APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Keratinocytes from APP/APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro Christina Siemes a,1 , Thomas Quast a,2 , Christiane Kummer a,3 , Sven Wehner a,4 , Gregor Kirfel a , Ulrike Müller b , Volker Herzog a,⁎ a

Institute of Cell Biology and Bonner Forum Biomedizin, University of Bonn, Ulrich-Haberlandstr. 61A, 53121 Bonn, Germany Max Planck Institute for Brain Research, Deutschordenstr. 46, 60528 Frankfurt, Germany and Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Growing evidence shows that the soluble N-terminal form (sAPPα) of the amyloid precursor

Received 4 January 2006

protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation,

Revised version received

migration and adhesion. APP is a member of a protein family including the two mammalian

22 February 2006

amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP

Accepted 23 February 2006

and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not

Available online 11 April 2006

display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by

Abbreviations:

about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro,

APP, β-amyloid precursor protein

which could be completely rescued by either exogenously added recombinant sAPPα, or by

APLP1, β-amyloid precursor

co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-

like protein 1

knockout keratinocytes revealed reduced migration velocity resulting from severely

APLP2, β-amyloid precursor

compromised cell substrate adhesion. Keratinocytes from double knockout mice died

like protein 2

within the first week of culture, indicating essential functions of APP-family members for

KGF, keratinocyte growth factor

survival in vitro. Our data indicate that sAPPα has to be considered as an essential epidermal

sAPPα, soluble form of APP

growth factor which, however, in vivo can be functionally compensated to a certain extent

wt, wild type

by other growth factors, e.g., factors released from dermal fibroblasts.

EGF, epidermal growth factor

© 2006 Elsevier Inc. All rights reserved.

TGFα, transforming growth factor-α FGF, fibroblast growth factor

Introduction The human epidermis represents the largest organ of the human organism covering an area of about 1.8 m2 and forms

the outer barrier, protecting the organism against a large variety of environmental stress factors such as physical, chemical and microbial noxious insults [1]. This protective role is accomplished by a series of cooperating mechanisms involving the

⁎ Corresponding author. Fax:+49 228 735302. E-mail address: [email protected] (V. Herzog). 1 Current address: Institute for Cell Biology, ETH Zürich, Hönggerberg, HPM D27, 8039 Zürich, Switzerland. 2 Current address: Institute of Molecular Physiology and Developmental Biology, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany. 3 Current address: The Scripps Research Institute, Department of Cell Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 4 Current address: Department of Surgery, Medical School, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.02.025

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constant renewal of the epidermis by a continuous cycle of proliferation, differentiation and shedding of keratinocytes [2], cornified envelope formation [3] and the epidermal resistance against physical stress based on the specific cytoskeleton of keratinocytes [4] as well as characteristic cell–cell [5] and cell– matrix interactions [6]. Important regulatory functions are mediated by ions, cytokines and growth factors [7] such as epidermal growth factor (EGF) [8] derived from platelets, transforming growth factor-α (TGFα) from basal keratinocytes [8], keratinocyte growth factor (KGF) from dermal fibroblasts [9] and activin [10]. More recently, sAPPα, the soluble N-terminal secreted fragment of the amyloid precursor protein (APP), has been described as an epidermal growth factor due to its mitogenic [11,12] and motogenic [13,14] properties. APP is a member of a larger protein family including the two amyloid precursor-like proteins APLP1 and APLP2 from mammals [15]. Both APLPs are highly homologous to APP and proteolytically processed in a similar way, leading to the secretion of the large ectodomains (sAPPα and sAPLP) [16,17]. Using in situ hybridization and RT-PCR analysis, it has been demonstrated that APP and APLP2 are expressed ubiquitously in largely overlapping patterns during embryonic development and in adult tissue [17]. In contrast, APLP1 is found primarily in the nervous system [18]. Besides its function as a growth factor, multiple other functions have been proposed for APP, mainly based on in vitro experiments (for review see [19]). According to its structural properties, APP has been suggested to serve as a cell surface receptor involved in signal transduction [20]. Moreover, the cytoplasmic domain which is released by γ-secretase cleavage may function as a transcription factor [21]. APP has also been recognized as a matrixbinding protein interacting with heparin [22], perlecan [23], laminin [24], collagen type IV [25] and entactin [23]. It has been shown that APP is a copper binding protein regulating the homeostasis of Cu (II) and its reduction to Cu (I) [26]. APP has also been proposed to function as a membrane receptor for kinesin-I, thereby mediating the axonal vesicular transport in neuronal cells [27]. Recent observations suggest, however, that APP does not directly bind kinesin-1 [28]. To address the physiological functions of APP in APPdeficient null mutants [29,30] and mice carrying a hypomorphic mutant of APP (APPΔ) [31] have been generated. The phenotypes of these mutants suggested that APP may play a role in neurite outgrowth and the formation of forebrain commissures, postnatal somatic growth and neurobehavioral development, locomotor activity and grip strength, copper homeostasis and the susceptibility to epileptic seizures and excitotoxic agents [29,30,32]. Whereas APP knockout (APP−/−) and APLP2 knockout (APLP2−/−) mice showed only minor abnormalities, double mutants obtained by crossing APP−/− mice to APLP2−/− mice were perinatally lethal, suggesting functional redundancy [33,34]. It has also been shown that APP family members serve essential, at least partially redundant functions by demonstrating early postnatal lethality for both APLP2−/−/APLP1−/− and APLP2−/−/APP−/− double knockout mutants. In addition, genetic evidence for distinct physiological roles of APP and APLP2 has been provided by showing that crosses of the respective single knockouts with APLP1−/− mice result in double mutants of clearly different phenotypes, being either lethal (APLP2−/−/APLP1−/−), or viable (APP−/−/APLP1−/−) [34].

