Received Date : 27-Oct-2013 Accepted Date : 08-May-2014 Article type

: Regular Article

Title Page: Expression of Decorin throughout the Murine Hair Follicle Cycle: Hair Cycle-Dependence and Anagen Phase Prolongation

Authors: Jing Jing1, Ph.D., M.D, Xian-jie Wu1, Ph.D., M.D., Yun-ling Li2, Ph.D., M.D., Sui-Qing Cai1, Ph.D., M.D., Min Zheng1, Ph.D., M.D.*, Zhong-Fa Lu, Ph.D., M.D.*,1 1

Zhejiang University School of Medicine, Dermatology, Second Affiliated Hospital,

Hangzhou, China


Department of Dermatology, Children’s Hospital, School of Medicine, Zhejiang

University, Hangzhou, China

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/exd.12441 This article is protected by copyright. All rights reserved.

Author’s email: Jing Jing ([email protected]); Xian-jie Wu ([email protected]); Yun-ling Li ([email protected]); Sui-Qing Cai ([email protected]); Min Zheng ([email protected]); Zhong-Fa Lu ([email protected]).

* corresponding authors. Address correspondence to Zhongfa Lu, [email protected] (corresponding author) and Min Zheng, [email protected] (co-corresponding author). Corresponding author’s address: No. 88, Jiefang Road, Hangzhou, 310009, China. Tel: +86 571 87783520, Fax: +86 571 87022776.

This work was supported by grant 2009R10045 from Qianjiang talent plan of Zhejiang Province of China and grant 81171521 from the National Natural Science Foundation of China (NSFC).

Abstract Decorin is a prototypical member of the small leucine-rich proteoglycan (SLRP) family, which is involved in numerous biological processes. The role of decorin, as a representative SLRP, in hair follicle morphogenesis has not been elucidated. We

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present our initial findings on decorin expression patterns during induced murine HF cycles. It was found that: decorin expression is exclusively restricted to the epidermis, outer root sheath, and sebaceous glands during the anagen phase, which correlates with the upregulation of decorin mRNA and protein expression in depilated murine dorsal skin. Furthermore, we used a functional approach to investigate the effects of recombinant human decorin (rhDecorin) via cutaneous injection into HFs at various murine hair cycle stages. The local injection of rhDecorin (100 μg/ml) into the hypodermis of depilated C57BL/6 mice at anagen delayed catagen progression. In contrast, rhDecorin injection during the telogen phase caused the premature onset of anagen, as demonstrated by assessment of the following parameters: (1) hair shaft length, (2) follicular bulbar diameter, (3) hair follicle cycling score, and (4) follicular phase percentage. Taken together, our results suggest that decorin may modulate follicular cycling and morphogenesis. In addition, this study also provides insight into the molecular control mechanisms governing hair follicular epithelial-mesenchymal interactions.

Keywords Decorin, hair follicle cycle, outer root sheath, dermal papilla.

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Introduction The human hair follicle (HF) is a unique miniorgan that is capable of recycling from the resting phase (telogen) to the growth phase (anagen) with rapid follicular keratinocyte proliferation as well as hair shaft elongation and thickening, followed by a regression phase (catagen) during life [1]. Epithelial-mesenchymal signaling plays a pivotal role in hair follicle cycling, leading to the identification of several important biomolecules that control epithelial morphogenesis and growth. Interestingly, a set of extracellular matrix proteoglycans (PGs), such as versican and syndecan, regulates epithelial-mesenchymal interactions during HF cycling [2, 3]. We focused on the role of decorin, a ubiquitous SLRP family member involved in the hair cycle. Decorin consists of a core protein and a covalently linked glycosaminoglycan chain, which can be either chondroitin sulfate or dermatan sulfate [4]. In addition to participating in matrix assembly and angiogenesis, decorin regulates essential signaling cascades by interacting with a range of cell surface receptors, cytokines, and growth factors (GFs) that also affect the HF cycle, such as transforming growth factor-β (TGF-β), insulin-like growth factor (IGF), and epidermal growth factor (EGF). Considerable attention has been given to the interaction between the decorin core protein and TGF-β [5, 6], a multifunctional bioregulator with the capacity to induce potent catagen development in murine and human anagen HF phases via proliferation inhibition and apoptotic induction in hair matrix keratinocytes [7, 8]. As a natural inhibitor of TGF-β1, decorin prevents TGF-β from

