Proteomic identification of cathepsin B and nucleophosmin as novel UVA-targets in human skin fibroblasts

Photochem Photobiol. Nov-Dec 2010;86(6):1307-17. doi: 10.1111/j.1751-1097.2010.00818.x. Epub 2010 Oct 14.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylcysteine / pharmacology
  • Antioxidants / pharmacology
  • Cathepsin B / metabolism*
  • Cell Line
  • Dipeptides / pharmacology
  • Fibroblasts / metabolism
  • Fibroblasts / radiation effects
  • Gene Expression Profiling
  • Humans
  • Lipofuscin / metabolism
  • Lysosomes / metabolism
  • Nuclear Proteins / metabolism*
  • Oxidative Stress
  • Proteomics
  • Skin / metabolism*
  • Skin / radiation effects*
  • Skin Aging / genetics
  • Skin Aging / physiology
  • Ultraviolet Rays / adverse effects*

Substances

  • Antioxidants
  • CA 074 methyl ester
  • Dipeptides
  • Lipofuscin
  • Nuclear Proteins
  • nucleophosmin
  • Cathepsin B
  • Acetylcysteine