Cathepsin F is a potential marker for senescent human skin fibroblasts and keratinocytes associated with skin aging

Abstract

Cellular senescence is characterized by cell cycle arrest and the senescence-associated secretory phenotype (SASP) and can be triggered by a variety of stimuli, including deoxyribonucleic acid (DNA) damage, oxidative stress, and telomere exhaustion. Cellular senescence is associated with skin aging, and identification of specific markers of senescent cells is essential for development of targeted therapies. Cathepsin F (CTSF) has been implicated in dermatitis and various cancers and participates in cell immortalization through its association with Bcl family proteins. It is a candidate therapeutic target to specifically label and eliminate human skin fibroblasts and keratinocytes immortalized by aging and achieve skin rejuvenation. In this study, we investigated whether CTSF is associated with senescence in human fibroblasts and keratinocytes. In senescence models, created using replicative aging, ionizing radiation exposure, and the anticancer drug doxorubicin, various senescence markers were observed, such as senescence-associated β-galactosidase (SA-β-gal) activity, increased SASP gene expression, and decreased uptake of the proliferation marker BrdU. Furthermore, CTSF expression was elevated at the gene and protein levels. In addition, CTSF-positive cells were abundant in aged human epidermis and in some parts of the dermis. In the population of senescent cells with arrested division, the number of CTSF-positive cells was significantly higher than that in the proliferating cell population. These results suggest that CTSF is a candidate for therapeutic modalities targeting aging fibroblasts and keratinocytes.

Keywords: Cathepsin; Cellular senescence; Fibroblast; Keratinocytes; Senescence-associated secretory phenotype; Skin aging.

Fig. 1

CTSF is upregulated in aged human skin fibroblasts. a SA-β-gal staining. Bar = 50 µm. b BrdU incorporation in proliferating and senescent cells. c Immunostaining of proliferating and senescent cells for CTSF and IL-6. Bar = 20 µm. d Gene expression of CTSF and SASP factors in proliferating and senescent cells. e Expression of CTSF protein in proliferating and senescent cells. *P < 0.05. CTSF, cathepsin F; DS, doxorubicin-induced senescence; ReS, replicative senescence; RS, radiation-induced senescence; SASP, senescence-associated secretory phenotype

Fig. 2

CTSF is upregulated in aged human skin keratinocytes. a SA-β-gal staining. Bar = 50 µm. b BrdU incorporation in proliferating and senescent cells. c Immunostaining of proliferating and senescent cells for CTSF and IL-6. Bar = 20 µm. d Gene expression of CTSF and SASP factors in proliferating and senescent cells. e Expression of CTSF protein in proliferating and senescent cells. *P < 0.05. CTSF, cathepsin F; DS, doxorubicin-induced senescence; ReS, replicative senescence; RS, radiation-induced senescence; SASP, senescence-associated secretory phenotype

Fig. 3

Expression of CTSF in human skin. In young skin tissue, only some cells in the epidermal layer are CTSF positive, but in old skin, many CTSF-positive cells are observed in all layers of the epidermis and dermis. Red arrows: CTSF-positive cells. Bar = 100 µm. CTSF, cathepsin F

Fig. 4

Sorting of senescent cells using an anti-CTSF antibody. a Sorting of CTSF-positive cells in proliferating and senescent human skin fibroblasts. b Comparison of the number of CTSF-positive fibroblasts. c Sorting of CTSF-positive cells in proliferating and senescent human skin keratinocytes. d Comparison of the number of CTSF-positive keratinocytes. Each data point represents a young (n = 3 total) or ReS (n = 3 total) subject; horizontal lines indicate the mean values. **P < 0.01. CTSF, cathepsin F; ReS, replicative senescence