EXPERIMENTAL CELL RESEARCH 312 (2006) 1939– 1949 available at www.sciencedirect.com www.elsevier.com/locate/yexcr Research Article Keratinocytes from APP/APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro Christina Siemesa,1, Thomas Quasta,2, Christiane Kummera,3, Sven Wehnera,4, Gregor Kirfela, Ulrike Müllerb, Volker Herzoga,⁎ aInstitute of Cell Biology and Bonner Forum Biomedizin, University of Bonn, Ulrich-Haberlandstr. 61A, 53121 Bonn, Germany bMax 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 ARTICLE INFORMATION ABSTRACT 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,orby 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 outer barrier, protecting the organism against a large variety of environmental stress factors such as physical, chemical and The human epidermis represents the largest organ of the microbial noxious insults [1]. This protective role is accom- human organism covering an area of about 1.8 m2 and forms plished 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 1940 EXPERIMENTAL CELL RESEARCH 312 (2006) 1939– 1949 constant renewal of the epidermis by a continuous cycle of A pharmacological approach to analyze selectively the proliferation, differentiation and shedding of keratinocytes [2], function of sAPPα has become possible by the application of cornified envelope formation [3] and the epidermal resistance hydroxamic-acid-based zinc metalloproteinase inhibitors against physical stress based on the specific cytoskeleton of known to block the release of sAPPα [35]. This inhibition keratinocytes [4] as well as characteristic cell–cell [5] and cell– resulted in a 50–60% reduction of keratinocyte proliferation matrix interactions [6]. Important regulatory functions are and the normalization of the increased growth rates of mediated by ions, cytokines and growth factors [7] such as psoriatic keratinocytes [12]. Corresponding results have been epidermal growth factor (EGF) [8] derived from platelets, reported with progenitor cells of the subventricular zone of the transforming growth factor-α (TGFα) from basal keratinocytes lateral brain ventricle showing a significant decrease in their [8], keratinocyte growth factor (KGF) from dermal fibroblasts [9] growth rates [36]. and activin [10]. More recently, sAPPα, the soluble N-terminal Despite these findings, a potential epidermal phenotype secreted fragment of the amyloid precursor protein (APP), has due to the lack of APP or APLP2 has not been analyzed in detail. been described as an epidermal growth factor due to its In this study, we have, therefore, investigated the epidermis of mitogenic [11,12] and motogenic [13,14] properties. APP/APLP2-deficient mice in situ and in vivo, as well as APP is a member of a larger protein family including the isolated keratinocytes derived from these mice with respect to two amyloid precursor-like proteins APLP1 and APLP2 from their growth rate and motile characteristics. We provide mammals [15]. Both APLPs are highly homologous to APP and evidence for decreased rates in keratinocyte proliferation, proteolytically processed in a similar way, leading to the reduced adhesion to the extracellular matrix and defects in secretion of the large ectodomains (sAPPα and sAPLP) [16,17]. cell migration as a consequence of APP deficiency. These Using in situ hybridization and RT-PCR analysis, it has been functional deficits could be rescued by the addition of demonstrated that APP and APLP2 are expressed ubiquitously exogenous sAPPα. Thus, our observations strongly support in largely overlapping patterns during embryonic develop- the concept that sAPPα acts as an epidermal growth factor ment and in adult tissue [17]. In contrast, APLP1 is found involved in wound healing, reepithelialization and epidermal primarily in the nervous system [18]. Besides its function as a morphogenesis. 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 Materials and methods suggested to serve as a cell surface receptor involved in signal transduction [20]. Moreover, the cytoplasmic domain which is Animals released by γ-secretase cleavage may function as a transcrip- − − − − − − tion factor [21]. APP has also been recognized as a matrix- Single and double knockout mice (APP / , APLP2 / , APP / / − − binding protein interacting with heparin [22], perlecan [23], APLP2 / ) [34] (Max Planck Institute for Brain Research, laminin [24], collagen type IV [25] and entactin [23]. It has been Frankfurt, Germany) and corresponding wild type control shown that APP is a copper binding protein regulating the mice with the same genetic background as the knockout mice homeostasis of Cu (II) and its reduction to Cu (I) [26]. APP has were used. The animals were housed in a special pathogen- also been proposed to function as a membrane receptor for free unit kept under optimal hygiene conditions. Timed kinesin-I, thereby mediating the axonal vesicular transport in matings were accomplished in the later afternoon followed neuronal cells [27]. Recent observations suggest, however, that by plug check on the next morning. The point of a detected APP does not directly bind kinesin-1 [28]. plug was accounted as embryonic day 0.5 (E0.5). To address the physiological functions of APP in APP- deficient null mutants [29,30] and mice carrying a hypomorphic Materials mutant of APP (APPΔ) [31] have been generated. The pheno- types of these mutants suggested that APP may play a role in Defined serum-free keratinocyte medium (SFM) was obtained neurite outgrowth and the formation of forebrain commis- from Invitrogen Corporation (Karlsruhe, Germany), Dulbecco's sures, postnatal somatic growth and neurobehavioral devel- minimal essential medium (DMEM), keratinocyte growth opment, locomotor activity and grip strength, copper medium (KGM) and trypsin/EDTA solutions were from Cam- homeostasis and the susceptibility to epileptic seizures and brex (Verviers, Belgium). The primary polyclonal rabbit anti- − − excitotoxic agents [29,30,32]. Whereas APP knockout (APP / ) sAPPα antibody 2189 was from Chemicon International − − and APLP2 knockout (APLP2 / ) mice showed only minor (Hofheim, Germany) and the monoclonal anti-actin antibody − − abnormalities, double mutants obtained by crossing APP / (clone C4) was from ICN (Eschwege, Germany). The secondary − − mice to APLP2 / mice were perinatally lethal, suggesting 5-(4.6-dichlorothiazin-2-yl)-amino-fluorescein hydrochloride functional redundancy [33,34]. It has also been shown that (DTAF) or peroxidase-labeled anti-mouse or anti-rabbit anti- APP family members serve essential, at least partially redun- bodies were from Dianova (Hamburg, Germany). dant functions by demonstrating early postnatal lethality for − − − − − − − − both APLP2 / /APLP1 / and APLP2 / /APP / double knockout PCR genotyping