A pharmacological approach to analyze selectively the function of sAPPα has become possible by the application of hydroxamic-acid-based zinc metalloproteinase inhibitors known to block the release of sAPPα [35]. This inhibition resulted in a 50–60% reduction of keratinocyte proliferation and the normalization of the increased growth rates of psoriatic keratinocytes [12]. Corresponding results have been reported with progenitor cells of the subventricular zone of the lateral brain ventricle showing a significant decrease in their growth rates [36]. Despite these findings, a potential epidermal phenotype due to the lack of APP or APLP2 has not been analyzed in detail. In this study, we have, therefore, investigated the epidermis of APP/APLP2-deficient mice in situ and in vivo, as well as isolated keratinocytes derived from these mice with respect to their growth rate and motile characteristics. We provide evidence for decreased rates in keratinocyte proliferation, reduced adhesion to the extracellular matrix and defects in cell migration as a consequence of APP deficiency. These functional deficits could be rescued by the addition of exogenous sAPPα. Thus, our observations strongly support the concept that sAPPα acts as an epidermal growth factor involved in wound healing, reepithelialization and epidermal morphogenesis.

Materials and methods Animals Single and double knockout mice (APP−/−, APLP2−/−, APP−/−/ APLP2−/−) [34] (Max Planck Institute for Brain Research, Frankfurt, Germany) and corresponding wild type control mice with the same genetic background as the knockout mice were used. The animals were housed in a special pathogenfree unit kept under optimal hygiene conditions. Timed matings were accomplished in the later afternoon followed by plug check on the next morning. The point of a detected plug was accounted as embryonic day 0.5 (E0.5).

Materials Defined serum-free keratinocyte medium (SFM) was obtained from Invitrogen Corporation (Karlsruhe, Germany), Dulbecco's minimal essential medium (DMEM), keratinocyte growth medium (KGM) and trypsin/EDTA solutions were from Cambrex (Verviers, Belgium). The primary polyclonal rabbit antisAPPα antibody 2189 was from Chemicon International (Hofheim, Germany) and the monoclonal anti-actin antibody (clone C4) was from ICN (Eschwege, Germany). The secondary 5-(4.6-dichlorothiazin-2-yl)-amino-fluorescein hydrochloride (DTAF) or peroxidase-labeled anti-mouse or anti-rabbit antibodies were from Dianova (Hamburg, Germany).

PCR genotyping The APP−/−, APLP2−/− and APP−/−/APLP2−/− mice were generated and genotyped by PCR as described before [34]. The PCR was performed on DNA obtained from tail biopsies as previously described [31]. For the APP knockout, a 3-primer PCR was used

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with the primers UM44 (5′-GAGACGAGGACGCTCAGTCCTAGGG-3′) and UM42 (5′-ATCACTGGTTCTAATCAGAGGCCC3′) against a 650 bp fragment of the wild type allele flanking exon 17. For the mutant allele (430 bp fragment), the primers UM42 located in intron 17 and P3-hygro (5′-CGAGATCAGCAGCCTCTGTTCCACA-3′) derived from the PGK-Hygrocassette were applied. The amplification of the APLP2 knockout was performed using the primers APLP2 [1] (5′GCCAAGCTTGAGTCGGTGTATCCGTGCT-3′) and APLP2 [2] (5′GCGACCGGAGGAGACGCACATCGGGAGCTCGCC-3′, 400 bp fragment) as well as the primers APLP2 [2] and APLP2 [3] (5′CCATTGCTCAGCGGTGCTG-3′, 350 bp fragment). The DNA was amplified for 38 cycles at 94°C for 45 s, at 55°C for 45 s and at 72°C for 1 min. PCR products were size-separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.

