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Partial Loss of Epithelial Phenotype in Kindlin-1–Deficient Keratinocytes

Published:February 10, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.01.005
      Kindlin-1 is an adaptor protein that is expressed by most epithelial cells and has been implicated in integrin bidirectional signaling. Mutations in the gene encoding kindlin-1 are associated with Kindler syndrome, a recessively inherited disorder that is characterized by fragile skin. Functionally, a loss of kindlin-1 impairs the adhesion of basal keratinocytes to the extracellular matrix both in vivo and in vitro. In this study, we show that the phenotype of mutant keratinocytes deficient in kindlin-1 is characterized by the modification of the cortical actin network and increased plasticity of the plasma membrane. At the molecular level, expression of several proteins associated with an epithelial phenotype, such as α6β4 integrin, collagen XVII, E-cadherin, and desmoglein-3, is strongly reduced, whereas, surprisingly, laminin 332 is synthesized in larger amounts than in control keratinocytes. In contrast, mesenchymal markers such as vimentin and fibronectin are increased in keratinocytes lacking kindlin-1. The switch in cell plasticity and protein expression was confirmed by siRNA-mediated down-regulation of kindlin-1 in HaCaT epithelial cells. Furthermore, there was up-regulation of matrix metalloproteinases and pro-inflammatory cytokines in kindlin-1–deficient keratinocytes. These results provide new insights into the pathogenic mechanisms that take place in Kindler syndrome. Moreover, the constellation of molecular defects associated with the loss of kindlin-1 may explain the higher incidence of skin cancer observed in patients affected with this disorder.
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      Genetic mutations in any of these proteins result in severe blistering disorders (epidermolysis bullosa), characterized by trauma-induced detachment of the epidermis from the underlying dermis. The most severe forms of the disorders, junctional epidermolysis bullosa, are caused by absence of either α6β4 integrin or its ligand LM-332.
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      Unique and redundant functions of integrins in the epidermis.
      This establishes definitively that α6β4 integrin is absolutely required to maintain anchorage of the epidermis to the dermis, and that a major property of β4 integrins and binding partners is to mediate the stable anchorage of basal keratinocytes and to resist mechanical stress applied to skin. It indicates also that there is no spontaneous activation of compensation mechanisms for this precise function, despite the presence of α2β1 and α3β1 integrins and their cognate collagen IV and LM-332 ligands, respectively. Contrary to the well-established role of β4 integrins, the in vivo function of β1 integrins in basal keratinocytes is poorly understood. In vitro studies and murine genetics have linked several biological processes to α3β1 integrin binding to LM-332, including regulation of keratinocyte adhesion, migration and survival, deposition and assembly of LM-332, and promotion of epithelial cell migration during wound healing.
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      and human
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      Integrin α3 subunit regulates events linked to epithelial repair, including keratinocyte migration and protein expression.
      keratinocytes. Similarly, the function of α2β1 integrin is still not fully elucidated. Mice with genetic ablation of the integrin α2 subunit do not show any overt skin phenotype, although α2β1 integrin is the only collagen receptor expressed by keratinocytes, and it is absolutely required for keratinocyte adhesion to collagens in vitro.
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      The Kindler syndrome is a rare skin fragility disorder caused by loss-of-function mutation in the FERMT1 gene coding for Kdl-1.
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      The disease is characterized by trauma-induced skin microblistering and photosensitivity in infancy, with improvement later in life, whereas poikiloderma, skin atrophy, and aggressive periodontal disease develop in adulthood.
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      At the ultrastructural level, there are discontinuities in the cutaneous basement membrane, and sometimes duplication of the lamina densa and hypoplastic hemidesmosomes.
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      Development of squamous cell carcinomas at an early age was reported for several individuals affected with the Kindler syndrome, suggesting a higher risk of muco-cutaneous malignancy.
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      Mice with silencing of the gene coding Kdl-1 die a few days after birth from ulcerative colitis.
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      Loss of Kindlin-1 causes skin atrophy and lethal neonatal intestinal epithelial dysfunction.
      In these mice, intestinal epithelial cells detach from the basement membrane, presumably because of reduced integrin activation, followed by a destructive inflammatory response.
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      Loss of Kindlin-1 causes skin atrophy and lethal neonatal intestinal epithelial dysfunction.
      In agreement with the notion that Kdl-1 is important for β1 integrin activation in focal adhesions, we previously reported that primary epidermal keratinocytes from patients affected with the Kindler syndrome display defects in β1 integrin-mediated in vitro processes such as adhesion to extracellular matrix proteins in short-term assays.
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      • Herz C.
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      • Qu H.Y.
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      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      However, keratinocytes from the patients show also loss of cohesion and shape abnormalities, suggesting that Kdl-1 has a role to play beyond β1 integrin activation and outside focal adhesions. Moreover, it is unclear how microblisters develop in the epidermis of patients with the Kindler syndrome, and how Kdl-1, a focal adhesion protein, controls epidermal anchorage to the dermis. As firmly established by human and mouse genetics of the dermal–epidermal junction, this process is mediated by β4 integrin–associated hemidesmosomes.
      • Aumailley M.
      • Has C.
      • Tunggal L.
      • Bruckner-Tuderman L.
      Molecular basis of inherited skin-blistering disorders, and therapeutic implications.
      • Margadant C.
      • Charafeddine R.A.
      • Sonnenberg A.
      Unique and redundant functions of integrins in the epidermis.
      Here we report that loss of Kdl-1 is associated with deregulated expression and distribution of several proteins of the dermal–epidermal junction, including β4 integrins. Moreover, keratinocytes lacking Kdl-1 acquire an exaggerated plasticity and a constellation of molecular defects that may favor the development of skin cancer in patients affected with the Kindler syndrome.

      Materials and Methods

      Cell Culture and Phase Contrast Microscopy

      Keratinocytes derived from the skin of a healthy individual and from a patient affected with the Kindler syndrome were immortalized with the HPV18 E6 and E7 genes as described previously.
      • Has C.
      • Herz C.
      • Zimina E.
      • Qu H.Y.
      • He Y.
      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.
      Spontaneously immortalized human skin keratinocytes HaCaT were kindly provided by Dr. N. Fusenig (DKFZ, Heidelberg, Germany). HaCaT present the phenotype of normal keratinocytes and are suited for in vitro studies of regulatory mechanisms.
      • Boukamp P.
      • Petrussevska R.T.
      • Breitkreutz D.
      • Hornung J.
      • Markham A.
      • Fusenig N.E.
      Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte line.
      All keratinocytes were cultivated in keratinocyte growth medium under low CaCl2 conditions (<100 μmol/L), containing 100 μg/mL human epidermal growth factor, 500 ng/mL hydrocortisone, 0.5 mg/mL insulin and 0.4% bovine hypophyse extract, at 37°C in humidified air containing 5% CO2. Cell morphology was analyzed on phase contrast photographs captured with an inverted microscope equipped with a camera (Nikon TE2000-U, Düsseldorf, Germany).