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binding to its receptors, thereby interfering with the Smad and non-Smad pathways downstream of the TGF-β receptors in keratinocytes, fibroblasts, and other cell types [9, 10]. In addition, decorin’s analog biglycan regulates the Wnt/β-catenin pathway, which is vital for HF induction [11]. Accumulating evidence suggests a role for decorin in several cellular processes, including proliferation, differentiation, and apoptosis during rapid growth, as observed in regenerating tissues and tumors [12-14]. However, the functional role of decorin in HF physiology remains poorly understood. Two decades ago, Couchman observed low expression of decorin in dermal papilla extracellular matrix (ECM) [15]. Decorin was recently implicated in the regulation of the human anagen HF bulge and follicular organogenesis [16, 17]. In addition, data suggest that decorin skin knockouts display reduced skin thickness and shortened hair follicles, while decorin-deficient matrix affects keratinocyte function [18, 19]. Furthermore, decorin has been identified in cytoplasm of differentiating keratinocytes located in supra-basal layers of human epidermis, thus indicating a role in keratinocyte terminal differentiation during follicular morphogenesis [20]. However, the exact localization and role of decorin in HFs remain poorly understood. These findings prompted us to investigate whether decorin is involved in hair cycling transitions. First, we investigated decorin expression and localization in the depilation-induced synchronized hair cycle of female C57BL/6 mice by immunochemistry, in situ hybridization (ISH), Western blot, and reverse transcription

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polymerase chain reaction (RT-PCR) analyses. Second, we tested the effect of rhDecorin on murine HF cyclic transitions.

Methods Experimental animals and skin collection Syngeneic, 6-8 week old female C57BL/6 mice (Animal Center, Chinese Academy of Sciences, Hangzhou, China) in the telogen phase of the hair growth cycle were selected. All methods were approved by the ethical committee of the Second Affiliated Hospital, School of Medicine, Zhejiang University. We ensure that this study met internationally accepted ethical standards, the guidelines published by Blackwell Publishing Ltd., and the guidelines adopted by the British Medical Association. Anagen was induced in the dorsal skin of telogen mice by applying liquid rosin under anesthesia according to previously described methods [21]. Skin specimens were obtained at days 0, 3, 5, 12, and 18 post-depilation (p.d.), representing the telogen, anagen II, anagen IV, anagen VI, and catagen phases, respectively [22-24]. Three mice per time point were sacrificed, and dorsal skin samples (0.3 cm × 0.5 cm) harvested from the depilated areas were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) or immediately frozen in liquid nitrogen at −80°C.

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Histology and immunohistochemistry The dorsal skin in 4% paraformaldehyde was paraffin-embedded and cut into 5-μm-thick sections for hematoxylin and eosin (H&E) staining or immunohistochemistry. For the immunohistochemical analysis, skin sections were dewaxed, microwaved in citrate-buffered saline, and incubated with rabbit monoclonal anti-mouse decorin antibody (Abcam, MA, USA) at 4°C overnight as previously described [25, 26]. After washing, sections were labeled with horseradish peroxidase (HRP) -conjugated secondary antibodies (Zhongshan Goldbridge Biotechnology, Beijing, China) for 1-2 h at room temperature and visualized with DAB peroxidase substrate (Zhongshan Goldbridge Biotechnology). Finally, sections were counterstained with hematoxylin (Zhongshan Goldbridge Biotechnology). The antibody specificity was demonstrated using PBS as a negative control.

Western blot analysis Western blot analysis was performed as previously described [27, 28]. Total protein extracted from each tissue sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were blocked with milk containing 1% Tween-20, washed, and cut into strips. Strips were probed with an anti-decorin antibody or an internal control anti-β-actin antibody (1:2000, Santa Cruz, USA) overnight at 4°C in milk containing 1% Tween-20. Then, the This article is protected by copyright. All rights reserved.

blots were incubated with a HRP-conjugated goat polyclonal anti-rabbit IgG antibody (1:5000, Jackson ImmunoResearch, West Grove, PA, USA). Immunoreactivity was detected using an enhanced chemiluminescent (ECL) plus reagent kit (Millipore) before exposure for at least 3 minutes using the chemiluminescence imaging system (BIO-RAD Laboratories, Richmond, CA, USA). Molecular weights were estimated using prestained SDS-PAGE standards (BIO-RAD Laboratories).

