Ann Dermatol.  2011 May;23(2):138-143. 10.5021/ad.2011.23.2.138.

Sox9 Increases the Proliferation and Colony-forming Activity of Outer Root Sheath Cells Cultured In Vitro

• Shi G
• Sohn KC
• Kim SY
• Ryu EK
• Park YS
• Lee Y
• Seo YJ
• Lee JH
• Kim CD

• KMID: 2156653
• DOI: http://doi.org/10.5021/ad.2011.23.2.138

Abstract

BACKGROUND
beta-catenin plays a pivotal role in hair follicle development and hair growth cycle.
OBJECTIVE
The aim of this study was to identify beta-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells.
METHODS
Primary cultured ORS cells were transduced with recombinant adenovirus expressing N-terminal truncated beta-catenin (constitutive active form), and beta-catenin-regulated genes were identified.
RESULTS
Overexpression of the constitutively active form of beta-catenin led to induction of Sox9 expression at both mRNA and protein levels. To investigate the potential role of Sox9, we made the recombinant adenovirus expressing green fluorescent protein-tagged Sox9, and then transduced into cultured ORS cells. Interestingly, Sox9 induced the expression of keratin 15, increased the proliferation of ORS cells in vitro, and enhanced colony-forming activity.
CONCLUSION
Our results suggest that Sox9 is a beta-catenin-regulated gene in ORS cells, and has potential importance in the regulation of hair follicle homeostasis.

Keyword

beta-catenin; Outer root sheath cells; Sox9

MeSH Terms

Adenoviridae
beta Catenin
Electrolytes
Hair
Hair Follicle
Homeostasis
Humans
Keratin-15
RNA, Messenger
Electrolytes
Keratin-15
RNA, Messenger
beta Catenin

Figure

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  Fig. 1 (A) Structure of β-catenin. The N-terminus has several phosphorylation sites, which are important for regulating the stability of β-catenin. Central domain consists of 12 arm repeats, which allow for overlapping interaction with multiple binding partners. The C-terminus functions as a transcriptional activation domain. N-terminal 87-amino acid truncated β-catenin (ΔN87β Cat) is depicted under the wild-type β-catenin. (B) Outer root sheath cells were transduced with adenovirus expressing ΔN87βCat at 10 multiplicity of infection. The mRNA level was verified by reverse transcription-polymerase chain reaction. (C) After adenoviral transduction, protein level was verified by Western blot. Upper band represents the endogenous β-catenin, while lower band represents the N-truncated β-catenin (arrow). Adenovirus expressing lacZ was used as negative control.

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  Fig. 2 Immunohistochemical staining of Sox9 (×100). Paraffin section of scalp specimen was incubated with anti-Sox9 antibody, and then sequentially incubated with HRP-conjugated secondary antibody. Intense expression of Sox9 is detected in the outer layer of outer root sheath in middle portion of hair follicle.

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  Fig. 3 (A) Expression of exogenous Sox9 in outer root sheath (ORS) cells. ORS cells were transduced with adenovirus expressing GFP-tagged Sox9 (GFP-Sox9) at 10 multiplicity of infection (MOI). Adenovirus expressing GFP was used as negative control. Twenty-four hours after adenoviral transduction, cells were observed under the fluorescent microscopy. (B) Effect of Sox9 on the expression of stem cell marker in ORS cells. Cells were transduced with adenovirus expressing GFP-Sox9 at the indicated MOI. The mRNA level was verified by reverse transcription-polymerase chain reaction. (C) Cellular extracts were prepared, and the protein level for stem cell marker was verified by Western blot. Actin was used as a loading control.

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  Fig. 4 Effect of Sox9 on cell proliferation. (A) Cells were transduced with adenovirus expressing GFP-Sox9 at the indicated multiplicity of infection. The proliferation marker PCNA was examined by Western blot. (B) Thymidine uptake assay. After adenoviral transduction, cells were received [3H]thymidine-containing medium. After a 3 day incubation, cells were lysed and radioactivity was measured using liquid scintillation counter. Data are expressed as % control and SEM from three independent experiments. *p<0.01.

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  Fig. 5 Effect of Sox9 on clonogenicity. ORS cells were transduced with adenovirus (10 multiplicity of infection). After 2 day incubation, cells were detached from the culture dish and 1,000 cells were re-seeded onto new culture dish. After 2 week incubation, colony formation was verified by staining with Crystal violet.

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