title: Autophagic-lysosomal dysregulation downstream of cathepsin B inactivation in human skin fibroblasts exposed to UVA.
abstract: Recently, using 2D-DIGE proteomics we have identified cathepsin B as a novel target of UVA in human Hs27 skin fibroblasts. In response to chronic exposure to noncytotoxic doses of UVA (9.9 J cm(-2), twice a week, 3 weeks), photooxidative impairment of cathepsin B enzymatic activity occurred with accumulation of autofluorescent aggregates colocalizing with lysosomes, an effect mimicked by pharmacological antagonism of cathepsin B using the selective inhibitor CA074Me. Here, we have further explored the mechanistic involvement of cathepsin B inactivation in UVA-induced autophagic-lysosomal alterations using autophagy-directed PCR expression array analysis as a discovery tool. Consistent with lysosomal expansion, UVA upregulated cellular protein levels of the lysosomal marker glycoprotein Lamp-1, and increased levels of the lipidated autophagosomal membrane constituent LC3-II were detected. UVA did not alter expression of beclin 1 (BECN1), an essential factor for initiation of autophagy, but upregulation of p62 (sequestosome 1, SQSTM1), a selective autophagy substrate, and α-synuclein (SNCA), an autophagic protein substrate and aggresome component, was observed at the mRNA and protein level. Moreover, UVA downregulated transglutaminase-2 (TGM2), an essential enzyme involved in autophagolysosome maturation. Strikingly, UVA effects on Lamp-1, LC3-II, beclin 1, p62, α-synuclein, and transglutaminase-2 were mimicked by CA074Me treatment. Taken together, our data suggest that UVA-induced autophagic-lysosomal alterations occur as a consequence of impaired autophagic flux downstream of cathepsin B inactivation, a novel molecular mechanism potentially involved in UVA-induced skin photodamage.
authors.affiliation: Department of Pharmacology and Toxicology, College of Pharmacy & University of Arizona, Arizona Cancer Center, 1515 North Campbell Avenue, Tucson, AZ 85724, USA.
authors.forename: Sarah D
authors.initials: SD
authors.lastname: Lamore
authors.forename: Georg T
authors.initials: GT
authors.lastname: Wondrak
mesh_terms: D001343:Autophagy; D002401:Cathepsin B / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology / Q000528:radiation effects*; D005434:Flow Cytometry; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D016133:Polymerase Chain Reaction; D012867:Skin / Q000166:cytology / Q000201:enzymology / Q000528:radiation effects*; D014466:Ultraviolet Rays
chemical_list: D002401:Cathepsin B