BALB/C mice were purchased from Orient Bio (Seongnam, Korea) and maintained in the barrier system with regulated light (700 to 1,900 h), temperature (23±1℃), humidity (50%±5%), and ventilation (10 to 12 times/h). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea and were carried out in accordance with the Guidelines for Animal Experimentation.

Mouse dorsal skins of BALB/C mice at postnatal days 10 (P10), P14, P17, P21 and P28 were gathered as previously described21. All skin samples were harvested from the same region (2×2 cm) of the upper back skin. Six-micrometer-thick paraffin sections were prepared. Hematoxylin and eosin (H&E) staining was carried out using the standard method, and the stained sections were observed with an optical microscope (Olympus, Tokyo, Japan).

Total RNA was extracted from the dorsal skins of BALB/C mice at P10, P12, P14, P17, P21, P28, and P35 using TRIZOL following the manufacturer's instructions (Invitrogen, Camarillo, CA, USA). Two micrograms of RNA were utilized to synthesize single stranded cDNAs using the PrimeScript 1st strand cDNA Synthesis kit following the manufacturer's instructions (TaKaRa Bio Inc., Shiga, Japan).

Polymerase chain reaction (PCR) was performed using Thermal Cycler-100 (MJ Research Inc., Waltham, MA, USA). PCR conditions were as follows: initial denaturation for 2 minutes at 95℃ followed by 28~30 cycles of 15 seconds at 94℃, 15 seconds at 62℃, and 15 seconds at 72℃. The final extension was performed for 10 minutes at 72℃. Real-time PCR was performed with the cDNAs prepared from the total RNAs of the skin at various time points (See the above section) using SYBR Premix Ex Taq (TaKaRa Bio Inc.) in an Mx3000P (Stratagene, La Jolla, CA, USA). Specific primer sequences for each gene are listed in Table 1. The cycling conditions were as follows; initial heating for 2 minutes at 95℃ followed by 45 cycles of 15 seconds at 94℃, 15 seconds at 62℃, and 15 seconds at 72℃, and the final extension was performed for 10 minutes at 72℃. Gene expression levels were determined by the comparative ΔΔCt method using the expression level of Gapdh as control22. Relative mRNA expression levels were determined based on Real-time PCR performed in duplicate using various numbers of independent samples for each point (number of sample for each point; P10=3, P12=4, P14=7, P17=4, P21=5, P28=5, P35=4).

The 888 bp of Sfrp2 coding sequence (CDS) were amplified by PCR using Expand High Fidelity enzyme (Roche Diagnostics, Basel, Switzerland) from skin cDNAs of BALB/C mice. Forward and reverse primer sequences are listed in Table 1. PCR products were subcloned into pcDNA 3.1 using EcoRI cloning sites (Invitrogen).

Mouse keratinocyte cells (PAM212 cell line) were cultured in DMEM (Invirogen) containing 10% FBS with 5% CO₂ in a 37℃ incubator. Transfection experiments were performed using polyethyleneimine (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. A total of 8×10⁵ cells were plated in 60 mm dishes in triplicate. Following this, either 500 ng of pcDNA 3.1/ Sfrp2 CDS or only pcDNA 3.1 plasmid was transfected into the cells with 0.4 µg of pCMV3.1/β-gal. Transfection experiments were normalized against transfection efficiency determined by β-galactosidase activity. To ensure that Sfrp2 overexpression was well induced, we performed Real-time PCR and found a 27118-fold increase in the Sfrp2 mRNA level compared with that of pcDNA 3.1 transfection. After incubation for 48 hours at 37℃, the transfected cells were observed using a microscope (Olympus) or harvested for extraction of total RNAs.

SFRP2 protein was purchased from R&D system and reconstituted in sterilized phosphate buffered saline (PBS). A total of 5×10⁵ PAM212 cells were plated on a 100 mm culture dish in DMEM media and were treated with SFRP2 the following day by replacing media with the media containing SFRP2 (50 ng/ml or 100 ng/ml). The treatment was continued for 3 days by changing the media every day.

