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

Alisa VespaDagger §, Alison J. Darmon, Christopher E. Turner||, Sudhir J. A. D'SouzaDagger **, and Lina DagninoDagger Dagger Dagger §§

From the Departments of Dagger  Physiology and Pharmacology and of Dagger Dagger  Paediatrics, Child Health Research Institute and Lawson Health Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada and the  Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210

Received for publication, August 14, 2002, and in revised form, January 23, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The epidermis is formed by keratinocytes at different stages of differentiation (1, 2). Undifferentiated keratinocytes reside in the basal cell layer and are attached to a basement membrane that separates them from the dermis (3-5). Proliferation, adhesion, and motility of undifferentiated keratinocytes are regulated by integrins and associated proteins, which form focal adhesions (6). 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 (6-9). Epidermal integrity and barrier function depend on these cell-cell junctions.

Originally identified as a beta 1 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 (10-12). Such interactions are essential for the formation of proper attachment sites of muscle fibers in Caenorhabditis 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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Cell Culture and Infections-- Primary keratinocytes were isolated from 1-2-day-old CD-1 mice and cultured in medium containing 0.05 mM Ca2+ as described (13). 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 mM Ca2+ 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.

Recombinant Adenoviruses and Plasmids-- cDNAs encoding V5-tagged wild-type ILK and the kinase mutant ILK E359K (14) were isolated to generate the corresponding recombinant adenoviruses using the pAdEasy system (15). Viral stocks were amplified and titered by dilution assay in HEK293 cells.

Anti-ILK Antibodies-- The mouse monoclonal anti-ILK antibody (611803) from Transduction Laboratories (hereafter termed ILK(TL)) was obtained using as immunogen the C terminus of ILK (12). The polyclonal goat anti-ILK antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (sc7516, denoted as ILK(SC)) was raised against a peptide sequence in the C terminus of ILK. The two rabbit polyclonal anti-ILK antibodies (06-550 and 06-552) from Upstate Biotechnology, Inc. (Lake Placid, NY) (ILK(USB)) were obtained using a purified, bacterially produced GST fusion containing the entire ILK protein as immunogen (16).

Other Antibodies and Chemicals-- Anti-beta 1-integrin (610468) and anti-E-cadherin (610405) antibodies were purchased from Transduction Laboratories. Anti-V5 (R96025) and anti-beta -catenin (C2206) antibodies were purchased, respectively, from Invitrogen and Sigma. The monoclonal E7 anti-beta -tubulin antibody was from the Iowa State University Hybridoma Bank. Rabbit anti-GAPDH (4699-9555) was purchased from Biogenesis. The anti-caveolin 1 (610057) and anti-paxillin (610051) antibodies were from Transduction Laboratories. Secondary antibodies conjugated to fluorescein isothiocyanate, Cy3, or Cy5 were purchased from Jackson Immunochemicals. All other chemicals were purchased from Sigma.

Cell Lysates, Immunoblotting, and Kinase Assays-- Total keratinocyte extracts were obtained by harvesting keratinocytes at indicated times after the addition of Ca2+, followed by suspension in lysis buffer T (20 mM Hepes, 450 mM NaCl, 4 M EDTA, 25% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml each leupeptin and aprotinin, 0.4 mM each NaF and Na3VO4), followed by three cycles of freeze-thawing. The lysates were cleared by centrifugation (12,000 × g, 15 min) and used immediately or stored at -80 °C. To obtain fractionated extracts, keratinocytes were harvested and suspended in ice-cold lysis buffer A (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 0.1% beta -mercaptoethanol, 0.4 mM NaF and sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 µg/ml each leupeptin, pepstatin, and aprotinin) and sonicated for 10 s on ice. After centrifugation (100,000 × g, 1 h), the supernatant (cytosolic fraction) was removed. The pellet was suspended in buffer A containing 1.2% Triton X-100, followed by centrifugation (12,000 × g, 15 min). After removal of the Triton X-soluble supernatant, the pellet was suspended in SDS buffer (50 mM Tris, pH 6.8, 1% SDS, 10% glycerol). Gel electrophoresis and immunoblotting were conducted with 18-50 µg of protein/sample, as described (13). Immunoblots were also probed for beta -tubulin or GAPDH to normalize for protein loading. No substantial differences in loading were noticed between samples in a given immunoblot using this method. The results shown in the immunoblots are representative of experiments conducted at least three times.

In vitro kinase activity in ILK immunoprecipitates was determined using in each sample 100 µg of protein from keratinocyte lysates as described (16), with the following modifications. Cell lysates were precleared by incubation with a mixture of protein A- and protein G-Sepharose beads for 2 h, prior to ILK immunoprecipitation using the mouse monoclonal anti-ILK antibody from Transduction Laboratories. Kinase assays were conducted for 30 min at 30 °C in the presence of 5 µg of MBP and 10 µCi of [gamma -32P]ATP. Kinase assay complexes were resolved by SDS-PAGE, and phosphorylated products were visualized by autoradiography.

