Amyloid ␤ peptide (A␤), the principal proteinaceous component of amyloid plaques in brains of Alzheimer's disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative ␣-secretase within the A␤ sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPs␣ into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated ␣-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous ␣-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and A␤ fragments confirm the correct ␣-secretase cleavage site and demonstrate a dependence on the substrate's conformation. Our results provide evidence that ADAM 10 has ␣-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer's disease.The amyloid precursor protein (APP) is a type I transmembrane glycoprotein constitutively expressed in many types of mammalian cells. APP is the precursor of the amyloid ␤ peptide (A␤), the principal proteinaceous component of amyloid plaques in brains of AlzheimerЈs disease patients (1, 2). The A␤ sequence includes 28 amino acids of the extracellular and 12-15 residues of the membrane-spanning region of APP. A␤ is derived by proteolytic processing of the precursor protein from as yet not identified proteases, the ␤-secretase cleaving at the N terminus, and the ␥-secretase cleaving at the C terminus.The major proteolytic pathway of APP is the constitutive secretory pathway that involves cleavage by a putative ␣-secretase within the A␤ sequence at the cell surface (3-5) and in the trans-Golgi network (6-9). Soluble N-terminal APP (APPs␣) fragments of 105-125 kDa (10) are released into the extracellular medium. The membrane-bound 10-kDa C-terminal fragment (p10) produced by ␣-secretase cleavage of APP contains only part of the amyloidogenic A␤ and can be further cleaved by the ␥-secretase to yield a secreted 3-kDa fragment (p3) (11). Because this pathway does not produce intact A␤, it is nonamyloidogenic and cannot lead to Alzheimer's disease pathology.There has been intensive interest in the secretase enzymes that cleave APP in relation to the pathology of AlzheimerЈs disease. The putative ␣-secretase cleavage site has been precisely determined (12): in human embryonic kidney cells (HEK 293) transfected with various constructs of APP, the soluble form ends at Gln-15 of A␤, and the N terminus of the cleaved C-terminal fragment begins at Leu-17. It ...

Skin is important for the absorption and metabolism of exposed chemicals such as cosmetics or pharmaceuticals. The Seventh Amendment to the EU Cosmetics Directive prohibits the use of animals for cosmetic testing for certain endpoints, such as genotoxicity; therefore, there is an urgent need to understand the xenobiotic metabolizing capacities of human skin and to compare these activities with reconstructed 3D skin models developed to replace animal testing. We have measured Phase I enzyme activities of cytochrome P450 (CYP) and cyclooxygenase (COX) in ex vivo human skin, the 3D skin model EpiDermÔ (EPI-200), immortalized keratinocyte-based cell lines and primary normal human epidermal keratinocytes. Our data demonstrate that basal CYP enzyme activities are very low in whole human skin and EPI-200 as well as keratinocytes. In addition, activities in monolayer cells differed from organotypic tissues after induction. COX activity was similar in skin, EPI-200 and NHEK cells, but was significantly lower in immortalized keratinocytes. Hence, the 3D model EPI-200 might represent a more suitable model for dermatotoxicological studies. Altogether, these data help to better understand skin metabolism and expand the knowledge of in vitro alternatives used for dermatotoxicity testing.Abbreviations: CYP, cytochrome P450-monooxygenase; COX, cyclooxygenase; PGE 2, prostaglandin E2; 3-MC, 3-methylcholanthrene; EPI-200, reconstituted epidermis model EpiDermÔ (MatTek); NHEK, normal human keratinocytes; HLM, human liver microsomes.