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• W2017209369 abstract "Cell migration depends on cells being able to create and disassemble adhesive contacts. Hemidesmosomes are multiprotein structures that attach epithelia to basal lamina and disassemble during migration and carcinoma invasion. Phosphorylation of the β4 integrin, a hemidesmosome component, induces disassembly. Although kinases involved in β4 phosphorylation have been identified, little is known about phosphatases countering kinase action. Here we report that calcineurin, a serine-threonine protein phosphatase, regulates β4 phosphorylation. Calcineurin inhibitor cyclosporin A (CsA) and calcineurin-siRNA increase β4 phosphorylation, induce hemidesmosome disassembly, and increase migration in HaCat keratinocytes, suggesting that calcineurin negatively regulates β4 phosphorylation. We found no direct dephosphorylation of β4 by calcineurin or association between β4 and calcineurin, suggesting indirect regulation of β4 phosphorylation. We therefore assessed calcineurin influence on MAPK and PKC, known to phosphorylate β4. CsA increased MAPK activity, whereas MAPK inhibitors reduced CsA-induced β4 phosphorylation, suggesting that calcineurin restricts β4 phosphorylation by MAPK. Calcineurin is activated by calcium. Increased [Ca2+]i reduces β4 phosphorylation and stabilizes hemidesmosomes, effects that are reversed by CsA, indicating that calcineurin mediates calcium effects on β4. However, MAPK activation is increased when [Ca2+]i is increased, suggesting that calcineurin activates an additional mechanism that counteracts MAPK-induced β4 phosphorylation. Interestingly, in some squamous cell carcinoma cells, which have reduced hemidesmosomes and increased β4 phosphorylation, an increase in [Ca2+]i using thapsigargin, bradykinin, or acetylcholine can increase hemidesmosomes and reduce β4 phosphorylation in a calcineurin-dependent manner. These findings have implications in calcineurin-inhibitor induced carcinoma, a complication of immunosuppressive therapy. Cell migration depends on cells being able to create and disassemble adhesive contacts. Hemidesmosomes are multiprotein structures that attach epithelia to basal lamina and disassemble during migration and carcinoma invasion. Phosphorylation of the β4 integrin, a hemidesmosome component, induces disassembly. Although kinases involved in β4 phosphorylation have been identified, little is known about phosphatases countering kinase action. Here we report that calcineurin, a serine-threonine protein phosphatase, regulates β4 phosphorylation. Calcineurin inhibitor cyclosporin A (CsA) and calcineurin-siRNA increase β4 phosphorylation, induce hemidesmosome disassembly, and increase migration in HaCat keratinocytes, suggesting that calcineurin negatively regulates β4 phosphorylation. We found no direct dephosphorylation of β4 by calcineurin or association between β4 and calcineurin, suggesting indirect regulation of β4 phosphorylation. We therefore assessed calcineurin influence on MAPK and PKC, known to phosphorylate β4. CsA increased MAPK activity, whereas MAPK inhibitors reduced CsA-induced β4 phosphorylation, suggesting that calcineurin restricts β4 phosphorylation by MAPK. Calcineurin is activated by calcium. Increased [Ca2+]i reduces β4 phosphorylation and stabilizes hemidesmosomes, effects that are reversed by CsA, indicating that calcineurin mediates calcium effects on β4. However, MAPK activation is increased when [Ca2+]i is increased, suggesting that calcineurin activates an additional mechanism that counteracts MAPK-induced β4 phosphorylation. Interestingly, in some squamous cell carcinoma cells, which have reduced hemidesmosomes and increased β4 phosphorylation, an increase in [Ca2+]i using thapsigargin, bradykinin, or acetylcholine can increase hemidesmosomes and reduce β4 phosphorylation in a calcineurin-dependent manner. These findings have implications in calcineurin-inhibitor induced carcinoma, a complication of immunosuppressive