E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions
2005; Springer Nature; Volume: 24; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7600605
ISSN1460-2075
AutoresJudith A Tunggal, Iris Helfrich, Annika Schmitz, Heinz Schwarz, Dorothee Günzel, Michael Fromm, Rolf Kemler, Thomas Krieg, Carien M. Niessen,
Tópico(s)Wound Healing and Treatments
ResumoArticle3 March 2005free access E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions Judith A Tunggal Judith A Tunggal Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Search for more papers by this author Iris Helfrich Iris Helfrich Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Search for more papers by this author Annika Schmitz Annika Schmitz Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Search for more papers by this author Heinz Schwarz Heinz Schwarz Max Planck Institute for Developmental Biology, Tuebingen, Germany Search for more papers by this author Dorothee Günzel Dorothee Günzel Department of Clinical Physiology, Charité, Campus Benjamin Franklin, Berlin, Germany Search for more papers by this author Michael Fromm Michael Fromm Department of Clinical Physiology, Charité, Campus Benjamin Franklin, Berlin, Germany Search for more papers by this author Rolf Kemler Rolf Kemler Department of Molecular Embryology, Max Planck Institute for Immunobiology, Freiburg, Germany Search for more papers by this author Thomas Krieg Thomas Krieg Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Carien M Niessen Corresponding Author Carien M Niessen Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Search for more papers by this author Judith A Tunggal Judith A Tunggal Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Search for more papers by this author Iris Helfrich Iris Helfrich Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Search for more papers by this author Annika Schmitz Annika Schmitz Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Search for more papers by this author Heinz Schwarz Heinz Schwarz Max Planck Institute for Developmental Biology, Tuebingen, Germany Search for more papers by this author Dorothee Günzel Dorothee Günzel Department of Clinical Physiology, Charité, Campus Benjamin Franklin, Berlin, Germany Search for more papers by this author Michael Fromm Michael Fromm Department of Clinical Physiology, Charité, Campus Benjamin Franklin, Berlin, Germany Search for more papers by this author Rolf Kemler Rolf Kemler Department of Molecular Embryology, Max Planck Institute for Immunobiology, Freiburg, Germany Search for more papers by this author Thomas Krieg Thomas Krieg Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Carien M Niessen Corresponding Author Carien M Niessen Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Search for more papers by this author Author Information Judith A Tunggal1,‡, Iris Helfrich1,‡, Annika Schmitz1, Heinz Schwarz2, Dorothee Günzel3, Michael Fromm3, Rolf Kemler4, Thomas Krieg1,5 and Carien M Niessen 1 1Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany 2Max Planck Institute for Developmental Biology, Tuebingen, Germany 3Department of Clinical Physiology, Charité, Campus Benjamin Franklin, Berlin, Germany 4Department of Molecular Embryology, Max Planck Institute for Immunobiology, Freiburg, Germany 5Department of Dermatology, University of Cologne, Cologne, Germany ‡These authors contributed equally to this work *Corresponding author. Center for Molecular Medicine (ZMMK), University of Cologne, LFI, 05, room 59, Joseph Stelzmannstrasse 9, 50931 Cologne, Germany. Tel.: +221 4787738; Fax: +221 4784836; E-mail: [email protected] The EMBO Journal (2005)24:1146-1156https://doi.org/10.1038/sj.emboj.7600605 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell–cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E-cadherin in stratified epithelium of the epidermis, we have conditionally inactivated its gene in mice. Here we show that loss of E-cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, our results indicate that E-cadherin is specifically required for tight junction, but not desmosome, formation and this appears to involve signalling rather than cell contact formation. Introduction E-cadherin is important for tissue morphogenesis and polarity (Larue et al, 1994). Upon establishing cell–cell adhesion, cadherins cluster in specialized cell junctions, known as adherens junctions, which associate with the actin cytoskeleton (Gottardi et al, 2002). By thus coupling cell adhesion to the cytoskeleton, E-cadherin can create a transcellular network that enables groups of cells or tissues to coordinate their behaviour. The association of E-cadherin with the actin cytoskeleton is mediated by α-catenin that is linked to the cadherin via β-catenin (Rimm et al, 1995). The latter molecule is also a key player in the Wnt signalling pathway (Gottardi et al, 2002). Another catenin, p120ctn, also binds directly to the cadherin and regulates the stability of the cadherin complex at the cell surface (Reynolds and Roczniak-Ferguson, 2004). The catenin plakoglobin can functionally replace β-catenin in the adherens junctions, but is also a structural component of desmosomes. These cell structures contain nonclassical cadherins and associate with the intermediate filament cytoskeleton. Desmosomes provide tissues with stable and strong cell–cell adhesion (Getsios et al, 2004). Next to this core cadherin–catenin complex, many other scaffolding, signalling and cytoskeletal molecules are associated with the adherens junctions (Perez-Moreno et al, 2003). For example, the tight junctional marker ZO-1 binds directly to α-catenin and this is considered an intermediate step in the formation of tight junctions (Itoh et al, 1997). Tight junctions act as selective permeability barriers but also form a fence that physically separates the apical membrane domain from the basolateral domain in simple epithelial cells (Anderson et al, 2004). The size and ion selectivity of tight junctions is variable within different tissues and depends on the type and levels of expression of claudins, four span transmembrane molecules, which constitute the tight junctional paired strands (Inai et al, 1999; Colegio et al, 2003). In vitro studies have shown that E-cadherin is not only necessary for adherens junction formation but its adhesive activity is also crucial for the assembly of other junctional complexes such as desmosomes, gap junctions and tight junctions (Gumbiner et al, 1988; Musil et al, 1990; Watabe et al, 1994). This junctional complex assembly coincides with the establishment of cell polarity and enrichment at the cell junctions of several protein complexes known to be essential for polarity, suggesting an intimate relationship between junction formation and polarity (Nelson, 2003). Indeed, experiments in Drosophila have shown that mutations in junctional proteins not only affect junction formation but also disturb epithelial polarity, whereas polarity mutants have a profound effect on junction formation as well (Cox et al, 1996; Muller and Wieschaus, 1996; Tanentzapf et al, 2000). Together, these data have led to a model in which initial cell–cell adhesion mediated by the cadherin complex is a key step in setting up other cell junctions and epithelial polarity. One of these polarity complexes consists of the Par3/Par6/atypicalPKC (aPKC) complex, which localizes to the apical junctional complex in Drosophila and to tight junctions in mammalian epithelial cells (Nelson, 2003). The activity of the Par3/Par6/aPKC polarity protein complex has been implicated in the formation of tight junctions in simple epithelia (Macara, 2004). Its activation depends on the formation of cell–cell contacts (Yamanaka et al, 2001) and Armadillo, the fly β-catenin homologue, is important for proper localization of this complex in Drosophila (Bilder et al, 2003; Tanentzapf and Tepass, 2003). Activation of the Par3/Par6/aPKC complex is achieved by binding to the small GTPase Cdc42, resulting in the phosphorylation and activation of aPKC (Joberty et al, 2000; Lin et al, 2000). The epidermis is a stratifying, squamous differentiating multilayered epithelium that serves as the first barrier with the outside environment. Not only does it protect against outside challenges such as microbes or toxic substances, it also prevents the unnecessary loss of water from the organism. Cell–cell adhesion within the epidermis is mediated by both adherens junctions and desmosomes. Two classical cadherins are expressed within the epidermis: P-cadherin, which is confined to the basal layer, and E-cadherin found in all epidermal cell layers (Jensen et al, 1997). The epidermis is not a classical polarized epithelium, such as intestinal epithelium, which has a clear apical and basolateral membrane domain separated by the apical junctional complex. Nevertheless, the epidermis shows polarization in a broader sense in that the specific layers of the epidermis express a unique set of differentiation and junctional markers (Watt, 2001). For example, hemidesmosomes are restricted to the basal layer, whereas desmosomes are found in all layers but their number and protein composition vary between the different layers (Getsios et al, 2004). Tight junctions, previously thought to be absent in the epidermis, are exclusively found in the granular layer (Morita et al, 1998; Pummi et al, 2001; Brandner et al, 2002). Their importance in maintaining the epidermal barrier was underscored by the observations that in vivo