Cell culture Keratinocytes from embryonic mouse skin were prepared 1 day before birth (day E19.5). The skin was freed of excess dermal tissue, cut into pieces of 5–10 mm2 and washed with serum- and EGF/BPE-free KGM (Cambrex). The skin pieces were digested with 10 mg/ml dispase-II (Roche MolecularBiochemicals, Mannheim, Germany) in KGM for 15 h at 4°C, before epidermis and dermis were separated. Care was taken that both the stratum basale of the isolated epidermis and the dermal papillae of the dermis remained intact, which was controlled by scanning electron microscopy (not shown). For separation of basal cells, the epidermis was dissociated with 0.05% trypsin for 6 min at 37°C. The cells were dispersed by pipetting, washed in completely supplemented SFM (Invitrogen) containing 10% FCS and 10 μg/ml tetracycline hydrochloride (Sigma-Aldrich Fine Chemicals, Taufkirchen, Germany), separated from the fragments by filtration with a cell-strainer (70 μm pore size Nylon gauze; Becton Dickinson Labware, Heidelberg, Germany), suspended in freshly prepared medium and seeded in culture dishes coated with 5 μg/ml collagen IV from human placenta (Sigma-Aldrich). The keratinocytes were cultured in SFM with 10% FCS and 10 μg/ml tetracycline hydrochloride for 24 h. After this time, the medium was replaced by serumand tetracycline-hydrochloride-free SFM, which was changed every 2 days. Under these conditions, the cells maintained their characteristic morphology and their normal proliferation activity. Because of the decline of the proliferation activity and the increased differentiation of keratinocytes from APP−/− and APLP2−/− skin after several passages, all experiments were performed in the zero passages. Fibroblasts were isolated from whole mount preparations of the separated dermis. For this purpose, isolated dermis pieces of the embryonic mouse skin were placed on 6-well tissue dishes (TPP Tissue Culture Labware, Trasadingen, CH) and cultured in DMEM with 10% FCS until the fibroblasts moved out of the dermis pieces. Subsequently, the dermis was removed and the fibroblasts were cultured until they were used for experiments. For experiments, unsupplemented, serum- and EGF/BPEfree DMEM (DMEM*) or KGM* was used.

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Keratinocyte and fibroblast co-cultures For co-culture, wild type and knockout keratinocytes were seeded at a density of 1 × 106 cells on 15 mm cover slips coated with 5 μg/ml collagen IV from human placenta (Sigma-Aldrich) and cultured for 3 days in SFM as described above. Fibroblasts were seeded with a density of 1 × 106 cells in 6-well tissue culture plates (TPP) and also cultured for 3 days in DMEM. Thereafter, the medium of the fibroblasts was changed to KGM and the cover slips with the keratinocytes, placed on tissueculture inserts (25 mm, LAB-TEK Nalge Nunc International, Naperville, USA) with a specific polycarbonate membrane (pore size of 3 μm), were put onto the cultured fibroblasts in the 6-well tissue culture plates. Care was taken that the keratinocytes on the cover slips were completely immersed in the medium. This co-culture system was incubated for 24 or 48 h before the proliferation competent keratinocytes were stained by BrdU assay as described below.

Preparation of recombinant sAPPα RNA was isolated from human brain tissue and reversely transcribed. APP cDNAs corresponding to the APP751 and APP770 isoforms were amplified by PCR using appropriate primers containing a His-tag at the N-terminus. The sAPPαisoforms were expressed in EBNA293 cells (Invitrogen, Groningen, The Netherlands) and cultured in serum-free DMEM, using the episomal pCEP4 expression vector system (Invitrogen) which included the signal peptide sequence from osteonectin obtained from Dr. Rupert Timpl (Max-Planck Institute for Biochemistry, Martinsried, Germany). The supernatants of cells were collected and His-tagged sAPPα was purified using Talon™ metal affinity chromatography (Clontech, Heidelberg, Germany). The bound His-tagged isoform was eluted by the application of a pH shift (50 mM Na2HPO4, pH 5.0, in 300 mM NaCl) as described before [37] and fractions were tested for purity by Coomassie-stained SDS-PAGE and gel-chromatography using standard procedures. The fractions with highest degree of purity showing only a single band in the SDS-PAGE were combined and dialyzed overnight against PBS prior to use.