therapy. Hemidesmosomes (HD) 2The abbreviations used are: HDhemidesmosome(s)HD-LShemidesmosome-like structuresBKbradykininBPAGbullous pemphigoid antigenCNcalcineurinCsAcyclosporin AIFimmunofluorescence analysisPPPserine-threonine protein phosphatasesSCCsquamous cell carcinomaBAPTA-AM1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)THGthapsigarginAbantibody. are specialized multiprotein complexes that mediate attachment of epithelial cells to the underlying basement membrane (1Borradori L. Sonnenberg A. Hemidesmosomes: roles in adhesion, signaling, and human diseases.Curr. Opin. Cell Biol. 1996; 8: 647-656Crossref PubMed Scopus (198) Google Scholar, 2Green K.J. Jones J.C. Desmosomes and hemidesmosomes: structure and function of molecular components.FASEB J. 1996; 10: 871-881Crossref PubMed Scopus (296) Google Scholar, 3Margadant C. Frijns E. Wilhelmsen K. Sonnenberg A. Regulation of hemidesmosome disassembly by growth factor receptors.Curr. Opin. Cell Biol. 2008; 20: 589-596Crossref PubMed Scopus (83) Google Scholar). These structures provide stability and mechanical strength to epithelia and are disassembled in migrating cells during wound healing and carcinoma invasion (3Margadant C. Frijns E. Wilhelmsen K. Sonnenberg A. Regulation of hemidesmosome disassembly by growth factor receptors.Curr. Opin. Cell Biol. 2008; 20: 589-596Crossref PubMed Scopus (83) Google Scholar, 4Gipson I.K. Spurr-Michaud S. Tisdale A. Elwell J. Stepp M.A. Redistribution of the hemidesmosome components α6β4 integrin and bullous pemphigoid antigens during epithelial wound healing.Exp. Cell Res. 1993; 207: 86-98Crossref PubMed Scopus (78) Google Scholar, 5Herold-Mende C. Kartenbeck J. Tomakidi P. Bosch F.X. Metastatic growth of squamous cell carcinomas is correlated with up-regulation and redistribution of hemidesmosomal components.Cell Tissue Res. 2001; 306: 399-408Crossref PubMed Scopus (38) Google Scholar). HD are composed of several transmembrane and cytoplasmic proteins that connect the cytokeratin network to the extracellular matrix. Tissue fragility and blistering disease develop when hemidesmosomal components are genetically altered, indicating the significance of HD in maintaining epithelial tissue integrity (6Borradori L. Sonnenberg A. Structure and function of hemidesmosomes: more than simple adhesion complexes.J. Invest. Dermatol. 1999; 112: 411-418Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 7Christiano A.M. Uitto J. Molecular complexity of the cutaneous basement membrane zone. Revelations from the paradigms of epidermolysis bullosa.Exp. Dermatol. 1996; 5: 1-11Crossref PubMed Scopus (183) Google Scholar). The main organizer and nucleator of HD is the a6β4 integrin, a laminin receptor (8Nievers M.G. Schaapveld R.Q. Sonnenberg A. Biology and function of hemidesmosomes.Matrix Biol. 1999; 18: 5-17Crossref PubMed Scopus (164) Google Scholar, 9Mercurio A.M. Laminin receptors: achieving specificity through cooperation.Trends Cell Biol. 1995; 5: 419-423Abstract Full Text PDF PubMed Scopus (200) Google Scholar, 10Rabinovitz I. Mercurio A.M. The integrin α6β4 and the biology of carcinoma.Biochem. Cell Biol. 1996; 74: 811-821Crossref PubMed Scopus (103) Google Scholar). This integrin recruits the HD component plectin, which then facilitates further association of the integrin with components BPAG1 and BPAG2 (8Nievers M.G. Schaapveld R.Q. Sonnenberg A. Biology and function of hemidesmosomes.Matrix Biol. 1999; 18: 5-17Crossref PubMed Scopus (164) Google Scholar, 11Koster J. Geerts D. Favre B. Borradori L. Sonnenberg A. Analysis of the interactions between BP180, BP230, plectin, and the integrin α6β4 important for hemidesmosome assembly.J. Cell Sci. 2003; 116: 387-399Crossref PubMed Scopus (173) Google Scholar). The cytokeratin network connects the HD through BPAG1 and plectin (11Koster J. Geerts D. Favre B. Borradori L. Sonnenberg A. Analysis of the interactions between BP180, BP230, plectin, and the integrin α6β4 important for hemidesmosome assembly.J. Cell Sci. 