Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation

Abstract

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases.

Keywords: aging; collagen; extracellular matrix; fibronectin; granzyme B; matrix metalloproteinase; photoaging; skin; ultraviolet light.

Figure 1

GzmB deficiency protects against wrinkle formation. (A) Representative images of wild-type (WT) and GzmB-knockout mice (KO) exposed to chronic low-grade UV irradiation (UVR) for 20 weeks. Nonirradiated controls were included for comparison. (B) Three independent scorers blinded to time, genotype, and treatment assessed each mouse, and results are presented as median scores for each scorer (*P < 0.05, Wilcoxon signed rank).

Figure 2

Increased GzmB expression and mast cells in UV-irradiated skin. (A) Dorsal skin sections from nonirradiated (WT-C) and UV-irradiated (WT-UVR) mice immunostained for GzmB. Insert – magnified view of degranulating cell with GzmB immunopositivity. Scale bars = 100 μm. (B) Dorsal skin sections from both nonirradiated (C) and UV-treated (UVR), wild-type (WT), and granzyme B-knockout (KO) mice were stained with TBO, and the number of mast cells was counted (mean ± SEM; ***P < 0.001 Tukey's multiple comparison). Scale bars = 60 μm. (C) Direct colocalization of GzmB immunopositivity with mast cells. Sections were first stained with TBO (pH = 2), and the same sections were then immunostained for GzmB following image capture. Scale bars = 60 μm.

Figure 3

GzmB deficiency protects against loss of dermal collagen density and fibronectin in UV-irradiated skin. (A) Dorsal skin sections were stained with picrosirius red and visualized under polarized light. Quantification of collagen was achieved by color segmentation and intensity analysis of stained sections. Scale bars = 100 μm. (B) Second-harmonic generation signals of collagen from intact ex vivo skin samples were quantified as a measure of collagen density. Scale bars = 20 μm (*P < 0.05; **P < 0.01; ***P < 0.001 Tukey's multiple comparison). (C) Fibronectin was assessed in tissue homogenates via Western blot. Results are presented as a percentage of wild-type nonirradiated control (WT-C) (mean ± SEM; *P < 0.05 Dunnett's multiple comparison).

Figure 4

GzmB causes fibroblast detachment and fibronectin fragmentation in vitro, and GzmB-mediated FN fragments induce MMP-1 expression in fibroblasts. (A) Mouse fibroblasts plated overnight in complete medium were treated with the indicated concentrations of GzmB ± Inhibitor (I; 50 μm) for 7 h (in serum-free conditions). Adherent cells were assessed by MTS assay after washing once in PBS to remove nonadherent cells (mean ± SEM from quadruplicate wells; ***P < 0.001 Dunnett's multiple comparison). (B) Supernatants collected from (A) were assayed for fibronectin by Western blot. Closed arrowhead = full-length fibronectin; open arrowhead = fibronectin fragments. Although the same blot, the dotted line represents sections presented at different brightness/contrast so that fragments can be clearly observed at different kDa. (C) Primary fibroblasts were added to GzmB-mediated FN fragments, and MMP-1 release was assayed in the supernatants after 20 h by Western blot. GAPDH probed from cell lysates of the same wells served as loading controls. Results are presented as a percentage of intact fibronectin control (mean ± SEM from quadruplicate wells, **P < 0.01 t-test). (D) Dorsal skin sections from mice were stained with MMP-1 antibody, and intensity of staining in the dermis (excluding hair follicles) was measured by detecting the number of positive pixels above a set threshold, normalized to area. Results are expressed as a percentage of wild-type control (WT-C) (mean ± SEM). Scale bars = 60 μm.

Figure 5

GzmB-mediated decorin cleavage renders collagen fibrils more susceptible to degradation by MMP-1. (A) Decorin cleavage assay. Recombinant decorin (0.4 μg) was treated with GzmB (100 nm) ± Inhibitor (I; 50 μm) for 8 h, 37°C. (B) Dorsal skin sections from control and UV-irradiated mice were stained with decorin antibody. Intensity of staining in the dermis (excluding hair follicles) was measured by detecting the number of positive pixels above a set threshold, normalized to area. Results are expressed as a percentage of wild-type control (WT-C) (mean ± SEM; *P < 0.05; ***P < 0.001 Tukey's multiple comparison). Scale bars = 60 μm. (C) Collagen fibrils ± decorin were treated with or without GzmB (100 nm) ± Compound 20 (I, 50 μm) as indicated. Loss of intact collagen (closed arrowhead) was assessed by SDS-PAGE electrophoresis followed by Coomassie blue staining. Open arrowhead = bovine serum albumin. Results are expressed as a percentage of intact collagen control (mean ± SEM of three independent experiments, *P < 0.05; ***P < 0.001 Tukey's multiple comparison).

Figure 6

Summary of potential mechanisms of action for GzmB in UV-irradiated skin. After UV irradiation, GzmB is expressed and released into the extracellular space by a number of cells (keratinocytes, mast cells). GzmB can cleave fibronectin to form fragments that increase the expression and release of MMPs from fibroblasts. GzmB cleavage of decorin renders collagen fibrils more susceptible to MMP-mediated degradation. Overall, there is a loss of collagen density and organization leading to a phenotype of aged skin.