inactivation of the tight junctional membrane protein claudin-1 results in epidermal water loss and ultimately in neonatal death of the mice (Furuse et al, 2002). In addition, claudin-6 overexpression in the skin also disturbs barrier function (Turksen and Troy, 2002). To study the role of E-cadherin in the morphogenesis and function of stratifying epithelium, we inactivated its gene specifically in the developing epidermis of mice using the Cre-loxP system. We show that E-cadherin is essential for epidermal barrier function. Mice lacking E-cadherin in the epidermis die shortly after birth because of dehydration. Closer molecular examination revealed that key tight junctional components are improperly localized, resulting in altered tight junctional architecture. Indeed, biotin penetrance of the granular layer and altered resistance using impedance measurements of the E-cadherin-negative epidermis revealed impaired tight junction function. Surprisingly, desmosomes are still normally formed, indicating that, unlike previously thought, E-cadherin is specifically required for tight junction formation. Since cell contacts are not majorly altered, E-cadherin may influence tight junction formation via signalling molecules. Potential candidates are the small GTPase Rac and atypical PKC, which both show an altered distribution upon loss of E-cadherin in the epidermis. Results Generation of mice lacking E-cadherin in the epidermis To study how cadherins contribute to junction formation and morphogenesis in stratifying epithelia, we specifically inactivated E-cadherin in developing mouse epidermis by crossing mice carrying two floxed E-cadherin alleles with mice expressing the Cre-recombinase enzyme under the human Keratin 14 promoter (Hafner et al, 2004) in combination with either one floxed E-cadherin allele or one deleted E-cadherin allele (Boussadia et al, 2002). This resulted in either K14-Cre/EcadFl/Fl or K14-Cre/EcadFl/− mice (Figure 1A) that both died within 1 day after birth. Since we have found no phenotypic difference between the two E-cadherin mutant lines, only data on the K14-Cre/EcadFl/− mice are described. Figure 1.(A) PCR analysis of offspring from E-cadherinFl/Fl mice crossed with a deleter K14-Cre/E-cadherin+/− line. Indicated is the DNA fragment generated by PCR reaction. (B) RT–PCR analysis of RNA isolated from skin biopsies of control (ct) and K14-Cre/EcadFl/− (−/−) mice. M: marker. (C) Immunohistochemical analysis of newborn and embryonic day 15.5 (E15.5) skin sections for E-cadherin (green). Nuclei were counterstained with propidium iodide (red). The different epidermal layers are also indicated: SB: stratum basale or basal layer; SS: stratum spinosum or spinal layer; SG: stratum granulosum or granular layer; SC: stratum corneum or cornified layer. The dotted white line indicates the epidermal–dermal junction. (D) Western blot analysis of total newborn mouse skin for E-cadherin. Download figure Download PowerPoint The E-cadherin gene was efficiently deleted from the epidermis of K14-Cre/EcadFl/− mice, as no RNA expression was observed (Figure 1B). Indeed, staining for E-cadherin was negative in the newborn epidermis of mutant mice, whereas E-cadherin staining was found as expected in all layers of the epidermis in the skin of newborn control mice (Figure 1C). In addition, E-cadherin was already almost completely absent in the epidermis of 15.5-day-old embryos (E15.5), indicating efficient deletion during development of the epidermis (Figure 1C). Western blot analysis of total skin lysates confirmed the absence of E-cadherin protein in knockout but not control skin (Figure 1D). Cell–cell contacts are maintained in the absence of E-cadherin Macroscopic examination of the K14-Cre/EcadFl/− mice did not reveal any blistering of the skin, even after rubbing, suggesting no major changes in cell contact formation. We therefore examined the expression and localization of other adherens junction molecules. P-cadherin expression is upregulated (Figure 2B) but was still mainly found in the basal layer of the epidermis of E-cadherin mutant mice (Figure 2A), suggesting that P-cadherin only compensates for the loss of E-cadherin in the basal layer. Absence of E-cadherin resulted in loss or largely reduced staining in the suprabasal layers for the cadherin-associated molecules α-catenin, β-catenin and p120ctn (Figure 2A). These results suggested that the absence of E-cadherin from the suprabasal layers was not compensated by the expression of other classical cadherins. Catenin protein expression in the skin was not substantially altered (Figure 2B), most likely because of the upregulation of P-cadherin expression in the basal layer. Figure 2.Adherens junctions expression is altered in