2003; 116: 387-399Crossref PubMed Scopus (173) Google Scholar). hemidesmosome(s) hemidesmosome-like structures bradykinin bullous pemphigoid antigen calcineurin cyclosporin A immunofluorescence analysis serine-threonine protein phosphatases squamous cell carcinoma 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) thapsigargin antibody. Although HD provides a stable anchor, its components undergo a rapid turnover, indicating a dynamic equilibrium (12Geuijen C.A. Sonnenberg A. Dynamics of the α6β4 integrin in keratinocytes.Mol. Biol. Cell. 2002; 13: 3845-3858Crossref PubMed Scopus (87) Google Scholar, 13Tsuruta D. Hopkinson S.B. Jones J.C. Hemidesmosome protein dynamics in live epithelial cells.Cell Motil. Cytoskeleton. 2003; 54: 122-134Crossref PubMed Scopus (43) Google Scholar). This rapid turnover might allow epithelial cells to efficiently switch between a stationary and a migratory state by quickly changing the balance toward disassembly. Disassembly of HD becomes important during wound healing to allow cell migration and re-epithelialization (4Gipson I.K. Spurr-Michaud S. Tisdale A. Elwell J. Stepp M.A. Redistribution of the hemidesmosome components α6β4 integrin and bullous pemphigoid antigens during epithelial wound healing.Exp. Cell Res. 1993; 207: 86-98Crossref PubMed Scopus (78) Google Scholar). Importantly, disassembly of HD is also seen during carcinoma invasion, where a reduction of HD can predict metastatic potential (5Herold-Mende C. Kartenbeck J. Tomakidi P. Bosch F.X. Metastatic growth of squamous cell carcinomas is correlated with up-regulation and redistribution of hemidesmosomal components.Cell Tissue Res. 2001; 306: 399-408Crossref PubMed Scopus (38) Google Scholar). Most of the efforts to understand the regulation of HD disassembly have focused on the main organizer of the HD, the a6β4 integrin. The phosphorylation of the β4 integrin subunit has been shown to be an important mechanism to induce HD disassembly (Refs. 14Dans M. Gagnoux-Palacios L. Blaikie P. Klein S. Mariotti A. Giancotti F.G. Tyrosine phosphorylation of the β4 integrin cytoplasmic domain mediates Shc signaling to extracellular signal-regulated kinase and antagonizes formation of hemidesmosomes.J. Biol. Chem. 2001; 276: 1494-1502Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15Mariotti A. Kedeshian P.A. Dans M. Curatola A.M. Gagnoux-Palacios L. Giancotti F.G. EGF-R signaling through Fyn kinase disrupts the function of integrin α6β4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.J. Cell Biol. 2001; 155: 447-458Crossref PubMed Scopus (36) Google Scholar, 16Rabinovitz I. Toker A. Mercurio A.M. Protein kinase C-dependent mobilization of the α6β4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.J. Cell Biol. 1999; 146: 1147-1160Crossref PubMed Scopus (187) Google Scholar, 17Rabinovitz I. Tsomo L. Mercurio A.M. Protein kinase C-α phosphorylation of specific serines in the connecting segment of the β4 integrin regulates the dynamics of type II hemidesmosomes.Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar, 18Germain E.C. Santos T.M. Rabinovitz I. Phosphorylation of a novel site on the β4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar, 19Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Serine phosphorylation of the integrin β4 subunit is necessary for epidermal growth factor receptor-induced hemidesmosome disruption.Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar, 20Frijns E. Sachs N. Kreft M. Wilhelmsen K. Sonnenberg A. EGF-induced MAPK signaling inhibits hemidesmosome formation through phosphorylation of the integrin β4.J. Biol. Chem. 2010; 285: 37650-37662Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 21Frijns E. Kuikman I. Litjens S. Raspe M. Jalink K. Ports M. Wilhelmsen K. Sonnenberg A. Phosphorylation of threonine 1736 in the C-terminal tail of integrin β4 contributes to hemidesmosome disassembly.Mol. Biol. Cell. 2012; 23: 1475-1485Crossref PubMed Scopus (39) Google Scholar and reviewed in Ref. 3Margadant C. Frijns