Wnt3a promotes melanin synthesis of mouse hair follicle melanocytes

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Abstract

Although the importance of Wnt3a in melanocyte development has been well recognized, the effect of Wnt3a in normal HF melanocytes has not been clearly elucidated yet. Thus, we sought to examine the presence and location of Wnt3a in HF during hair cycle. By using melanocyte-targeted Dct-LacZ transgenic mice, we found that Wnt3a signaling is activated in mouse HF melanocytes during anagen of hair cycle. To further explore the potential functions of Wnt3a in mouse melanocytes, we infected melan-a cells with AdWnt3a to serve as the production source of Wnt3a protein. We demonstrated that Wnt3a promoted melanogenesis through upregulation of MITF and its downstream genes, tyrosinase and TRP1, in melanocytes. In vivo, AdWnt3a rescued the effects of AdsimMITF on HF melanocytes and promoted melanin synthesis. Our results suggest that Wnt3a plays an important role in mouse HF melanocytes homeostasis.

Highlights

► We examine the expression of Wnt3a in mouse hair follicles during hair cycle. ► We examine the expression of Wnt3a in mouse hair follicle melanocytes. ► We examine Wnt/β-catenin signaling in mouse hair follicle melanocytes. ► Wnt3a promotes the melanin synthesis of melanocytes though upregulation of MITF and its downstream genes, tyrosinase and TRP1. ► Wnt3a promotes the melanogenesis of hair follicle melanocytes in vivo.

Introduction

Melanocytes play important roles in skin and hair pigmentation by producing melanin [1], [2]. Melanocytes originate from neural crest-derived melanoblasts. During embryogenesis, melanoblasts migrate through the dermis into the epidermis. In the hairy region of mammalian skin, melanoblasts further enter the newly developing hair follicles (HFs) where they finally become localized [3], [4], [5]. Henceforward, melanoblasts are separated into two subpopulations: differentiated melanocytes, localized in the hair bulb, expresses MITF, DCT, TRP1, and tyrosinase, responsible for hair pigmentation; and melanocyte stem cells (McSCs), localized in the bulge region, expresses only DCT, responsible for the repopulation of the melanocyte system in subsequent hair cycles [6], [7]. Melanocytes in HFs repeatedly proliferate and differentiate during every hair cycle [8]. During active growth (anagen) of HFs, melanocytes that are located in the hair bulb proliferate and differentiate to produce pigment for the hair shaft [2], [9]. Melanocytes begin to shut down melanogenesis in late anagen and regression phase (catagen), and die by apoptosis from the hair bulb in rest phase (telogen), but reappear at the same location when HFs reenter anagen [10], [11], [12].

Wnt3a has been reported to act through the canonical pathway and has important roles in the development of melanocytes. Mutant mice deficient in Wnt1 and Wnt3a are almost completely devoid of pigment cells [13]. In vitro studies found that neural crest cells depleted of Wnt1 and Wnt3a become neurons rather than melanocytes [14], while treatment with Wnt3a caused them to become pigment cells rather than neurons and glial cells [15]. Wnt3a acts on melanoblasts along with EDN3 and KITL to maintain MITF expression and promote melanoblasts to differentiate to become melanocytes [16].

Although the importance of Wnt3a in melanocyte development has been well recognized, the effect of Wnt3a in normal HF melanocytes has not been clearly elucidated yet. To address this issue, we used Dct-LacZ transgenic mice [17] to examine the expression of Wnt3a in HF melanocytes and adenoviral gene delivery of Wnt3a to investigate the effects of Wnt3a in mouse melanocyte cell line (melan-a) and primary cultured mouse melanocytes.

Section snippets

Animals and skin samples

Dct-LacZ transgenic mice were kindly provided by Prof. Ian Jackson [17]. The generated colony was backcrossed with C57BL/6J. Mice were housed in the community cages at the animal facility of Third Military Medical University. All the animal-related procedures were in strict accordance with the approved institutional animal care and maintenance protocols.

X-gal staining and immunohistochemistry

The harvested skins were first fixated at 4 °C in 4% paraformaldehyde for 2 h, then stained with X-gal staining solution (Beyotime, China) at

Wnt3a is expressed in melanocyte stem cells and melanocytes during anagen

By using Dct-LacZ transgenic mice, X-gal staining was used to label LacZ-positive melanocytes in HFs. The results showed that melanocyte stem cells (McSCs) were located in the bulge of the outer root sheath and melanocytes were located in the hair bulb and above the dermal papilla (Fig. S1).

In anagen, high expression of Wnt3a protein was detected in cells at the epidermis, bulge, inner root sheath (IRS) precursors and hair bulb, including melanocytes and McSCs (Fig. 1A). In catagen, the

Discussion

Despite the well-known role for Wnt3a in the development of melanocytes, the effect of Wnt3a signaling in HF melanocytes remains undetermined. Recent studies have shown that postnatally, Wnt3a mRNA and protein are expressed in the IRS precursors of HFs and basal keratinocytes of epidermis [28], [29]. However, whether HF melanocytes express Wnt3a or whether Wnt3a expression correlates with HF melanocyte proliferation, differentiation, and apoptosis are unknown. To address these questions, we

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 30872711). We thank Dr. T-C. He (Chicago University) for adenovirus production and technical assistance. We thank Prof. Dorothy C Bennett (University of London) and Dr. Zhixiu Lin (The Chinese University of Hong Kong) for providing melan-a cells; We thank Prof. Ian Jackson (MRC Human Genetics Unit) for the Dct-LacZ transgenic mice. We are also grateful to Cheng-Ming Chuong (University of Southern California) for

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