K14-Cre/EcadFl/− mice. (A) Immunohistochemical analysis of newborn control and K14-Cre/EcadFl/− (−/−) skin sections stained for adherens junction markers. Nuclei are counterstained with propidium iodide. (B) Western blot analysis of total newborn skin lysates for the indicated adherens junction markers. Download figure Download PowerPoint In vitro studies have shown that E-cadherin is essential for desmosome formation, and we therefore asked the question if in vivo loss of E-cadherin affects desmosomal components. As described previously (Young et al, 2003; Tinkle et al, 2004), no difference in localization of desmosomal markers like plakoglobin, desmocollin 2 and desmoglein 1/2 was observed (Figure 3A). Indeed, ultrastructural analysis showed the presence of well developed desmosomes in the K14-Cre/EcadFl/− mice (Figure 3B). Desmosomal cadherin expression was upregulated (Figure 3C), suggesting compensation for the loss of classical cadherins in the suprabasal layers. Thus, E-cadherin is not essential for desmosome formation in vivo. Importantly, histological and ultrastructural analysis revealed no obvious defects in cell contacts in skin sections of control and K14-Cre/EcadFl/− mice (Figure 4 and Supplementary Figure 1). Differentiation markers and proliferation appeared normal (Supplementary Figure 2). The upregulation of P-cadherin in the basal layer in combination with an increase in desmosomal cadherins may explain why no obvious loss of cell–cell cohesion is observed in the K14-Cre/EcadFl/− mice. Figure 3.Desmosomes are formed in the absence of E-cadherin. (A) Immunohistochemical analysis of newborn control and K14-Cre/EcadFl/− (−/−) skin sections stained for desmosomal markers. Desmoglein 1/2 is red with nuclei counterstained with dapi (blue) whereas desmocollin 2 and plakoglobin are green with nuclei counterstained with propidium iodide (red). (B) Ultrastructural analysis of desmosomes in newborn control and K14-Cre/EcadFl/− (−/−) skin. (C) Western blot analysis of total newborn skin lysates for plakoglobin and desmoglein 1/2. Download figure Download PowerPoint Figure 4.Normal structure and cell–cell contacts in K14-Cre/EcadFl/− mice. Ultrastructural analysis of newborn control and K14-Cre/EcadFl/− (−/−) skin. Download figure Download PowerPoint Early deletion of E-cadherin in the epidermis results in perinatal death due to water loss The K14-Cre/EcadFl/− mice die perinatally around 7–12 h after birth. The mutant skin appears more red and shiny than that of their control littermates, and has a parchment-like resemblance. In all K14-Cre/EcadFl/− mice, scaling of the upper ventral side occurs after a few hours (Figure 5A). This macroscopic appearance of the mice suggested a perturbed water barrier. Weighing experiments showed that E-cadherin mutant mice lost around 9% of their body weight during their short lifespan in comparison to an average 3% in control mice (Figure 5B). An increased haematocrit of mutant mice was also observed (Figure 5C), indicating loss of fluids from the K14-Cre/EcadFl/− mice. Indeed, a considerable increase in transepidermal water loss (TEWL) was measured in the K14-Cre/EcadFl/− mice compared to control (Figure 5D). Thus, the lethal phenotype of the E-cadherin mutant mice most likely results from water loss as a result of a disturbance in epidermal barrier function. Figure 5.K14-Cre/EcadFl/− mice die of water loss. (A) Macroscopic appearance of newborn control E-cadherin and K14-Cre/EcadFl/− mice. The inset shows close-up of the ventral side. (B) Weight time-course analysis of newborn control and K14-Cre/EcadFl/− mice. (C) Haematocrit analysis of blood isolated from control (n=35) and mutant mice (n=13). (D) TEWL measurements of control and E-cadherin mutant mice. Download figure Download PowerPoint The outside-in barrier is functional in K14-Cre/EcadFl/− mice To examine if the observed TEWL was due to a nonfunctional stratum corneum, we assessed the permeability of the newborn epidermal barrier from the outside using the dye lucifer yellow. Only staining of the surface of the cornified layer was observed in both control and K14-Cre/EcadFl/− newborn mice (Figure 6A), indicating the presence of a functional stratum corneum. Barrier formation may be delayed in the K14-Cre/EcadFl/− mice and we therefore performed toluidine blue penetration assays of E18.5 and E16.5 embryos. The skin was impermeable for both control and K14-Cre/EcadFl/− E18.5 embryos (Figure 6B), indicating that the outside-in barrier is functional at this stage in the E-cadherin mutant embryos. Barrier formation starts around E16 in a patterned fashion (Hardman et al, 1998). Indeed, partial penetration of toluidine blue is observed around the paws and the ventral side in control E16.5 embryos