For immunocytochemistry, PAM212 cells either treated with SFRP2 or transfected with pcDNA 3.1/Sfrp2 were washed three times with cold PBS and fixed using 4% paraformaldehyde for 10 minutes at room temperature. The cells were then treated with 0.5% Triton X-100 for 10 minutes and washed three times with PBS. After blocking with 3% bovine serum albumin for 1 hour, the cells were incubated with antibodies against Ki67 (Thermo Scientific, Hudson, NH, USA; 1:200) or Involucrin (Covance, Berkeley, CA, USA; 1:200) overnight. After washing, Alexa Fluor 488 goat anti-rabbit secondary antibody was applied for 3hr (Invitrogen, 1:500), and 4 g/ml of Hoechst 33342 (Sigma-Aldrich) was also applied for 1 minute to stain the nuclei. Fluorescence signal was observed with a Fluorescent microscope (Olympus).

p-values were calculated using Student's t-test. p<0.05 was regarded as statistically significant. Analyses were performed using SPSS for Windows version 12.00 (SPSS Inc., Chicago, IL, USA).

To determine the role of Sfrp2 in HFs, first we investigated the relative expression level of Sfrp2 compared to those of other Wnt inhibitors using reverse transcription-polymerase chain reaction (RT-PCR) and Real-time PCR. Among the Wnt inhibitors with relatively high expression at P10, Sfrp2 was the only one whose expression was dramatically increased at P17. Furthermore, the expression level of Sfrp2 was 5.9-fold higher than that of the second most expressed Wnt inhibitor, wif1 (Fig. 1). These results suggest that Sfrp2 is the most abundantly expressed Wnt inhibitor in HFs at the catagen phase.

Relative expression level of Sfrp2 mRNA was determined at different hair cycle stages by RT-PCR as well as Real-time PCR analyses. We found that Sfrp2 was highly expressed during catagen, which was maintained until the early telogen phase (Fig. 2A). During anagen, Sfrp2 expression was weaker than those of other stages. After this, the expression level increased significantly by 2.4~3.6 fold as the stages progressed into the catagen and early telogen phases. As the next anagen phase began, Sfrp2 expression started to decline and then decreased again to a low level that is similar to that of the first anagen phase by P35 (Fig. 2B). These Sfrp2 mRNA expression patterns suggest that Sfrp2 may function during the catagen phase.

During the catagen phase, HFs undergo degeneration through cessation of proliferation and subsequent differentiation of keratinocytes. Because Sfrp2 expression increased at the catagen phase as shown in Fig. 2, we investigated whether Sfrp2 is involved in the regulation of proliferation and/or differentiation using the PAM212 mouse keratinocyte cell line. Cells were treated with SFRP2 at 50 and 100 ng/ml for 48 hours, and the number of live cells was counted using the trypan blue exclusion method. We found that SFRP2 significantly reduced the number of cells by 20% compared to the mock-treated control at both 50 and 100 ng/ml (Fig. 3A, B).

To investigate whether this decrease was caused by inhibition of cell proliferation, we used immunofluorescent staining for the Ki67 proliferation marker23. Immunocytochemistry showed decreased Ki67 expression in cells treated with SFRP2 and in cells overexpressing SFRP2 (Fig. 3C), indicating that SFRP2 inhibits keratinocyte proliferation.

To investigate whether SFRP2 treatment would affect the differentiation status of keratinocytes, we performed immunocytochemistry for Involucrin expression, a keratinocyte differentiation marker24, in SFRP2 treated or Sfrp2-overexpressed PAM212 cells. Unlike Ki67, we found no difference in Involucrin expression between control and SFRP2-treated cells (Fig. 4A).

RT-PCR and Real-time PCR analyses also revealed no significant difference in the Involucrin mRNA level between control and SFRP2-overexpressed cells (Fig. 4B, C). These results indicate that Sfrp2 does not affect Involucrin expression and suggest that Sfrp2 is not involved in keratinocyte differentiation.

SFRP2 function in the Wnt pathway is controversial. SFRP2 was reported to be inhibitory in the Wnt pathway12, whereas it was also shown to activate Wnt signaling through the canonical activation of β-catenin in intestinal epithelial cells and canine mammary glands25-28. To determine SFRP2 function in the Wnt pathway of HFs, we investigated the expression of the wnt target genes Ccnd1 and C-myc on the back skin of mice. Real-time PCR analysis revealed an inverse relationship between the Ccnd1 and C-myc expression pattern and that of Sfrp2 throughout the HF cycle (Fig. 5), suggesting that Sfrp2 may function as a Wnt inhibitor in HFs.