      Antibodies

      Mouse monoclonal antibodies used in this study were directed against fibronectin (clone 1932, Millipore, Molsheim, France), integrin α3A and α6A subunits (clones 29A3 and 1A10, respectively, kindly donated by Dr. Arnoud Sonnenberg, the Netherlands Cancer Institute, Amsterdam, the Netherlands), E-cadherin (Zymed Laboratories, Carlsbad, CA), laminin α3 chain (BM165; 17), tubulin (Sigma-Aldrich, Taufkirchen, Germany), Cdc42 (BD Transduction Laboratories, Heidelberg, Germany), Rac1 (Upstate Biotechnology, Lake Placid, NY), RhoA, Slug (both from Santa Cruz Biotechnology, Heidelberg Germany), integrin-linked kinase (BD Biosciences, Heidelberg, Germany), paxillin (clone 349, BD Transduction Laboratories), BD Slug, vinculin (F-VII, a gift from Dr. Marina A. Glukhova, Institut Curie, Paris, France), and kindlin-2 (a gift from Dr. Rheinhard Fässler, Max-Planck Institut für Biochemie, Martinsried, Germany). Rabbit polyclonal antibodies used in this study were directed against ezrin (Chemicon, Hofheim, Germany), myosin IIA (Abcam, Cambridge, UK), focal adhesion kinase (Upstate Biotechnology, Lake Placid, NY), collagen XVII (NC16), ZEB2 (Sigma-Aldrich), and Kdl-1 (kind gifts from Dr. Leena Bruckner-Tuderman and Dr. Cristina Has, University of Freiburg, Germany), LG2 domain of laminin α3 chain,
      • Wen T.
      • Zhang Z.
      • Yu Y.
      • Qu H.
      • Koch M.
      • Aumailley M.
      Integrin α3 subunit regulates events linked to epithelial repair, including keratinocyte migration and protein expression.
      cofilin (ACFL02, Tebu-Bio, Offenbach, Germany). Other antibodies included rat monoclonal antibody against integrin β4 subunit (clone CD104, Millipore), goat polyclonal antibody against laminin γ2 chain (sc7652, Santa Cruz Biotechnology), guinea pig polyclonal antibody GP53 against vimentin (Progen Biotechnik GmbH, Heidelberg, Germany).

      Cell Adhesion Assays

      LM-111 was purified from the mouse EHS tumor, and LM-332 was purified from the cell culture medium of human keratinocytes by affinity chromatography as previously reported.
      • Rousselle P.
      • Aumailley M.
      Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.
      • Tasanen K.
      • Tunggal L.
      • Chometon G.
      • Bruckner-Tuderman L.
      • Aumailley M.
      Keratinocytes from patients lacking collagen XVII display a migratory phenotype.
      Collagen and fibronectin were from Biochrom (Berlin, Germany). The proteins were used to coat tissue culture wells on 96-well plates (Costar). After saturation of the wells with 1% BSA (Fraction V, Serva, Heidelberg, Germany), equal number of cells were seeded in triplicate wells for 30 minutes. At the end of the experiment, adherent cells were quantified as previously reported
      • Rousselle P.
      • Aumailley M.
      Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.
      and photographed with a digital camera (PowerShot G5, Canon, Tokyo, Japan) mounted on a phase-contrast microscope (Axiovert 100; Carl Zeiss, Göttingen, Germany).

      siRNA Transfection

      Small interfering RNAs were designed and purchased from Eurogentec (Sart-Tilman, Belgium). Two siRNA duplexes for Kdl-1 were used (sequence 1: 5′-GAAGUUACUACCAAAAGCU-3′ and sequence 2: 5′-ACUUGCAGAUAAUCUCAAA-3′). Scramble oligonucleotides were used as control. Semiconfluent cells were transfected with the duplexes (100 mmol/L) using HiPerFect (Qiagen, Hilden, Germany), as previously described.
      • Has C.
      • Herz C.
      • Zimina E.
      • Qu H.Y.
      • He Y.
      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.

      Time-Lapse Video Microscopy

      Live keratinocytes in tissue culture dishes were monitored by time-lapse video-microscopy as previously described.
      • Zhang Z.G.
      • Bothe I.
      • Hirche F.
      • Zweers M.
      • Gullberg D.
      • Pfitzer G.
      • Krieg T.
      • Eckes B.
      • Aumailley M.
      Interactions of primary fibroblasts and keratinocytes with extracellular matrix proteins: contribution of α2β1 integrin.
      • Chometon G.
      • Zhang Z.G.
      • Rubinstein E.
      • Boucheix C.
      • Mauch C.
      • Aumailley M.
      Dissociation of the complex between CD151 and laminin-binding integrins permits migration of epithelial cell.
      Briefly, the culture plates were placed on an inverted phase contrast microscope (Axiovert S100TV Zeiss) equipped with a heated stage, 5% CO2 supply, and a CCD camera (Xillix MicroImager). Images of the cells captured automatically and stored with Openlab software (Improvision, Heidelberg, Germany) were converted to Quick Time movies. Migration tracks of single cells (20 cells per video) were recorded with the Dynamic Image Analysis System (DIAS) software (Solltech Inc., Oakdale, IA), and used to calculate cell velocity, net and total path lengths, directionality (ratio between net and total path lengths; with 0 and 1 indicating absolute random and straight path, respectively) and persistence. Extent of cell shape changes was determined by measuring on successive frames the areas involved in protrusion and retraction of the plasma membrane as previously described.
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      Alternatively, phase contrast images of successive frames were transferred to PowerPoint (Microsoft Corporation, Redmond, WA) and cell contours were manually delineated with different colors and then merged as previously described.
      • Wen T.
      • Zhang Z.
      • Yu Y.
      • Qu H.
      • Koch M.
      • Aumailley M.
      Integrin α3 subunit regulates events linked to epithelial repair, including keratinocyte migration and protein expression.
      At least 10 cells were analyzed for each condition. Student's t-test was used for statistical analysis of the data.

      Protein Analysis, Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis, and Immunoblotting

      Cell culture media were collected and kept frozen at −20°C after addition of a cocktail of protease inhibitors. The cell monolayers were either directly resuspended in Laemmli buffer or lysed in 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, containing 1% Triton-X 100, 1 mmol/L each MgCl2, MnCl2, and CaCl2, protease inhibitors (10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride), and phosphatase inhibitors (1 mmol/L sodium vanadate, 10 mmol/L sodium fluoride), as described previously.
      • Zhang Z.
      • Chometon G.
      • Wen T.
      • Qu H.
      • Mauch C.
      • Krieg T.
      • Aumailley M.
      Migration of epithelial cells on laminins: rhoA antagonizes directionally persistent migration.
      After protein solubilization for 1 hour at 4°C, the soluble and insoluble fractions were separated by centrifugation and mixed with Laemmli buffer. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, followed by immunoblotting with specific primary antibodies, and horseradish peroxidase–conjugated, species-specific secondary antibodies. Bound secondary antibodies were revealed using ECL detection system (GE Healthcare, Freiburg, Germany). To allow comparison of protein levels between control and Kdl-1-null keratinocytes, equivalent amounts of proteins were loaded as verified by both Ponceau staining and immunoblotting of tubulin or actin. In the case of proteins secreted into the culture medium, care was taken that the media to be analyzed were conditioned by equivalent numbers of cells. This was verified by immunoblotting of cellular proteins that expression did not change (tubulin or actin). The membranes were re-used several times to detect proteins of different electrophoretic mobilities and/or with primary antibodies raised in different species and to control sample loading. Band intensity on blots was measured with ImageJ software (http://rsbweb.nih.gov/ij).
      For zymography, SDS-PAGE was performed on gelatin-containing gels as described.
      • Kurschat P.
      • Wickenhauser C.
      • Groth W.
      • Krieg T.
      • Mauch C.
      Identification of activated matrix metalloproteinase-2 (MMP-2) as the main gelatinolytic enzyme in malignant melanoma by in situ zymography.

      Immunofluorescence Staining

      Cells were grown on uncoated glass coverslips for 3 to 4 days. Fixation was with 2% paraformaldehyde in PBS for 15 minutes, followed by permeabilization with ice-cold 0.2% Triton X-100 in PBS. Fixed cells were incubated with primary antibodies diluted in PBS for 1 hour, followed by Cy-2 or Cy-3 conjugated antibodies for 45 minutes. Fibrillar actin was stained with fluorescein isothiocyanate–conjugated phalloidin (Sigma-Aldrich). The coverslips were mounted on histoslides in DAKO medium (DakoCytomation, Hamburg, Germany). Staining was observed with a laser scanning confocal microscope (Leica Instruments, Heidelberg, Germany).

      Cytokine Expression Profile

      Cytokine expression profiles were determined using RayBio human cytokine antibody array V (RayBiotech, Hoelzel Diagnostika GmbH, Cologne, Germany) according to manufacturer's instructions. The signals were revealed by ECL. To optimize quantification, X-ray films were successively exposed for different time. The intensity of signals was measured with ImageJ software. For standardization, the average intensity of the positive controls was calculated and set as 1, and the signal intensity of the different cytokines was calculated by comparison to it.