and this pattern is similar for K14-Cre/EcadFl/− E16.5 embryos (Figure 6B). We therefore conclude that maturation of the cornified layer developed normal in the absence of E-cadherin, resulting in a properly functioning outside-in barrier. Figure 6.Epidermal loss of E-cadherin does not affect the outside-in barrier. (A) Lucifer yellow dye penetration assay of newborn control and E-cadherin mutant mice. No penetration of lucifer yellow across the stratum corneum is observed when comparing control mice to K14-Cre/EcadFl/− mice. (B) Toluidine blue penetration assay of E18.5- and E16.5-day-old control and K14-Cre/EcadFl/− mice. Download figure Download PowerPoint Increased permeability of tight junctions in the absence of E-cadherin Interestingly, mice deficient for the tight junctional protein claudin-1 also show extensive water loss, appear red, shiny and wrinkled, and die within the same time frame as our mutant mice negative for E-cadherin in the epidermis (Furuse et al, 2002). These mice also have an apparently normal cornified envelope. To examine if the observed TEWL is a result of increased inside-out permeability due to leaky tight junctions, which are present in the stratum granulosum (Brandner et al, 2002; Furuse et al, 2002), we injected newborn mice intradermally with biotin and assessed its diffusion into the epidermis. In control skin, biotin moves up to the stratum granulosum, where it is halted due to the presence of tight junctions, marked by occludin staining, between the cells of the granular layer (Furuse et al, 2002). However, in K14-Cre/EcadFl/− epidermis, biotin penetrates the granular layer and is able to reach the stratum corneum (Figure 7A, left panel). This suggested that tight junctions are either absent or are no longer properly functioning in E-cadherin mutant skin. Figure 7.Tight junctions are functionally impaired in K14-Cre/EcadFl/− mice. (A) Inside-out permeability assay. Skin sections of control and mutant newborn mice intradermally injected with biotin were stained with streptavidin to follow the penetration of biotin (green) and counterstained with occludin to mark the tight junctions present in the granular layer. (B) Impedance measurements of ventral skin biopsies isolated from newborn control and E-cadherin mutant mice. Typical experiments are depicted in Nyquist plots; numerical values represent mean±s.e.m. *P-value=0.03. N=8 for control and N=4 for K14-Cre/EcadFl/− (−/−) mice. Download figure Download PowerPoint Tight junctions restrict paracellular diffusion of ions, and this function can be determined by measuring transepithelial resistance. We performed impedance analysis on skin biopsies from control and K14-Cre/EcadFl/− mice in order to measure simultaneously the resistance of epidermal and dermal tissues (Fromm et al, 1985). A three-fold lower epidermal resistance was observed in mutant skin compared to control skin, whereas the dermal resistance was not significantly affected (Figure 7B). Together, these results strongly indicate that the water loss seen in K14-Cre/EcadFl/− mice is directly related to increased tight junctional permeability. Altered tight junction formation in K14-Cre/EcadFl/− mice Since the permeability of tight junction is affected in K14-Cre/EcadFl/− mice, we examined the localization of different tight junctional components. Occludin is specifically concentrated on the membrane in the granular layer of control mice (Morita et al, 1998; Furuse et al, 2002). Occludin is still enriched at the membrane in the granular layer of K14-Cre/EcadFl/− mice (Figure 8A), implying that tight junctions are not completely disturbed in the absence of E-cadherin. This is also suggested by the observation of tight junction-like structures in the granular layer of K14-Cre/EcadFl/− mice by conventional electron microscopy (Supplementary Figure 3). Figure 8.Improper formation of tight junctions in K14-Cre/EcadFl/− mice. (A) Immunohistochemical analysis of skin sections for the tight junctional markers (green) occludin, claudin-4, claudin-1 and ZO-1. Nuclei were counterstained with propidium iodide (red). For claudin-1, both × 400 and × 1000 images are shown. White vertical lines indicate absence of claudin-1 staining from granular layer. White bars=25 μM. (B) Western blot of total skin lysates isolated from newborn control and K14-Cre/EcadFl/− (−/−) mice using antibodies against the indicated tight junctional proteins. Download figure Download PowerPoint Since claudins are essential for the size and ion specificity of the tight junctional barrier, we next addressed the localization of these proteins. Like occludin, claudin-4 is found at the membrane in the granular layer of the epidermis in control mice (Furuse et al, 2002). Although its