Proliferation assays In vitro BrdU assay To determine the proliferation rate of keratinocytes in culture, incorporation of 10 mg/ml BrdU (Sigma-Aldrich) in DMEM without FCS was allowed for 3 h [38]. Thereafter, the cells were fixed by immersion in absolute methanol for 10 min at 4°C and air-dried. The DNA was denatured by incubating the cells in 2N HCl (Roth GmbH, Karlsruhe, Germany) for 60 min at 37°C followed by the neutralization of the acid by immersing the slides for 3 × 10 min in 0.1 M borate buffer (pH 8.5, SigmaAldrich) and 3 × 10 min in PBS. The cells were stained with the monoclonal anti-BrdU antibody (6 μg/ml, Roche-Diagnostics GmbH, Mannheim, Germany) and subsequently with the secondary DTF-labeled anti-mouse IgG antibody each for 60 min at 37°C. After washing in PBS (3 × 5 min), the cells were mounted in the presence of anti-fade reagent (Biomeda Corporation, Foster City, CA) and viewed with a

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fluorescence microscope (Axiophot, Zeiss, Jena, Germany). The proliferation-index was calculated from the ratio of BrdUpositive cells and the total number of cells. For morphometric quantification the imaging software ImageProPlus (Media Cybernetics, Silver Spring MD, USA) was applied.

the DTAF-labeled goat anti-mouse or anti-rabbit IgG antibody in PBS for 60 min at 37°C. After washing 3 × 5 min with PBS, the preparations were mounted with anti-fade reagent (Biomeda) and viewed using a LSM510 (Zeiss, Jena, Germany).

In vivo BrdU assay

Hematoxylin and eosin staining

To analyze the proliferation rate of keratinocytes in vivo, pregnant APP−/−/APLP2−/− and wild type mice were injected 1 day before birth and 4 and 2 h before preparation subcutaneously with 0.5 ml BrdU solution (8 mg/ml in 0.9% NaCl) [39]. In this assay, a transfer of the BrdU from the pregnant mouse to the embryonic DNA was taken advantage of. After 4 h of BrdU incubation, the embryos were prepared and 7-μm cryostat sections of the isolated skin were immunofluorescence labeled and quantitated as described above. To exclude regional differences in proliferative activity, only skin samples derived from the same areas of the back were excised.

Cell adhesion assay For the analysis of cell-substrate adhesion, freshly isolated mouse keratinocytes were seeded at a density of 1 × 105 cells per well in a 12-well uncoated tissue culture plate (TPP, Techno Plastic Products, Trasadingen, Switzerland), each well containing a final volume of 500 μl KGM without FCS and EGF/ BPE. After incubation at 37°C for the indicated times, the plates were swirled three times and the media with the unattached keratinocytes were collected. The number of cells was determined using a Bürker counting chamber (Marienfeld GmbH, Laboratory Glassware, Lauda-Königshofen, Germany). For each time point, two samples were assayed and each experiment was repeated three times.

Immunoelectrophoretic blot analysis To analyze the expression of APP in keratinocytes, celllysates were separated on 10% polyacrylamide SDS-gel and blotted onto a nitrocellulose membrane (Protan-Nitrocellulose, Schleicher-Schuell, Dassel, Germany). Subsequently, the proteins were detected using the rabbit anti-APP antiserum 2189, visualized by chemiluminescence (ECL, Amersham, Braunschweig, Germany), documented on XAR-5 films (Kodak, Stuttgart, Germany) and evaluated by digital image analysis.

Immunocytochemistry To investigate the expression of APP in the epidermis of the embryonic mouse skin and in cultured keratinocytes as well as to characterize the APP knockout, the cryostat sections of the epidermis (7 μm thick) or the cells were fixed in 4% paraformaldehyde in PBS (45 min at room temperature) followed by treatment with 0.2% Triton X-100 (5 min at room temperature) and blocking with 3% BSA in PBS (1 h at 37°C). APP was detected by the polyclonal anti-APP antiserum 2189 (90 min at 37°C). To identify the basal lamina, the cryostat sections were also stained with a polyclonal antilaminin-5 antibody (Chemicon). For immunofluorescence visualization, the sections or the cells were incubated with

To characterize differences in the morphology of the embryonic mouse epidermis by comparing the APP−/− and APLP2−/− with the wild type skin, the isolated pieces of the skin were fixed in 4% paraformaldehyde in PBS for 2 h at room temperature, dehydrated in ethanol and embedded in paraffin. Seven-micrometer sections were stained with hematoxylin and eosin using standard procedures.

Computer-assisted analysis of keratinocyte migration velocity and lamella dynamics (live cell imaging) SACED assay Freshly isolated keratinocytes were placed at a density of 1 × 105 cells/cm2 in two chambered cover glass culture dishes (LAB-TEK Nunc) coated with 5 μg/ml collagen IV from human placenta (Sigma-Aldrich) and cultured for 24 h as described above. To analyze the effects of the knockout of APP and APLP2 on lamella dynamics, the keratinocytes were cultured another 24 h in KGM* before live cell imaging was started. Microscopy of living keratinocytes was performed using the standard software of an inverted LSM510 (Zeiss) equipped with a 63 × 1.4 NA plan apochromat oil immersion objective and an incubation chamber in which a constant temperature of 37°C was kept. Phase contrast images of motile keratinocytes were taken at an interval of 2 s for 5 min and the lamellopodia protrusion velocity was analyzed by SACED as described before [40]. Lines extending from inside the cell body to the substrate, crossing the lamella transversally to the cell edge, were placed over the phase contrast image of a motile keratinocyte. Along these lines, distinct gray values of structures with a width of one pixel visible in phase contrast in the area of interest were digitally recorded. A time space plot based on 150 images resulted in a stroboscobic image, which allowed the analysis of lamellopodia protrusion velocity. The assay was performed in triplicate by analyzing 5 cells in each experiment.