      Results

      Lack of Kdl-1 Prevents Keratinocytes from Growing in Colonies and Forming a Cortical Actin Network

      Multiple and variable shapes were the hallmark of primary keratinocytes isolated from the skin of patients with Kindler syndrome, in contrast to the regular cobblestone morphology of keratinocytes isolated from healthy epidermis.
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      Because detailed studies are limited by the finite lifespan of primary cells, keratinocytes isolated from a patient with the Kindler syndrome were immortalized,
      • Has C.
      • Herz C.
      • Zimina E.
      • Qu H.Y.
      • He Y.
      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.
      allowing further molecular analysis of the phenotype. The abnormal cellular shapes persisted in immortalized Kdl-1–deficient (Kld-1def) keratinocytes.
      • Has C.
      • Herz C.
      • Zimina E.
      • Qu H.Y.
      • He Y.
      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.
      The phenotype is particularly exacerbated in sparse cultures, and under these conditions most Kdl-1def keratinocytes are elongated and bipolar, like fibroblasts, in contrast to the polygonal shape of controls (Figure 1, A–C). Moreover, whereas control keratinocytes have a marked propensity to grow as cohesive colonies, less than 10% of Kdl-1def keratinocytes form coherent groups (Figure 1D). This is consistent with our previous observation that siRNA-mediated down-regulation of Kdl-1 in HaCaT keratinocytes induces loosening of intercellular adhesions.
      • Has C.
      • Herz C.
      • Zimina E.
      • Qu H.Y.
      • He Y.
      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.
      Finally, staining of fibrillar actin revealed that the cell shape phenotype is associated with disorganization of the cortical actin network in Kdl-1def keratinocytes (Figure 1, E and F). Importantly, and in agreement with other reports,
      • Lai-Cheong J.E.
      • Ussar S.
      • Arita K.
      • Hart I.R.
      • McGrath J.A.
      Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome.
      • He Y.
      • Esser P.
      • Heinemann A.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 and -2 have overlapping functions in epithelial cells implications for phenotype modification.
      lack of Kdl-1 did not affect Kdl-2 expression and cellular localization (see Supplemental Figure S1 at http://ajp.amjpathol.org). Furthermore, adhesion to various extracellular matrix proteins do not rescue the shape phenotype of Kdl-1def keratinocytes (Figure 2). Disruption of the subcortical actin network and impaired intercellular adhesions observed for Kdl-1def keratinocytes are features highly reminiscent of the effects of an antibody against β1 integrins, which induced dissociation of keratinocyte colonies and perturbation of the circumferential organization of actin microfilaments.
      • Larjava H.
      • Peltonen J.
      • Akiyama S.K.
      • Yamada S.S.
      • Gralnick H.R.
      • Uitto J.
      • Yamada K.M.
      Novel function for β1 integrins in keratinocyte cell-cell interactions.
      Thus the absence of Kdl-1 in keratinocytes is associated with cell shape changes, loss of the typical cortical actin network, and altered intercellular cohesion, arguing for an additional function of Kdl-1 outside the focal adhesions, nevertheless still related to β1 integrins.
      Figure thumbnail gr1
      Figure 1Kdl-1def keratinocytes display morphological changes compared with control keratinocytes. Sparse cultures of control (A) and Kdl-1def keratinocytes (B) grown under low calcium conditions were photographed with phase contrast microscopy. Kdl-1def keratinocytes adopt an elongated shape and fail to cluster like control keratinocytes. Cell shape was defined as the ratio between short and long cell axis (C). The ratio is significantly lower for Kdl-1def compared with control (Co) keratinocytes. Cell clustering was determined by counting the number of cells forming a group of three or more cells (D). For both C and D, more than 500 cells were counted for each population. Mean and SD are shown. Values for Kdl-1def and control keratinocytes are significantly different (*P < 0.05). Staining of fibrillar actin in control (E) and Kdl-1def (F) keratinocytes revealed disruption of the cortical actin network in Kdl-1def keratinocytes compared with controls.
      Figure thumbnail gr2
      Figure 2Adhesion and spreading of control and Kdl-1def keratinocytes to extracellular matrix proteins. A: Equal numbers of control (open bars) and Kdl-1def (filled bars) keratinocytes were seeded on triplicate wells coated with collagen I (Col I), collagen IV (Col IV), fibronectin (Fn), LM-111, and LM-332, as indicated. Adhesion was measured after 30 minutes of incubation on the coats. Data are mean ± SD of triplicate measurements in one representative experiment out of at least five experiments showing the same results. B: At the end of the adhesion assay, adhering keratinocytes were photographed under phase contrast microscopy. Note extremely low spreading of Kdl-1def keratinocytes compared with controls.

      Keratinocyte Plasticity Is Enhanced on Kdl-1 Deficiency

      The dynamics of the cytoskeleton and focal adhesions directly influence plasma membrane movement and cell motility.
      • Wehrle-Haller B.
      • Imhof B.A.
      Actin, microtubules and focal adhesion dynamics during cell migration.
      To determine whether alterations in cell shape and in the network of cortical actin have functional consequences, Kdl-1def keratinocytes were monitored by time-lapse video microscopy and compared with controls. Analysis of cell contours on successive frames of the videos indicated increased movement of the plasma membrane of Kdl-1def keratinocytes in comparison to controls (Figure 3A). A similar enhancement of plasma membrane plasticity was observed for primary cells lacking Kdl-1,
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      and for HaCaT keratinocytes transfected with siRNAs down-regulating Kdl-1 expression (Figure 3B). Computer-assisted quantification of cell shape changes indicated a threefold increase in the extent of plasma membrane protrusion and retraction of Kdl-1def keratinocytes compared with controls (Figure 3C). In addition, detailed quantification of migratory parameters, indicated that Kdl-1def cells move slightly faster and cover longer distances than control cells (Figure 3, D–F). This is in contrast to the reduced velocity observed for primary cells lacking Kdl-1,
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      which might have been caused by their reduced adhesion, survival, and proliferation capacities. Analysis of single cell tracks on video recordings was not compatible with the low migratory capacity of HaCaT cells under standard culture conditions.
      • Chometon G.
      • Zhang Z.G.
      • Rubinstein E.
      • Boucheix C.
      • Mauch C.
      • Aumailley M.
      Dissociation of the complex between CD151 and laminin-binding integrins permits migration of epithelial cell.
      Figure thumbnail gr3
      Figure 3Increased plasticity of Kdl-1def keratinocytes. Control (Co) and Kdl-1def keratinocytes (A), as well as HaCaT cells transfected with scramble and Kdl-1–specific siRNAs (B), were monitored by time-lapse video microscopy. Cell contours were manually delineated with different colors on successive frames of the videos and then merged. For control and Kdl-1def keratinocytes, 50-minute video were used, with contours delineated on frames at 5-minute intervals. For HaCaT cells, which have very low motility, the videos were for 180 minutes with contours on frames at 18-minute intervals. Computer-assisted cell tracking on video recordings of control (Co) and Kdl-1def keratinocytes was used to extract migration parameters: protrusion and retraction (C), total path length (D), directionality (E), and velocity (F). Mean and SD are shown for control (Co) and Kdl-1def keratinocytes (n ≥ 10 cells for each population). **P < 0.01.