Immunoprecipitation-- Keratinocytes were cultured, infected with ILK-encoding adenovirus, and induced to differentiate with 1.0 mM Ca2+ for 24 h. The monolayers were rinsed with ice-cold PBS, harvested, and lysed in immunoprecipitation buffer (10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% Nonidet P-40, 10% glycerol, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM Na3VO4). Samples containing 500 µg of protein were precleared with protein A/G-Sepharose for 2 h at 4 °C. Following centrifugation, lysates were incubated overnight at 4 °C with anti-V5 antibodies (1 µg/sample). Immune complexes were isolated by the addition of protein A/G-Sepharose (2 h, 4 °C) and washed five times with immunoprecipitation buffer. Proteins bound to the Sepharose beads were released by boiling in SDS-PAGE sample buffer for 5 min, resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies indicated in individual experiments.

Indirect Immunofluorescence and Confocal Microscopy-- Keratinocytes were rinsed once in PBS prior to fixation. For the experiments shown in Figs. 5, 7, and 8, cells were permeabilized (0.2% Triton X-100 in PBS, 4 °C, 15 min) and fixed with 2% PFA (4 °C, 40 min), followed by quenching with glycine (100 mM in PBS). In indicated experiments, cells were fixed with cold methanol (-20 °C, 15 min) in place of Triton X-100 permeabilization and PFA fixation. After two PBS washes, cells were blocked in PBS containing 5% nonfat dry milk and 5% goat serum (16 h, 4 °C), followed by incubation with primary antibody (1 h, 22 °C). After three 20-min PBS washes, the cells were probed with the appropriate Cy3- or Cy5-labeled secondary antibody, at 22 °C for 1 h. Following removal of the secondary antibody, the cells were rinsed twice, incubated with Hoescht 33258 (10 µg/ml) in PBS for 5 min at 22 °C, rinsed five times, and mounted in anti-fade medium (Dako). Photomicrographs were obtained with a Leica DMIRBE microscope equipped with an Orca II digital camera (Hamamatsu), using Openlab 3.0 software (Improvision). The results shown are representative of experiments conducted on duplicate samples at least three times.

Confocal microscopy was conducted with a Zeiss LSM metalaser-scanning microscope. For the analysis of undifferentiated keratinocytes shown in Fig. 6, cells were fixed with 4% PFA, 0.1% Triton X-100 at 22 °C for 15 min. Blocking and incubation with antibodies was conducted as described above, and confocal images were obtained with a Zeiss 63 × 1.25 Plan-Neofluor oil immersion lens. The Ca2+-treated keratinocytes shown in Fig. 6 were first permeabilized (0.2% Triton X-100 in PBS, 4 °C, 15 min) and then fixed with 2% PFA (4 °C, 40 min) prior to blocking and incubation with antibodies. Differentiated keratinocyte confocal images were obtained with a Zeiss 100 × 1.3 Plan-Neofluor oil immersion lens.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Identification of ILK in Keratinocyte Lysates-- ILK has been reported as a protein with a molecular mass of 59 kDa in a variety of cell types when detected on immunoblots using rabbit polyclonal antibodies raised against the entire protein (16). However, ILK has also been detected as a protein with molecular mass of 51 kDa in lysates from multiple rat tissues, mouse fibroblasts, and epithelial HeLa and COS-7 cells, using monoclonal antibodies raised against the C terminus of ILK (12). Thus, we first sought to determine the apparent molecular mass of ILK in mouse keratinocyte extracts analyzed with the available anti-ILK antibodies. Using either the mouse monoclonal antibody (ILK(TL)) raised against the C terminus of ILK or a goat polyclonal antibody raised against a synthetic peptide in the C terminus of ILK(ILK(SC)), we observed a single species of about 51 kDa on immunoblots, corresponding to endogenous ILK (Fig. 1). We also included in this analysis cell lysates from keratinocytes infected with recombinant adenoviruses encoding wild-type or E359K mutant V5-tagged ILK. We observed that the exogenous ILK exhibited a slightly lower mobility than the endogenous protein and an apparent mass of ~56 kDa. The mobility of the exogenously expressed ILK was confirmed using antibodies directed against the V5 epitope tag (Fig. 1). In all of the blots probed with the mouse and goat antibodies, the 56-kDa protein from infected cells overlapped with that of the exogenously expressed ILK forms, identified with the anti-V5 antibody (Fig. 1, V5 panel), indicating that the presence of the V5 tag slightly reduces the mobility of ILK on SDS-PAGE. Detection of both endogenous and exogenous ILK proteins was inhibited when the blots were probed with the goat polyclonal ILK(SC) antibody in the presence of the corresponding blocking peptide (Fig. 1), thus confirming the specificity of this antibody. These observations are consistent with the notion that, in mouse keratinocytes, endogenous ILK is expressed as a polypeptide of apparent molecular mass of 51 kDa. In contrast, when we probed those immunoblots with the rabbit polyclonal antibody ILK(UB)), raised against a purified, bacterially produced glutathione S-transferase fusion protein containing the entire ILK sequence, we were barely able to detect the V5-tagged exogenous ILK at 56 kDa and could not visualize the endogenous ILK protein at 51 kDa (Fig. 1). Rather, the ILK(UB) antibody detected a doublet at about 60 kDa, which does not correspond to any of the ILK species detected by the other antibodies and appears to represent an as yet unidentified protein (Fig. 1, complex A). As a result of these experiments, subsequent studies were conducted with either the mouse monoclonal or the goat polyclonal antibody, which specifically recognize ILK in primary mouse keratinocytes.


