Xiaohua Lian

Adenovirus-Mediated Wnt10b Overexpression Induces Hair Follicle Regeneration

Hair follicles periodically undergo regeneration. The balance between activators and inhibitors may determine the time required for telogen hair follicles to reenter anagen. We previously reported that Wnt10b (wingless-type mouse mammary tumor virus integration site family member 10b) could promote the growth of hair follicles in vitro. To unveil the roles of Wnt10b in hair follicle regeneration, we established an in vivo mouse model using intradermal injection. On the basis of this model, we found that Wnt10b could induce the biological switch of hair follicles from telogen to anagen when overexpressed in the skin. The induced hair follicles expressed structure markers and could cycle normally into catagen. Conversely, anagen onset was abrogated by the knockdown of Wnt10b with small interfering RNA (siRNA). The Wnt10b aberrant expression data suggest that it is one of the activators of hair follicle regeneration. The β-catenin protein is translocated to the nucleus in Wnt10b-induced hair follicles. The biological effects of Wnt10b were abrogated when β-catenin expression was downregulated with siRNA. These data revealed that Wnt10b might induce hair follicle regeneration in vivo via the enhanced activation of the canonical Wnt signaling pathway. To our knowledge, our data provide previously unreported insights into the regulation of hair follicle cycling and provide potential therapeutic targets for hair follicle-related diseases.

Wnt10b promotes growth of hair follicles via a canonical Wnt signalling pathway

Background.  Wnt10b (wingless‐related mouse mammary tumour virus integration site 10b) plays various roles in a wide range of biological actions, including hair‐follicle development.

Conditional Immortalization Establishes a Repertoire of Mouse Melanocyte Progenitors with Distinct Melanogenic Differentiation Potential

