Ann Dermatol.  2014 Feb;26(1):79-87. 10.5021/ad.2014.26.1.79.

Expression of Sfrp2 Is Increased in Catagen of Hair Follicles and Inhibits Keratinocyte Proliferation

Affiliations
  • 1Department of Medical Lifesciences, The Catholic University of Korea, School of Medicine, Seoul, Korea. sjkyoon@catholic.ac.kr

Abstract

BACKGROUND
Hair follicles undergo cycles of repeated growth and regression. The Wnt pathway plays an important role in the regeneration and differentiation of hair follicles. Sfrp2, a Wnt inhibitor, is involved in the developmental and disease processes of various cells and tissues by modulating the Wnt pathway.
OBJECTIVE
The aim of this study was to understand the role of Sfrp2 in hair follicles through investigation of the Sfrp2 expression pattern in the skin and its effect on keratinocytes.
METHODS
We investigated Sfrp2 mRNA expression and the expression of the wnt target genes, Ccnd1 and C-myc, at various mouse hair follicle developmental stages using Real-time polymerase chain reaction. We also investigated the effect of SFRP2 on the proliferation and differentiation of mouse keratinocyte cells by adding SFRP2 protein or overexpressing Sfrp2 using an in vitro culture system.
RESULTS
Sfrp2 expression peaked in the catagen phase and remained high until telogen, and then declined at the beginning of the next anagen. An inverse relationship to Sfrp2 expression was found for the expression of the Wnt target genes, C-myc and Ccnd1. In addition, we also observed inhibited proliferation of mouse keratinocytes in the presence of SFRP2.
CONCLUSION
These results suggest that Sfrp2 may play a role in the catagen phase by inhibiting the proliferation of keratinocyte and functioning as a Wnt inhibitor in keratinocytes.

Keyword

Catagen; Keratinocyte; Proliferation; SFRP2; Wnt signaling pathway

MeSH Terms

Animals
Genes, myc
Hair Follicle*
Hair*
Keratinocytes*
Mice
Real-Time Polymerase Chain Reaction
Regeneration
RNA, Messenger
Skin
Wnt Signaling Pathway
RNA, Messenger

Figure

  • Fig. 1 Expression levels of Sfrp2 compared with those of other Wnt inhibitors including Dkk1, Dkk2, Dkk4, Sfrp1 and Wif1. Reverse transcription-polymerase chain reaction (A) and Real-time polymerase chain reaction (B) revealed that Sfrp2 is the most abundantly expressed Wnt inhibitor in hair follicles, especially at the phase of catagen. P: postnatal day.

  • Fig. 2 Sfrp2 is mainly expressed during the catagen phase in the hair cycle. To investigate Sfrp2 expression, total RNA of BABL/C mice was prepared from the dorsal skin at each time point. Reverse transcription-polymerase chain reaction (A) and Real-time polymerase chain reaction (B) analyses showed expression patterns of Sfrp2 mRNA from P10 until P35 after birth. The data are normalized against Gapdh mRNA expression levels. The value is the average of the relative expression levels of three independent mice cDNAs measured in duplicate polymerase chain reaction reactions. *p<0.05. P: postnatal day.

  • Fig. 3 Effect of SFRP2 on keratinocyte proliferation. SFRP2 treatment on PAM212 cells decreased the number of live cells compared to the mock-treatment. After 48 hour treatment with 50 ng/ml of SFRP2, the cells were observed using a microscope (A) and counted using the trypan blue exclusion method (B). Three independent experiments were carried out in duplicate for cell count. The values are mean±standard deviation. *p<0.05. (C) SFRP2 inhibits keratinocyte proliferation. PAM212 cells were treated with SFRP2 or transfected with 500 ng of pCDNA3.1/ Sfrp2 CDS vector. The cells were then used for immnucytochemistry for Ki67. 4',6-diamidino-2-phenylindole (DAPI) staining (blue) indicates nuclei. The numbers under the figures represent relative intensity of florescence (A: ×10; C: ×100).

  • Fig. 4 Effect of SFRP2 on the expression of keratinocyte differentiation markers. (A) PAM212 cells were treated with 50 ng/ml of SFRP2 or transfected with 500 ng of pCDNA3.1/ Sfrp2 CDS vector. The cells were then used for immnucytochemistry for involucrin. 4',6-diamidino-2-phenylindole (DAPI) staining (blue) indicates nuclei. The numbers under figures represent the relative intensity of florescence (immunohistochemistry, ×100). (B, C) Expression of Involucrin mRNA detected by reverse transcription-polymerase chain reaction (B) and Real-time polymerase chain reaction (C). The data are normalized against Gapdh mRNA expression.

  • Fig. 5 Expression of Wnt target genes, Ccnd1 (A) and C-myc (B) in the mouse skin. Relative expression levels of Ccnd1 and C-myc were determined using Real-time polymerase chain reaction with total RNAs extracted from the dorsal skins of BALB/C at P10~P28. The expression of these genes was inversely proportional to that of Sfrp2. The data are normalized against Gapdh mRNA expression. The values are the average of the relative expression levels determined in three mice, each measured in duplicate. *p<0.05. P: postnatal day.


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