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Fig. 1.   Identification of ILK as a 51-kDa protein in epidermal keratinocytes. Lysates were prepared from uninfected undifferentiated primary mouse keratinocytes (U) or from keratinocytes infected 24 h prior to harvesting with a recombinant adenovirus encoding V5-tagged wild-type (Wt) or E359K mutant (Mt) ILK. Whole-cell lysates (50 µg/lane) were resolved by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The blots were probed with the following antibodies: ILK(TL), Transduction Laboratories mouse monoclonal anti-ILK (611803); ILK(SC), Santa Cruz Biotechnology goat polyclonal anti-ILK (sc7516), in the absence or presence of blocking peptide, as indicated; V5, Invitrogen anti-V5 (R96025); and ILK(UB), Upstate Biotechnology rabbit polyclonal anti-ILK (06-550). The positions of exogenous V5-tagged ILK and endogenous ILK are indicated. Complex A denotes the ~59-kDa species exclusively detected by the ILK(UB) antibody.

Regulation of ILK Protein Expression during Keratinocyte Differentiation-- Primary keratinocytes are a useful model of epidermal development. When cultured in medium containing low Ca2+ concentration (0.05-0.07 mM), these cells remain undifferentiated and retain proliferative capacity. Increases in extracellular Ca2+ (>= 0.1 mM) elicit terminal differentiation within 24 h, characterized by changes in gene expression and cytoskeletal organization, as well as formation of E-cadherin-containing adherens junctions involved in intercellular contact (17, 18).

Undifferentiated keratinocytes express various integrins, including alpha 6beta 4 and alpha 5beta 1, and signaling through these integrins is necessary for normal keratinocyte proliferation (19, 20). Induction of differentiation in keratinocytes in vivo or in culture is accompanied by integrin down-regulation and exit from the cell cycle (21). Given the association between ILK and beta 1 integrin function, we first investigated whether ILK expression was regulated during Ca2+-induced keratinocyte maturation. We found that cellular ILK protein levels were unaltered throughout the time course of keratinocyte differentiation (Fig. 2A), although beta 1 integrin expression was substantially reduced by 24 h following the addition of Ca2+ (Fig. 2B).


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Fig. 2.   ILK protein expression during keratinocyte differentiation. A, primary keratinocytes cultured in 0.05 mM Ca2+ were induced to differentiate by increasing extracellular Ca2+ levels to 1.0 mM. Cell lysates (50 µg/lane) were prepared at the indicated times after Ca2+ addition, resolved by SDS-PAGE, and probed with ILK(TL) or beta -tubulin antibodies. B, lysates from undifferentiated keratinocytes (lane 1) or from cells differentiated by culture in 1.0 mM Ca2+ for 24 h (lane 2) were resolved by SDS-PAGE, and immunoblots were probed with the indicated antibodies. C, adherent keratinocytes were maintained as undifferentiated cells (lane 1), were induced to differentiate with 1.0 mM Ca2+ (lane 2), or were trypsinized and cultured in suspension at low cell density in normal growth medium (0.05 mM Ca2+) containing 1.45% methyl-cellulose for 24 h (lane 3). Cell lysates were prepared, resolved by SDS-PAGE, and transferred to membranes, which were probed with the indicated antibodies. beta -Tubulin and GAPDH signals were used to normalize for differences in loading.

Culturing keratinocytes as a single-cell suspension in the presence of low extracellular Ca2+ has a dual effect. It initially prevents integrin ligation and subsequently results in differentiation and reduction in integrin abundance (22, 23). To determine whether ILK protein levels are regulated by integrin ligation, we also cultured keratinocytes in suspension for 24 h in medium containing 0.05 mM Ca2+. Similar to the Ca2+ treatments, culture in suspension for 24 h did not appreciably alter cellular ILK levels (Fig. 2C). Thus, ILK protein expression in cultured epidermal keratinocytes is not associated with differentiation status and is independent of integrin beta 1 expression or ligation.