TO THE EDITOR Epidermal melanocytes (MCs) are specialized melanin-producing cells (Slominski et al., 2004; Yamaguchi et al., 2007; Thomas and Erickson, 2008; Park et al., 2009) that synthesize melanin within the melanosome (Slominski et al., 2004) and protect individuals from harmful UV rays (Slominski et al., 2004; Yamaguchi et al., 2007; Thomas and Erickson, 2008; Park et al., 2009). Defects in or a lack of MCs can lead to melanoma, pigment disorders, and auditory defects. MC proliferation and differentiation in skin is tightly linked to hair regeneration cycles. MCs in vertebrates are derived from neural crest (Thomas and Erickson, 2008). In hair follicles, melanoblasts are segregated into hair matrix MCs (for hair pigmentation) and melanocyte stem cells (MCSCs). The discovery of MCSCs and induced pluripotent (iPS) cells provides important resources to elucidate mechanisms underlying melanogenesis and pathogenesis of MC-related disorders (Lin and Chuong, 2011; Nishikawa-Torikai et al., 2011; Nishimura, 2011; Ohta et al., 2011; Yang et al., 2011). Although MCSCs and iPS cells are important MC precursors for mechanistic studies, their isolation and expansion are technically challenging. Here we investigate whether MCs can be immortalized without compromising melanogenic potential. By using primary MCs isolated from newborn mouse skin, we engineered >100 clones by introducing SV40 T antigen (SV40T), which is flanked with FRT sites (Supplementary Figure S1A online; Westerman and Leboulch, 1996). We found that although primary MCs grew slower after passage 5 immortalized MC (iMC) cells acquired high proliferative activity (Supplementary Figure S1B online); however, wide variations in proliferation were observed among clones. For instance, iMC23 grew faster than iMC65 (Figure 1a). Figure 1 Characterization of SV40T-immortalized melanocytes (iMCs) We analyzed melanogenic markers in iMC clones. More than half of the analyzed clones expressed progenitor marker c-kit, whereas MC progenitor markers Pax3, Sox10, and MITF-m were readily detected in most clones (Supplementary Figure S2A online). Dopachrome tautomerase and tyrosinase-related protein 1 (TRP-1) were highly expressed in most iMC clones, whereas tyrosinase was highly expressed in about half of the clones (Supplementary Figure S2B and C online). It is conceivable that early melanogenic iMCs should express c-kit and early melagonic markers but not late markers (e.g., tyrosinase). Three iMC clones were chosen for further characterization: iMC23 (melanoblast progenitor-like) (Figure 1aA), iMC65 (late-stage melanocyte-like) (Figure 1aB), and iMC37 (intermediately differentiated melanoblast-like). These results indicate that SV40T-mediated immortalization can create a repertoire of iMCs with varied melanogenic potential. We also tested whether immortalization phenotypes were reversible by flippase recombination enzyme (FLP). By using adenovirus FLP (AdFLP) that co-expresses green fluorescent protein (GFP; He et al., 1998; Luo et al., 2007), we found that iMCs were effectively transduced by AdFLP or AdGFP, and SV40T expression was reduced in FLP-transduced iMCs (Figure 1bA and bB). iMC23 infected with AdFLP grew slower (Figure 1bC and bD), suggesting that the proliferative activity of iMCs may be reversed by removing SV40T. By using reporter pTyr-Gluc-expressing Gaussia luciferase (GLuc) driven by a 2.0-kb mouse tyrosinase promoter, we found that GLuc activity was increased by dexamethasone and was potentiated by removing SV40T with FLP (Supplementary Figure S3A and B online). We further analyzed spontaneous differentiation of iMCs by assessing endogenous melanin and tyrosinase activity. Cell pellets from iMCs exhibited varied amounts of melanin. iMC65 and iMC61 exhibited the highest level of melanin and iMC23 produced the lowest level, whereas a majority of iMC clones produced low-to-modest levels of melanin (Figure 1cA). Accordingly, quantitative analysis revealed that iMC65 and iMC61 exhibited high tyrosinase activity, and seven clones including iMC23 had low tyrosinase activity, whereas iMC37 exhibited modest-to-high levels of tyrosinase activity (Figure 1cB), consistent with the qualitative results shown in Figure 1cA. We characterized the early and late markers c-kit and HMB45, respectively, in iMC23, iMC37, and iMC65. HMB45 is a widely used mAb detecting melanocytic tumors (Gown et al., 1986). iMC23 expressed a high level of c-kit, whereas iMC37 and iMC65 were strongly stained with HMB45 (Figure 1dA). Fontana–Masson staining revealed the highest level of melanin in iMC65 and a modest level in iMC37, whereas a negligent level of melanin was observed in iMC32 (Figure 1dB). Thus, these clones may represent different stages of melanogenic differentiation. We further analyzed the melanogenic potential of iMC23. When iMC23 was treated with dexamethasone, tyrosinase activity increased in a dose-dependent manner (Figure 2aA). Dexamethasone induced tyrosinase and TRP-1 expression (Figure 2aB). Dose-dependent melanin production was visible in culture and cell pellet (Figure 2bA and bB), which was further confirmed by Fontana–Masson staining (Figure 2bC). These results indicate that iMC23 exhibits melanogenic potential. Figure 2 Melanogenic potential of immortalized melanocytes (iMCs) Finally, we determined whether iMC immortalization phenotypes were reversible in vivo. iMC23 and iMC65 tagged with firefly luciferase were infected with AdFLP or AdGFP, collected for subcutaneous injection into athymic mice, and monitored by Xenogen bioluminescence imaging (Caliper Life Sciences, Hopkinton, MA). AdGFP-transduced iMC23 yielded a stronger signal than that of AdFLP-transduced iMC65, whereas within the same lines AdFLP-transduced cells produced no signal compared with the AdGF-transduced iMC65 (Figure 2cA), suggesting that removing SV40T may reduce proliferation and survival of iMCs in vivo. Pigmentation of injected iMCs was visible through skin, as iMC65 exhibited a higher level of pigmentation than iMC23. AdFLP-transduced iMC23 yielded more pigmentation than AdGFP-transduced iMC23 (Figure 2cB). Histologically, iMC23 injection sites had higher cellularity than iMC65 sites, whereas in both lines cellularity was lower in AdFLP-transduced cells (Supplementary Figure S3C online). Production of melanin was more pronounced in AdFLP-transduced iMCs as indicated by HMB45 immunostaining and Fontana–-Masson staining (Figure 2dA and dB). Overall, the in vivo results are consistent with these clones’ in vitro features. SV40T-immortalized MCs are non-tumorigenic under our experimental conditions. Interestingly, melanoma phenotypes have been reported by enabling neoplastic transformation of primary human MCs with SV40 early region, which encodes both SV40 T and t antigens, in conjunction with hTERT (Gupta et al., 2005). Nevertheless, we established a repertoire of conditionally immortalized MCs with varied melanogenic potential, ranging from melanoblast-like to well-differentiated phenotypes. Such repertoire of iMCs should be useful for understanding MC biology and raveling molecular pathogenesis of pigment cell disorders, including melanoma. An efficient method to isolate and expand cutaneous MCs is needed, as it enables us to better understand melanogenesis. Here we demonstrate that SV40T-mediated immortalization of MCs is simplistic, effective, and reversible. This approach should be considered an important alternative to the isolation and characterization of melanogenic stem cells.