E. Wilhelmsen K. Sonnenberg A. Regulation of hemidesmosome disassembly by growth factor receptors.Curr. Opin. Cell Biol. 2008; 20: 589-596Crossref PubMed Scopus (83) Google Scholar). Growth factors that are secreted during wound healing or carcinoma invasion can induce the phosphorylation of the β4 integrin on serine and tyrosine residues and promote HD disassembly (16Rabinovitz I. Toker A. Mercurio A.M. Protein kinase C-dependent mobilization of the α6β4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.J. Cell Biol. 1999; 146: 1147-1160Crossref PubMed Scopus (187) Google Scholar, 17Rabinovitz I. Tsomo L. Mercurio A.M. Protein kinase C-α phosphorylation of specific serines in the connecting segment of the β4 integrin regulates the dynamics of type II hemidesmosomes.Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar, 22Mainiero F. Pepe A. Yeon M. Ren Y. Giancotti F.G. The intracellular functions of α6β4 integrin are regulated by EGF.J. Cell Biol. 1996; 134: 241-253Crossref PubMed Scopus (169) Google Scholar). Most of the phosphorylation on the β4 integrin occurs on serine residues of the connecting segment domain (17Rabinovitz I. Tsomo L. Mercurio A.M. Protein kinase C-α phosphorylation of specific serines in the connecting segment of the β4 integrin regulates the dynamics of type II hemidesmosomes.Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar). A cluster of serines at the beginning of the connecting segment, Ser1356-Ser1360-Ser1364, as well as at a nearby site, Ser1424 (17Rabinovitz I. Tsomo L. Mercurio A.M. Protein kinase C-α phosphorylation of specific serines in the connecting segment of the β4 integrin regulates the dynamics of type II hemidesmosomes.Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar, 18Germain E.C. Santos T.M. Rabinovitz I. Phosphorylation of a novel site on the β4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar, 19Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Serine phosphorylation of the integrin β4 subunit is necessary for epidermal growth factor receptor-induced hemidesmosome disruption.Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar, 20Frijns E. Sachs N. Kreft M. Wilhelmsen K. Sonnenberg A. EGF-induced MAPK signaling inhibits hemidesmosome formation through phosphorylation of the integrin β4.J. Biol. Chem. 2010; 285: 37650-37662Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), or a threonine at the C-tail (21Frijns E. Kuikman I. Litjens S. Raspe M. Jalink K. Ports M. Wilhelmsen K. Sonnenberg A. Phosphorylation of threonine 1736 in the C-terminal tail of integrin β4 contributes to hemidesmosome disassembly.Mol. Biol. Cell. 2012; 23: 1475-1485Crossref PubMed Scopus (39) Google Scholar), have been shown to play an important role in HD stability. Mutation on some of these serine residues to alanine to impede phosphorylation reduces the disruptive activity of EGF on HD, increases the interaction of β4 and plectin, and can inhibit cell migration by stabilizing HD, indicating a function of β4 serine phosphorylation in HD regulation (17Rabinovitz I. Tsomo L. Mercurio A.M. Protein kinase C-α phosphorylation of specific serines in the connecting segment of the β4 integrin regulates the dynamics of type II hemidesmosomes.Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar, 18Germain E.C. Santos T.M. Rabinovitz I. Phosphorylation of a novel site on the β4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar, 19Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Serine phosphorylation of the integrin β4 subunit is necessary for epidermal growth factor receptor-induced hemidesmosome disruption.Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar, 20Frijns E. Sachs N. Kreft M. Wilhelmsen K. Sonnenberg A. EGF-induced MAPK signaling inhibits hemidesmosome formation through phosphorylation of the integrin β4.J. Biol. Chem. 2010; 285: 37650-37662Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In contrast, mutation of these sites into aspartate, which mimics phosphorylation, increases HD disruption and plectin dissociation. Importantly, some of these sites are found hyperphosphorylated in invasive squamous cell carcinoma (SCC), suggesting that β4 phosphorylation plays a role in carcinoma invasion (23Kashyap T. Germain E. Roche M. Lyle S. Rabinovitz I. Role of β4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration.Lab. Invest. 