Ca2+-dependent Localization of Integrin-linked Kinase to Cell Junctions in Differentiating Keratinocytes

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca2+-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca2+ as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes. Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca2+-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca2+ as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes. integrin-linked kinase glyceraldehyde-3-phosphate dehydrogenase myelin basic protein phosphate-buffered saline paraformaldehyde The epidermis is formed by keratinocytes at different stages of differentiation (1Zinkel S. Fuchs E. Semin. Cancer Biol. 1994; 5: 77-90Google Scholar, 2Fuchs E. Curr. Opin. Cell Biol. 1990; 2: 1028-1035Google Scholar). Undifferentiated keratinocytes reside in the basal cell layer and are attached to a basement membrane that separates them from the dermis (3Jones P.H. Watt F.M. Cell. 1993; 73: 713-724Google Scholar, 4Adams J.C. Watt F.M. J. Cell Biol. 1991; 115: 829-841Google Scholar, 5Hertle M.D. Adams J.C. Watt F.M. Development. 1991; 112: 193-206Google Scholar). Proliferation, adhesion, and motility of undifferentiated keratinocytes are regulated by integrins and associated proteins, which form focal adhesions (6Fuchs E. Raghavan S. Nat. Rev. Genet. 2002; 3: 199-209Google Scholar). Upon receiving appropriate signals, basal keratinocytes initiate terminal differentiation, characterized by loss of proliferative capacity, decrease in integrin expression, detachment from the basement membrane, and migration upward to form postmitotic suprabasal keratinocyte layers. In culture, primary keratinocytes can be induced to differentiate by elevating the extracellular Ca2+concentration. Induction of differentiation by Ca2+ is accompanied by loss of integrin expression and formation of epithelial sheets. Intercellular adhesion in differentiated keratinocytes occurs through tight junctions, cadherin-mediated formation of adherens junctions, and desmosomes (6Fuchs E. Raghavan S. Nat. Rev. Genet. 2002; 3: 199-209Google Scholar, 7Jamora C. Fuchs E. Nat. Cell Biol. 2002; 4: 101-108Google Scholar, 8Jensen P.J. Wheelock M.J. Exp. Cell Res. 1995; 219: 322-331Google Scholar, 9Braga V.M. Hajibagheri N. Watt F.M. Cell Adhes. Commun. 1998; 5: 137-149Google Scholar). Epidermal integrity and barrier function depend on these cell-cell junctions. Originally identified as a β1 integrin-binding protein, ILK1 also interacts with paxillin, actopaxin, and affixin in focal adhesions. Accumulating evidence from a variety of cell types indicates that ILK can function as a kinase and as a scaffold protein that mediates interactions between integrins and the actin cytoskeleton through interaction with multiple proteins (10Zervas C.G. Gregory S.L. Brown N.H. J. Cell Biol. 2001; 152: 1007-1018Google Scholar, 11Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Google Scholar, 12Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Google Scholar). Such interactions are essential for the formation of proper attachment sites of muscle fibers inCaenorhabditis elegans and Drosophila melanogaster. The regulation and function of ILK have yet to be explored in keratinocytes. Given that keratinocyte differentiation involves marked changes in both integrin signaling and cytoskeletal organization, we postulate that ILK must be involved in these processes. We now show that ILK kinase activity, but not abundance, is substantially reduced during Ca2+-induced keratinocyte differentiation. Further, we report a novel link between ILK and the actin cytoskeleton in areas of intercellular contact, which involves distribution of ILK to areas adjacent to cell borders specifically in differentiated keratinocytes. Finally, and in contrast to its effects on other epithelial cell types, exogenous ILK expression in differentiated keratinocytes does not induce epithelial-mesenchymal transition through loss of E-cadherin expression and cell-cell adhesion. Primary keratinocytes were isolated from 1–2-day-old CD-1 mice and cultured in medium containing 0.05 mm Ca2+ as described (13D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Google Scholar). To induce differentiation, keratinocytes were cultured in medium with 1.0 mm Ca2+ for 24 h. Viral infections were