Regulation of ILK-associated Kinase Activity in Differentiated Keratinocytes-- ILK exhibits weak kinase activity and has been reported to phosphorylate substrates such as protein kinase B/Akt and myosin light chain in some cell types (24, 25). ILK kinase activity increases upon integrin stimulation by fibronectin (16) and upon phosphoinositide 3-kinase stimulation (11). Whereas Ca2+-induced keratinocyte differentiation triggers integrin down-regulation, it also induces activation of phosphoinositide 3-kinase (17). Therefore, we examined whether kinase activity associated with ILK is altered in Ca2+-treated cells. We immunoprecipitated ILK from undifferentiated or differentiated keratinocyte lysates and observed that ILK immunoprecipitates from undifferentiated keratinocyte lysates efficiently phosphorylated MBP in vitro (Fig. 3A). In contrast, the kinase activity in ILK immunoprecipitates from differentiated cells was substantially reduced, although the total ILK protein levels were comparable in all lysates (Fig. 3, B and C). Thus, differentiation in keratinocytes triggers a reduction in kinase activity associated with ILK immunoprecipitates, suggesting kinase-independent roles for ILK in this cell type.


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Fig. 3.   Regulation of ILK-associated kinase activity during keratinocyte differentiation. A, phosphorylation of MBP by ILK immunoprecipitates from keratinocytes. ILK was immunoprecipitated from lysates (100 µg of protein/sample) obtained from keratinocytes cultured in 1.0 mM Ca2+ for the indicated times. Immune complexes were assayed for kinase activity using MBP as substrate and [gamma -32P]ATP. Phosphorylated MBP was resolved by SDS-PAGE and visualized by autoradiography. B, to verify the levels of ILK present in the immunoprecipitates used for kinase assays, replicate samples were prepared, resolved by SDS-PAGE, and immunoblotted with ILK antibodies. ILK migrates just under the IgG heavy chain, as indicated on the blot. C, a darker exposure of the ILK signal shown in B.

Distribution of ILK in Fractionated Keratinocyte Lysates-- In addition to its function as a kinase, ILK localizes to focal and fibrillar adhesions, interacting with several proteins and serving as a cytoskeletal adapter between integrins and the actin cytoskeleton (10, 11). To address the possibility of a scaffolding role for ILK in keratinocytes, we first examined its subcellular localization using fractionated cell extracts. We found that ILK is abundant in cytoplasmic fractions, irrespective of the differentiation status of the cells (Fig. 4A). ILK was also present in the Triton X-100-soluble fraction obtained after extraction of the cytoplasmic component as well as in the Triton X-100-insoluble fraction (Fig. 4A). To verify the efficiency of our fractionation, we sequentially probed our blots with marker proteins for each component (Fig. 4A). We observed that >= 95% of the cytosolic marker 14-3-3beta was present in the cytoplasmic fractions, in agreement with its reported distribution in a variety of cell types (26). Similarly, we used Na+/K+ ATPase as a marker for Triton X-100-soluble proteins (26) and found that it was exclusively present in this fraction. We also probed the membranes with alpha -actinin and caveolin 1, which are two proteins that generally segregate to Triton X-100-insoluble fractions (27, 28). We found that caveolin 1 was present exclusively in the Triton X-100-insoluble portion of extracts from both undifferentiated and differentiated keratinocytes. We also observed that, whereas alpha -actinin was exclusively present in Triton-insoluble fractions in undifferentiated keratinocyte lysates, a small proportion of alpha -actinin was present in cytosolic and Triton X-100-soluble fractions from differentiated cell extracts (Fig. 4A). We conclude that different pools of ILK are present in primary murine keratinocytes, which suggests that this protein probably fulfills multiple cellular functions in these cells.


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Fig. 4.   ILK subcellular fractionation and association with paxillin. A, primary mouse keratinocytes were cultured for 24 h in medium containing 0.05 mM Ca2+ (Low) or 1.0 mM Ca2+ (High). Cell lysates containing cytoplasmic (C), Triton X-100-soluble (S), or Triton X-100-insoluble (I) fractions were prepared, resolved by SDS-PAGE (50 µg of protein/lane), and transferred to a polyvinylidene difluoride membrane. The membrane was sequentially probed with the indicated antibodies. The positions of molecular mass markers are indicated on the left. The membrane was probed for alpha -actinin just prior to probing for Na+/K+ ATPase. The signal corresponding to alpha -actinin was still visible when the membrane was probed with the Na+/K+ ATPase antibody, as indicated. B, keratinocytes infected with an ILK-encoding adenovirus were cultured in the presence of 0.05 mM Ca2+ (lane 1) or 1.0 mM Ca2+ (lane 2) for 24 h. Cells were harvested and lysed, and paxillin was immunoprecipitated (IP) with a specific antibody. Immune complexes were resolved by SDS-PAGE, blotted, and analyzed for the presence of ILK. Control samples included cell lysates incubated with Sepharose beads in the absence of anti-paxillin antibody (lane 3) or 50 µg of whole cell lysate from undifferentiated cells to confirm the presence of ILK in the extracts (lane 4). C, keratinocytes expressing exogenous V5-tagged ILK were cultured in the presence of 0.05 mM Ca2+ (lane 1) or 1.0 mM Ca2+ (lane 2) for 24 h. Cells were harvested and lysed, and ILK was immunoprecipitated using an anti-V5 antibody. Immune complexes were resolved by SDS-PAGE, blotted, and analyzed for the presence of paxillin. Control samples included cell lysates incubated with protein A- and protein G-Sepharose in the absence of anti-V5 antibody (lane 3) and 50 µg of whole cell lysate from undifferentiated cells as positive control for paxillin (lane 4).

