Photochemistry and Photobiology, 2003, 78(2): 000–000 p38 Mitogen-activated Protein Kinase Activation by Ultraviolet A Radiation in Human Dermal Fibroblasts { Rozen Le Panse, Louis Dubertret and Bernard Coulomb* INSERM Unite ´ 532, Institut de Recherche sur la Peau, Pavillon Bazin, Ho ˆ pital Saint-Louis, Paris Cedex, France Received 26 September 2002 ABSTRACT UVA radiation penetrates deeply into the skin reaching both the epidermis and the dermis. We thus investigated the effects of naturally occurring doses of UVA radiation on mitogen- activated protein kinase (MAPK) activities in human dermal fibroblasts. We demonstrated that UVA selectively activates p38 MAPK with no effect on extracellular-regulated kinases (ERK1–ERK2) or JNK–SAPK (cJun NH 2 -terminal kinase– stress-activated protein kinase) activities. We then investigated the signaling pathway used by UVA to activate p38 MAPK. L-Histidine and sodium azide had an inhibitory effect on UVA activation of p38 MAPK, pointing to a role of singlet oxygen in transduction of the UVA effect. Afterward, using prolonged cell treatments with growth factors to desensitize their signal- ing pathways or suramin to block growth factor receptors, we demonstrated that UVA signaling pathways shared elements with growth factor signaling pathways. In addition, using emetine (a translation inhibitor altering ribosome functioning) we detected the involvement of ribotoxic stress in p38 MAPK activation by UVA. Our observations suggest that p38 activation by UVA in dermal fibroblasts involves singlet oxygen–dependent activation of ligand–receptor signaling pathways or ribotoxic stress mechanism (or both). Despite the activation of these two distinct signaling mechanisms, the selective activation of p38 MAPK suggests a critical role of this kinase in the effects of UVA radiation. INTRODUCTION For a long time, UVA (320–400 nm) radiation was wrongly considered less noxious than UVB (290–320 nm), which can directly damage proteins and nucleic acids. However, UVA penetrates more deeply into the skin than UVB, reaching not only the epidermis but also the dermis. Moreover, after its absorption by endogenous chromophores, UVA leads to the formation of reactive oxygen species, which can alter protein and nucleic acid structure (1–3). UVA can thus modify cell–matrix interactions (4), disorganize the cytoskeleton (5) and alter different cellular functions (6,7). Among the cellular functions, UVA can activate transcription factors such as AP1, NF-kB or TCF and thus modulates the expression of various genes (6,8,9). However, how these UVA effects are transduced at the intracellular level is unclear. Mitogen-activated protein kinase (MAPK) cascades are essential intracellular transduction pathways that are conserved from yeasts to mammalian cells. The extracellular-regulated kinase (ERK) cascade, involving ERK1 and ERK2, was the first to be discovered and is characterized by its ability to be strongly activated by various mitogenic signals such as growth factors and hormones (10,11). Two other MAPK cascades were subsequently identified, namely, the cJun NH 2 -terminal kinase–stress-activated protein kinase (JNK–SAPK) (12) and the p38-reactivating kinase (p38 MAPK) (13). These two groups of MAPK are also called SAPK because they are mainly activated by various cellular stresses such as inflammatory cytokines, UV radiation, osmotic shock and heat shock (14). Most studies of the effects of UV radiation on MAPK activities have been carried out with UVC radiation (200–290 nm), which does not reach the earth’s surface. UVC activates all the MAPK so far studied, albeit with a more marked effect on JNK–SAPK and p38 MAPK than on ERK1 and ERK2 (12,15,16). Moreover, these studies using UVC have provided a good deal of informa- tion on the different actors in MAPK cascades. Concerning UVA effects on skin cells, it can also stimulate ERK, JNK–SAPK and p38 MAPK activities in keratinocytes (8,17,18). Using foreskin fibroblasts, Klotz et al. (19) observed that high doses of UVA radiation activated JNK–SAPK and p38 MAPK but not ERK. However, high doses of UVA radiation can be toxic to fibroblasts, especially when cultured within a collagen matrix, an environment that confers characteristics close to those observed in vivo (20). In this study we analyzed the effects of naturally occurring doses of UVA radiation on MAPK activities in human dermal fibroblasts in monolayer culture. We observed a selective activation of p38 MAPK by UVA and investigated parts of the UVA signaling path- way leading to this kinase activation. Our experiments demonstrated the involvement of different signaling pathways in p38 MAPK activation by UVA radiation. {Posted on the website on *To whom correspondence should be addressed at: INSERM Unite ´ 532, Institut de Recherche sur la Peau, Ho ˆpital Saint-Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France. Fax: 33-1-5372-2051; e-mail: bcoulomb@chu-stlouis.fr Abbreviations: ATP, adenosine triphosphate; EDTA, ethylenediamine tetraacetic acid; EGF, epidermal growth factor; EMEM, Earle modified Eagle medium; FCS, fetal calf serum; HBIB, HEPES binding buffer; HBSS, Hanks balanced salt solution; HEPES, hydroxythylpiperazine-N9- 2-ethane-sulfonic acid; GST, glutathione S-transferase; Hsp27, heat shock protein 27; JNK–SAPK, c-Jun NH 2 -terminal kinase–stress-acti- vated protein kinase; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; TBST, Tris-buffered saline Tween-20. Ó 2003 American Society for Photobiology 0031-8655/03 $5.00 þ0.00