conducted by incubating keratinocytes with appropriate adenovirus at a multiplicity of infection of 10 for 5 h in serum-free medium, followed by 24 h in the presence or absence of 1.0 mmCa2+ prior to cell processing. For Ca2+withdrawal experiments, cells were incubated for 24 h in medium containing 1.0 mm Ca2+. The cells were washed in Ca2+-free PBS three times and cultured for an additional 12 h in medium containing 0.05 mm Ca2+. In these experiments, control cultures were incubated in 0.05 or 1.0 mm Ca2+ for a total of 24 h. In experiments in which the cytoskeleton was disrupted, cells were incubated with cytochalasin D (8 μm final concentration) 15 min prior to Ca2+ addition and a further 24 h prior to processing for immunofluorescence microscopy. Human HEK293 cells were purchased from the American Type Culture Collection and cultured with Dulbecco's modified minimal essential medium containing 8% fetal bovine serum. cDNAs encoding V5-tagged wild-type ILK and the kinase mutant ILK E359K (14Novack A. Hsu S.C. Leung-Hagesteijn C. Radeva G. Papkoff J. Montesano R. Roskelley C. Grosschedl R. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4374-4379Google Scholar) were isolated to generate the corresponding recombinant adenoviruses using the pAdEasy system (15He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Google Scholar). Viral stocks were amplified and titered by dilution assay in HEK293 cells. The mouse monoclonal anti-ILK antibody (611803) from Transduction Laboratories (hereafter termed ILK(TL)) was obtained using as immunogen the C terminus of ILK (12Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Google Scholar). The polyclonal goat anti-ILK antibody from Santa Cruz Biotechnology, as was a in the C terminus of The polyclonal anti-ILK and from Biotechnology, were obtained using a containing the ILK protein as immunogen Leung-Hagesteijn C. L. Radeva G. J. J.C. Dedhar S. Scholar). and were purchased from Transduction and were from and The monoclonal antibody was from the was purchased from The and were from Transduction to or were purchased from other were purchased from keratinocyte were obtained by keratinocytes at times the addition of followed by in mm mm mm mm and and followed by three of The were by 15 and or at To keratinocytes were and in A mm mm mm and mm and and for 10 on the was The was in A containing followed by 15 of the the was in mm and were conducted with of as described (13D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Google Scholar). were also for or to for protein in were between in a using The in the are of experiments conducted at three In kinase activity in ILK immunoprecipitates was using in of protein from keratinocyte as described Leung-Hagesteijn C. L. Radeva G. J. J.C. Dedhar S. with the Cell were by with a of protein and protein for prior to ILK using the mouse monoclonal anti-ILK antibody from Transduction were conducted for min at in the presence of 5 of and 10 of assay complexes were by and were by were with and induced to differentiate with 1.0 mm Ca2+ for 24 h. The were with and in mm mm mm 10 mm mm containing of protein were with protein for h at were incubated at with complexes were isolated by the addition of protein and washed times with to the were by in for 5 by to and with in were in PBS prior to For the experiments in and cells were in 15 and with followed by with mm in In experiments, cells were with 15 in of and PBS cells were in PBS containing and goat followed by with primary antibody three PBS the cells were with the appropriate or at for h. of the the cells were incubated with in PBS for 5 min at and in medium were obtained with a with an using The are of experiments conducted on at three localization to cell-cell borders requires an intact actin cytoskeleton. primary keratinocytes were cultured for the times in medium containing 1.0 mm Ca2+. To the actin cultures were incubated in the presence of cytochalasin D μm final concentration) for 15 min prior to the addition of mm final concentration) and cultured for 24 h. ILK was using a goat anti-ILK and actin was using containing cells with wild-type adenovirus and cultured in medium with 1.0 mmCa2+ for or 24 as and for immunofluorescence but in the presence of in of primary anti-ILK localization and adjacent to cell-cell borders requires extracellular Primary