Integrins are essential for keratinocyte migration, adhesion, and proliferation (20, 29, 30). In undifferentiated keratinocytes, integrins concentrate in focal adhesions, connecting the actin cytoskeleton with the extracellular matrix. Focal adhesions also associate with the scaffolding protein paxillin, which interacts with ILK and various other proteins in nonepithelial cell types (12). The presence of ILK in Triton X-100-insoluble fractions, together with the known interactions between ILK and paxillin at focal adhesions, prompted us to investigate whether ILK and paxillin associate in undifferentiated keratinocytes. Thus, we isolated ILK immunoprecipitates from undifferentiated keratinocytes and probed them for the presence of paxillin. We observed that paxillin is a component of ILK immunoprecipitates from undifferentiated keratinocytes. Conversely, we were also able to demonstrate the presence of ILK in paxillin immunoprecipitates (Fig. 4, B and C), demonstrating the existence in undifferentiated keratinocytes of complexes containing both ILK and paxillin.

As mentioned above, differentiation induces marked decreases in integrin levels and focal adhesions in keratinocytes. Consequently, we investigated whether integrin down-regulation is associated with loss of ILK-paxillin interactions in differentiating keratinocytes. Using similar co-immunoprecipitation assays as those described for undifferentiated cells, we found that the interaction between ILK and paxillin is maintained in differentiated keratinocytes (Fig. 4, B and C), indicating its independence from integrin levels and differentiation status in these cells.

ILK Localizes to Cell-Cell Borders in Differentiated Keratinocytes-- To further investigate the regulation of ILK during keratinocyte maturation, we focused on the subcellular distribution of ILK, inducing expression of ILK by adenovirus-mediated gene transfer in undifferentiated and differentiated cells. We then visualized the exogenous protein by immunofluorescence microscopy. Undifferentiated keratinocytes cultured in 0.05 mM Ca2+ exhibit abundant focal adhesions but do not form adherens junctions (31). We observed that ILK immunofluorescence was detectable throughout the cell (Fig. 5) in samples fixed with cold methanol. Methanol fixation interferes with proper detection of several structures associated with the cytoskeleton, including focal adhesions. When we fixed the cells with PFA in the presence of Triton X-100 to better preserve cytoskeletal components, we observed that wild-type ILK exhibited a punctate pattern throughout the cell in undifferentiated keratinocytes (Fig. 6).


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Fig. 5.   Localization of ILK to cell-cell borders in differentiated keratinocytes. A, keratinocytes were infected with adenovirus encoding wild-type ILK (WT) 24 h prior to induction of differentiation by culture in medium containing 1.0 mM Ca2+. Cells were fixed with cold methanol and processed for immunofluorescence using an anti-ILK antibody at measured intervals following the addition of Ca2+, as indicated in individual micrographs. B, keratinocytes infected with a recombinant adenovirus encoding a V5-tagged E359K ILK mutant were induced to differentiate by culture in medium containing 1.0 mM Ca2+. The cells were processed to visualize ILK immunofluorescence using Triton X-100 permeabilization, followed by fixation in 2% PFA, as described under "Materials and Methods." The numbers in individual micrograph panels indicate the time of fixation after the addition of Ca2+. The No Ab panels represent negative controls containing infected cells cultured in medium with 1.0 mM Ca2+ for 0 or 24 h, as indicated, and processed for immunofluorescence microscopy excluding incubation with anti-V5 antibodies.


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Fig. 6.   Ca2+-induced changes in distribution of ILK and actin filaments. Primary mouse keratinocytes were infected with V5-tagged ILK-encoding adenovirus, and cultured in growth medium containing 0.05 µM (Low Ca2+) or 1.0 mM Ca2+ (High Ca2+) for 24 h prior to processing for confocal laser-scanning microscopy. Actin and exogenous ILK were visualized, respectively, using Cy3- or fluorescein isothiocyanate-labeled phalloidin and an anti-V5 antibody. Insets represent higher magnification images of the areas outlined in the High Ca2+ panel. Scale bars, 8 µm.

Increases in extracellular Ca2+ to 1-2 mM in keratinocyte cultures trigger the formation of intercellular junctions, which involves homotypic interactions of E-cadherin molecules localized on adjacent cells (18). In this system, there is an initial stage of adherens junction formation within 1 h of Ca2+ addition, followed by maturation and stabilization of the cell contacts within 20 h. Cultured keratinocytes infected with the ILK-encoding adenovirus in 1 mM Ca2+ for 24 h exhibited striking changes in ILK distribution to regions juxtaposed to points of cell-cell contact, which in some cells comprised the entire cell diameter (Fig. 5). Localization to cell-cell borders in Ca2+-treated keratinocytes was observed with exogenously expressed ILK (e.g. Figs. 5 and 6), as well as with the endogenous protein (e.g. Figs. 7 and 8). Importantly, ILK localization to cell-cell borders was not apparent during the first 12 h after Ca2+ addition but was readily detected in the mature junctions present after 24 h (Fig. 5). These results indicate that ILK movement to areas associated with intercellular junctions is not an immediate response to increased extracellular Ca2+ or initial formation of adherens junctions. Rather, ILK becomes associated with cell borders with kinetics comparable to those of junction maturation.