Time lapse microscopy To determine differences in the migration velocity between knockout and wild type keratinocytes, the isolated cells were seeded in two-chambered cover slip culture dishes and cultured as described above. After incubation for 24 h in serum- and EGF/BPE-free KGM, phase contrast images of motile keratinocytes were captured at an interval of 10 s for 1 h. The migration velocity of the cells was quantified by adding the images on top of each other and measuring the distances between the cell nucleus using the imaging software ImageProPlus (Media Cybernetics, Silver Spring, MD, USA).

Statistical analysis The results were analyzed for statistical differences by a oneway analysis of variance (one-way ANOVA). In general, the

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Despite their postnatal lethality, the APP−/−/APLP2−/− embryos exhibited no specific phenotype. About 5–10% of these embryos revealed a dysplasia of the brain (exencephalia), but they were also viable until birth [34]. Furthermore, APP−/−/ APLP2−/− embryos showed no differences concerning the thickness and morphology of the skin compared to the wild type embryos (data not shown), indicating that the knockout of APP and APLP2 may be compensated for by other growth factors and, thus be morphogenetically not essential.

Decreased keratinocyte proliferation in APP−/−/APLP2−/− epidermis in vivo Fig. 1 – Genotyping of APP−/−/APLP2−/− mice by genomic PCR of tissue from tail biopsies of the wild type and knockout alleles. KO1 and KO2 (lanes 1 and 2) were heterozygous for APP, showing the wild type (upper lane) and the APP knockout allele (lower lane) and homozygous for APLP2 (APP+/−/APLP2−/−; lanes 5 and 6). KO3, an APP and APLP2 double knockout mouse (APP−/−/APLP2−/−), exhibited only the APP and APLP2 knockout allele (lane 3 and 7) whereas KO4 was an APLP2 single knockout mouse (APP+/+/APLP2−/−; lanes 4 and 8).

differences were assumed to be statistically significant at P < 0.05.

Results In this study, the mitogenic and motogenic effects of APP and sAPPα on keratinocytes were determined using APP−/− and APLP2−/− knockout mice [29,33]. To obtain APP/APLP2 double knockout (APP−/−/APLP2−/−) mice, animals homozygous for APLP2 and heterozygous for APP (APP+/−/APLP2−/−) were intercrossed and expected to yield 25% offspring deficient for both proteins. Because of early postnatal lethality, embryos were obtained from the pregnant mice 1 day before birth, at day E19.5 and upon tail biopsy genotyped by PCR as APP−/−/ APLP2−/−, APP+/−/APLP2−/− and APP+/+/APLP2−/− (Fig. 1) as described before [34].

Potential differences in the proliferation rate of the APP−/−/ APLP2−/− keratinocytes were analyzed by an in vivo BrdU assay after subcutaneous injection of the pregnant mice at day E19.5 and sacrifice of pregnant females 4 h later. BrdU-positive cells were detected in cryostat sections of the isolated embryonic skin by anti-BrdU specific antibodies (Fig. 2). To localize the basal cell layer, the basal lamina was immunolabeled with anti-laminin-5 antibodies. A semi-quantitative analysis of immunofluorescence micrographs revealed a reduction by about 40% in the number of BrdU-positive keratinocytes in the skin of APP−/−/APLP2−/− mice (green, Fig. 2b) as compared to wild type skin (green, Fig. 2a). As this result points to a role of APP in the regulation of keratinocyte proliferation, it was of particular interest to study the proliferation rate of isolated keratinocytes. Despite optimal conditions during the separation, isolation and cultivation of the keratinocytes [41], the APP−/−/APLP2−/− cells showed clearly reduced proliferation and died during the first week of culture, which made reproducible experiments with these double knockout keratinocytes impossible. These observations may be explained by the early apoptosis of keratinocytes in culture and the absence of sAPPα as reported previously [42]. Therefore, all further studies were performed on isolated keratinocytes from APP−/− or APLP2−/− mice. These cells showed apparently normal growth upon plating and reached confluence after 7 to 10 days. Differences in cell morphology between knockout and wild type keratinocytes were not observed at this stage (Figs. 3a–c). Upon trypsinization and replating (first passage), knockout keratinocytes adhered,

Fig. 2 – In vivo BrdU assay of wild type and APP−/−/APLP2−/− mice. The incorporation of BrdU in the embryonic skin resulted from the injection of pregnant mice at day E19.5. (a, b) Immunocytochemical detection of cryostat section showed a clearly reduced labeling of keratinocyte nuclei in the basal layer (BL) indicating a reduced proliferation rate in the double knockout skin (b) compared to the wild type skin (a). Semi-quantitative analysis of the proliferation competent keratinocytes in the sections revealed a reduction of proliferation by about 40% of the APP−/−/APLP2−/− skin compared to the wild type skin (c) (E, epidermis; D, dermis; green, BrdU; red, laminin-5; n = 10 sections; N = 3 animals; scale bar, 50 μm).

