Expression and functional role of Sox9 in human epidermal keratinocytes

Abstract

In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.

Figure 1. Expression of Sox9 in epidermal keratinocytes.

(A) Immunohistochemical staining of Sox9 in scalp skin. Intense nuclear staining is seen in outer root sheath (black arrows) and sebaceous gland (red arrowheads) (left), and interfollicular epidermis (right). (B) Cultured human epidermal keratinocytes were treated with 1.2 mM calcium for the indicated time points. The mRNA level for Sox was decreased in a time-dependent manner during calcium-induced keratinocyte differentiation. Involucrin was used as a positive control for induction of keratinocyte differentiation. Cyclophilin was used as a loading control. (C) The protein level for Sox9 was detected by Western blot analysis. Involucrin and actin were used for positive control and loading control, respectively.

Figure 2. Overexpression of Sox9 inhibits keratinocyte differentiation.

(A) Expression of exogenous Sox9 in keratinocytes cultured in vitro. Cells were transduced with adenovirus expressing GFP-tagged Sox9 (Ad/GFP-Sox9) at 10 multiplicity of infection (MOI) for overnight. Cells were replenished with fresh medium and incubated for 2 d. The expression of exogenous Sox9 was observed under the fluorescent microscopy. Adenovirus expressing GFP (Ad/GFP) was used as a negative control. (B) Effect of Sox9 on the expression of differentiation markers. Keratinocytes were transduced with adenovirus expressing GFP-Sox9 at the 10 MOI for overnight. Cells were replenished with fresh medium and incubated for the indicated time points, in a low calcium concentration (<0.1 mM) or a high calcium concentration (1.2 mM). The protein level was verified by Western blot. Involucrin and loricrin are the markers for keratinocyte differentiation. Sox9 bands represent the exogenously expressed GFP-Sox9 fusion protein of ∼82 kDa. (C) The effect of Sox9 on involucrin and loricrin promoter activity. Keratinocytes were transduced with involucrin-luc or loricrin-luc reporter adenovirus together with GFP-Sox9 expressing adenovirus for overnight. Next day, cells were replenished with low calcium (0.01 mM) or high calcium (1.8 mM) medium and incubated for indicated time points. Cells were lysed and assayed for luciferase activity. Data are presented as fold induction and SEM, measured from three independent experiments.

Figure 3. Effect of Sox9 on the growth of keratinocytes.

(A) Keratinocytes were transduced with 10 MOI of adenovirus expressing GFP-Sox9 for overnight, washed twice with PBS, and incubated with fresh medium containing [³H]thymidine. At the indicated time points, cells were lysed and radioactivity was measured by scintillation counter. Control is non-virus transduced group. (B) Keratinocytes were transduced with adenovirus expressing GFP-Sox9 at the indicated MOI for overnight. Cells were replenished with fresh medium and incubated for 2 d. The protein level for cell cycle modulators were verified by Western blot.

Figure 4. Ectopic expression of Sox9 in rat skin.

Female Sprague Dawley (SD) rats were intradermally injected with 50 µl of Sox9 expressing adenovirus (10⁹ particles). After 10 days, skin specimens were harvested and stained with hematoxylin and eosin. Epidermal thickness of Sox9-injected rat is greater than that of GFP-injected rat. CTL, non-injected; PBS, phosphate-buffered saline-injected. Sequentially, paraffin-embedded tissue sections were stained with anti-GFP, anti-loricrin, anti-keratin 10 (K10), and anti-PCNA antibodies. Expression of the early differentiation marker K10 and the late differentiation marker loricrin show that Sox9 can inhibit keratinocyte differentiation. While, ectopic expression of Sox9 increases the cell proliferation in terms of PCNA positivity.

Figure 5. Sox9 protects UVB- induced keratinocyte apoptosis.

(A) Keratinocytes were treated with UVB at the indicated doses. Cells were refed with fresh medium and incubated for a further 24 h. Sox9 protein level was increased in a dose-dependent manner. (B) Effect of Sox9 overexpression on UVB-induced keratinocyte apoptosis. Keratinocytes were transduced with 10 MOI of adenovirus for overnight, washed twice with PBS, and incubated with fresh medium for 2 d. Then, cells were UVB-irradiated at the dose of 20 mJ/cm², then further incubated for 24 h. Apoptosis was detected by PARP cleavage. In Sox9 overexpressed group, cleaved PARP (lower band) is significantly reduced, indicating that Sox9 protects UVB-induced keratinocyte apoptosis. In Western blot with anti-Sox9 antibody, a band represents the exogenously expressed GFP-Sox9 fusion protein (∼82 kDa). In Western blot with anti-GFP antibody, upper band (arrow) indicates the GFP-fused Sox9 (∼82 kDa) and lower band (arrow head) indicates the GFP protein (∼26 kDa). (C) Knockdown of Sox9 by microRNA (miR). Keratinocytes were transduced with 10 MOI of adenoviruses expressing miR for Sox9 for overnight. After washing, cells were incubated for a further 2 d, and expression of Sox9 was detected by Western blot. Scrambled (Scr) miR was used for negative control. (D) Keratinocytes were transduced with adenovirus, then UVB-irradiated. Cell apoptosis was analyzed by TUNEL assay. Sox9 overexpression protects UVB-induced apoptosis, while Sox9 knockdown enhances UVB-induced apoptosis.

Figure 6. Expression of Sox9 in skin diseases.

Paraffin-embedded tissue sections of several skin diseases were stained with anti-Sox9 antibody. (A) psoriasis. (B) basal cell carcinoma (BCC). (C) keratoacanthoma (KA). (D) squamous cell carcinoma (SCC).