Original Contribution IMPAIRMENT OF PROTEASOME FUNCTION UPON UVA- AND UVB- IRRADIATION OF HUMAN KERATINOCYTES ANNE-LAURE BULTEAU,* MARIELLE MOREAU, † CARINE NIZARD, † and BERTRAND FRIGUET* *Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite ´ Paris, Paris 7-Denis Diderot, France; and † LVMH branche Parfums et Cosme ´tiques, Laboratoires Recherche et De ´veloppement, Saint Jean de Braye, France (Received 6 November 2001; Revised 19 February 2002; Accepted 7 March 2002) Abstract—The major environmental influence for epidermal cells is sun exposure and the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Therefore, the effects of UV-irradiation on proteasome have been investigated in human keratinocyte cultures. Human keratino- cytes were irradiated with 10 J/cm 2 of UVA and 0.05 J/cm 2 of UVB and proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidase activities were decreased as early as 1 h and up to 24 h after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit degradation by the proteasome, demonstrating the presence of endogeneous inhibitors, including 4-hydroxy-2-nonenal modified proteins, generated upon UV-irradiation. © 2002 Elsevier Science Inc. Keywords—UV-irradiation, Proteasome, Keratinocytes, Photoaging, Protein oxidation, Protein modification, 4-Hy- droxy-2-nonenal, Free radicals INTRODUCTION Exposure of human skin to UV-irradiation over a period of years results in chronic photodamage, also called photoaging [1]. Photoaging induces morphological changes such as wrinkling and sagging due to general alteration of all the epidermal and dermal components of the skin including the cutaneous cells [2]. UVA radiation (320 – 400 nm) constitutes more than 90% of the terres- trial UV solar energy and penetrates more efficiently than UVB (280 –320 nm) in the proliferative basal layers of the epidermis [3]. Although it is well accepted that low levels of reactive oxygen species (ROS) are contin- uously produced in vivo and are involved in physiolog- ical processes, there is accumulating evidence for the damaging effects of higher concentrations of ROS (hy- droxyl radical, superoxide anion, and singlet oxygen) generated in vivo after UVA- and UVB-irradiation of the skin [4 – 6]. Generation of ROS following irradiation with UVA [7] and UVB [8] requires the absorption of photons by endogenous photosensitizer molecules [9]. The UVA-induced skin damage is mainly related to cell photosensitization involving endogenous sensitizers such as NAD(P)H flavines, or porphyrins [10 –12]. The pho- tosensitized reactions produce ROS such as singlet oxy- gen and oxygen radicals, which react with DNA, result- ing in transient and permanent genetic damage, and with lipids and proteins, resulting in the activation of cyto- plasmic signal transduction pathways that are related to growth, differentiation, and replicative senescence [5,9, 13,14]. The peroxidation of lipids and oxidation of glu- tathione have been shown to be the immediate conse- quences of UVA-irradiation of human skin [15–17]. UVB-dependent generation of hydroxyl radicals and lip- ids peroxides has been demonstrated in murine skin and Address correspondence to: Bertrand Friguet, Laboratoire de Biolo- gie et Biochimie Cellulaire du Vieillissement, Universite ´ Paris 7-Denis Diderot, 2 Place Jussieu, 75251, Paris Cedex 05, France; Tel: +33 (1) 44 27 82 34; Fax: +33 (1) 44 27 82 34; E-Mail: bfriguet@paris7. jussieu.fr. Free Radical Biology & Medicine, Vol. 32, No. 11, pp. 1157–1170, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter PII S0891-5849(02)00816-X 1157