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Fig. 3 – Keratinocyte culture of wild type and of APP−/− and of APLP2−/− mice. In the zero passages, (P0) keratinocytes of the wild type (a), APLP2−/− (b) and APP−/− (c) mice showed no morphological differences. After the first passages (P1) of the APLP2−/− (e) and APP−/− (f) keratinocytes, the cells revealed a high degree of differentiation and only small areas of proliferation active keratinocytes with a high nucleus/cytoplasm ratio compared to the wild type keratinocytes were detectable (yellow arrows and dotted lines) (scale bars, 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

but the number of proliferating keratinocytes with a high nucleus/cytoplasm percentage was clearly reduced (Figs. 3e–f) compared to the wild type keratinocytes (Fig. 3d). Since it was not possible to separate the proliferation active keratinocytes from the differentiated cells, all experiments were conducted in the zero passages.

of sAPPα. Hence, both APP and APLP2 exert mitogenic effects on keratinocytes with APLP2 being a slightly weaker mediator. It should be pointed out that the effect of rec-sAPPα is not due to its His-tag as untagged sAPPα has been shown to induce proliferation as well [43].

Compensation of the APP−/− keratinocytes by fibroblasts −/−

Reduced proliferation of APP by addition of rec-sAPPα

keratinocytes is complemented

Keratinocytes isolated from APP−/− or APP2−/− embryos were characterized by Western blot analysis (Fig. 4a) and by immunofluorescence microscopy (Figs. 4b–d) using the antibody 2189, which allows to distinguish between APP and APLP2. In APP−/− keratinocytes (Fig. 4c), no endogenous APP was detectable, whereas in wild type (Fig. 4b) and APLP2−/− keratinocytes (Fig. 4d) it was mainly found in the perinuclear region displaying the typical staining pattern of the Golgi complex [11]. The proportion of proliferation competent keratinocytes was determined by BrdU assay (Fig. 4e). A significant reduction (40%; P = 0.005) of BrdU-positive cells was observed in APP−/− and APLP2−/− keratinocytes as compared to wild type cells. This decrease could be abolished when the isolated keratinocytes of the APP−/−, APLP2−/− or wild type embryos were incubated in the presence of 10 nM rec-sAPPα for 24 h. APP−/− and APLP2−/− keratinocytes showed significant increases in the proliferation rates after application of rec-sAPPα, reaching statistically indistinguishable maximum levels, approximately 20% above the level reached by wild type cells in the absence

To investigate possible compensatory effects of dermal growth factors, we co-cultured APP−/− keratinocytes with wild type or APP−/− fibroblasts (Fig. 5a). In this special coculture system, the keratinocytes are only in contact with the medium and the substances released by the fibroblasts. The quantitation of the BrdU assay showed a significant increase of the proliferation rate of the APP−/− keratinocytes in the presence of wild type fibroblasts (Fig. 5b; P = 0.001). The proliferation of the knockout keratinocytes was almost identically increased in the presence of APP−/− fibroblasts (P = 0.001), demonstrating that the effect of the APP knockout on the proliferation of keratinocytes in vivo might be compensated at least in part by other factors released by fibroblasts.

Effects of APP−/− or APLP2−/− on migration velocity and lamellipodia dynamics To determine the potential motogenic effect of the APP knockout on keratinocytes, we analyzed different parameters of the migratory process comprising migration velocity and lamellipodia dynamics, i.e., their actin assembly-driven protrusion velocity at the leading edge and their persistence which

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APLP2−/− cells was reduced either but the difference to wild type cells was smaller than for APP−/− cells (Fig. 6c; P = 0.057). This SACED assay [13,40] allows to simultaneously analyze lamellipodia protrusion velocity (data not shown) and lamellipodia persistence (Fig. 7). Quantification of lamellipodia protrusion velocity revealed no significant differences between the APP−/−, APLP2−/− or wild type keratinocytes. In contrast to lamellipodia protrusion velocity, lamellipodia persistence was reduced in APP−/− keratinocytes, albeit the difference did not reach statistical significance. The lack of APLP2 had apparently no effect on the lifetime of membrane protrusions (Fig. 7). In contrast to wild type keratinocytes, APPdeficient cells formed several lamellar regions and displayed a more random, i.e., less directed, migration.

