Eur. J. Cell Biol. 83 (2004); 701 ± 708 http: // www.elsevier.de/ejcb Cytoprotective function of sAPPa in human keratinocytes Sven Wehner 2)3) , Christina Siemes 3) , Gregor Kirfel, Volker Herzog 1) Institute of Cell Biology and Bonner Forum Biomedizin, University of Bonn, Bonn, Germany AKT; Amyloid precursor protein; Apoptosis; Cell adhesion; Cytoprotection; Staurosporine; UV-B sAPPa, the soluble form of the b-amyloid precursor protein, has been shown to act as a potent epidermal growth factor by stimulating keratinocyte proliferation and migration. In this report we provide evidence for a cytoprotective role of sAPPa. As a model we used HaCaT cells and normal human keratinocytes (NHK) cultured in the absence of fetal calf serum and bovine pituitary extract. Under these conditions keratinocytes began to undergo apoptosis at increasing rates after 96 h of culture. Surprisingly, keratinocytes were protected from apoptosis by the addition of 50 nM recombinant sAPPa. Subsequent experiments were performed to elucidate the regulatory basis of the cytoprotective role of sAPPa. We found that recombinant sAPPa facilitated the substrate adhesion of keratinocytes in the first 30 minutes after seeding. The basis for this adhesion-promoting function was shown by the ability of recombinant sAPPa to continuously coat the culture dish thereby promoting the ability to bind keratinocytes. A second mechanism explaining the cytoprotective role was found in the significant inhibition of apoptosis by recombinant sAPPa. In HaCaT cells moderate UV-B irradiation was sufficient to induce apoptosis. In contrast, induction of apoptosis in NHK required additionally the depletion of endogenous sAPPa suggesting that sAPPa mediates protection against UV-B irradiation. Staurosporine-induced apoptosis rates were sig- nificantly reduced by about 59% after addition of recombinant sAPPa. These results show that sAPPa exerts a pronounced cytoprotective effect and that this effect is mediated by facilitated cell adhesion and by the antiapoptotic function of sAPPa. Abbreviations. APP Amyloid precursor protein ± NHK Normal human keratinocytes ± PKC Protein kinase C ± sAPPa Soluble form of APP Introduction The human epidermis represents the largest organ of the humanorganismcoveringanareaofabout1.8 m 2 andformsthe outer barrier, protecting the organism against a large variety of physical, chemical and microbiological disturbances and other possible environmental impairments (Holbrook, 1994). This protective role is accomplished by a series of cooperating mechanisms which involve the constant renewal of the epider- mis through proliferation of keratinocytes and shedding (Jones et al., 1985), cornified envelope formation (Kalinin et al., 2002) and the epidermal resilience against physical stress through the specificcytoskeletonofkeratinocytes(CowinandBurke,1996) as well as the characteristic cell-cell (Braga, 2002) and cell- matrix interactions (Jamora and Fuchs, 2002; Madison, 2003). Important regulatory roles in these functions are facilitated by ions, cytokines and growth factors (Fuchs and Raghavan, 2002; Werner and Grose, 2003). Epidermal growth factor (EGF) (Nanney et al., 1996) derived from platelets, transforming growth factor-a (TGF-a) from basal keratinocytes (Nanney et al., 1996; Rappolee et al., 1988), keratinocyte growth factor (KGF) from dermal fibroblasts (Werner et al., 1992), and activin (Grose and Werner, 2002; Munz et al., 1999) are key regulators of central epidermal functions. More recently sAPPa, the soluble N-terminal form of the amyloid precursor protein (APP), has been described as a new epidermal growth factor due to its mitogenic (Hoffmann et al., 2000; Saitoh et al., 1989; Siemes et al., 2004) and motogenic (Kirfel et al., 2002) activities. Some growth factors have been shown to exert in addition to these functions a cytoprotective effect. Thus, KGF has been identifiedasapotentsurvivalfactorforkeratinocytes(Aufdem Keller et al., 2004) and for different types of other epithelial cells (Werner, 1998), e.g. lung epithelial cells, which are protected from hyperoxide-induced cell death in the presence of this growth factor (Ray et al., 2003). These effects can be 0171-9335/04/083/11-12-701$30.00/0 1) Corresponding author: ProfessorDr.VolkerHerzog,InstituteofCell Biology, University of Bonn, Ulrich-Haberland-Str. 61A, D-53121 Bonn, Germany, e-mail: herzog@uni-bonn.de, Fax:  49228735302. 2) Present address: Department of Surgery, Medical School, Sigmund- Freud-Str. 25, D-53105 Bonn, Germany 3) Both authors contributed equally to this work.