      Kdl-1 Deficiency Affects Expression of Specific Epithelial Proteins

      To determine the molecular mechanisms possibly associated with modifications in cell shape and plasticity, we examined the expression levels of transmembrane and intracellular proteins required for the integrity of the cytoskeleton and of cell–cell and cell–matrix adhesions. Keratinocytes form two types of cell–matrix adhesions, namely focal adhesions, nucleated around β1 integrins and linked to the actin cytoskeleton, and stable adhesion complexes, or hemidesmosome-like structures, associated with β4 integrins and keratins.
      • Carter W.G.
      • Kaur P.
      • Gil S.G.
      • Gahr P.J.
      • Wayner E.A.
      Distinct functions for integrins α3β1 in focal adhesions and α6β4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes.
      • Krieg T.
      • Aumailley M.
      The extracellular matrix of the dermis: flexible structures with dynamic functions.
      The two types of molecular assemblies can be distinguished by their solubility in Triton X-100-containing buffers.
      • Carter W.G.
      • Kaur P.
      • Gil S.G.
      • Gahr P.J.
      • Wayner E.A.
      Distinct functions for integrins α3β1 in focal adhesions and α6β4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes.
      • Franke W.W.
      • Schmid E.
      • Osborn M.
      • Weber K.
      The intermediate-sized filaments in rat kangaroo PtK2 cells II. Structure and composition of isolated filaments.
      SDS-PAGE and immunoblotting analysis of proteins in cell lysates was therefore performed either directly or after separation of proteins into detergent-soluble and insoluble fractions. Interestingly, Kdl-1 was associated with both fractions (Figure 4A). Most focal adhesion and actin cytoskeleton–associated proteins, including paxillin, vinculin, integrin linked kinase, cofilin, Rac1, tubulin, actin, and myosin IIA, were expressed at the same levels in control and Kdl-1def keratinocytes (Figure 4B). In contrast, transmembrane proteins such as integrin α6 and β4 subunits and collagen XVII, which are components of stable adhesion complexes, as well as E-cadherin and desmoglein-3 associated with intercellular junctions, all being classical epithelial markers, were distinctly decreased in Kdl-1def keratinocytes compared with controls (Figure 4B). Conversely, there was an approximately fourfold increase in vimentin (Figure 4B), an intermediate filament protein of mesenchymal cells.
      • Hynes R.O.
      • Destree A.T.
      10 nm filaments in normal and transformed cells.
      • Franke W.W.
      • Schmid E.
      • Winter S.
      • Osborn M.
      • Weber K.
      Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates.
      Interestingly, there was a remarkable correlation between the reduced expression of α6β4 integrin, collagen XVII, and desmoglein-3 in the insoluble protein fraction and the loss of Kdl-1 (Figure 4). In addition, ezrin, focal adhesion kinase, Cdc42, and RhoA were also reduced in Kdl-1def keratinocytes compared with controls (Figure 4B). Although these proteins are not regarded as specific epithelial proteins, their relative levels are lower in fibroblasts than in keratinocytes (M. Pesch and M. Aumailley, unpublished data). Expression of collagen XVII, α6β4 integrin, RhoA, and Cdc42 were also reduced on siRNA-mediated down-regulation of Kdl-1 in HaCaT epithelial cells (see Supplemental Figure S2 at http://ajp.amjpathol.org). Thus, in the absence of Kdl-1, there is a distinct reduction in transmembrane proteins involved in cell–cell and cell–matrix adhesions that are required for cohesion and stable anchorage of quiescent basal keratinocytes. Moreover, lack of Kdl-1 is associated not only with reduced levels of active Rac1 and RhoA,
      • Has C.
      • Herz C.
      • Zimina E.
      • Qu H.Y.
      • He Y.
      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.
      but also with reduced expression levels of RhoA and Cdc42 (Figure 4), thus with markedly deregulated Rho GTPases.
      Figure thumbnail gr4
      Figure 4Modification in the expression of cellular proteins in Kdl-1def compared with control keratinocytes. Control (C) and Kdl-1def (K-) keratinocytes were suspended either directly in Laemmli buffer (whole cell lysates, Total), or in 1% Triton X-100–containing lysis buffer followed by homogenization, stirring, and separation of the detergent-soluble (Sol) and -insoluble (Insol) pools. All samples were subjected in duplicate to SDS-PAGE under reducing conditions, and immunoblotted with antibodies against the indicated proteins. Representative blots are shown (A). Band intensity determined on blots from three independent experiments was used to calculate mean average of protein expression in whole cell lysates (B). Quantification is shown for proteins which expression in Kdl-1def keratinocytes is altered at least 1.5-fold compared with controls. Bars indicate SD.

      Absence of Kdl-1 Is Associated with Dispersal of β4 Integrin and LM-332

      In the epidermal basement membrane, α6β4 integrins are essential to anchor basal keratinocytes to LM-332 in the underlying connective tissue.
      • Aumailley M.
      • Has C.
      • Tunggal L.
      • Bruckner-Tuderman L.
      Molecular basis of inherited skin-blistering disorders, and therapeutic implications.
      • Margadant C.
      • Charafeddine R.A.
      • Sonnenberg A.
      Unique and redundant functions of integrins in the epidermis.
      In vitro, cultured keratinocytes assemble LM-332 and integrin β4-enriched protein complexes into characteristic and overlapping patterns easily viewed by indirect immunofluorescence staining of both proteins and microscopy observation focused at the cell–matrix interface.
      • deHart G.W.
      • Healy K.E.
      • Jones J.C.
      The role of α3β1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.
      • Tasanen K.
      • Tunggal L.
      • Chometon G.
      • Bruckner-Tuderman L.
      • Aumailley M.
      Keratinocytes from patients lacking collagen XVII display a migratory phenotype.
      • Zhang Z.
      • Chometon G.
      • Wen T.
      • Qu H.
      • Mauch C.
      • Krieg T.
      • Aumailley M.
      Migration of epithelial cells on laminins: rhoA antagonizes directionally persistent migration.
      Observation of the staining by laser scanning microscopy showed that both β4 integrin and LM-332 form the well-recognizable pattern of roughly concentric rings at the basal surface of control keratinocytes (Figure 5). On the contrary, β4 integrin and LM-332 displayed disorganized, lace-like patterns that were scattered at the ventral surface of Kdl-1def keratinocytes (Figure 5).
      Figure thumbnail gr5
      Figure 5Organization patterns of LM-332 and β4 integrin in Kdl-1def and control keratinocytes. Keratinocytes grown on glass coverslips were stained by indirect immunofluorescence with antibody against LM-332 (green; A, B, E, F) or the integrin β4 chain (red; G, H). Staining was observed by laser scanning confocal microscopy focused and the cell-matrix interface. C and D are phase contrast pictures of the cells shown in A and B. Scale bars: 80 μm (A–D); 20 μm (E–H).

      Kdl-1 Deficiency Causes Increased Expression of Secreted Extracellular Matrix Proteins

      The morphological, functional, and molecular changes reported above suggest that a reprogramming toward a mesenchymal phenotype is taking place in Kdl-1def keratinocytes. To further appreciate the extent of molecular reprogramming, we analyzed the expression of extracellular matrix proteins secreted in the culture medium of keratinocytes, including LM-332, a protein exclusively synthesized by basal keratinocytes, and other, more ubiquitous proteins such as thrombospondin-1, BM40/SPARC, and fibronectin. The amount of LM-332 in the medium of Kdl-1def keratinocytes was markedly increased compared with controls, as shown by immunoblotting of the processed α3 chain (165 kDa) and its released C-terminal fragment (40–50 kDa), as well as of the unprocessed (155 kDa) and processed (105 kDa) γ2 chain (Figure 6A). The amount of fibronectin, BM-40 and thrombospondin-1 were also significantly increased in the culture medium of Kdl-1def keratinocytes compared with that of control keratinocytes (Figure 6A). In contrast, the amount of the ectodomain shed from collagen XVII and released in the culture medium of Kdl-1def keratinocytes was drastically reduced compared with controls (Figure 6A), in agreement with the diminished expression of the full-length form of collagen XVII observed in cell lysates (Figure 4A). Levels of cell associated LM γ2 chain and fibronectin are shown in Figure 6B.
      Figure thumbnail gr6
      Figure 6Expression of extracellular matrix proteins by Kdl-1def and control keratinocytes. Proteins secreted in the culture medium (A) or associated with the cell layers (B) were analyzed by SDS-PAGE and immunoblotting, with antibodies against proteins indicated. Representative blots, out of at least three independent analysis showing the same results, are shown. Samples were loaded in duplicate, and loading was adjusted to the amount of tubulin in the cell layer.

