Dynamic Wnt5a expression in murine hair follicle cycle and its inhibitory effects on follicular in vivo

De-Ren Fang, Zhong-Fa Lv, Gang Qiao

1Department of Dermatology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China

2Department of Dermatology, the Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China

3Department of Dermatology, the Third People’s Hospital of Hanghzhou, Hanghzhou, 310009 China

Dynamic Wnt5a expression in murine hair follicle cycle and its inhibitory effects on follicular in vivo

De-Ren Fang1*, Zhong-Fa Lv2, Gang Qiao3

1Department of Dermatology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China

2Department of Dermatology, the Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China

3Department of Dermatology, the Third People’s Hospital of Hanghzhou, Hanghzhou, 310009 China

Objective: To analyze the dynamic expression of Wnt family member 5A (Wingless-type MMTV integration Wnt site family, member 5a) in murine hair cycle and its inhibitory effects on follicle in vivo. Methods: Situ hybridization in full-thickness skin was used to observe the change of mouse protein expression in different growth stages, and Ad-Wnt5a was injected after defeathering to observe the hair follicle growth in vivo. Results: The Wnt5a mRNA was expressed at birth, and was firstly increased then decreased along with the progress of the hair cycle. It reached the peak in advanced stage of growth cycle (P<0.05). Rhoa and β-catenin expression levels were significantly decreased in three groups. Rac2 expression was significantly up-regulated, and the expression level of Wnt5a, Shh and Frizzled2 was increased, but less significantly than group 2. Conclusions: The expression of Wnt5a mRNA is consistent with change of murine follicle cycle, and has obvious inhibitory effects on the growth of hair follicle in vivo, indicating that it is antagonistic to Wnts pathway and interferes the growth of follicle together.

ARTICLE INFO

Article history:

Received 10 December 2013

Received in revised form 15 January 2014

Accepted 15 March 2014

Available online 20 April 2014

Wnt5a

1. Introduction

Wingless-type MMTV integration Wnt site family, member 5a is a non-classical Wnt signaling pathway which is closely related to the differentiation of skin tissue[1,2]. The theory that classical Wnt pathway can induce the morphological change of follicle and maintain the cycle has been verified universally[3-5]. However, report about the effects of non-classical Wnt pathway on follicle is lack. We analyzed the dynamic expression of Wnt 5a in murine hair follicle cycle and its inhibitory effects on folliclein vivoto explore the mechanism of Wnt signaling pathway in follicle cycle signal regulation network.

2. Materials and Methods

2.1. Experimental materials

115 C57BL/6J mice were purchased from Institute of Laboratory Animal, Chinese Academy of Medical Sciences, 15 mice were used for follicle cycle research and the rest 100 were used for the inhibitory effect analysis on folliclein vivo. Experimental reagents such as 40% paraformaldehyde solution, 0.01 M PBS solution, 0.75 mol/L sodium citrate, prehybridization solution, 2×SDS loading buffer, 30% (w/v) gel preservation liquid, Tris-HCl (1.5 mol/L and 1.0 mol/L), 11% electrophoresis separation gel, 4% electrohpresis condense gel, 5×Tris-Glycine electrophoresis buffer, 10% SDS, 10% APS, Electricity transfer buffer, 0.5M EDTA, Tris buffer, 80% glycerin, PBST, and sample loading buffer were prepared by ourselves according to related references[6-13].

2.2. Experimental methods

2.2.1. Wnt5a dynamic expression experiment

Five μL plasmid DNA was obtained for plasmidestablishment and probe preparation[14,15], 15 mice were divided into two groups according to body development: Group A (Advanced stage), Group B (early stage of growth), group C (advanced stage of growth), group D (regression stage) and group E (stationary stage) with 3 mice in each. Hybridization in situ in full-thickness skin, immunohistochemistry, Semi-quantitative RT-PCR, immunoblotting were applied on the middle of back skin according to methods in related references[16-18] to observe the difference of back follicles among different groups after treatment.

2.2.2. Inhibition of follicle in vivo

One hundred stationary stage mice were selected. Twenty were set as control, 80 were applied by mixture of rosin and beewax at back to defeather and induce the follicles to enter intermediate growth stage and establish hair follicle cycle synchronization model. The above 100 mice were divided into three groups, the control group as group 1 with 20 mice, group 2 and group 3 with 40 in either. Group 1 received no special treatment; group 2 had only defeathering treatment; group 3 had Ad-Wnt5a subcutaneous injection after defeathering. Skin phenotypic changes, HE staining, gene microarrays and immunohistochemistry were compared 7 days after treatment. The testing methods were referred to related references[19-21].

2.3. Statistical analysis

All date in our study were analyzed by SPSS13.0. Enumeration data was analyzed byChi-square test and measurement data was analyzed byttest. The test level was set as α=0.05. The difference was considered as statistically significant when P＜0.05.

