Aging Fibroblasts Adversely Affect Extracellular Matrix Formation via the Senescent Humoral Factor Ependymin-Related Protein 1

Abstract

Skin senescence is characterized by a decrease in extracellular matrix and the accumulation of senescent fibroblasts in the dermis, and their secretion of humoral factors. Ependymin-related protein 1 (EPDR1) is involved in abnormal fibroblast metabolism and collagen deposition, however, its relation to skin aging is unclear. We investigated whether and how EPDR1 is involved in age-related dermal deterioration. When young dermal fibroblasts and senescent cells were co-cultured in a semipermeable membrane separation system, the young fibroblasts showed decreased gene expression of collagen type I α1 chain (COL1A1) and elastin, and increased expression of matrix metalloproteinase (MMP)1 and MMP3. Senescence marker expression and EPDR1 production were increased in the culture medium of senescent cells. Treatment of young fibroblasts with recombinant EPDR1, enhanced matrix-related gene expression and suppressed COL1A1 expression, whereas EPDR1 knockdown had the opposite effects. EPDR1 gene and protein expression were increased in aged skin, compared to young skin. These results suggest that senescent cells affect nearby fibroblasts, in part through EPDR1 secretion, and exert negative effects on matrix production in the dermis. These results may lead to the discovery of potential candidate targets in the development of skin anti-aging therapies.

Keywords: ependymin-related protein 1; fibroblast; senescence; senescence-associated secretory phenotype; skin aging.

Figure 1

Expression of extracellular matrix-related genes in senescent dermal fibroblasts. (A) SA-β-gal staining of proliferating and senescent cells. Bar = 100 µm. (B) BrdU absorption in proliferating and senescent cells. (C) RT-qPCR analysis of extracellular matrix-related gene expression in young and senescent cells. GAPDH was used as a reference gene. (D) Western blot analysis of extracellular matrix-related protein expression in young and senescent cells. The expression levels of each protein were normalized to the expression level of GAPDH. * p < 0.05. All assays were conducted in experimental triplicate.

Figure 2

EPDR1 expression in aged dermal fibroblasts. (A) Immunostaining of p16ink4a and EPDR1 in young (proliferating) and senescent cells. Merge is a combined fluorescence image of DAPI, p16ink4a, and EPDR1. Bar = 20 µm. (B) RT-qPCR analysis of p16ink4a and EPDR1 gene expression. GAPDH was used as a reference gene. (C) Western blot analysis of EPDR1 expression in young and senescent cells. GAPDH was used as a loading control. (D) ELISA of EPDR1 protein levels in culture medium of senescent cells. (E) Cell viability after recombinant EPDR1 treatment. (F) RT-qPCR analysis of changes in gene expression following the administration of recombinant EPDR1. GAPDH was used as a reference gene. (G) Western blot analysis of protein expression after recombinant EPDR1 treatment. The expression levels of each protein were normalized to the expression level of GAPDH. (H) RT-qPCR analysis of changes in gene expression after EPDR1 knockdown. GAPDH was used as a reference gene. (I) Western blot analysis of protein expression after EPDR1 knockdown. The expression levels of each protein were normalized to the expression level of GAPDH. * p < 0.05. All assays were conducted in experimental triplicate.

Figure 2

EPDR1 expression in aged dermal fibroblasts. (A) Immunostaining of p16ink4a and EPDR1 in young (proliferating) and senescent cells. Merge is a combined fluorescence image of DAPI, p16ink4a, and EPDR1. Bar = 20 µm. (B) RT-qPCR analysis of p16ink4a and EPDR1 gene expression. GAPDH was used as a reference gene. (C) Western blot analysis of EPDR1 expression in young and senescent cells. GAPDH was used as a loading control. (D) ELISA of EPDR1 protein levels in culture medium of senescent cells. (E) Cell viability after recombinant EPDR1 treatment. (F) RT-qPCR analysis of changes in gene expression following the administration of recombinant EPDR1. GAPDH was used as a reference gene. (G) Western blot analysis of protein expression after recombinant EPDR1 treatment. The expression levels of each protein were normalized to the expression level of GAPDH. (H) RT-qPCR analysis of changes in gene expression after EPDR1 knockdown. GAPDH was used as a reference gene. (I) Western blot analysis of protein expression after EPDR1 knockdown. The expression levels of each protein were normalized to the expression level of GAPDH. * p < 0.05. All assays were conducted in experimental triplicate.

Figure 2

EPDR1 expression in aged dermal fibroblasts. (A) Immunostaining of p16ink4a and EPDR1 in young (proliferating) and senescent cells. Merge is a combined fluorescence image of DAPI, p16ink4a, and EPDR1. Bar = 20 µm. (B) RT-qPCR analysis of p16ink4a and EPDR1 gene expression. GAPDH was used as a reference gene. (C) Western blot analysis of EPDR1 expression in young and senescent cells. GAPDH was used as a loading control. (D) ELISA of EPDR1 protein levels in culture medium of senescent cells. (E) Cell viability after recombinant EPDR1 treatment. (F) RT-qPCR analysis of changes in gene expression following the administration of recombinant EPDR1. GAPDH was used as a reference gene. (G) Western blot analysis of protein expression after recombinant EPDR1 treatment. The expression levels of each protein were normalized to the expression level of GAPDH. (H) RT-qPCR analysis of changes in gene expression after EPDR1 knockdown. GAPDH was used as a reference gene. (I) Western blot analysis of protein expression after EPDR1 knockdown. The expression levels of each protein were normalized to the expression level of GAPDH. * p < 0.05. All assays were conducted in experimental triplicate.

Figure 3

EPDR1 expression in aged human skin. (A) Immunostaining of EPDR1 in human skin dermis. (B) RT-qPCR analysis of gene expression using total RNA extracted from human skin dermis. GAPDH was used as a reference gene. (C) Western blot analysis using proteins extracted from human skin dermis. * p < 0.05. All assays were conducted in experimental triplicate.