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Fig. 7.   ILK localization to cell-cell borders requires an intact actin cytoskeleton. A, primary keratinocytes were cultured for the indicated times in medium containing 1.0 mM Ca2+. To disrupt the actin cytoskeleton, some cultures were incubated in the presence of cytochalasin D (+cyto; 8 µM final concentration) for 15 min prior to the addition of Ca2+ (1.0 mM final concentration) and cultured for 24 h. Endogenous ILK was visualized using a goat anti-ILK antibody, and actin was visualized using fluorescein isothiocyanate-labeled phalloidin. B, controls containing cells infected with wild-type ILK-encoding adenovirus and cultured in medium with 1.0 mM Ca2+ for 0 or 24 h, as indicated, and processed for immunofluorescence microscopy, but in the presence of IgG in place of primary anti-ILK antibody.


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Fig. 8.   ILK localization and permanence adjacent to cell-cell borders requires high extracellular Ca2+ levels. Primary keratinocytes were cultured in medium containing 0.05 mM Ca2+ (A) or 1.0 mM Ca2+ (B) for 24 h prior to processing for fluorescence microscopy. Alternatively, the cells were cultured for 24 h in medium containing 1.0 mM Ca2+, washed thoroughly, and cultured for an additional 12 h in medium containing 0.05 mM Ca2+ (C). Endogenous ILK and E-cadherin were detected with the corresponding antibodies, whereas actin was visualized using fluorescein isothiocyanate-labeled phalloidin. ILK and actin were detected by double labeling of a single set of cells, whereas E-cadherin was visualized using replicate cell samples.

ILK localization to focal adhesions and interaction with paxillin requires a region in ILK that encompasses its putative kinase domain (32). Indeed, an E359K mutant of ILK that behaves as a dominant negative form in some cells but still exhibits kinase activity in vitro neither associates with paxillin nor localizes to focal adhesions (16, 32-34). To begin to explore the molecular determinants for ILK localization in keratinocytes, we investigated whether an intact kinase domain is required for normal distribution, analyzing the subcellular localization of the E359K ILK mutant. In undifferentiated keratinocytes, ILK E359K exhibited a diffuse distribution throughout the cell, distinct from the punctate pattern shown by the wild-type protein, indicating a requirement for the kinase domain in the normal distribution of this protein (Fig. 5). In contrast, this mutant was able to localize to cell-cell junctions in keratinocytes induced to differentiate with Ca2+ as efficiently as wild-type ILK (Fig. 5). This indicates that the mechanisms responsible for ILK association with paxillin and characteristic subcellular distribution in undifferentiated keratinocytes are dissociable from those that induce ILK localization to intercellular borders in differentiated cells. ILK localization to cell borders in Ca2+-treated cells does not appear to depend on the presence of a fully functional kinase domain or on direct association with paxillin.

ILK Subcellular Distribution and Actin Filament Organization-- Undifferentiated primary keratinocytes form actin networks of stress fibers that appear as thin cobweb-like structures (Fig. 6). F-actin stress fibers in undifferentiated keratinocytes can end at focal adhesion sites or participate in the formation of filopodia or leading edges in migrating cells (18, 35). In these cells, we observed areas of endogenous ILK localization in close proximity to the edges of actin bundles in addition to the previously described punctate distribution throughout the cell (Fig. 6). The formation of intercellular junctions in keratinocytes triggered by increases in extracellular Ca2+ also requires actin reorganization and polymerization to seal cell-cell borders (18). This results in the formation of cortical actin fibers that run parallel to the plasma membrane (Fig. 6). When we examined the relationship between these cortical actin filaments and ILK located along intercellular junctions, we observed that ribbons of ILK immunoreactivity occur along the cell membrane, running parallel and in close apposition to the actin fibers (Fig. 6). The localization patterns of ILK and actin fibers prompted us to examine whether ILK would still segregate to cell borders in cultures in which the cytoskeleton was disrupted by treatment with cytochalasin D. We found that disassembly of F-actin filaments in cells cultured in the presence of cytochalasin D and 1 mM Ca2+ was accompanied by loss of ILK from the cell periphery (Fig. 7), indicating that actin polymerization is an essential requirement for ILK redistribution in differentiated keratinocytes.