keratinocytes were cultured in medium containing 0.05 mm Ca2+ or 1.0 mm Ca2+ for 24 h prior to processing for microscopy. the cells were cultured for 24 h in medium containing 1.0 washed and cultured for an additional 12 h in medium containing 0.05 mm ILK and E-cadherin were with the corresponding actin was using ILK and actin were by of a of cells, E-cadherin was using cell was conducted with a For the of undifferentiated keratinocytes in cells were with at for 15 and with was conducted as described and were obtained with a The keratinocytes in were in 15 and with prior to and with keratinocyte were obtained with a ILK as a protein with a of in a variety of cell types when on using polyclonal the protein Leung-Hagesteijn C. L. Radeva G. J. J.C. Dedhar S. Scholar). ILK also as a protein with of in from multiple mouse and epithelial and cells, using monoclonal the C terminus of ILK (12Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Google Scholar). Thus, we to the of ILK in mouse keratinocyte with the anti-ILK the mouse monoclonal antibody the C terminus of ILK or a goat polyclonal antibody a in the C terminus of we observed a of on corresponding to ILK We also in cell from keratinocytes with recombinant adenoviruses encoding wild-type or E359K mutant V5-tagged We observed that the exogenous ILK a the protein and an of The of the expressed ILK was using the In of the with the mouse and goat the protein from cells with that of the expressed ILK identified with the antibody that the presence of the the of ILK on of both and exogenous ILK proteins was when the were with the goat polyclonal antibody in the presence of the corresponding the of are with the in mouse keratinocytes, ILK is expressed as a of of In contrast, when we with the polyclonal antibody a protein containing the ILK we were to the V5-tagged exogenous ILK at and not the ILK protein at the antibody a at which does not to of the ILK by the other and to an as yet protein a of these experiments, were conducted with the mouse monoclonal or the goat polyclonal which specifically ILK in primary mouse keratinocytes. Primary keratinocytes are a of epidermal cultured in medium containing Ca2+ these cells remain undifferentiated and proliferative in extracellular Ca2+ terminal differentiation 24 characterized by changes in expression and cytoskeletal organization, as well as formation of adherens junctions involved in intercellular Rev. Biol. Scholar, C. Fuchs E. Cell. Scholar). Undifferentiated keratinocytes and and signaling through these integrins is necessary for keratinocyte proliferation R. S. Watt F.M. J. 2001; Scholar, C. A. J. Scholar). Induction of differentiation in keratinocytes in or in is accompanied by integrin down-regulation and from the cell V.M. Watt F.M. Cell Adhes. Commun. 1995; 3: Scholar). Given the between ILK and β1 integrin we ILK expression was regulated during Ca2+-induced keratinocyte We that ILK protein levels were the of keratinocyte differentiation β1 integrin expression was substantially reduced by 24 h the addition of Ca2+ keratinocytes as a in the presence of extracellular Ca2+ a integrin and in differentiation and reduction in integrin L. J. Scholar, L. S. Watt F.M. Biol. Cell. Scholar). To ILK protein levels are regulated by integrin we also cultured keratinocytes in for 24 h in medium containing 0.05 mm Ca2+. to the in for 24 h not ILK levels Thus, ILK protein expression in cultured epidermal keratinocytes is not associated with differentiation and is independent of integrin or ILK kinase activity and to as protein kinase and in cell types C. J. Biol. Chem. 2001; 276: Scholar, S. S. N. J. J. Dedhar S. J. Biol. Chem. 2001; 276: Scholar). ILK kinase activity integrin by Leung-Hagesteijn C. L. Radeva G. J. J.C. Dedhar S. Scholar) and C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Google Scholar). Ca2+-induced keratinocyte differentiation integrin also of Rev. Biol. Scholar). we kinase activity associated with ILK is altered in cells. We ILK from undifferentiated or differentiated keratinocyte and observed that ILK immunoprecipitates from undifferentiated keratinocyte In contrast, the kinase activity in ILK immunoprecipitates from differentiated cells was substantially the total ILK protein levels were in Thus, differentiation in keratinocytes a reduction in kinase activity