2011; 91: 1414-1426Crossref PubMed Scopus (18) Google Scholar). In vitro, SCC cells show an increase in β4 phosphorylation when compared with keratinocytes. Interestingly, β4 seems to be preferentially phosphorylated in certain areas of the cells, such as trailing edges and retraction fibers, where previous studies have shown that HD disassemble as the cell moves forward (13Tsuruta D. Hopkinson S.B. Jones J.C. Hemidesmosome protein dynamics in live epithelial cells.Cell Motil. Cytoskeleton. 2003; 54: 122-134Crossref PubMed Scopus (43) Google Scholar, 18Germain E.C. Santos T.M. Rabinovitz I. Phosphorylation of a novel site on the β4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar, 23Kashyap T. Germain E. Roche M. Lyle S. Rabinovitz I. Role of β4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration.Lab. Invest. 2011; 91: 1414-1426Crossref PubMed Scopus (18) Google Scholar). This distinct distribution of β4 phosphorylation suggests that local factors such as traction forces or calcium fluxes may be regulating β4 phosphorylation (24Almeida E.A. Huovila A.P. Sutherland A.E. Stephens L.E. Calarco P.G. Shaw L.M. Mercurio A.M. Sonnenberg A. Primakoff P. Myles D.G. White J.M. Mouse egg integrin α6β1 functions as a sperm receptor.Cell. 1995; 81: 1095-1104Abstract Full Text PDF PubMed Scopus (471) Google Scholar). An important aspect of protein function regulation through phosphorylation is its reversibility. Although several kinases, such as PKC and the MAPK ERK1/2 and RSK2, have been shown to be involved in serine phosphorylation of β4 (16Rabinovitz I. Toker A. Mercurio A.M. Protein kinase C-dependent mobilization of the α6β4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.J. Cell Biol. 1999; 146: 1147-1160Crossref PubMed Scopus (187) Google Scholar, 17Rabinovitz I. Tsomo L. Mercurio A.M. Protein kinase C-α phosphorylation of specific serines in the connecting segment of the β4 integrin regulates the dynamics of type II hemidesmosomes.Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar, 18Germain E.C. Santos T.M. Rabinovitz I. Phosphorylation of a novel site on the β4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar, 19Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Serine phosphorylation of the integrin β4 subunit is necessary for epidermal growth factor receptor-induced hemidesmosome disruption.Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar, 20Frijns E. Sachs N. Kreft M. Wilhelmsen K. Sonnenberg A. EGF-induced MAPK signaling inhibits hemidesmosome formation through phosphorylation of the integrin β4.J. Biol. Chem. 2010; 285: 37650-37662Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), little is known about the pathways that lead to dephosphorylation. Based on mutational studies, it can be asserted that dephosphorylation of the β4 integrin is necessary to stabilize HD (17Rabinovitz I. Tsomo L. Mercurio A.M. Protein kinase C-α phosphorylation of specific serines in the connecting segment of the β4 integrin regulates the dynamics of type II hemidesmosomes.Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar, 18Germain E.C. Santos T.M. Rabinovitz I. Phosphorylation of a novel site on the β4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar, 19Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Serine phosphorylation of the integrin β4 subunit is necessary for epidermal growth factor receptor-induced hemidesmosome disruption.Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar, 20Frijns E. Sachs N. Kreft M. Wilhelmsen K. Sonnenberg A. EGF-induced MAPK signaling inhibits hemidesmosome formation through phosphorylation of the integrin β4.J. Biol. Chem. 2010; 285: 37650-37662Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). There is little information about serine-threonine protein phosphatases (PPP) that might be involved in this process. PPP inhibitors, such as okadaic acid and calyculin, have been shown to increase β4 phosphorylation as well as destabilize HD, implicating protein phosphatase 2a and or protein phosphatase 1 (18Germain E.C. Santos T.M. Rabinovitz I. Phosphorylation of a novel site on the β4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar, 19Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Serine phosphorylation of the integrin β4 subunit is necessary for epidermal growth factor receptor-induced hemidesmosome disruption.Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). In the present study we addressed whether another major PPP, calcineurin (CN), is involved in the dephosphorylation and function of the β4 integrin. CN is a heterodimeric serine/threonine phosphatase, and it is the only PPP known to become highly stimulated in response to Ca2+ signals because of the direct binding of Ca2+ and calmodulin (25Rusnak F. Mertz P. Calcineurin: form and function.Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1100) Google Scholar). CN is one of the key players directing the flow of information from local or global calcium signals to effectors that control immediate cellular responses and alter gene transcription. This may be of particular relevance to β4 function given that local factors seem to regulate β4 phosphorylation (18Germain E.C. Santos T.M. Rabinovitz I. Phosphorylation of a novel site on the β4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly.Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar). Interestingly, CN inhibitors that are commonly used in immunosuppressive therapy, such as cyclosporin A (CsA), are known to increase the incidence of SCC (26Euvrard S. Kanitakis J. Claudy A. Skin cancers after organ transplantation.N. Engl. J. Med. 2003; 348: 1681-1691Crossref PubMed Scopus (1305) Google Scholar), a type of tumor that, as mentioned above, shows increased migration through β4 phosphorylation and HD disassembly (23Kashyap T. Germain E. Roche M. Lyle S. Rabinovitz I. Role of β4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration.Lab. Invest. 2011; 91: 1414-1426Crossref PubMed Scopus (18) Google Scholar). Therefore, a study of the relationship between CN and β4 phosphorylation should improve our understanding of CN inhibitor-induced SCC. (24Almeida E.A. Huovila A.P. Sutherland A.E. Stephens L.E. Calarco P.G. Shaw L.M. Mercurio A.M. Sonnenberg A. Primakoff P. Myles D.G. White J.M. Mouse egg integrin α6β1 functions as a sperm receptor.Cell. 1995; 81: 1095-1104Abstract Full Text PDF PubMed Scopus (471) Google Scholar) In this study, we have found that CN negatively regulates β4 phosphorylation and increases HD stability, whereas reducing cell migration. The effect of CN on β4 phosphorylation is indirect and occurs through two different pathways. In one case, CN inhibits the MAPK pathway, where ERK1/2 has been shown to directly phosphorylate β4. A second route is independent of MAPK, where CN seems to positively promote β4 dephosphorylation, probably regulating a downstream phosphatase. We also show that calcium flows affect β4 phosphorylation through CN and calmodulin and that membrane receptors modulating calcium, such as the bradykinin and acetylcholine receptors, can also regulate β4 phosphorylation and HD stability through CN. Interestingly, the activation of Ca/CN pathway can reduce the hyperphosphorylation of β4 in SCC cells and increase HD stability as well as reduce migration. Our results show that CN plays an important role in HD function through regulation of β4 phosphorylation and may be of particular relevance in SCC carcinoma induced by CN inhibitors. HaCat keratinocytes were obtained from Dr. S. La Flamme (Albany Medical College, Albany, NY). Squamous cell carcinoma cell lines were obtained as follows: A431 cells (vulvar SCC