Original Contribution Extracellular generation of hydrogen peroxide is responsible for activation of EGF receptor by ultraviolet A radiation Claudia von Montfort, Niklas S. Fernau, Juliane I. Beier, Helmut Sies, Lars-Oliver Klotz ⁎ Institut für Biochemie und Molekularbiologie I, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 22.03, Ebene 2, D-40225 Düsseldorf, Germany Received 4 November 2005; revised 10 July 2006; accepted 8 August 2006 Available online 11 August 2006 Abstract Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) have been proposed to be activated in cells exposed to ultraviolet A (UVA) radiation (320–400 nm) and to be involved in photocarcinogenesis. Singlet oxygen and hydrogen peroxide are being discussed as mediators of the activation of signal transduction pathways by UVA. It is demonstrated here that EGFR is not activated in cells exposed to UVA in the absence of extracellular photosensitizers. Rather, UVAwas capable of activating the EGFR and the related ErbB2 receptor tyrosine kinase in HeLa cells and human keratinocytes only under conditions that allowed for the extracellular photochemical generation of H 2 O 2 , such as when cells were covered with cell culture medium during exposure to UVA. Pretreatment of cells with vanadate was required for UVA- induced EGFR activation, pointing to the involvement of protein tyrosine phosphatases. Unlike H 2 O 2 , photochemically generated singlet oxygen did not activate EGFR but instead impaired the activation of EGFR by its ligand, EGF. In summary, extracellularly generated H 2 O 2 mediates UVA-induced activation of the EGFR and of ErbB2, whereas intracellular generation of reactive oxygen species upon exposure of cells to UVA is not sufficient for activation of the receptor. © 2006 Elsevier Inc. All rights reserved. Keywords: Ultraviolet radiation; Epidermal growth factor receptor; Protein tyrosine phosphatase; Stress signaling; Reactive oxygen species; Hydrogen peroxide; Singlet oxygen; Free radicals Epidermal growth factor (EGF) receptor-dependent signaling cascades promote cell division as well as the expression of genes coding for proteins known to foster metastasis, such as matrix metalloproteinases [1]. EGFR is either overexpressed or present in a mutant, more active form in a multitude of human cancers [2,3], and chemotherapeutic regimens based on anti-EGFR drugs such as neutralizing antibodies or small-molecule inhibitors are being developed [4,5]. Although predisposition to carcinogenesis may be due to high basal cellular EGFR expression levels or to the presence of a mutant form of the receptor, it may also be based on the continuous exposure to stressful stimuli that causes the ligand-independent activation of EGFR. Several stressful conditions, including exposure to reactive oxygen species [6,7], quinones [8–10], or chemother- apeutic drugs [11], were reported to cause activation of EGFR. This is also true for ultraviolet (UV) radiation: photo-oxidative stress is known to be capable of activating cellular signaling processes that are involved in photoaging and skin carcinogen- esis [12]. EGFR activation was demonstrated by several laboratories to occur in response to treatment with UV of wavelengths <320 nm, i.e., UVB (280–320 nm) [13] or UVC (<280 nm) [14], or after treatment with broadband UV composed of UVB and UVA (320–400 nm) [15]. In murine skin, UV-induced proliferation of keratinocytes, epidermal hyperplasia, and tumorigenesis were clearly blocked in the presence of EGFR tyrosine kinase inhibitors [15,16]. Although there is no doubt that UV radiation in general is capable of activating cellular signaling cascades, including EGFR-depen- dent signaling, the contributions of the UVA spectral region—a Free Radical Biology & Medicine 41 (2006) 1478 – 1487 www.elsevier.com/locate/freeradbiomed Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; JNK, c-Jun N- terminal kinase; MAPK, mitogen-activated protein kinase; NDP, 3,3′-(1,4- naphthylidene) dipropionate; NDPO 2 , 1,4-endoperoxide of NDP; PTPase, protein tyrosine phosphatase; RB, Rose Bengal; ROS, reactive oxygen species; UVA, ultraviolet A (320–400 nm). ⁎ Corresponding author. Fax: +49 211 8113029. E-mail address: LarsOliver.Klotz@uni-duesseldorf.de (L.-O. Klotz). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.08.005