion E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar), and differentiation E. C. Dev. 1995; PubMed Scopus Google Scholar). in to in cells, tyrosine phosphorylation plays a role in of cell adhesion in differentiating keratinocytes R.W. J. Mol. 2007; PubMed Scopus Google Scholar). Ca2+o activates Ca2+i signaling and E-cadherin-mediated cell-cell adhesion that to differentiation. the we the of CaR in the activation of E-cadherin signaling. CaR expression blocked the Ca2+o-induced formation of the association of PI3K with the E-cadherin-catenin and expression of late differentiation the Ca2+o-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and Fyn blocked in the keratinocytes. that the CaR regulates the E-cadherin/PI3K pathway a Src family tyrosine kinase-mediated signaling and on keratinocyte differentiation. membrane Ca2+ acid-AM 2-aminoethoxydiphenyl borate and Src kinase and its from of these in other from used in of to the for and from The antibody for CaR, was the to of the human keratinocyte CaR (6Tu C.L. Chang W. Bikle D.D. J. Biol. Chem. 2001; 276: 41079-41085Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). and E-cadherin, α-, β-, γ-, and p120-catenin, and the of PI3K, from was from was from Cell human keratinocytes from human and in keratinocyte (17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar). keratinocytes from human by and cultures in 0.07 mm keratinocytes in 0.03 mm and used in the of and of the antisense human CaR cDNA and the an (8Tu C.L. Chang W. Bikle D.D. J. Investig. Dermatol. 2007; 127: 1074-1083Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). and an and used to inactivation of the CaR, with the in 0.03 mm and for to to mm for to induce formation of AJ for to induce differentiation. the was with 0.03 mm was the cells with an expression of late differentiation was by with in 0.03 mm and for to mm for then the to the of by to was on cDNA and from of late differentiation markers in keratinocytes to for cultures on with cells in 0.03 mm for 2 in cells by in apoptosis to the cells and endogenous is by with in the of at for and then with with the with for to the the and of and a The of apoptosis was the of cells cells in the was used for of Ca2+i responses to Ca2+i was a Y. Tu C.L. Chang W. L. Mauro T. Bikle D.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). keratinocytes with an the antisense human CaR cDNA a on a in 0.03 mm to cells with in in mm mm mm mm 0.07 mm then and in 0.03 mm Ca2+ to 2 mm The cells with and and the at was at The signals from to cells for was by the of by mm Ca2+ concentration was from the of at the on the at and for for Ca2+ is Cell and cell and membrane proteins from to adenoviral Keratinocytes in mm and for on in mm mm with mm and cell for at and the was Keratinocyte membrane the to the The in the cell and membrane by the of through reducing and with in mm mm mm the at with E-cadherin, and p120-catenin at a of β- and at a of Fyn and at a of and at a of the with for at The antibody was the and to cell of by of β-, γ-, and p120-catenin, and and membrane proteins by to E-cadherin and by in of at with in and then by on with for at and with in for with in cells with of at for cells with the antibody at for cells with at for in on and examined with a Inhibition of CaR Ca2+i to and Ca2+o-induced Keratinocyte the role of CaR in activation of E-cadherin signaling and Ca2+o-induced we inhibited CaR expression by human keratinocytes with an a CaR antisense cDNA and that blocked the endogenous CaR with the cells with a To whether inhibition of CaR expression altered Ca2+ signaling, we examined its impact on Ca2+i response to with studies (6Tu C.L. Chang W. Bikle D.D. J. Biol. Chem. 2001; 276: 41079-41085Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 8Tu C.L. Chang W. Bikle D.D. J. Investig. Dermatol. 2007; 127: 1074-1083Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), of CaR inhibited the Ca2+o-induced increase in Ca2+i. in Ca2+o from 0.03 to 2 mm induced an increase in Ca2+i in keratinocytes with the from Keratinocytes with the Ca2+i but a in the rise of Ca2+i in response to 2 mm of CaR inhibited but not the ability of Ca2+o to increase Ca2+i, other calcium of CaR to a role in mediating Ca2+o-induced Ca2+i To whether the in Ca2+ signaling in CaR expression affected cell we the apoptosis in keratinocytes with to that in cells with by of cells that of keratinocytes apoptosis in 0.03 mm whereas the of cells in the keratinocytes and in 0.03 and mm an role of CaR in cell To whether the in CaR expression affected we examined its impact on Ca2+o-induced expression of terminal differentiation by in keratinocytes with in of in mm Ca2+ the of terminal differentiation markers and in keratinocytes with and but to in the cells with Ca2+o CaR a for keratinocytes. results demonstrated that CaR regulates calcium signaling, cell and differentiation in keratinocytes. Inhibition of CaR E-cadherin-mediated and of PI3K to the Cell of the cellular responses to Ca2+o in keratinocytes is the formation of the E-cadherin-catenin complex at cell-cell and the of core adhesive structure to the actin To whether CaR is required for Ca2+o activation of E-cadherin signaling, we examined the impact of CaR on the assembly of the E-cadherin-catenin