      Kdl-1 Deficiency Is Associated with Modification in the Expression of MMPs, Cytokines, and the Transcription Factor Slug

      The multiple cellular and molecular changes reported in the preceeding paragraphs for the Kdl-1def keratinocytes evoke an epithelial-to-mesenchymal transition. Additional molecular changes are usually associated with the process, including modifications in the expression of MMPs, cytokines, and transcription factors. Analysis of the culture media by zymography showed that pro-MMP-2 and pro–MMP-9 were distinctly increased in Kdl-1def keratinocytes compared with controls, whereas MMP-9 activation was only marginally changed (Figure 7, A and B). As long as MMP activation is not increased, enhanced MMP expression may have no consequence in cell culture. However, such an increase in pro-MMP-2 and pro-MMP-9 might have very significant effects in vivo, because the proteases could be activated by neighboring fibroblasts. We then used antibody arrays to examine the expression of cytokines and growth factors in the media from control and Kdl-1def keratinocytes. Among the 79 tested cytokines, interleukin-6, angiogenin, and monokine induced by interferon-γ were significantly increased in Kdl-1def keratinocytes compared with controls, whereas there was a 4.7-fold reduction of IGFBP-2 (Figure 7C). There was no obvious difference in the expression of several other cytokines and growth factors, including TGF-β1, -β2, or -β3 (see Supplemental Figure S3 at http://ajp.amjpathol.org). Finally, we determined the expression level of transcription factors that could be involved in epithelial-to-mesenchymal transition. Slug was distinctly increased in Kdl-1def keratinocytes, compared with controls, whereas ZEB2 was unchanged (Figure 7D).
      Figure thumbnail gr7
      Figure 7Expression of MMPs, cytokines, and transcription factors in Kdl-1def and control keratinocytes. A: Cell culture media of control and Kdl-1def keratinocytes were assessed by gelatin zymography. Pro-MMP-9, active MMP-9, and pro-MMP-2 are indicated. B: Pro-MMP-9, active MMP-9, and pro-MMP-2 were quantified from three independent experiments. Data represent the relative amount of MMPs in control (open bars) and Kdl-1def keratinocytes (filled bars). Values for controls were set as 1. C: Expression profile of 79 different cytokines was analyzed and quantified as described in Materials and Methods. Graph shows only the cytokines with expression that is changed by a factor of at least 2 compared with control. Each column represents the average from two independent experiments. Bars indicate SD. D: Expression of slug and ZEB2 was determined by SDS-PAGE and immunoblotting of lysates of control and Kdl-1def keratinocytes as indicated. Actin was used to control sample loading.

      Discussion

      By binding to the cytoplasmic tail of integrin β subunits, kindlins are involved in the bidirectional signaling taking place in focal adhesions.
      • Larjava H.
      • Plow E.F.
      • Wu C.
      Kindlins: essential regulators of integrin signalling and cell-matrix adhesion.
      • Meves A.
      • Stremmel C.
      • Gottschalk K.
      • Fässler R.
      The kindlin protein family: new members to the club of focal adhesion proteins.
      • Bouaouina M.
      • Calderwood D.A.
      Kindlins.
      In agreement with this notion, we and others have shown that integrin-associated cellular activities such as adhesion, spreading, and proliferation are profoundly altered in primary keratinocytes isolated from the skin of patients or mice with null mutations in the gene coding Kdl-1.
      • Ussar S.
      • Moser M.
      • Widmaier M.
      • Rognoni E.
      • Harrer C.
      • Genzel-Boroviczeny O.
      • Fässler R.
      Loss of Kindlin-1 causes skin atrophy and lethal neonatal intestinal epithelial dysfunction.
      • Has C.
      • Herz C.
      • Zimina E.
      • Qu H.Y.
      • He Y.
      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      In this report, we present evidence that, in addition to a role in focal adhesions, Kdl-1 is important to maintain intercellular adhesion and hemidesmosome stability in keratinocytes, and that loss of Kdl-1 expression recapitulates some critical steps of epithelial to mesenchymal transition. These data extend our mechanistic understanding of epithelial tissue fragility caused by loss of Kdl-1. They also provide new insights on the molecular pathways potentially associated with the increased risk of cutaneous malignancy in the Kindler syndrome.

      Important Role of Kdl-1 in Cell Shape and Plasma Membrane Plasticity

      In healthy skin basal keratinocytes have a regular cuboid shape, although they look distorted in the skin of patients with the Kindler syndrome.
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      A change in shape is also a hallmark of Kdl-1 deficiency ex vivo, whether the keratinocytes are isolated from patients with the Kindler syndrome, or had been treated with specific siRNAs to down-regulate the protein.
      • Has C.
      • Herz C.
      • Zimina E.
      • Qu H.Y.
      • He Y.
      • Zhang Z.G.
      • Wen T.T.
      • Gache Y.
      • Aumailley M.
      • Bruckner-Tudermann L.
      Kindlin-1 is required for RhoGTPase mediated lamellipodia formation in keratinocytes.
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      The immortalized Kdl-1def keratinocytes that are investigated in the present work also have this characteristic, as well as a reduced ability to cluster in colonies. Together it strongly suggests that Kdl-1 is required to maintain the shape of and intercellular adhesions between keratinocytes, and probably some other epithelial cells, implying that Kdl-1 has a role to play beyond activation of β1 integrin–mediated inside–out signaling and beyond β1 integrin–mediated cell adhesion. It has been suggested that when cadherins do not interact under low-calcium conditions, β1 integrins are needed in areas of epithelial cell–cell contacts for circumferential organization of subcortical actin microfilaments and keratinocyte aggregation into colonies.
      • Larjava H.
      • Peltonen J.
      • Akiyama S.K.
      • Yamada S.S.
      • Gralnick H.R.
      • Uitto J.
      • Yamada K.M.
      Novel function for β1 integrins in keratinocyte cell-cell interactions.
      Furthermore, it has been proposed that integrin-mediated assembly of the subcortical actin cytoskeleton facilitates association of the cadherin:catenin complex with the cytoskeleton.
      • Wang Z.
      • Symons J.M.
      • Goldstein S.L.
      • McDonald A.
      • Miner J.H.
      • Kreidberg J.A.
      α3β1 integrin regulates epithelial cytoskeletal organization.
      The dis-assembly of the subcortical actin network observed when Kdl-1 is absent or down-regulated favors the notion that Kdl-1 is crucial to connect β1 integrins with the subcortical network of actin microfilaments at the level of intercellular adhesions. At this location, and in contrast to focal adhesions, the β1 integrin platforms lack talin,
      • Kaiser H.W.
      • Ness W.
      • Offers M.
      • O'Keefe E.J.
      • Kreysel H.W.
      Talin: adherens junction protein is localized at the epidermal-dermal interface in skin.
      • Hentula M.
      • Peltonen J.
      • Peltonen S.
      Expression profiles of cell-cell and cell-matrix junction proteins in developing human epidermis.
      and they should therefore fulfill their functions with a mechanism different from that taking place in focal adhesions. By interacting with integrin-linked kinase and migfilin, Kdl-1 acts as a cytoskeletal linker protein.
      • Larjava H.
      • Plow E.F.
      • Wu C.
      Kindlins: essential regulators of integrin signalling and cell-matrix adhesion.
      • Meves A.
      • Stremmel C.
      • Gottschalk K.
      • Fässler R.
      The kindlin protein family: new members to the club of focal adhesion proteins.
      • Bouaouina M.
      • Calderwood D.A.
      Kindlins.
      We suggest that the cytoskeletal linker function of Kdl-1 is crucial to transduce outside–in signals between β1 integrin complexes and the actin cytoskeleton at sites of cell–cell contacts. Interestingly, irregular shape and molecular defects in the composition of the cell membrane-associated skeleton characterize Kdl-3 deficiency in erythrocytes, which do not have integrins.
      • Krüger M.
      • Moser M.
      • Ussar S.
      • Thievessen I.
      • Luber C.A.
      • Forner F.
      • Schmidt S.
      • Zanivan S.
      • Fässler R.
      • Mann M.
      SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function.
      Here we show that ezrin is reduced in Kdl-1 keratinocytes, suggesting that kindlins share a role in plasma membrane organization, probably through their cytoskeletal linker function. Future studies based on mutagenesis experiments should lead to a detailed understanding of the cytoskeletal linker function of kindlins-1 and give insights into the underlying molecular mechanisms.