associated with ILK for ILK in cell In addition to its function as a ILK to focal and with proteins and as a cytoskeletal between integrins and the actin cytoskeleton (10Zervas C.G. Gregory S.L. Brown N.H. J. Cell Biol. 2001; 152: 1007-1018Google C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Google Scholar). To the of a for ILK in keratinocytes, we its subcellular localization using cell We that ILK is in of the differentiation of the cells ILK was also in the obtained of the component as well as in the To the of we with proteins for component We observed that of the was in the in with its distribution in a variety of cell types J. Biol. Chem. Scholar). we as a for proteins J. Biol. Chem. Scholar) and that was in We also the with and which are proteins that to K. J. J. Biol. Chem. 2002; Scholar, J. Biol. 2001; Scholar). We that was in the of from both undifferentiated and differentiated keratinocytes. We also observed was in in undifferentiated keratinocyte a of was in and from differentiated cell We that different of ILK are in primary murine keratinocytes, which that protein multiple in these cells. are essential for keratinocyte adhesion, and proliferation C. A. J. Scholar, K. Watt F.M. Exp. Scholar, Watt F.M. Proc. Natl. Acad. Sci. U. S. A. Scholar). In undifferentiated keratinocytes, integrins in focal the actin cytoskeleton with the extracellular adhesions also with the protein paxillin, which interacts with ILK and other proteins in cell types (12Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Google Scholar). The presence of ILK in with the interactions between ILK and at focal to ILK and in undifferentiated keratinocytes. Thus, we isolated ILK immunoprecipitates from undifferentiated keratinocytes and them for the presence of We observed that is a component of ILK immunoprecipitates from undifferentiated keratinocytes. we were also to the presence of ILK in immunoprecipitates and the in undifferentiated keratinocytes of complexes containing both ILK and differentiation marked in integrin levels and focal adhesions in keratinocytes. we integrin down-regulation is associated with loss of interactions in differentiating keratinocytes. as described for undifferentiated cells, we that the interaction between ILK and is in differentiated keratinocytes and its from integrin levels and differentiation in these cells. To further the regulation of ILK during keratinocyte we on the subcellular distribution of expression of ILK by in undifferentiated and differentiated cells. We the exogenous protein by immunofluorescence microscopy. Undifferentiated keratinocytes cultured in 0.05 mm Ca2+ focal adhesions but not form adherens junctions Fuchs E. Curr. Opin. Cell Biol. 2001; Scholar). We observed that ILK immunofluorescence was the cell in with with proper of associated with the focal adhesions. we the cells with in the presence of to cytoskeletal we observed that wild-type ILK a the cell in undifferentiated keratinocytes in extracellular Ca2+ to mm in keratinocyte cultures the formation of intercellular junctions, which involves interactions of E-cadherin on adjacent cells C. Fuchs E. Cell. Scholar). In is an of adherens formation h of followed by and of the cell h. keratinocytes with the adenovirus in mm Ca2+ for 24 h changes in ILK distribution to to of cell-cell contact, which in cells the cell to cell-cell borders in keratinocytes was observed with expressed ILK as well as with the protein ILK localization to cell-cell borders was not during the 12 h Ca2+ addition but was in the junctions 24 h that ILK to areas associated with intercellular junctions is not an to extracellular Ca2+ or formation of adherens junctions. ILK associated with cell borders with to of ILK localization to focal adhesions and interaction with requires a in ILK that its kinase S.N. Turner C.E. J. Biol. Chem. 2002; Scholar). an E359K mutant of ILK that as a form in cells but kinase activity in with to focal adhesions Leung-Hagesteijn C. L. Radeva G. J. J.C. Dedhar S. Scholar, S.N. Turner C.E. J. Biol. Chem. 2002; Scholar, C. L. J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: Scholar, C. J. Cell Biol. Scholar). To to the for ILK localization in keratinocytes, we an intact kinase is for the subcellular localization of the E359K ILK In undifferentiated keratinocytes, ILK E359K a