that the of E-cadherin-mediated cell-cell AJ formation the Ca2+o was from 0.03 to 2 mm in keratinocytes Ca2+o the co-localization of E-cadherin with β- and p120-catenin actin in AJ in whereas inhibition of CaR expression by blocked the ability of Ca2+o to induce the formation of then whether inhibition of CaR expression the of the of the E-cadherin signaling on the cell from keratinocytes with and with 2 mm Ca2+ E-cadherin, α-, β-, γ-, and and the of PI3K. CaR the Ca2+ altered the expression of these proteins not To the we whether CaR the cell membrane of these on the membrane demonstrated that of Ca2+ of E-cadherin, α-, β-, γ-, p120-catenin, and to the cell membrane in whereas the Ca2+o-induced membrane association of E-cadherin, catenins, and in the cells was used a to membrane The of the membrane was the antibody the membrane but not the antibody with the membrane to whether CaR the complex formation of E-cadherin, catenins, and PI3K. Keratinocytes with then with 2 mm Ca2+ for membrane with a monoclonal antibody E-cadherin and with The then for the of E-cadherin, α-, β-, γ-, p120-catenin, and by in Ca2+o the complex formation of E-cadherin, α-, β-, γ-, and p120-catenin, and in but it to in the was used a for membrane for Inhibition of CaR expression suppressed the complex formation of E-cadherin, catenins, and PI3K at the cell membrane, a key role for CaR in the Ca2+o activation of E-cadherin/PI3K Src the Activation of E-cadherin to an increase in Ca2+i, the of keratinocyte differentiation is with tyrosine kinase (17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar, E. C. Dev. 1995; PubMed Scopus Google Scholar). To whether the activation of E-cadherin pathway by Ca2+o is a of Ca2+i the activation of tyrosine we keratinocytes with a for the Src family tyrosine and that the Ca2+o-induced increase in Ca2+i, then examined impact on AJ formation. in that the intercellular E-cadherin-mediated adhesive structure a of 2 mm Ca2+ in normal keratinocytes with keratinocytes with the Ca2+i the Ca2+ and inositol 1,4,5-trisphosphate receptor for on the Ca2+o-induced formation of the E-cadherin-catenin complex at the other of keratinocytes with blocked the formation of cell-cell adhesion The of and on the Ca2+o-induced increase in Ca2+i was by the of Ca2+i in response to the of whereas the Ca2+o-induced rise in Ca2+i results indicate that Ca2+i is not a major that regulates cell-cell of the membrane for E-cadherin and demonstrated that blocked the Ca2+o-induced of PI3K to E-cadherin, whereas the the of affected the of PI3K by E-cadherin studies that affected the Ca2+o-induced formation of complex not results indicate that Src family tyrosine kinase-mediated signaling, but not Ca2+i, plays a major role in the Ca2+o activation of E-cadherin/PI3K signaling. of keratinocytes that tyrosine phosphorylation of β-, γ-, and p120-catenin is essential for association with E-cadherin and the activation of PI3K, and that Fyn tyrosine kinase is in process E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar, E. M. C. Y. M. Y. Li J. J. Cell Biol. 2002; PubMed Scopus Google Scholar). To whether CaR regulates the Ca2+o activation of E-cadherin-mediated cell-cell adhesion tyrosine kinase-mediated signaling, we examined the impact of CaR on tyrosine phosphorylation of β-, γ-, and p120-catenin, and PI3K. Keratinocytes with and to 2 mm Ca2+ for to induce cell junction formation. cell and with an antibody then by to β-, γ-, p120-catenin, and tyrosine kinases Src and in Ca2+o the tyrosine phosphorylation of β-, γ-, p120-catenin, and but not in whereas the of tyrosine phosphorylation of these proteins by Ca2+o was in the was used a for for the cell by β-, γ-, p120-catenin, and and by for Our results that CaR the Ca2+o-induced cell-cell adhesion tyrosine kinase-mediated signaling. To the role of Src and Fyn in the Ca2+o-induced cell-cell adhesion, we whether these proteins with E-cadherin and PI3K at the cell on membrane of and keratinocytes demonstrated that Ca2+o the membrane of not to the cell membrane in but to in cells with of cell that the expression of Src and Fyn not by Ca2+ CaR demonstrated that Ca2+o induced of Fyn to the complex at the cell membrane in keratinocytes the Ca2+o of the association of Fyn with E-cadherin and PI3K was blocked CaR expression was inhibited by association of Src with E-cadherin PI3K in the cell membrane was by the not results indicate that CaR with the Ca2+o activation of E-cadherin-mediated cell adhesion to tyrosine kinase signaling that was mediated by the Fyn tyrosine E-cadherin-mediated intercellular adhesion is critical for maintaining the of and for differentiation of epidermal keratinocytes. keratinocyte Ca2+o stimulates the interaction of E-cadherin with the actin cytoskeleton the binding of to actin and of other the AJ and to C. Fuchs E. Dev. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). adhesion, E-cadherin signaling and keratinocytes that crucial for cell survival and differentiation (16Calautti E. Li J. Saoncella S. Brissette J.L. Goetinck P.F. J. Biol. Chem. 2005; 280: 32856-32865Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, Z. Bikle D.D. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). E-cadherin interacts with the kinase PI3K at the Phosphatidylinositol 3,4,5-triphosphate by the PI3K activates its which promotes differentiation and keratinocytes from apoptosis (16Calautti E. Li J. Saoncella S. Brissette J.L. Goetinck P.F. J. Biol. Chem. 2005; 280: 32856-32865Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Phosphatidylinositol 3,4,5-triphosphate also binds to and activates PLCγ1 (17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar), which is essential for maintaining the Ca2+o-induced increase in Ca2+i and keratinocyte differentiation (18Xie Z. Bikle D.D. J. Biol. Chem. 1999; 274: 20421-20424Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). of E-cadherin in cell L. M. J. Kemler R. U. S. 1994; PubMed Scopus Google and in affected differentiation and cell O. S. Kemler R. Dev. 2002; PubMed Scopus Google Scholar). of the E-cadherin gene in led to loss of AJ and altered epidermal differentiation (14Young P. Boussadia O. Halfter H. Grose R. Berger P. Leone D.P. Robenek H. Charnay P. Kemler R. Suter U. EMBO J. 2003; 22: 5723-5733Crossref PubMed Scopus (114) Google Scholar, C.L. T. Fuchs E. U. S. PubMed Scopus Google Scholar). we demonstrated that CaR in the Ca2+o activation of E-cadherin-mediated signaling. of CaR expression in keratinocytes in the intercellular adhesion, blocked Ca2+o-induced and activation of PI3K, inhibited Ca2+i signaling, cell and inhibited terminal differentiation. Ca2+o-induced increase in Ca2+i is critical for of Ca2+o-induced E-cadherin complex formation and cell-cell adhesion not a increase in Ca2+i, blocked results that CaR regulates cell-cell interactions a Src family signaling, which is mediated by AJ tyrosine kinase β-, and p120-catenin, tyrosine Ca2+ stimulation, with the of intercellular and the onset of E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar). phosphorylation of β- and increases the association of with E-cadherin, the of cell adhesion to the ability of the complex and the actin cytoskeleton E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar). Src family tyrosine kinases an part of the Ca2+o-induced E-cadherin signaling Src family tyrosine kinases blocked formation of Ca2+o activation of PI3K E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar), and suppressed expression of differentiation markers in keratinocytes (17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar). cells, Src is required for the of PI3K to that in human Ca2+o by the membrane of and induced association of Fyn with E-cadherin and PI3K, the of Fyn in the Ca2+o activation of the E-cadherin/PI3K Our results with the in Fyn was during differentiation E. C. Dev. 1995; PubMed Scopus Google induced by Ca2+o and was to with E-cadherin at the cell-cell E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar). tyrosine phosphorylation of β-, γ-, and p120-catenin and cell adhesion in keratinocytes Fyn E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar). Fyn is not the kinase to in E-cadherin signaling, a of and is required to the Ca2+o-induced PI3K activation and keratinocyte differentiation (17Xie Z. Singleton P.A. Bourguignon L.Y. Bikle D.D. Mol. Biol. Cell. 2005; 16: 3236-3246Crossref PubMed Scopus (85) Google Scholar). the of with a the of with a of the and tyrosine phosphorylation of the and p120-catenin and impaired cell adhesion, the of the Src kinase family E. S. M. N. J. Cell Biol. PubMed Scopus Google Scholar). The CaR to proteins in signaling to the of C. M. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and extracellular kinase O. R. M. T. T. J. Physiol. 2001; 280: PubMed Google Scholar), to and phospholipase C. N. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and to PI3K M. H. J. 2003; 23: PubMed Google Scholar). the we Src family tyrosine kinase-mediated signaling of CaR of CaR in keratinocytes blocked the Ca2+o-induced activation and association of Fyn with E-cadherin and PI3K, the tyrosine phosphorylation of β-, γ-, and and inhibited cell adhesion and E-cadherin/PI3K signaling. the by which the CaR regulates Src family kinases is Fyn is a of in intercellular adhesion, endogenous inhibited Ca2+o-induced Fyn tyrosine phosphorylation of β-, γ-, and and AJ formation E. M. C. Y. M. Y. Li J. J. Cell Biol. 2002; PubMed Scopus Google Scholar). studies demonstrated the ability of CaR to signaling by to C. Z. J. Physiol. PubMed Scopus Google Scholar, O. S.H. J. L. E. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google M. Tu T. 2002; PubMed Scopus Google Scholar). activation of the interaction of the of CaR with the H. C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, O. N. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). interacts with a of C. C. Y. Cell Biol. PubMed Scopus Google to and M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). a by which the CaR regulates keratinocyte cell-cell Ca2+o regulates cell survival and induces keratinocyte differentiation by at least activation of CaR by Ca2+o stimulates the pathway to increase Ca2+i and kinase C and signaling a Ca2+o induces the formation of E-cadherin-mediated a for and activating other signaling molecules, PI3K, and which is critical for differentiation. of CaR pathway results in differentiation in although other also in cellular for and the Cell and of for human epidermal keratinocytes.