      Novel Role of Kdl-1 in Regulation of Hemidesmosome Formation and Stability

      In the skin, α6β4 integrins anchor the epidermis to the underlying dermis by connecting LM-332 in the extracellular space to hemidesmosomal proteins and keratin intermediate filaments of basal keratinocytes.
      • Aumailley M.
      • Has C.
      • Tunggal L.
      • Bruckner-Tuderman L.
      Molecular basis of inherited skin-blistering disorders, and therapeutic implications.
      • Margadant C.
      • Charafeddine R.A.
      • Sonnenberg A.
      Unique and redundant functions of integrins in the epidermis.
      • Krieg T.
      • Aumailley M.
      The extracellular matrix of the dermis: flexible structures with dynamic functions.
      In healthy skin, immunofluorescence staining of LM-332, α6β4 integrin, and collagen XVII shows a similar thin and linear decoration along the dermal–epidermal junction. In contrast, LM-332 and α6β4 integrin co-localize as a thick and fragmented pattern in the skin of patients affected with the Kindler syndrome, whereas collagen XVII is depolarized and distributes all around basal keratinocytes, including at the apical plasma membrane and in the cytoplasm.
      • Herz C.
      • Aumailley M.
      • Schulte C.
      • Schlotzer-Schrehardt U.
      • Bruckner-Tuderman L.
      • Has C.
      Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.
      • Lai-Cheong J.E.
      • Parsons M.
      • Tanaka A.
      • Ussar S.
      • South A.P.
      • Gomathy S.
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      • Techanukul T.
      • Almaani N.
      • Clements S.E.
      • Hart I.R.
      • McGrath J.A.
      Loss-of-function FERMT1 mutations in Kindler syndrome implicate a role for fermitin family homolog-1 in integrin activation.
      In addition, hemidesmosomes are hypoplastic and reduced in numbers.
      • Lai-Cheong J.E.
      • Parsons M.
      • Tanaka A.
      • Ussar S.
      • South A.P.
      • Gomathy S.
      • Mee J.B.
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      • Almaani N.
      • Clements S.E.
      • Hart I.R.
      • McGrath J.A.
      Loss-of-function FERMT1 mutations in Kindler syndrome implicate a role for fermitin family homolog-1 in integrin activation.
      The reduced expression of collagen XVII and α6β4 integrin as well as the scattered distribution of α6β4 integrin and its LM-332 ligand in Kdl-1def keratinocytes, are consistent with defective anchorage of basal keratinocytes in the skin of patients with the Kindler syndrome, and with both the increased migration and exaggerated plasticity displayed by Kdl-1def keratinocytes in vitro. Interestingly, skin blistering, abnormal basement membrane, and distorted hemidesmosomes characterize mice deficient in integrin α3 subunit
      • DiPersio C.M.
      • Hodivala-Dilke K.M.
      • Jaenisch R.
      • Kreidberg J.A.
      • Hynes R.O.
      α3β1 Integrin is required for normal development of the epidermal basement membrane.
      or with keratinocyte-restricted deletion of integrin-linked kinase
      • Lorenz K.
      • Grashoff C.
      • Torka R.
      • Sakai T.
      • Langbein L.
      • Bloch W.
      • Aumailley M.
      • Fässler R.
      Integrin-linked kinase is required for epidermal and hair follicle morphogenesis.
      or integrin α3 or β1 subunits.
      • Margadant C.
      • Raymond K.
      • Kreft M.
      • Sachs N.
      • Janssen H.
      • Sonnenberg A.
      Integrin α3β1 inhibits directional migration and wound re-epithelialization in the skin.
      • Brakebusch C.
      • Grose R.
      • Quondamatteo F.
      • Ramirez A.
      • Jorcano J.L.
      • Pirro A.
      • Svensson M.
      • Herken R.
      • Sasaki T.
      • Timpl R.
      • Werner S.
      • Fässler R.
      Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.
      Reduced levels of α6β4 integrins have also been observed in mice with a keratinocyte–restricted deletion of integrin β1 subunit
      • Brakebusch C.
      • Grose R.
      • Quondamatteo F.
      • Ramirez A.
      • Jorcano J.L.
      • Pirro A.
      • Svensson M.
      • Herken R.
      • Sasaki T.
      • Timpl R.
      • Werner S.
      • Fässler R.
      Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.
      or as a consequence of siRNA-mediated down-regulation of integrin α3 subunit in human keratinocytes.
      • Wen T.
      • Zhang Z.
      • Yu Y.
      • Qu H.
      • Koch M.
      • Aumailley M.
      Integrin α3 subunit regulates events linked to epithelial repair, including keratinocyte migration and protein expression.
      Finally, keratinocytes lacking the integrin α3 subunit have an increased migration.
      • Margadant C.
      • Raymond K.
      • Kreft M.
      • Sachs N.
      • Janssen H.
      • Sonnenberg A.
      Integrin α3β1 inhibits directional migration and wound re-epithelialization in the skin.
      • Wen T.
      • Zhang Z.
      • Yu Y.
      • Qu H.
      • Koch M.
      • Aumailley M.
      Integrin α3 subunit regulates events linked to epithelial repair, including keratinocyte migration and protein expression.
      Collectively, these data indicate that Kdl-1 and α3β1 integrin are functionally linked, and the data strongly suggest that both proteins are required for the interplay between focal adhesions and hemidesmosomes. It is thought that β1 integrin–containing focal adhesions are a prerequisite to, and promote the nucleation of, β4 integrin–containing hemidesmosomes.
      • Carter W.G.
      • Kaur P.
      • Gil S.G.
      • Gahr P.J.
      • Wayner E.A.
      Distinct functions for integrins α3β1 in focal adhesions and α6β4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes.
      • Litjens S.H.
      • de Pereda J.M.
      • Sonnenberg A.
      Current insights into the formation and breakdown of hemidesmosomes.
      Thus, skin blistering in the Kindler syndrome and in mice lacking α3 or β1 integrins may be due to reduced levels of α6β4 integrins and impaired hemidesmosome formation, caused by dysfunctional focal adhesions in the absence of Kdl-1 or β1 integrin, rather than being a direct consequence of Kdl-1 or β1 integrin deficiency. Alternatively or in addition, Kdl-1 may play a dual role in focal adhesions and in hemidesmosomes, as has been shown for plectin, which binds both fibrillar actin and keratin intermediate filaments or for CD151, which has affinity for both integrin α3 and α6 subunits.
      • Litjens S.H.
      • de Pereda J.M.
      • Sonnenberg A.
      Current insights into the formation and breakdown of hemidesmosomes.
      • Ozawa T.
      • Tsuruta D.
      • Jones J.C.
      • Ishii M.
      • Ikeda K.
      • Harada T.
      • Aoyama Y.
      • Kawada A.
      • Kobayashi H.
      Dynamic relationship of focal contacts and hemidesmosome protein complexes in live cells.
      Although, as yet, there is no direct evidence that Kdl-1 interacts with components of hemidesmosomes, the protein is present in both the detergent-soluble fraction together with actin and focal adhesion proteins, and the detergent-insoluble fraction together with intermediate filaments and components of hemidesmosomes, such as collagen XVII and α6β4 integrins. Interestingly, structural modeling of Kdl-1 suggests the presence of a canonical phosphotyrosine-binding domain.
      • Larjava H.
      • Plow E.F.
      • Wu C.
      Kindlins: essential regulators of integrin signalling and cell-matrix adhesion.
      • Kloeker S.
      • Major M.B.
      • Calderwood D.A.
      • Ginsberg M.H.
      • Jones D.A.
      • Beckerle M.C.
      The kindler syndrome protein is regulated by transforming growth factor-β and involved in integrin-mediated adhesion.
      Such domains are quite typical of adaptor proteins and they allow interactions with a host of receptor proteins containing the consensus NxxY binding sequence, including integrins.
      • Calderwood D.A.
      • Fujioka Y.
      • de Pereda J.M.
      • García-Alvarez B.
      • Nakamoto T.
      • Margolis B.
      • McGlade C.J.
      • Liddington R.C.
      • Ginsberg M.H.
      Integrin β cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling.
      • Uhlik M.T.
      • Temple B.
      • Bencharit S.
      • Kimple A.J.
      • Siderovski D.P.
      • Johnson G.L.
      Structural and evolutionary division of phosphotyrosine binding (PTB) domains.
      Indeed, it has been shown that a direct interaction is taking place between Kdl-1 and the NPKY motif of the intracellular domain of integrin β1 subunit.
      • Harburger D.S.
      • Bouaouina M.
      • Calderwood D.A.
      Kindlin-1 and -2 directly bind the C-terminal region of β integrin cytoplasmic tails and exert integrin specific activation effects.
      Moreover, screens of proteome-derived NPXY peptide arrays revealed that the NHSY motif in the atypical intracellular domain of the integrin β4 subunit is a binding site for at least eight different phosphotyrosine-binding domain–containing proteins.
      • Smith M.J.
      • Hardy W.R.
      • Murphy J.M.
      • Jones N.
      • Pawson T.
      Screening for PTB domain binding partners and ligand specificity using proteome-derived NPXY peptide arrays.
      This makes an interaction between integrin β4 subunit and Kdl-1 quite plausible; a hypothesis that should be tested in future studies.