Ca2+ Dependence of ILK Localization to Intercellular Junctions-- The development of adherens junctions in keratinocytes cultured in 1-2 mM Ca2+ also requires the accumulation at cell-cell borders of E-cadherin complexes associated with the actin cytoskeleton and that contain alpha - and beta -catenin (21, 36). Because homotypic E-cadherin interactions are Ca2+-dependent, maintenance of adherens junctions in differentiated keratinocytes requires the continuous presence of high extracellular Ca2+ levels. To further investigate the mechanisms directing ILK to intercellular junctions in differentiated keratinocytes, we determined the calcium requirements of this process. Thus, we first cultured cells for 24 h in 1 mM extracellular Ca2+ to induce ILK migration to areas of cell-cell contact (Fig. 8, A and B). Subsequently, we switched the culture medium from 1.0 to 0.05 mM Ca2+ and cultured the cells for an additional 12-h period. As shown in Fig. 8C, Ca2+ withdrawal in differentiated keratinocytes induced the loss of E-cadherin localization to cell-cell borders, disrupting the previously formed adherens junctions. Decreases in extracellular Ca2+ levels also altered the cortical actin fibers assembled parallel to the membrane, although other cytoskeletal actin fibers throughout the cell remained assembled (Fig. 8C). Ca2+ withdrawal also resulted in the disappearance or substantial reduction of ILK immunoreactivity from cell borders, similar to that observed for the cortical actin filaments. These results demonstrate that the patterns of localization of F-actin, ILK, and E-cadherin along intercellular borders require the continuous presence of high extracellular Ca2+ concentrations. When we conducted these experiments in cells exogenously expressing ILK by adenovirus-mediated gene transfer, we found that the increased ILK levels in differentiated keratinocytes did not interfere with formation of cell junctions or with expression and localization of E-cadherin and beta -catenin to cell borders in response to elevated extracellular Ca2+ (e.g. Fig. 6 and data not shown), indicating that elevated ILK does not induce mesenchymal transformation in differentiated epidermal keratinocytes. Together, our results demonstrate that ILK is tightly regulated at multiple post-translational levels during keratinocyte differentiation and suggest a potential novel role for this protein in the genesis of cell-cell contacts in differentiated keratinocytes and in epidermal barrier formation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Modulation of ILK Abundance and Kinase Activity during Keratinocyte Differentiation-- The epidermis is a complex epithelial tissue, which consists of undifferentiated, proliferative cells and their terminally differentiated progeny. A notable characteristic of primary murine epidermal keratinocytes is their ability to undergo terminal differentiation in response to increases in extracellular Ca2+ concentrations. Differentiation in primary cultured keratinocytes is accompanied by loss of integrin expression and focal adhesions. Because ILK is an important component of integrin signaling and focal adhesion complexes, we investigated if ILK is also regulated during Ca2+-induced keratinocyte differentiation. We found that, whereas ILK protein levels do not appreciably change in differentiated keratinocytes, kinase activity in ILK immunoprecipitates is substantially decreased. The ILK immune complexes we isolated from keratinocytes also contained other cellular proteins and, consequently, it remains to be determined whether the kinase activity we measured in the keratinocyte extracts arises from ILK itself or from another protein present in ILK immunoprecipitates. Regardless, the decreased ILK-associated kinase activity triggered by Ca2+ treatment in keratinocytes is significant, since it demonstrates the existence of tight regulatory mechanisms for this pathway in response to differentiation signals in epidermal tissue.

ILK as a Cytoskeletal Adapter in Undifferentiated Keratinocytes-- ILK appears to have dual functions as a kinase and as a scaffold in multiprotein complexes. Specifically, ILK localizes to focal and fibrillar adhesions, serving as a cytoskeletal adapter between integrins and the actin cytoskeleton, which is essential for muscle development in C. elegans and D. melanogaster (10, 11). Central for the adapter functions of ILK are its interactions with factors such as PINCH, paxillin, and actopaxin (12, 32, 37). In undifferentiated keratinocytes, ILK can be found in Triton X-100-insoluble fractions and in complexes that contain paxillin. In these cells, ILK is distributed with a punctate pattern. It remains to be determined whether ILK is directly associated with focal adhesions in undifferentiated keratinocytes. The different patterns of ILK cellular distribution in undifferentiated versus differentiated keratinocytes suggest that ILK may play multiple roles in adhesion, migration, and/or proliferation in these cells through a combination of signaling and scaffold functions.

ILK and Formation of Cell-Cell Junctions-- Primary keratinocytes cultured in 1-2 mM Ca2+ form stratified layers with extensive cell-cell contacts (38). Under these conditions, integrins do not play a major role in intercellular adhesion (8, 21). The formation of adherens junctions in keratinocytes cultured in 1-2 mM Ca2+ is accompanied by accumulation of E-cadherin complexes containing alpha - and beta -catenin. Such complexes are associated with the actin cytoskeleton and exhibit decreased solubility in Triton X-100 (21, 36). The development of adherens junctions requires reciprocal interactions between E-cadherin and the actin cytoskeleton. Specifically, initial intercellular ligation of E-cadherin dimers can directly induce actin assembly at the cell surface (39), but continuous actin reorganization and polymerization is required to maintain sealed cell borders, as evidenced by the disruption of cadherin-mediated cell-cell junctions in cytochalasin D-treated cells (18).