3. Results

3.1. Result of dynamic expression in different follicle cycle

3.1.1. Wnt5a mRNA expression in different cycles

Bluish violet color represents positive reaction. As shown in Figure 1, Wnt5a mRNA had high expression in group A follicle, especially condensed at part Mx (Figure 1A’). The part which migrates to corium layer; had significantly increased expression at part Mx in group B and decreased at part IRS of follicle (Figure 1B’). Expression was highest in group C. The positive signal trended to decrease in group D. There was only a little Wnt5a mRNA expression in group E.

3.1.2. Semi-quantitative RT-PCR results

The Wnt5a mRNA was expressed at birth. It was firstly increased then decreased along with the progress of the hair follicle cycle. It reached the peak at advanced stage of growth cycle (P<0.05) (Table 1 and Figure 2).

Figure 1. Wnt5a mRNA expression in different cycles.

Table 1 Comparison of Semi-quantitative RT-PCR results among different cycles

Table 1 Comparison of Semi-quantitative RT-PCR results among different cycles

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Figure 2. Semi-quantitative RT-PCR results in different cycles.

3.1.3. Immunoblotting results

Immunoblotting results in different groups were in accordance with Semi-quantitative RT-PCR results, as shown in Figure 3.

Figure 3. Immunoblotting results in different cycles.

3.2. Follicle growth in vivo

3.2.1. Model establishment.

Back skin of three groups showed pink color after defeathering, and turned to black after 7 days to effectively synchronize follicle cycles, as shown in Figure 4.

Figure 4. Model establishment in group 1.A. Right after defeathering; B.7 days after defeathering.

3.2.2. Phenotype change

Mice in group 1 and group 2 entered growth stage 14 days after defeathering, however, Ad-Wnt5a injection region in group 3 still stayed pink, as shown in Figure 5.

Figure 5. Phenotype change of nice in different groups 14 days after defeathering.

3.2.3. Differential gene analysis

Rhoa and β-catenin expression levels were significantly decreased in group 3, and Rac2 expression was significantly up-regulated. Wnt5a, Shh and Frizzled2 expression levels were increased, but less obvious than group 2, as shown in Table 2.

Table 2 Comparison of differential gene expression analysis among different groups after defeathering

Table 2 Comparison of differential gene expression analysis among different groups after defeathering

Note: group 1 was control group, its gene expression was set as standard.

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4. Discussion

Skin of mammal is the primary protective barrier of body, and follicle is the main accessory of skin. The growth of follicle includes growth, regression and stationary stages to ensure the recycling growth and regression process under the regulation of complicated signaling network[22,23]. Research has proved that classical Wnts pathway promotes the follicle growth cycle, however, there has been less research about non-classical Wnts pathway on follicle cycle. Veraitchet al[24,25] found that Wnt5a showed high expression in psoriasis patients accomplished with the cornification differentiation disorder. Thus, research on mechanism of Wnt5s may provide explanation for variety of skin diseases and guide clinical treatment.

We observed the dynamic change of Wnt5a in mouse follicle cycle and found that Wnt5a mRNA expression was closely related to the murine follicle cycle. Wnt5a mRNA showed obvious high expression in cell matrix at follicle growing stage. Wnt5a mRNA first increased and then decreased along with the hair follicle cycle, then follicle epithelial cells stopped proliferation and started to apoptosis, which was consistent with the trend of Wnt5a mRNA. This indicated that Wnt5a had dynamic expression model in follicle cycle and may have critical effects in the process of follicle growth. In follicle inhibition experimentin vivowe found that overexpression of Wnt5a could inhibit the normal growth of follicle, and significant decrease of β -catenin in group 3 implied that follicle growth ability in mice which accepted Ad-Wnt5a greatly decreased, this was also verified in phenotype experiment. As the representive of non-classical pathways, the effects of Wnt5a is exactly opposite to Wnts pathway, indicating that the two pathways keep follicle growth balancein vivoantagonisticly[26-28]. For example, Wnt5a can activate JNK and inhibit Wnts pathway, but Wnts can inhibit Camk-Ⅱ and promote the β-catenin formation to promote follicle growth cycle[29]. Besides, Mardaryevet al[30] found that Wnt5a could active PCP pathway signaling chain with Rac2 involved to some extent. The mechanism may be related to PCP signaling, this also explained the reason of Rac2 upregulation and Rhoa downregulation.

In conclusion, Wnt5a mRNA expression is in accordance with murine hair follicle cycle, and can inhibit growth of folliclein vivosignificantly, indicating that it is antagonistic to Wnts pathway and affect the growth of follicle together.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

The research was funded b by Science Research Foundation of Department of Education of Zhejiang Province, China(NO20061246 ) .

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ment heading

10.1016/S1995-7645(14)60039-0

*Corresponding author: De-Ren Fang, Department of Dermatology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.

Tel: 86-571-87236385

E-mail: fangdr@gmail.com

Foundation project: It is supported by Zhejiang University Medical Key Funds (No 201028382).

Hair follicle cycle

Iinhibition

Proliferation