Effects of APP−/− on the efficiency of adhesion Since the persistence of lamellipodia has been shown to depend on the efficiency of cell-substrate adhesion, we analyzed the adhesiveness of wild type and double knockout keratinocytes. After the indicated incubation times (30, 60, 120, 180 and 240 min), the number of the non-adherent cells in the supernatant was determined. Based on the seeded cell number, it was possible to deduce the number of adherent cells (Fig. 8). After 30 min, nearly 75% of the seeded wild type keratinocytes had adhered, whereas only 1% of the APP−/− cells became adherent. After 240 min, the number of adherent cells increased for all genotypes reaching almost identical levels. These results point to the importance of APP as an essential factor during the early stages of cell-substrate adhesion thus strongly supporting previous observations on the function of sAPPα in cell adhesion [42,45].

Discussion

−/−

−/−

Fig. 4 – APP- or APLP2-expression of APP and APLP2 keratinocytes. (a) APP expression was not detectable in the APP−/− mice (upper blot). Protein loading was monitored by an anti-actin antibody (lower blot). (b–d) Immunocytochemical staining of APP confirmed the knockout of APP (c) and demonstrated the localization of the endogenous APP in the perinuclear region of wild type (b) and APLP2−/− (d) keratinocytes (scale bar, 10 μm). Reduced proliferation of APP−/− keratinocytes is shown in panel e. Isolated APP−/−, APLP2−/− or wild type keratinocytes were incubated in serum- and EGF/BPE-free KGM with or without 10 nM rec-sAPPα for 24 h. The quantitation of the BrdU-positive keratinocytes showed a significant reduction in the knockout keratinocytes (light gray), which was compensated by the addition of 10 nM rec-sAPPα (dark gray) (P < 0.05).

represents a direct measure for the adhesiveness between the cell and the substrate [44]. Evaluation of the locomotion velocity revealed a significant reduction (P = 0.006) of cell migration for APP−/− keratinocytes (Fig. 6b) as compared to the wild type keratinocytes (Fig. 6a). The migration velocity of

It is well-established that the combined deficiency of APP and APLP2 in mice (APP−/−/APLP2−/−) is lethal clearly pointing to the vital importance of these proteins [33,34]. In the normal human [11] and murine [46] epidermis, APP and APLP2 are predominantly expressed in the basal keratinocyte layer, which represents the center of proliferation and of cell renewal and provides migratory cells during the process of epidermal wound healing. It has been shown that sAPPα in human keratinocytes acts as a potent growth factor due to its mitogenic activity [11,12] and is also involved in mediating cell adhesion [42] and stimulating keratinocyte migration [13]. Surprisingly, APP−/− or APLP2−/− mice do not exhibit any obvious morphogenetic epidermal defects. A detailed analysis of the epidermis in situ and of keratinocytes isolated from APP−/− and APLP2−/− mice appeared, therefore, necessary. The key finding of our study is that the two epidermal members of the APP family, APP and APLP2, play crucial roles in keratinocyte proliferation, adhesion and migration in culture by serving as a source for the release of sAPPα and sAPLP2 in vitro. Both sAPPα and sAPLP2 appear structurally closely related to each other and, hence, functionally redundant [47]. Despite these structural homologies, the biological roles of APP and APLP2 appear to differ as shown by double mutants of APLP2−/−/APLP1−/− which are lethal and of APP−/−/APLP1−/−

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Fig. 5 – Compensation of the reduced APP−/− keratinocyte proliferation by co-culture with fibroblasts. Keratinocytes from APP−/− mice were placed on tissue culture inserts and cultured with wild type or APP−/− fibroblasts for 4 h in serum- and EGF/BPE-free KGM (a). (b) The quantitation of the BrdU assay showed a significant increase in proliferation of the APP−/− keratinocytes cultured with either wild type or APP−/− fibroblasts as compared to the keratinocytes without fibroblasts indicating that other growth factors may overcome the proliferation block (n = 3; * = significant to keratinocytes cultured without fibroblasts; P < 0.05).

which are viable [34]. Here, we report also on differences in the biological roles between sAPPα and sAPLP2 in that sAPLP2 exerts in general the same but weaker physiological effects.

Fig. 6 – Diminished migration velocity of APP−/− keratinocytes grown in two chambered cover slip culture dishes coated with collagen IV. APP−/− keratinocytes formed several lamellipodia and showed a reduced directional migration (b, arrows) compared to wild type (wt) (a, arrows) and APLP2−/− keratinocytes (not shown). (c) Quantitation of the migrating cells revealed a significant reduction in the migration velocity of APP−/− and a strong but not significant diminishment of APLP2−/− keratinocytes compared to wild type cells (n = 3; * = significant to wild type keratinocytes; P < 0.05).