      Novel Role of Kdl-1 in Epithelial-to-Mesenchymal Transition

      Kdl-1def keratinocytes exhibit morphological and functional features of epithelial cells undergoing the transition to a mesenchymal phenotype. The molecular reprogramming of epithelial cells toward a mesenchymal phenotype is characterized by loss of polarity, with cell shape change from polygonal to bipolar, down-regulation of epithelial markers, de novo expression of mesenchymal markers, capacity to degrade basement membrane proteins and acquisition of a migratory phenotype.
      • Thiery J.P.
      • Acloque H.
      • Huang R.Y.
      • Nieto M.A.
      Epithelial-mesenchymal transitions in development and disease.
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      In addition to shape changes, loss of cohesion and faster migration, there are also several hints at the molecular level suggesting reprogramming of Kdl-1def keratinocytes toward a mesenchymal phenotype. First, there is decreased expression of epithelial proteins such as E-cadherin and desmoglein-3 involved in intercellular adhesions, as well as collagen XVII and α6β4 integrins required for hemidesmosome formation. Second, the mesenchymal marker vimentin is strongly expressed in Kdl-1def keratinocytes. Vimentin facilitates cell migration
      • Eckes B.
      • Dogic D.
      • Colucci-Guyon E.
      • Wang N.
      • Maniotis A.
      • Ingber D.
      • Merckling A.
      • Langa F.
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      • Delouvée A.
      • Koteliansky V.
      • Babinet C.
      • Krieg T.
      Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts.
      and its expression is often coupled with low level of E-cadherin.
      • Thiery J.P.
      • Acloque H.
      • Huang R.Y.
      • Nieto M.A.
      Epithelial-mesenchymal transitions in development and disease.
      Third, expression of extracellular matrix proteins typical of mesenchymal cells, such as fibronectin, thrombospondin-1, and BM40/SPARC, as well as MMP-2 and MMP-9, was higher in Kdl-1def keratinocytes than in controls. Finally, LM-332 which is specifically expressed by epithelial cells, but not mesenchymal cells, was significantly enhanced in Kdl-1def keratinocytes compared with controls. LM-332 has both adhesion-promoting and scatter factor properties, respectively.
      • Rousselle P.
      • Aumailley M.
      Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.
      • Zhang Z.
      • Chometon G.
      • Wen T.
      • Qu H.
      • Mauch C.
      • Krieg T.
      • Aumailley M.
      Migration of epithelial cells on laminins: rhoA antagonizes directionally persistent migration.
      • Kikkawa Y.
      • Umeda M.
      • Miyazaki K.
      Marked stimulation of cell adhesion and motility by ladsin, a laminin-like scatter factor.
      • Aumailley M.
      • El Khal A.
      • Knoss N.
      • Tunggal L.
      Laminin 332 processing and its integration into the ECM.
      • Hamill K.J.
      • Paller A.S.
      • Jones J.C.
      Adhesion and migration, the diverse functions of the laminin α3 subunit.
      By activating diverse signaling intermediates such as focal adhesion kinase, protein kinase C, phosphatidylinositol 3- kinase, Rac1, extracellular signal-related kinase 1/2, and nuclear factor–κB, LM-332 supports cell survival, proliferation, migration, and invasion.
      • Zhang Z.
      • Chometon G.
      • Wen T.
      • Qu H.
      • Mauch C.
      • Krieg T.
      • Aumailley M.
      Migration of epithelial cells on laminins: rhoA antagonizes directionally persistent migration.
      • Fukushima Y.
      • Ohnishi T.
      • Arita N.
      • Hayakawa T.
      • Sekiguchi K.
      Integrin α3β1-mediated interaction with laminin-5 stimulates adhesion, migration and invasion of malignant glioma cells.
      • Kariya Y.
      • Miyazaki K.
      The basement membrane protein laminin-5 acts as a soluble cell motility factor.
      • Nikolopoulos S.N.
      • Blaikie P.
      • Yoshioka T.
      • Guo W.
      • Puri C.
      • Tacchetti C.
      • Giancotti F.G.
      Targeted deletion of the integrin β4 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-κB, causing defects in epidermal growth and migration.
      • Marinkovich M.P.
      Tumour microenvironment: laminin 332 in squamous-cell carcinoma.
      Therefore the increase in LM-332 that has not been deposited possibly stimulates signaling as a soluble ligand, which may in part explain morphological and functional changes of Kdl-1def keratinocytes. In addition, deposition of LM-332 is perturbed in Kdl-1def keratinocytes. Correct deposition and supramolecular organization of LM-332 require both α3β1 integrin and RhoA-dependent control of myosin-mediated cytoskeleton contraction.
      • deHart G.W.
      • Healy K.E.
      • Jones J.C.
      The role of α3β1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.
      • deHart G.W.
      • Jones J.C.
      Myosin-mediated cytoskeleton contraction and Rho GTPases regulate laminin-5 matrix assembly.
      In the absence of Kdl-1, insufficient activation of α3β1 integrin, as well as decreased expression of RhoA are likely to impair LM-332 deposition and supramolecular organization. Interestingly, silencing of RhoA perturbs also intercellular contacts and β4 integrin localization.
      • Zhang Z.
      • Chometon G.
      • Wen T.
      • Qu H.
      • Mauch C.
      • Krieg T.
      • Aumailley M.
      Migration of epithelial cells on laminins: rhoA antagonizes directionally persistent migration.
      Thus, scattered LM-332 deposition could also be linked to aberrant targeting of β4 integrin, although the question remains open whether reduced β4 integrin causes scattered LM-332 deposition or vice versa. In any case, abnormal LM-332 deposition is very likely felt by the cells as a wound, triggering a healing response, including the release of pro-inflammatory cytokines and the subsequent development of a localized dermal inflammation. This notion is perfectly in line with the severe inflammatory reaction taking place in the intestine of mice with a disruption of the Kdl-1 coding gene
      • Ussar S.
      • Moser M.
      • Widmaier M.
      • Rognoni E.
      • Harrer C.
      • Genzel-Boroviczeny O.
      • Fässler R.
      Loss of Kindlin-1 causes skin atrophy and lethal neonatal intestinal epithelial dysfunction.
      or in the skin of mice with a conditional ablation of the integrin α6 subunit.
      • Niculescu C.
      • Ganguli-Indra G.
      • Pfister V.
      • Dupé V.
      • Messaddeq N.
      • De Arcangelis A.
      • Georges-Labouesse E.
      Conditional ablation of integrin alpha-6 in mouse epidermis leads to skin fragility and inflammation.
      In addition to the inflammatory reaction known to associate with the loss of Kdl-1, it would be interesting to see whether the changes in protein expression observed in Kdl-1–deficient cells are also seen in the skin from Kindler patients. However, the amount of biological material required for such investigations would be quite substantial, and beyond ethical rules.
      Epithelial cell reprogramming is a physiological event in the course of tissue response to injury, whereas inappropriate reactivation of the process in the context of chronic inflammation might become a pathological trigger for fibrosis and cancer progression.
      • Mantovani A.
      • Allavena P.
      • Sica A.
      • Balkwill F.
      Cancer-related inflammation.
      • Lopez-Novoa J.M.
      • Nieto M.A.
      Inflammation and EMT: an alliance towards organ fibrosis and cancer progression.
      In line with this concept, dermal fibrosis developing in adult patients with Kindler syndrome or in mice with a keratinocyte-restricted deletion of β1 integrins is thought to result from the chronic inflammatory reaction associated with repeated tissue injuries followed by repair.
      • Brakebusch C.
      • Grose R.
      • Quondamatteo F.
      • Ramirez A.
      • Jorcano J.L.
      • Pirro A.
      • Svensson M.
      • Herken R.
      • Sasaki T.
      • Timpl R.
      • Werner S.
      • Fässler R.
      Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.
      • Heinemann A.
      • He Y.
      • Zimina E.
      • Boerries M.
      • Busch H.
      • Chmel N.
      • Kurz T.
      • Bruckner-Tuderman L.
      • Has C.
      Induction of phenotype modifying cytokines by FERMT1 mutations.
      In particular, in the skin of Kindler patients, dermal fibroblasts acquire an activated phenotype likely via activation of paracrine signaling.
      • Heinemann A.
      • He Y.
      • Zimina E.
      • Boerries M.
      • Busch H.
      • Chmel N.
      • Kurz T.
      • Bruckner-Tuderman L.
      • Has C.
      Induction of phenotype modifying cytokines by FERMT1 mutations.
      Finally, chronic inflammation also facilitates the development of squamous cell carcinoma.
      • Mantovani A.
      • Allavena P.
      • Sica A.
      • Balkwill F.
      Cancer-related inflammation.
      • Lopez-Novoa J.M.
      • Nieto M.A.
      Inflammation and EMT: an alliance towards organ fibrosis and cancer progression.
      We suggest that the partial epithelial–mesenchymal transition caused by Kdl-1 deficiency, together with chronic inflammation, play an important role in the development at a rather young age of squamous cell carcinomas in patients affected by Kindler syndrome.
      • Ashton G.H.S.
      Kindler syndrome.
      • Natsuga K.
      • Nishie W.
      • Shinkuma S.
      • Nakamura H.
      • Matsushima Y.
      • Tatsuta A.
      • Komine M.
      • Shimizu H.
      Expression of exon-8-skipped kindlin-1 does not compensate for defects of Kindler syndrome.
      • Lotem M.
      • Raben M.
      • Zeltser R.
      • Landau M.
      • Sela M.
      • Wygoda M.
      • Tochner Z.A.
      Kindler syndrome complicated by squamous cell carcinoma of the hard palate: successful treatment with high-dose radiation therapy and granulocyte-macrophage colony-stimulating factor.
      • Emanuel P.O.
      • Rudikoff D.
      • Phelps R.G.
      Aggressive squamous cell carcinoma in Kindler syndrome.
      • Arita K.
      • Wessagowit V.
      • Inamadar A.C.
      • Palit A.
      • Fassihi H.
      • Lai-Cheong J.E.
      • Pourreyron C.
      • South A.P.
      • McGrath J.A.
      Unusual molecular findings in Kindler syndrome.
      It should be noted that overexpression of Kdl-1 has been reported for several types of cancers.
      • Weinstein E.J.
      • Bourner M.
      • Head R.
      • Zakeri H.
      • Bauer C.
      • Mazzarella R.
      URP1: a member of a novel family of PH and FERM domain-containing membrane-associated proteins is significantly over-expressed in lung and colon carcinomas.
      • Sin S.
      • Bonin F.
      • Petit V.
      • Meseure D.
      • Lallemand F.
      • Bièche I.
      • Bellahcène A.
      • Castronovo V.
      • de Wever O.
      • Gespach C.
      • Lidereau R.
      • Driouch K.
      Role of the focal adhesion protein kindlin-1 in breast cancer growth and lung metastasis.
      • Fan J.
      • Yan D.
      • Teng M.
      • Tang H.
      • Zhou C.
      • Wang X.
      • Li D.
      • Qiu G.
      • Peng Z.
      Digital transcript profile analysis with aRNA-LongSAGE validates FERMT1 as a potential novel prognostic marker for colon cancer.
      However, a clear distinction should be made between a loss of function by lack of Kdl-1 in Kindler syndrome and a gain of function by overexpression (or neo-expression) of Kdl-1 occurring on a substantially modified cancerous background in sporadic cancers. Such an opposite regulation of epithelial-to-mesenchymal transition depending on the tissue context has been reported for periostin.
      • Kim C.J.
      • Sakamoto K.
      • Tambe Y.
      • Inoue H.
      Opposite regulation of epithelial-to-mesenchymal transition and cell invasiveness by periostin between prostate and bladder cancer cells.
      In conclusion, our data shed new light on the multiple functions of Kdl-1 and on the mechanisms of the cellular responses associated with the Kindler syndrome. Because Kdl-1 is one piece of the multiprotein machinery working as a functional unit ensuring the anchorage of the epidermis to the dermis, we anticipate a certain degree of mechanistic similarity with some other skin fragility syndromes.