localization is still confined to the granular layer in K14-Cre/EcadFl/− mice, its distribution on the membrane is altered, showing a more punctate staining pattern. This appears to correlate with less protein expression in the K14-Cre/EcadFl/− mice (Figure 8B). Claudin-1 is not a specific marker for tight junctions in the epidermis but, as described previously (Furuse et al, 2002), is found at the membrane of all epidermal layers in control mice (Figure 8A). Interestingly, in K14-Cre/EcadFl/− mice, claudin-1 staining was absent from cells of the granular layer of the epidermis (Figure 8A), which form the functional tight junctions. A slight decrease of total claudin-1 protein was also observed (Figure 8B). How does loss of E-cadherin affect proper formation of tight junctions? The most direct link between adherens and tight junctions is ZO-1, which can directly interact with the cadherin complex via α-catenin (Itoh et al, 1997). This is suggested to be an intermediate step in tight junction formation. To examine if loss of E-cadherin affects recruitment of ZO-1 to the membrane, we stained control and mutant skin for this protein. ZO-1 membrane staining was still observed, indicating that the improper formation of epidermal tight junctions is not due to the inability to recruit ZO-1 to the membrane. However, we did see altered distribution of ZO-1 at the cell surface, showing a more punctate pattern in the epidermis of K14-Cre/EcadFl/− mice (Figure 8A). Inappropriate localization of ZO-1 may thus contribute to altered tight junction function. Overall, our results show that even though tight junction formation is not completely disturbed, key components are not properly incorporated, which likely underlies the functional impairment of tight junctions. Altered localization of atypical PKC and its activated forms in K14-Cre/EcadFl/− mice The activity of the Par3/Par6/aPKC polarity protein complex has been implicated in the formation of tight junctions in simple epithelia (Hirose et al, 2002; Macara, 2004). To examine if E-cadherin is important for the proper distribution of this complex in epidermis, we stained control and mutant skin for its different components. Par3 is found at cell–cell contacts in all suprabasal layers of the epidermis, and its distribution (Figure 9A) and amount of protein (Figure 9B) were not significantly altered in K14-Cre/EcadFl/− mice. Figure 9.Altered localization of activated atypical PKC and the small GTPase Rac in K14-Cre/EcadFl/− mice. (A) Immunohistochemical analysis of skin sections for the indicated polarity complex proteins and the small GTPases Rac and Cdc42 (green). Nuclei were counterstained with propidium iodide (red). (B) Western blot analysis of the indicated proteins in total skin lysates isolated from newborn control and K14-Cre/EcadFl/− (−/−) mice. (C) Rac activation assay on total skin lysates of control and K14-Cre/EcadFl/− mice. Download figure Download PowerPoint Although aPKC forms a complex with Par3, its overall distribution was not similar to that of Par3 but, instead, was highly concentrated at the epidermal–dermal junction in basal keratinocytes of control mice. Surprisingly, this basal staining pattern was disturbed upon loss of E-cadherin, resulting in a patchy pattern instead of the relatively regular lining at the basal side of these cells (Figure 9A). This phenotype was observed in 50% of the K14-Cre/EcadFl/− mice. No major difference in protein amounts was found (Figure 9B). To examine if E-cadherin also regulates epidermal aPKC activation, necessary for tight junction formation in simple epithelia (Suzuki et al, 2002), we studied the distribution of phosphorylated forms of aPKC, indicative of activated aPKC. In control mice, phospho-aPKC antibodies stained cell–cell contacts, next to the epidermal–dermal junction, showing that the predominant total protein localization does not necessarily reflect the distribution of active forms. More importantly, we found that phospho-aPKC was absent or largely diminished at cell–cell borders of the suprabasal layers in K14-Cre/EcadFl/− mice (Figure 9A). Thus, E-cadherin is essential for activation of aPKC at the appropriate localization or for recruitment of activated forms to sites of cell–cell contact. It is not necessary for phosphorylation of aPKC per se because phospho-aPKC was detected in total skin lysates of control and K14-Cre/EcadFl/− mice (Figure 9B). The absence of activated aPKC from sites of epidermal tight junction formation offers a likely explanation for the inability of K14-Cre/EcadFl/− mice to form functional tight junctions. The small GTPase Rac is lost from the membrane in K14-Cre/EcadFl/− mice How does E-cadherin affec
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