origin) were obtained from ATCC; Colo-16 (skin SCC origin) was obtained from Dr. N. Hail (University of Colorado, Denver, CO); and SCC-25 cells (head and neck SCC origin) were provided by Dr. A. M. Mercurio (University of Massachusetts Medical School, Worcester, MA). Cos-7 cells were provided by Dr. Pierre Kinet (Beth Israel Deaconess Medical Center, Boston, MA). Cells were maintained in DMEM with 10% fetal calf serum, except SCC25 cells, which were grown in DMEM/Ham's F12 (1:1) containing hydrocortisone (400 ng/ml) and 10% fetal bovine serum. Antibodies were as follows: 3E1 (β4, Chemicon); GoH3 (α6, Chemicon); rabbit anti-β4 (16Rabinovitz I. Toker A. Mercurio A.M. Protein kinase C-dependent mobilization of the α6β4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.J. Cell Biol. 1999; 146: 1147-1160Crossref PubMed Scopus (187) Google Scholar); anti-BPAG1 (27Hieda Y. Nishizawa Y. Uematsu J. Owaribe K. Identification of a new hemidesmosomal protein, HD1: a major, high molecular mass component of isolated hemidesmosomes.J. Cell Biol. 1992; 116: 1497-1506Crossref PubMed Scopus (201) Google Scholar); and anti-plectin (Santa Cruz Biotechnology). Affinity-purified phospho-specific rabbit polyclonal Ab (anti- phospho-Ser1356 Ab) was previously characterized (23Kashyap T. Germain E. Roche M. Lyle S. Rabinovitz I. Role of β4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration.Lab. Invest. 2011; 91: 1414-1426Crossref PubMed Scopus (18) Google Scholar). Anti-phospho ERK1/2 and anti-ERK1/2 Abs were from Cell Signaling Technology. Cyclosporin A, BAPTA-AM, thapsigargin, bradykinin, and acetylcholine were from Sigma. Inhibitors for PKC (Go6983), MEK (U0126), and calmodulin (W13) were from Enzo Biochemicals. Full-length cDNA calcineurin (PPP3CA, Open Biosystems) was used to produce a constitutively active form containing amino acids 1–390 (28Clipstone N.A. Crabtree G.R. Calcineurin is a key signaling enzyme in T lymphocyte activation and the target of the immunosuppressive drugs cyclosporin A and FK506.Ann. N.Y. Acad. Sci. 1993; 696: 20-30Crossref PubMed Scopus (113) Google Scholar) and fused to Myc using a Myc-pcDNA4 plasmid (Invitrogen). Transfection of the plasmid was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. ON-TARGETplus SMARTpool human PPP3CA (Dharmacon) was transfected using Lipofectamine® RNAiMAX, and cells were grown for 48 h before experiments. An AllStars negative control siRNA (Qiagen) was used to control siRNA transfections. Cells were stained as described previously (16Rabinovitz I. Toker A. Mercurio A.M. Protein kinase C-dependent mobilization of the α6β4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.J. Cell Biol. 1999; 146: 1147-1160Crossref PubMed Scopus (187) Google Scholar, 29Rabinovitz I. Mercurio A.M. The integrin α6β4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures.J. Cell Biol. 1997; 139: 1873-1884Crossref PubMed Scopus (202) Google Scholar). Briefly, cells grown on coverslips were extracted or not with detergent buffer containing 0.5% Triton X-100, 100 mm KCl, 200 mm sucrose, 10 mm EGTA, 2 mm MgCl2, and 10 mm PIPES at pH 6.8 for 1 min and then fixed using paraformaldehyde or methanol. Cells were rinsed, blocked, and stained with the indicated Abs and Cy2/Cy3-conjugated secondary antibodies. Slides were analyzed using fluorescence microscopy. Quantitative analysis of HD-like structures was performed as described previously (23Kashyap T. Germain E. Roche M. Lyle S. Rabinovitz I. Role of β4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration.Lab. Invest. 2011; 91: 1414-1426Crossref PubMed" @default.
• W2017209369 created "2016-06-24" @default.
• W2017209369 creator A5018764325 @default.
• W2017209369 creator A5061973648 @default.
• W2017209369 date "2012-09-01" @default.
• W2017209369 modified "2023-09-29" @default.
• W2017209369 title "The Calcium/Calcineurin Pathway Promotes Hemidesmosome Stability through Inhibition of β4 Integrin Phosphorylation" @default.
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