Constantly, significant results were obtained only for APP−/− keratinocytes. The lack of sAPPα in keratinocytes may explain the survival of keratinocytes for only a few days in culture. Hence, the supplementation with other growth factors including sAPLP2 can only partially compensate the lack of sAPPα. Our in situ observations show that keratinocytes in the epidermis of APP−/−/APLP2−/− mice exhibit a nearly 40% reduced rate of proliferation. Isolated keratinocytes deficient in both proteins, however, undergo cell death within the first week of culture whereas keratinocytes deficient either in APP or APLP2 were able to survive in vitro for prolonged periods of time. Their growth rates were, nevertheless, strongly reduced by about 35% in APP−/− and about 28% for APLP2−/− keratinocytes. Keratinocytes of single knockout mice were completely rescued in their growth rates by exogenously added recombinant sAPPα. Interestingly, the situation for double knockout keratinocytes is less severe in situ, as shown by the lack of a specific phenotype. A possible explanation is provided by the observation that APP−/− fibroblasts in co-culture are able to normalize the rate of proliferation in APP−/− keratinocytes,

Fig. 7 – Effect of APP deficiency on lamellipodia dynamics as analyzed by SACED assay. Lamellipodia persistence was markedly but not significantly reduced in APP−/− keratinocytes (n = 3; P < 0.05).

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migration in APP−/− keratinocytes was more random, i.e., less directed. This is in great agreement with recent observations that sAPPα is able to induce a directed movement thereby showing its chemotactic potency [13]. The epidermal morphogenetic balance between cell proliferation and cell death is of crucial importance and might be disturbed in APP−/−/APLP2−/− mice. A striking example of such an imbalance is the underlying cause of hyperproliferative skin diseases. Excessive production and exfoliation of epidermal cells are the hallmarks of such diseases, e.g., psoriasis. Psoriatic keratinocytes have been shown to have strongly increased growth rates which appear, at least in part, to be induced by the mitogenic overproduction of sAPPα [12].

Fig. 8 – Delayed adhesion of APP−/− keratinocytes compared to wild type keratinocytes. When keratinocytes were seeded on uncoated culture dishes in KGM without FCS and EGF/BPE, the wild type cells (–♦–) became rapidly adherent within the first 30 min after seeding (arrow and circles) whereas the APP−/− keratinocytes (–▲–) showed a delayed adhesion which began not until 30 to 60 min after seeding (n = 3; * = significant to wild type keratinocytes; P < 0.05).

suggesting reduced apoptosis, enhanced cell size or delayed differentiation in the epidermis of these mice. It remains to be determined which of these possible factors contribute to this effect. We can also not exclude that sAPLP2 is able to compensate in part for the lack of sAPPα. However, as discussed before, sAPLP2 exerts minor biological effects and may support the effect of sAPPα but does not appear to be essential for proliferation. Growth factors released by fibroblasts have been well characterized and include KGF and fibroblast growth factor 10 (FGF10). KGF is, similar to sAPPα, known to stimulate migration and to exert a strong cytoprotective effect in addition to its mitogenic effects on keratinocytes [48] as well as on a variety of other cell types, e.g., lung epithelial cells, which are protected from hyperoxide-induced cell death in the presence of this growth factor [49]. The lack of a specific phenotype in vivo in spite of prominent defects being observed ex vivo has also been demonstrated for other growth factors. Intriguingly, mice lacking KGF showed, similar to APP/APLP2 double knockouts, normal epidermal morphogenesis, e.g., during wound repair [50]. The cytoprotective role of sAPPα as shown by the use of exogenously added recombinant sAPPα appears to be mediated by supporting cell adhesion and by inhibition of apoptosis [42]. Besides the cytoprotective effects of sAPPα, the mechanisms involved in cell adhesion are well known to be essential for cell migration [13], in particular to establish contact between the newly formed lamellipodia and matrix constituents [51]. As a consequence, APP−/− keratinocytes which display reduced lamellipodia persistence exhibit a lowered migratory velocity, which in turn is presumably due to the strongly reduced sAPPα levels. These findings correspond to former observations on the stimulatory role of sAPPα on lamellipodia dynamics and migratory velocity in keratinocytes [13]. The importance of sAPPα as a motogenic growth factor is further supported by our findings that keratinocyte

Acknowledgments We would like to thank Dipl. Biol. Robert Torka for his help with the time lapse microscopy and Dr. Wolfgang Neumüller for critical reading of the manuscript. We also thank Deutsche Forschungsgemeinschaft (DFG Research Group Keratinocytes – Proliferation and Differentiation in the Epidermis“), the “Bonner Forum Biomedizin” and the “Verband der Chermischen Industrie” for generous financial support.

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