Ann Dermatol.  2018 Aug;30(4):432-440. 10.5021/ad.2018.30.4.432.

Possible Role of Single Stranded DNA Binding Protein 3 on Skin Hydration by Regulating Epidermal Differentiation

• Choi MR
• Shin JM
• Shin YA
• Chang YH
• Chang MY
• Lim CA
• Sohn KC
• Seo YJ
• Kim CD
• Lee JH
• Lee Y

• KMID: 2457520
• DOI: http://doi.org/10.5021/ad.2018.30.4.432

Abstract

BACKGROUND
Skin hydration is a common problem both in elderly and young people as dry skin may cause irritation, dermatological disorders, and wrinkles. While both genetic and environmental factors seem to influence skin hydration, thorough genetic studies on skin hydration have not yet been conducted.
OBJECTIVE
We used a genome-wide association study (GWAS) to explore the genetic elements underlying skin hydration by regulating epidermal differentiation and skin barrier function.
METHODS
A GWAS was conducted to investigate the genetic factors influencing skin hydration in 100 Korean females along with molecular studies of genes in human epidermal keratinocytes for functional study in vitro.
RESULTS
Among several single nucleotide polymorphisms identified in GWAS, we focused on Single Stranded DNA Binding Protein 3 (SSBP3) which is associated with DNA replication and DNA damage repair. To better understand the role of SSBP3 in skin cells, we introduced a calcium-induced differentiation keratinocyte culture system model and found that SSBP3 was upregulated in keratinocytes in a differentiation dependent manner. When SSBP3 was overexpressed using a recombinant adenovirus, the expression of differentiation-related genes such as loricrin and involucrin was markedly increased.
CONCLUSION
Taken together, our results suggest that genetic variants in the intronic region of SSBP3 could be determinants in skin hydration of Korean females. SSBP3 represents a new candidate gene to evaluate the molecular basis of the hydration ability in individuals.

Keyword

Cell differentiation; Genome-wide association study; Hydration; Keratinocytes; Single stranded DNA binding protein 3

MeSH Terms

Adenoviridae
Aged
Cell Differentiation
DNA Damage
DNA Replication
DNA, Single-Stranded*
DNA-Binding Proteins*
Female
Genome-Wide Association Study
Humans
In Vitro Techniques
Introns
Keratinocytes
Polymorphism, Single Nucleotide
Skin*
DNA, Single-Stranded
DNA-Binding Proteins

Figure

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  Fig. 1 Expression of SSBP3 in epidermal keratinocytes and epidermis. (A) Cellular extracts were prepared from cultured normal human epidermal keratinocytes (NHEKs) and HaCaT cells. The endogenous protein expression of SSBP3 was detected via western blot analysis. (B) Paraffin-embedded normal skin tissue was immunohistochemically stained with anti-SSBP3. Immunohistochemical staining shows nuclear SSBP3 staining in all epidermal layers and increased SSBP3 positive staining in the spinous layer (hematoxylin and DAB, ×200; scale bar=50 µm).

• 

  Fig. 2 Effect of SSBP3 overexpression on keratinocyte differentiation. (A) Keratinocytes were treated with 1.8 mM CaCl2 for indicated times. SSBP3 protein levels were detected via western blot analysis. Actin was used as a loading control. (B) Keratinocytes were transduced with recombinant adenoviruses expressing SSBP3 or LacZ control for 12 h. Cells were replenished with fresh medium and then cultured for 3 days. mRNA level of SSBP3 was determined by quantitative polymerase chain reaction (qPCR). (C) After overexpression of SSBP3 in keratinocytes, cells were further treated with 1.8 mM CaCl2 for 3 days. The mRNA levels of epidermal differentiation-related markers involucrin, loricrin, and cytokeratin 1 (K1) were measured using qPCR. (D) The effect of SSBP3 overexpression on loricrin and involucrin promoter activity. The involucrin or loricrin promoter-luciferase reporter adenoviruses were co-transduced with adenoviruses expressing SSBP3 or LacZ for 12 h. Cells were replenished with fresh medium and treated with 1.8 mM CaCl2. Cells were further cultured for 3 days and assayed for luciferase activity. (E) After overexpression of SSBP3 in keratinocytes, cells were further treated with 1.8 mM CaCl2 for 3 days. The protein expression of involucrin and loricrin were examined by western blot analysis. Actin was used as a loading control. Values are presented as mean±standard deviation of triplicate measurements (*p<0.01).

• 

  Fig. 3 Effect of SSBP3 knockdown on keratinocyte differentiation. (A) Keratinocytes were transduced with adenoviruses expressing microRNA (miR)-SSBP3 or scrambled control (miR-scr) for 12 h. Cells were replenished with fresh medium and then cultured for 3 days. The SSBP3 protein level was measured via western blot analysis. Actin was used as the loading control. (B) After knockdown of SSBP3 in keratinocytes, cells were further treated with 1.8 mM CaCl2 for 3 days. The mRNA levels of epidermal differentiation-related markers involucrin, loricrin, and cytokeratin 1 (K1) were measured using quantitative polymerase chain reaction. (C) The effect of SSBP3 knockdown on involucrin and loricrin promoter activity. The involucrin or loricrin promoter-luciferase reporter adenoviruses were co-transduced with adenoviruses expressing miR-SSBP3 or miR-scr for 12 h. Cells were replenished with fresh medium and treated with 1.8 mM CaCl2. Cells were further cultured for 3 days and assayed for luciferase activity. (D) After knockdown of SSBP3 in keratinocytes, cells were further treated with 1.8 mM CaCl2 for 3 days. The protein expression of involucrin and loricrin were examined by western blot analysis. Actin was used as a loading control. Values are presented as mean±standard deviation of triplicate measurements (*p<0.01).

• 

  Fig. 4 Immunohistochemical analysis of SSBP3 in epidermal differentiation-related skin disorders. (A) Psoriasis, (B) ichthyosis, and (C) atopic dermatitis. (A~C) Paraffin-embedded lesional tissues were immunohistochemically stained with anti-SSBP3 (hematoxylin and DAB, ×200, scale bars=50 µm). Positive staining of SSBP3 in epidermis was quantified. PSO: psoriasis, ICH: ichthyosis, AD: atopic dermatitis, N: normal skin.

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