Secreted Frizzled-related protein 4 inhibits the regeneration of hair follicles

Animals and skin samples

C57 BL/6 mice were obtained from and housed in the laboratory animal center of the Army Medical University. All the animal-related procedures were in strict accordance with the approved institutional animal care and maintenance protocols. All experimental protocols were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University. Permission number for producing animals: SCXK (YU)-20170002. Permission number for using animals: SYXK (YU)-20170002.

Human skin samples were obtained from 30 to 40 years old donors in the second affiliated Hospital of the Army Medical University. The use of the samples was consented verbally by the donors. The research was approved by the ethics committee of the Third Military Medical University.

Immunofluorescence

Immunostaining was performed on five μm sections from tissue samples. Sections were dewaxed, rehydrated, and boiled in citrate buffer solution. Then the sections were blocked with 10% goat serum, incubated with the following primary antibodies: goat anti-sFRP4 (1:100; Abcam, Shanghai, China), rabbit anti-K6 (1:50; Sangon Biotech, Songjiang, China), rabbit anti-K10 (1:50; Sangon Biotech, Songjiang, China), rabbit anti-K14 (1:50, Sangon Biotech, Songjiang, China), and rabbit anti-K19 (1:50, Sangon Biotech, Songjiang, China), and rabbit anti-β-catenin (1:200; Abcam, Cambridge, MA, USA). After washing, the sections were incubated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) or Cy3 (Beyotime, Haimen, China) labeled secondary antibodies. After that, sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclei visualization. At last, the cover slides were moved to microscope slides, mounted with antifade mounting medium (Beyotime, Haimen, China), and observed under fluorescent microscope.

Intradermal administration

To induce synchronized hair cycle, the hairs on the dorsal skin of 7-week-old C57 mice were depilated as described previously (Muller-Rover et al., 2001). A total of 25 μL recombinant sFRP4 (50 μg/mL, R&D Systems, Minneapolis, MN, USA) or phosphate buffer saline (PBS) (control) was administered intradermally into the dorsal skin after depilation. At 2, 3, or 4 days after the administration of sFRP4, the dorsal skin samples were harvested and prepared for analysis of histology or immunostaining.

Hematoxylin and eosin staining

The skin collected from sFRP4 or PBS administrated region were fixed with 4% paraformaldehyde overnight, gradually dehydrated in graded alcohol solutions, cleared in xylene, and subsequently embedded in paraffin. Sectioned samples were dewaxed, rehydrated, stained with hematoxylin (ZSGB-bio, Beijing, China) for 2 min and subsequently rinsed with water. The sections were later stained with eosin (ZSGB-bio, Beijing, China) for 2 min and rinsed with water thereafter. At last, the sections were dehydrated gradually, mounted with neutral gum (ZSGB-bio, Beijing, China) and observed under a microscope.

Statistical analysis

Data were presented as a mean ± SD of three independent experiments. Statistical analyses were evaluated using t-test and P < 0.05 was considered statistically significant.

sFRP4 is expressed in human and mouse hair follicles

At first, we detected the endogenous expression of sFRP4 in human scalp skin by immunofluorescence. In epidermis, sFRP4 was expressed in all the layers (Figs. 1A–1C). In the human hair follicle, sFRP4 was strongly expressed in the inner root sheath (IRS) and the outer root sheath (ORS), weaker in the hair shaft (HS) and pre-cortex (Figs. 1D–1I).

Then we detected the expression pattern of sFRP4 in mouse dorsal skin. Among all the hair cycle stages, sFRP4 was widely expressed in the hair follicle (Figs. 2A–2I). In anagen, nearly all the hair follicle structures can be observed. Double-labeling immunostaining further confirmed that sFRP4 was expressed at the ORS (K14 positive) and IRS (K10 positive) region in anagen hair follicles. The expression of sFRP4 was also detected in the IRS precursors and hair matrix region of mouse hair follicles (Figs. 2J–2Q).

sFRP4 inhibits the growth of hair follicle in vivo

The expression pattern of sFRP4 in hair follicle suggests that sFRP4 may play some roles in hair follicle cycle. To find out the roles of sFRP4 in hair follicle cycle and hair follicle regeneration, we induced synchronized hair growth by depilation and injected sFRP4 protein intradermally into the mouse dorsal skin (Fig. 3A). At 2 days after injection, the regeneration of the hair follicle was inhibited. However, the regeneration of the hair follicle was not blocked, it went on growing after the injection of sFRP4 stopped (Figs. 3C–3J). We also measured the length of hair follicles in the pictures of hematoxylin and eosin (H&E) staining with Image Pro Plus 6.0 software. Statistical analysis revealed that hair follicles in the sFRP4-injected group showed decreased hair length at 4 days after depilation (Fig. 3B).

sFRP4 does not affect the expression pattern of structure markers in hair follicles

K6, K10, K14, and K19 were expressed in the regenerated hair follicles, both in the PBS-treated group and sFRP4-treated group. K6 and K14 were expressed in both IRS and ORS. K10 was expressed in the IRS. K19 was expressed in the bulge region (Fig. 4). The expression patterns were similar between the two groups, but the regeneration of hair follicles in sFRP4-treated group was delayed.

sFRP4 inhibits the nuclear translocation of β-catenin in hair follicles

Beta-catenin expressed in the regenerated hair follicles, both in the PBS-treated group and sFRP4-treated group. In PBS-treated group, β-catenin was expressed in both nucleus and cell plasma (Figs. 5A and 5C). At 2 days after treatment, only few nuclear translocation of β-catenin was observed (Fig. 5A). At 4 days after treatment, more nuclear translocation of β-catenin was observed (Fig. 5C). In sFRP4-treated group, β-catenin was expressed in cell plasma (Figs. 5B and 5D).