distribution the from the by the wild-type protein, a for the kinase in the distribution of protein In contrast, mutant was to to cell-cell junctions in keratinocytes induced to differentiate with Ca2+ as as wild-type ILK indicates that the mechanisms for ILK with and subcellular distribution in undifferentiated keratinocytes are from that induce ILK localization to intercellular borders in differentiated cells. ILK localization to cell borders in cells does not to depend on the presence of a kinase or on with Undifferentiated primary keratinocytes form actin of fibers that as fibers in undifferentiated keratinocytes can at focal adhesion sites or in the formation of or in cells C. Fuchs E. Cell. Scholar, J. Cell Biol. 1998; Scholar). In these cells, we observed areas of ILK localization in close to the of actin in addition to the described distribution the cell The formation of intercellular junctions in keratinocytes by in extracellular Ca2+ also requires actin and to cell-cell borders C. Fuchs E. Cell. Scholar). in the formation of actin fibers that to the membrane we the between these actin and ILK along intercellular junctions, we observed that of ILK along the cell membrane, and in close apposition to the actin fibers The localization of ILK and actin fibers to ILK to cell borders in cultures in which the cytoskeleton was by with cytochalasin We that of in cells cultured in the presence of cytochalasin D and mmCa2+ was accompanied by loss of ILK from the cell that actin is an essential for ILK in differentiated keratinocytes. The of adherens junctions in keratinocytes cultured in mm Ca2+ also requires the at cell-cell borders of E-cadherin complexes associated with the actin cytoskeleton and that and V.M. Watt F.M. Cell Adhes. Commun. 1995; 3: E. A. Cell Adhes. Commun. 1998; Scholar). E-cadherin interactions are of adherens junctions in differentiated keratinocytes requires the presence of extracellular Ca2+ To further the mechanisms ILK to intercellular junctions in differentiated keratinocytes, we the of Thus, we cultured cells for 24 h in mm extracellular Ca2+ to induce ILK migration to areas of cell-cell A we the medium from 1.0 to 0.05 mm Ca2+ and cultured the cells for an additional in Ca2+ in differentiated keratinocytes induced the loss of E-cadherin localization to cell-cell the formed adherens junctions. in extracellular Ca2+ levels also altered the actin fibers to the membrane, other cytoskeletal actin fibers the cell Ca2+ also in the or reduction of ILK from cell to that observed for the actin that the of localization of and E-cadherin along intercellular borders the presence of extracellular Ca2+ we conducted these experiments in cells ILK by we that the ILK levels in differentiated keratinocytes not with formation of cell junctions or with expression and localization of E-cadherin and to cell borders in to extracellular Ca2+ and not that ILK does not induce transformation in differentiated epidermal keratinocytes. that ILK is regulated at multiple levels during keratinocyte differentiation and a novel for protein in the of cell-cell in differentiated keratinocytes and in epidermal barrier The epidermis is a epithelial which of proliferative cells and differentiated A of primary murine epidermal keratinocytes is to terminal differentiation in to in extracellular Ca2+ Differentiation in primary cultured keratinocytes is accompanied by loss of integrin expression and focal adhesions. ILK is an component of integrin signaling and focal adhesion we ILK is also regulated during Ca2+-induced keratinocyte differentiation. We ILK protein levels not in differentiated keratinocytes, kinase activity in ILK immunoprecipitates is substantially The ILK complexes we isolated from keratinocytes also other proteins to be the kinase activity we in the keratinocyte from ILK or from protein in ILK the kinase activity by Ca2+ in keratinocytes is since the of tight mechanisms for in to differentiation in epidermal ILK to have as a kinase and as a scaffold in Specifically, ILK to focal and as a cytoskeletal between integrins and the actin which is essential for muscle in C. elegans and (10Zervas C.G. Gregory S.L. Brown N.H. J. Cell Biol. 2001; 152: 1007-1018Google Scholar, 11Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Google Scholar). for the of ILK are its interactions