UVA causes dual inactivation of cathepsin B and L underlying lysosomal dysfunction in human dermal fibroblasts

Abstract

Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. 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 an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, 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 display ‘UVA-mimetic’ effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts.

The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death

“…8,38 Cells (1 × 10 6 ) were lysed in 0.5 ml of chilled lysis buffer. After 10 min incubation on ice, lysates were centrifuged at 10,000 g at 4 °C for 5 min and the supernatant fraction was retained for analysis.…”

“…8 Processing of samples and the protocol were identical to the CTSB activity assay as described above with the following modifications: CTSL determination: After lysis, 50 μL lysate were incubated with 50 μL of reaction buffer containing Ca074 (Sigma, C5732; 1 µM, 15 min) to irreversibly inhibit CTSB, thereby eliminating interference from CTSB-dependent cleavage of the substrate. 91,92 CTSL substrate (Ac-Phe-Arg-AFC, Abcam, ab157769; 200 μM final concentration) was then added and the mixture was incubated for 1 h at 37 °C followed by AFC detection (λex 400 nm, λem 505 nm).…”

“…[1][2][3] Dysregulation of autophagy has been implicated in a broad range of human pathologies including neurodegeneration and cancer, 4,5 and pathological alterations of autophagic-lysosomal function can also occur in response to exposure to environmental toxicants such as arsenic and solar UV radiation. [6][7][8] Recent evidence suggests that cancer cells may harness autophagic pathways as an adaptation to conditions associated with the tumor microenvironment including increased levels of oxidative and proteotoxic stress, hypoxia, and energy crisis. 5,9,10 Autophagy also occurs in response to exposure to major classes of chemotherapeutic agents (e.g., cisplatin and doxorubicin [Doxo]), a process thought to contribute to cancer cell chemoresistance.…”

Ultraviolet A Enhances Cathepsin L Expression and Activity via JNK Pathway in Human Dermal Fibroblasts

“…[12] Recent research shows that CatL activity in fibroblasts is significantly suppressed 1 h after four consecutive daily exposures to 9.9 J/cm 2 UVA, whereas its gene expression level remains unchanged. [13] However, repetitive UVA irradiation elevates CatL protein synthesis through increased transcript levels in fibroblasts. [12] Little is known about the molecular mechanisms, whereby UVA induces CatL expression and activity.…”

Genotoxic changes in peripheral lymphocytes after therapeutic exposure to crude coal tar and ultraviolet radiation

“…However, the results of recent studies have shown that the UVA can also be a potent inductor of DNA damage and may be associated with skin cancer 9 . Protracted exposure to UVA leads to lysosomal dysfunction in human fibroblasts 10 , malignant transformation in human cultured keratinocytes 11 and the formation of skin carcinomas in hairless mice in vivo 12 . Unlike the shorter-wavelength UVB photons, which are almost completely absorbed by the epidermis, the longerwave length UVA photons can reach deeper dermal layer of skin and its blood vessels.…”