RT-PCR Total RNA was extracted from depilated mice dorsal skin samples using Trizol, and reverse-transcribed into first-strand cDNA using a high-capacity RNA to cDNA kit (Takara, Kyoto, Japan), according to the manufacturer's protocol. SYBR Green assay reagents (Takara) were used to analyze the expression of murine decorin (NM_001190451.1) according to the manufacturer's instructions. The primers for decorin were 5′-TCTTGGGCTGGACCATTTGAA -3′ (forward) and 5′-CATCGGTAGGGGCACATAGA-3′ (reverse). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize mRNA levels using the following primers: 5′-TGACCACAGTCCATGCCATC-3′ and 5′-GACGGACACATTGGGGGTAG-3′. The relative expression levels were determined using the ΔΔCt method to compare the mRNA expression levels of target and housekeeping genes [29].

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Local injection of decorin Recombinant human decorin (R&D Systems Inc., Minneapolis, MN, USA) was dissolved in 0.1% sterile and toxin-free bovine serum albumin (BSA) in PBS and injected into the dorsal skin of C57BL/6 telogen mice (0 days after depilation) and C57BL/6 anagen VI mice (12 days after depilation) as previously reported [23, 30, 31]. Each mouse received only one treatment, and each group consisted of three mice from different litters. After injection, dorsal skin samples at the injection sites were collected shortly before entry into the next HF phase. Telogen HFs grew spontaneously and entered anagen between p.d. days 2 and 4, while anagen HFs regressed and entered catagen between p.d. days 17 and 19 [23, 32]. Control mice of the same age were injected with 100 μl of 0.1% BSA-PBS once daily for consecutive days following the same timing as that in the decorin-injected group. For the telogen-anagen transition study, three mice were administered a dorsal intradermal injection of 2 μg decorin dissolved in 100 μl of 0.1% BSA-PBS once daily on days 0, 1, and 2 after depilation (total 6 μg). All mice were sacrificed on day 3, and one skin sample per mouse was prepared. For the anagen-catagen transition study, three mice were administered a dorsal intradermal injection of 2 μg decorin dissolved in 100 μl of 0.1% BSA-PBS once daily beginning at p.d. day 12 for 6 consecutive days (total 12 μg). The mice were then sacrificed to evaluate HF growth on p.d. day 18. The skin samples resected from the injection site were fixed in 4%

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paraformaldehyde, paraffin-embedded, and H&E-stained for quantitative histomorphometry.

Quantitative histomorphometry and statistical analysis H&E-stained paraffin sections were screened for longitudinal HFs using a Nikon E-600 microscope (Nikon, Inc., Japan). HF growth was evaluated photometrically and histologically using the following four parameters: skin thickness corresponding to HF length, HF tissue size corresponding to the largest diameter of hair bulbs with clearly visible dermal papilla, HF percentage, and HF cycling score (HCS). These parameters allowed for assessment and comparison of the full range of anagen/catagen stages between the experimental groups. Skin thickness was defined as the distance from the epidermal granular layer to the top edge of the panniculus carnosus in all longitudinally cut H&E-stained sections [30]. To assess HF size, non-curved hairs with visible papillae were evaluated, and the largest follicle diameter was measured in digital images representing each stained section [33]. Skin thickness and size were evaluated by digital image analysis using Photoshop® software, and at least 50 longitudinally cut follicles per sample were counted. Then, the hair stage percentage and the HCS were classified and assessed by histomorphometry according to previously published hair cycle staging guidelines and morphological characteristics of the murine hair cycle [23, 34, 35]. For the anagen-catagen transition study, anagen VI HFs were assigned a score of 100, HFs in This article is protected by copyright. All rights reserved.

early catagen (catagen II-III) received a score of 200, while HFs in mid- and late catagen (catagen IV-V) were assigned a score of 300. For the telogen-anagen transition study, anagen I-IIIa HFs were assigned a score of 100, anagen IIIb-IIIc HFs were given a score of 200, and anagen IV-VI HFs were assigned a score of 300. HCS values were added within a group and divided by the number of staged follicles. Each HCS indicates the mean HF stage per group. Statistical analyses were performed using the independent sample t-test (SPSS version 13.0, SPSS Inc., IBM, Chicago, IL, USA). The results are expressed as the means

SD, and a p-value of less than 0.05

was considered statistically significant.