      Acknowledgments

      We are grateful to the colleagues who generously provided reagents.

      Supplementary data

      • Supplemental Figure S1

        Expression of Kindlin-2 in control and Kdl-1def keratinocytes. A: Control and Kdl-1def keratinocytes were lysed in 1% Triton X-100–containing buffer followed by homogenization, stirring, and separation of the detergent-soluble (Sol) and -insoluble (Ins) pools. The samples were submitted in duplicate to SDS-PAGE under reducing conditions, and immunoblotted with antibody against kindlin-2 (right). Ponceau staining of the proteins blotted onto the nitrocellulose membrane (left). Most of the kindlin-2 is found in the detergent-soluble fraction for both control Kdl-1def keratinocytes. B: Control and Kdl-1def keratinocytes were stained for Kindlin-2. For both type of cells, a large pool of kindlin-2 is located in the cytoplasm, whereas a fraction of the protein is targeted to tiny spots (arrowheads) at the cell periphery.

      • Supplemental Figure S2

        Consequences of RNA interference–mediated down-regulation of Kdl-1 in HaCaT keratinocytes. A: HaCaT cells transfected with siRNAs (sequence 1 and 2) specific for Kdl-1 were stained for fibrillar actin. Note changes in cell shape network of cortical actin. B: HaCaT keratinocytes transfected with control (C) and Kdl-1-specific (K) siRNAs were lysed either directly in Laemmli buffer (whole cell lysates, Total) or in 1% Triton X-100–containing buffer followed by separation of the detergent-soluble (Sol) and -insoluble (Insol) fractions. The samples were analyzed in duplicate by SDS-PAGE under reducing conditions, and immunoblotted with antibodies against the indicated proteins. Detection of β4 integrin in the insoluble fractions is shown with longer exposure time than the other lanes. Representative blots are shown. Numbers in parentheses indicate protein level determined by band densitometry of the blots obtained with the whole lysates (Total) of Kdl-1–down-regulated cells compared with controls set as 100.

      • Supplemental Figure S3

        RayBio human cytokine antibody arrays were incubated with the culture medium of control and Kdl-1def keratinocytes. To facilitate visual comparison of the results, the lane of the arrays were cut using Photoshop and pasted side-by-side (control and Kdl-1def keratinocytes). All cytokines are indicated. Boxed areas represent the cytokines or growth factors in which expression levels differed between control and Kdl-1def keratinocytes.

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