We have determined that, in differentiated keratinocytes, ILK is present in cytosolic, Triton X-100-soluble and -insoluble fractions. This contrasts with its primarily cytoskeletal distribution in other cell types (24, 34). Notably, induction of differentiation by Ca2+ in keratinocytes is accompanied by marked changes in subcellular location, characterized by ILK concentration to cell-cell borders. The keratinocyte response to Ca2+ may be tissue-specific, since ILK appears to be excluded from intercellular adhesion sites in other cell types (37). Ca2+ also triggers accumulation of E-cadherin and beta -catenin at intercellular junctions (18). We observed that beta -catenin shows only partial co-localization with ILK by immunofluorescence microscopy, but we have not detected beta -catenin in ILK immunoprecipitates,2 suggesting that ILK and beta -catenin may participate in the formation of distinct complexes associated with cell borders and/or the cytoskeleton.

It has been demonstrated that Ca2+ plays a dual role in keratinocyte adhesion; it stimulates homotypic cadherin engagement and activates a pathway that leads to actin polymerization at contact sites (18). Our experiments suggest that both events are necessary for ILK localization to cell junctions. This is evidenced by the loss of ILK from intercellular adhesion sites following either disruption of E-cadherin ligation by blocking antibodies2 or disruption of actin polymerization by cytochalasin D treatment. In keratinocytes induced to differentiate by Ca2+, groups of cortical actin bundles are formed in a Ca2+-dependent manner parallel to the cell borders (Fig. 6) in a process that coincides with the appearance of E-cadherin localization at intercellular junctions (41). We have identified ILK as an additional component in those multiprotein complexes found adjacent to the sealed cell borders in keratinocytes.

A tight relationship between ILK and the actin cytoskeleton is also indicated by the close localization of ILK to cortical actin fibers at cell-cell borders in differentiated keratinocytes (e.g. Fig. 6). Taken together with the known down-regulation of integrins that occurs upon keratinocyte differentiation (42), our data suggest that ILK may uniquely participate in the establishment and/or maintenance of cell-cell contacts in differentiated epidermal keratinocytes that is unrelated to integrin function.

In addition to cell-cell borders, we also observed ILK immunofluorescence in inner cell regions in differentiated keratinocytes. Our analysis of ILK immunoprecipitates from differentiated keratinocytes has shown the presence of paxillin and actopaxin in these complexes (Fig. 4B).2 However, the capacity of the mutant E359K to segregate to cell-cell borders despite its inability to interact with paxillin (12) suggests that the ILK-paxillin complexes detected in differentiated keratinocytes may serve a separate function. Suprabasal epidermal layers containing differentiated keratinocytes do not exhibit focal adhesions, although they form extensive cell-cell contacts. In these regions, paxillin is not found in foci corresponding to areas of intercellular contact (21).

ILK Does Not Induce Epithelial-Mesenchymal Transition in Differentiated Keratinocytes-- We observed E-cadherin formation of adherens junctions and ILK localization to cell-cell borders in Ca2+-treated keratinocytes, irrespective of whether the cells exogenously expressed ILK. The fact that in epidermal keratinocytes ILK overexpression does not induce epithelial-mesenchymal transitions through E-cadherin degradation and loss of intercellular adhesion contrasts sharply with the reported responses to ectopic ILK expression in mammary and in intestinal epithelial cell lines (40, 43). Indeed, our results are consistent with the proposal that ILK may serve a unique role in intercellular adhesion, cytoskeletal organization, and maintenance of barrier function in differentiated keratinocytes.

    ACKNOWLEDGEMENTS

We thank Drs. S. Dedhar for ILK and ILK E359K cDNA constructs, R. Kothary for the anti-beta -tubulin antibody, S. Ferguson for helpful comments on the manuscript, and L. Dale for help with confocal microscopy.

    FOOTNOTES

* This work was supported by grants from the Canadian Institutes of Health Research (to L. D.) and from the Kidney Foundation of Canada (to S. J. A. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of an Ontario Graduate Scholarship in Science and Technology.

|| Supported by National Institutes of Health Grant HL70244.

** To whom correspondence may be addressed: Dept. of Physiology and Pharmacology, Medical Sciences Bldg., University of Western Ontario, London, Ontario N6A 5C1, Canada. Fax: 519-661-3827; E-mail: sjdsouza@uwo.ca.

§§ A Cancer Research Society/CIHR Scholar. To whom correspondence may be addressed: Dept. of Physiology and Pharmacology, Medical Sciences Bldg., University of Western Ontario, London, Ontario N6A 5C1, Canada. Fax: 519-661-3827; E-mail: ldagnino@uwo.ca.

Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M208337200

2 A. Vespa, S. D'Souza, and L. Dagnino, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: ILK, integrin-linked kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MBP, myelin basic protein; PBS, phosphate-buffered saline; PFA, paraformaldehyde.

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