Identification of Cathepsin B and L as Novel Uva Targets Upstream of Cutaneous Lysosomal-Autophagic Dysregulation

Chronic exposure to solar UVA plays a causative role in skin photoaging and photocarcinogenesis. Guided by exploratory difference-in-gel-electrophoresis (DIGE)-proteomics, we identified the thiol-dependent cysteine-proteases cathepsin B and cathepsin L as novel UVA-targets undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction. In human skin fibroblasts, exposure to noncytotoxic doses of chronic UVA (9.9 J/cm ², twice a week, 3 weeks) caused pronounced photooxidative impairment of cathepsin B and L enzymatic activity suppressed by antioxidant intervention. Western blot analysis revealed extensive 4-hydroxy-2-trans-nonenal (4-HNE) modification of cathepsin B in UVA-exposed fibroblasts. Consistent with lysosomal impairment, accumulation of cellular autofluorescent material colocalizing with lysosomes was observed by confocal fluorescence microscopy, and extensive deposition of lipofuscin was detectable by transmission electron microscopy (TEM). Lysosomal expansion was further evidenced by increased immunodetection of lysosomal associated membrane protein-1 (Lamp-1) and Lysotracker-based flow cytometric analysis. While lysosomal membrane integrity remained intact, autophagic blockade was suggested by accumulation of cellular protein levels of LC3-II and p62 (sequestosome 1) in UVA-exposed fibroblasts. Furthermore, UVA-exposure modulated transcriptional levels of p62 (sequestosome 1, SQSTM1), α-synuclein (SNCA), and transglutaminase-2 (TGM2). Strikingly, pharmacological cathepsin B/L inhibition using CA074Me mimicked UVA-induced accumulation of lipofuscin and autophagic-lysosomal proteins (Lamp-1, LC3-II, and p62), as well as changes at the transcriptional levels. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring protein levels of Lamp-1, LC3-II, and p62, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not reproduce the UVA-induced phenotype. Similarly, TEM revealed massive accumulation of lipofuscin-containing lysosomal vesicles in fibroblasts only after CTSB/CTSL-double knockdown. Taken together, our data indicate for the first time that UVA impairs lysosomal function causing autophagic-lysosomal alterations downstream of cathepsin B/L enzymatic inactivation. This work provides evidence for a heretofore unrecognized 'double-hit' mechanism of UVA skin photodamage where primary photo-oxidative insult occurs simultaneously with impaired clearance of damaged molecules and organelles downstream of dual inactivation of cathepsin B and L.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Graduate College

Grantor dc:publisher

The University of Arizona.

Year dc:date.issued

2012

Author dc:creator

• Lamore, Sarah Diane

Advisor dc:contributor.advisor

• Wondrak, Georg T.

Committee members dc:contributor.committeemember

• Smith, Catharine
• Monks, Terrence
• Cherrington, Nathan
• Wondrak, Georg T.
• Jacobson, Elaine

• lysosome
• photodamage
• skin
• Ultraviolet A
• Pharmacology & Toxicology
• cathepsin
• fibroblast

Statement dc:rights

• Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10150/228173

OAI identifier oai:identifier

oai:repository.arizona.edu:10150/228173