Results Enhanced decorin expression in the HF epithelium and dermal papilla throughout the anagen phase During the early anagen phase (3 days post-depilation) (Fig. 1a, 1b, S1b-c), decorin was intensely expressed throughout the interfollicular epidermis and displayed strong cytoplasmic immunolabeling in the proliferating strand of keratinocytes in a cytoplamic pattern (Fig. 1a, 1b). Moderate expression was also noted in the dermal papilla (DP) and sebaceous glands (SGs) cells. During the anagen IV phase (5 days post-depilation) (Fig. 1c, 1d, S1d), moderate decorin immunoreactivity was widely detected in the epidermis and HF, which included the outer root sheath (ORS), inner root sheath (IRS), and cortex. In particular, vertically sectioned anagen IV HFs This article is protected by copyright. All rights reserved.

displayed significant decorin immunostaining in the isthmus proximal to the bulge (Fig. 1c, 1d). The positive-staining ORS also displayed a primarily cytoplasmic pattern during this stage. As the follicles approached the late stages of the anagen phase (12 days post-depilation) (Fig. 1e-g, S1e-h) and the catagen phase (18 days post-depilation) (Fig. 2a-d, S2a-d), the staining gradually retreated from the lower ORS, IRS, and hair bulb until only weak staining was observed in these regions, as well as in the bulbar matrix adjacent to the DP. A ubiquitous distribution was primarily observed in the middle and upper ORS, whereas mild staining was evident in the epidermis. During the telogen phase (0 days after depilation), when the HF regressed to a small dormant structure that appeared as a club hair, decorin was weakly localized in the epidermis, germ cells, and cap, whereas the bulge region adjacent to the SG showed relatively strong staining (Fig. 2e, S1a). The compacted DP displayed positive staining (Fig. 2f).

In general, decorin immunostaining was enhanced during the anagen IV phase compared with other phases of the HF cycle throughout the ORS, IRS, cortex, epidermal matrix, and DP. Decorin expression was enhanced in the follicles at anagen VI and catagen phases compared with the epidermis. Interestingly, decorin expression was persistent in the SG during the hair cycle. The negative control showed no contaminant, nonspecific staining in the sections (Fig. S2e-g).

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Upregulation of decorin mRNA and protein in anagen To explore decorin mRNA and protein expression, we performed RT-PCR (Fig. 2g) and Western blot analysis (Fig. 2f) respectively, on dorsal skin samples, which were collected during depilation-induced murine hair cycling. We observed that decorin mRNA and protein were expressed in all stages of HF morphogenesis and exhibited similar expression patterns. Decorin mRNA and protein were upregulated in the early to late anagen phases, and the levels were then reduced to the minimum levels during the catagen and telogen phases. Notably, although a greater than five-fold increase in decorin mRNA was observed during the middle and late anagen phases, decorin protein expression was stronger in the anagen phase overall (days 3, 5, and 12), especially in the early anagen phase.

Local injection of decorin in anagen HFs delays catagen progression To test whether decorin is required during anagen for cyclic transitions, we performed a functional study using decorin injections. Although HF groups treated with intradermal decorin did not show external pigmentation alterations (data not shown), histological sections representative of the decorin-injected group displayed an increased hair matrix (HM) volume and a more onion-like DP shape (Fig. 3b, 3c), which are characteristic of anagen VI development [34]. In contrast, the control group showed a diminished HM volume with a narrowed DP. Qualitative morphological image analyses revealed a prominent increase in skin thickness and a This article is protected by copyright. All rights reserved.

concomitant increase in the maximum bulb diameter (p

Expression of decorin throughout the murine hair follicle cycle: hair cycle dependence and anagen phase prolongation.

Decorin is a prototypical member of the small leucine-rich proteoglycan (SLRP) family, which is involved in numerous biological processes. The role of...
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