Proteomic Identification of Cathepsin B and Nucleophosmin as Novel UVA‐Targets in Human Skin Fibroblasts

Solar UVA exposure plays a causative role in skin photoaging and photocarcinogenesis. Here, we describe the proteomic identification of novel UVA‐targets in human dermal fibroblasts following a two‐dimensional‐difference‐gel‐electrophoresis (2D‐DIGE) approach. Fibroblasts were exposed to noncytotoxic doses of UVA or left untreated, and total protein extracts underwent CyDye‐labeling followed by 2D‐DIGE/mass‐spectrometric identification of differentially expressed proteins, confirmed independently by immunodetection. The protein displaying the most pronounced UVA‐induced upregulation was identified as the nucleolar protein nucleophosmin. The protein undergoing the most pronounced UVA‐induced downregulation was identified as cathepsin B, a lysosomal cysteine‐protease displaying loss of enzymatic activity and altered maturation after cellular UVA exposure. Extensive lysosomal accumulation of lipofuscin‐like autofluorescence and osmiophilic material occurred in UVA‐exposed fibroblasts as detected by confocal fluorescence microscopy and transmission electron microscopy, respectively. Array analysis indicated UVA‐induced upregulation of oxidative stress response gene expression, and UVA‐induced loss of cathepsin B enzymatic activity in fibroblasts was suppressed by antioxidant intervention. Pharmacological cathepsin B inhibition using CA074Me mimicked UVA‐induced accumulation of lysosomal autofluorescence and deficient cathepsin B maturation. Taken together, these data support the hypothesis that cathepsin B is a crucial target of UVA‐induced photo‐oxidative stress causatively involved in dermal photodamage through the impairment of lysosomal removal of lipofuscin.