SOX9 in Keratinocytes Regulates Claudin 2 Transcription during Skin Aging

Abstract

In order to prove that SOX9 in keratinocytes regulates claudin 2 transcription during skin aging, the skin of 8-week-old and 24-month-old mice is sequenced to obtain a differentially expressed gene SOX9. The gene is mainly expressed in keratinocytes, and it increases first and then decreases from newborn to aging. Six core sequences of SOX9 and claudin 2 are predicted from Jaspar. The double Luciferase Report shows that overexpression of SOX9 induces the full-length promoter of claudin 2 significantly and has no effect on the mutation and cleavage plasmid without SOX9 response. Claudin 2 is consistent with SOX9 in the skin of mice of different ages, and SOX9 is strongly positively correlated with claudin 2. Finally, overexpression of SOX9 and claudin 2 will delay PM2.5-induced keratinocyte senescence. The silencing of claudin 2 leads to the loss of SOX9 function. It is clearly evident that SOX9 can affect the transcription of claudin 2, which increases first and then decreases in the process of mice from newborn to aging. SOX9 inhibits proinflammatory mediators, increases antioxidant capacity, and restores keratin differentiation. It can effectively prevent melanin deposition and delay aging.

Figure 1

Differential gene expression in skin tissues of 8-week-old and 24-month-old mice: (a) library building and sequencing process; (b) volcano plot of differentially expressed genes; (c) heat map of the top 20 differential genes.

Figure 2

The expression of SOX9 in skin tissues of mice from birth to 24 months: (a) the mRNA expression of SOX9; (b) immunofluorescence for SOX9; (c) immunohistochemistry for SOX9.

Figure 3

Regulation of oxidative stress, differentiation, inflammation, and melanin deposition in NEK by SOX9: (a) expression of SOX9 in NEK; (b) Krt1, Krt10, Krt14, and Lor in NEK; (c) TNF-α, IL-1β, IL-6, and COX-2 in NEK; (d) Regulation of ROS by SOX9 in NEK; (e) EDN1 and PGE2 in NEK and NM. Groups represented by different letters have significant differences at P < 0.05, while the same letters indicate no significant differences.

Figure 4

SOX9 regulates claudin 2 promoter activity in HEK-293 cells: (a) SOX9 response element at the promoter; (b) dose relationship of SOX9 on claudin promoter activity; (c) schematic representation of the claudin 2 5′ upstream promoter region, and SOX9 binds to the claudin2 promoter binding site (pcDNA-SOX9, 100 ng).

Figure 5

Regulation of oxidative stress, differentiation, inflammation, and melanin deposition in NEK by claudin 2: (a) expression of claudin 2 in the skin of mice of different ages; (b) activation of claudin 2 in NEK; (c) inhibition of claudin 2 in NEK; (d) Krt1, Krt10, Krt14, and Lor in NEK; (e) regulation of ROS by claudin 2 in NEK; (f) TNF-α, IL-1β, IL-6, and COX-2 in NEK; (g) EDN1 and PGE2 in NEK and NM.

Figure 6

Differences in oxidative stress, keratinocyte differentiation, inflammation, and melanin deposition in the skin tissues of 8-week-old and 24-month-old mice: (a) scatter plot of correlation analysis between SOX9 and claudin 2 at 8 weeks, Y = 1.652∗X − 4.520, R² = 0.7453; (b) scatter plot of correlation analysis between SOX9 and claudin 2 at 24 months, Y = 0.7862∗X + 0.4914, R² = 0.7372; (c) oxidative stress-related factors MDA, GSH-Px, and SOD; (d) Skin keratin formation-related genes Krt1, Krt10, Krt14, and Lor; (e) inflammatory mediators TNF-α, IL-1β, IL-6, and COX-2; (f) melanogenesis-related factors EDN1 and PGE2.