with as paxillin, and (12Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Google Scholar, S.N. Turner C.E. J. Biol. Chem. 2002; Scholar, C. J. Cell Sci. 112: Scholar). In undifferentiated keratinocytes, ILK can be in and in complexes that In these cells, ILK is with a to be ILK is associated with focal adhesions in undifferentiated keratinocytes. The different of ILK distribution in differentiated keratinocytes that ILK multiple in adhesion, proliferation in these cells through a of signaling and scaffold Primary keratinocytes cultured in mm Ca2+ form with cell-cell Watt F.M. Exp. Cell Res. Scholar). these integrins not a in intercellular adhesion P.J. Wheelock M.J. Exp. Cell Res. 1995; 219: 322-331Google Scholar, V.M. Watt F.M. Cell Adhes. Commun. 1995; 3: Scholar). The formation of adherens junctions in keratinocytes cultured in mm Ca2+ is accompanied by of E-cadherin complexes containing and Such complexes are associated with the actin cytoskeleton and in V.M. Watt F.M. Cell Adhes. Commun. 1995; 3: Scholar, E. A. Cell Adhes. Commun. 1998; Scholar). The of adherens junctions requires interactions between E-cadherin and the actin cytoskeleton. Specifically, intercellular of E-cadherin can induce actin at the cell Curr. Biol. 2002; but actin and is to cell as by the of cadherin-mediated cell-cell junctions in cytochalasin cells C. Fuchs E. Cell. Scholar). We have in differentiated keratinocytes, ILK is in and with its cytoskeletal distribution in other cell types C. J. Biol. Chem. 2001; 276: Scholar, C. J. Cell Biol. Scholar). of differentiation by Ca2+ in keratinocytes is accompanied by marked changes in subcellular characterized by ILK to cell-cell The keratinocyte to Ca2+ be since ILK to be from intercellular adhesion sites in other cell types C. J. Cell Sci. 112: Scholar). Ca2+ also of E-cadherin and at intercellular junctions C. Fuchs E. Cell. Scholar). We observed that with ILK by immunofluorescence but we have not in ILK S. and L. that ILK and in the formation of complexes associated with cell borders the cytoskeleton. that Ca2+ a in keratinocyte and a that to actin at sites C. Fuchs E. Cell. Scholar). experiments that both are necessary for ILK localization to cell junctions. is by the loss of ILK from intercellular adhesion sites of E-cadherin by or of actin by cytochalasin D In keratinocytes induced to differentiate by of actin are formed in a to the cell borders in a that with the of E-cadherin localization at intercellular junctions A. C. Fuchs E. Cell. 2002; 3: Scholar). We have identified ILK as an additional component in complexes adjacent to the cell borders in keratinocytes. A tight between ILK and the actin cytoskeleton is also by the close localization of ILK to actin fibers at cell-cell borders in differentiated keratinocytes with the down-regulation of integrins that occurs keratinocyte differentiation A. Watt F.M. J. Cell Biol. 1995; that ILK in the of cell-cell in differentiated epidermal keratinocytes that is to integrin In addition to cell-cell we also observed ILK immunofluorescence in cell in differentiated keratinocytes. of ILK immunoprecipitates from differentiated keratinocytes the presence of and in these complexes the of the mutant E359K to to cell-cell borders its to with (12Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Google Scholar) that the complexes in differentiated keratinocytes a epidermal containing differentiated keratinocytes not focal they form cell-cell In these is not in corresponding to areas of intercellular V.M. Watt F.M. Cell Adhes. Commun. 1995; 3: Scholar). We observed E-cadherin formation of adherens junctions and ILK localization to cell-cell borders in keratinocytes, of the cells expressed The that in epidermal keratinocytes ILK overexpression does not induce epithelial-mesenchymal through E-cadherin and loss of intercellular adhesion with the to ILK expression in mammary and in intestinal epithelial cell A. A. Dedhar S. Roskelley J. Cell Sci. 2001; Scholar, C. Leung-Hagesteijn C. Radeva G. S. Dedhar S. J. Biol. Chem. 1998; Scholar). are with the that ILK a in intercellular adhesion, cytoskeletal organization, and of barrier function in differentiated keratinocytes. We S. Dedhar for ILK and ILK E359K R. for the S. for on the and L. for with microscopy.

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