Wnt3a promotes melanin synthesis of mouse hair follicle melanocytes

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.

Modulating hair follicle size with Wnt10b/DKK1 during hair regeneration.

Hair follicles have characteristic sizes corresponding to their cycle‐specific stage. However, how the anagen hair follicle specifies its size remains elusive. Here, we showed that in response to prolonged ectopic Wnt10b‐mediated β‐catenin activation, regenerating anagen hair follicles grew larger in size. In particular, the hair bulb, dermal papilla and hair shaft became enlarged, while the formation of different hair types (Guard, Awl, Auchene and Zigzag) was unaffected. Interestingly, we found that the effect of exogenous WNT10b was mainly on Zigzag and less on the other kinds of hairs. We observed dramatically enhanced proliferation within the matrix, DP and hair shaft of the enlarged AdWnt10b‐treated hair follicles compared with those of normal hair follicles at P98. Furthermore, expression of CD34, a specific hair stem cell marker, was increased in its number to the bulge region after AdWnt10b treatment. Ectopic expression of CD34 throughout the ORS region was also observed. Many CD34‐positive hair stem cells were actively proliferating in AdWnt10b‐induced hair follicles. Importantly, subsequent co‐treatment with the Wnt inhibitor, DKK1, reduced hair follicle enlargement and decreased proliferation and ectopic localization of hair stem cells. Moreover, injection of DKK1 during early anagen significantly reduced the width of prospective hairs. Together, these findings strongly suggest that Wnt10b/DKK1 can modulate hair follicle size during hair regeneration.

Gsdma3 is a new factor needed for TNF-α-mediated apoptosis signal pathway in mouse skin keratinocytes

Gsdma3, a newly found gene, is expressed restrictedly in mouse skin keratinocytes and gastrointestinal tract. But until now, there is little information on the regulation and the function of Gsdma3 in skin keratinocytes. In our previous study, we found that Gsdma3 mutation resulted in a decrease in catagen-associated apoptosis of hair follicle keratinocytes. Apoptosis of skin keratinocytes is strictly regulated by a series of signal pathways, among of which, tumor necrosis factor (TNF)-α-induced signal pathway has been extensively studied. To further investigate the role and the pathway of Gsdma3 involved in skin keratinocyte apoptosis, using immunofluorescence, RT-PCR, western blot and TUNEL analysis, we showed here that accompanying TNF-α-induced apoptosis and Caspase-3 expression in mouse skin keratinocytes in vivo and in vitro, Gsdma3 expression was significantly upregulated. After Gsdma3 gene mutation, TNF-α-induced apoptosis and Caspase-3 expression in skin keratinocytes were reduced. The injection of Gsdma3 expression plasmid could directly enhance the apoptosis and Caspase-3 expression in skin keratinocytes. These results, taken together, indicated that in mouse skin keratinocytes, Gsdma3 expression could be regulated by TNF-α. Gsdma3 was not only involved in but also necessary for the TNF-α-induced apoptosis pathway by directly enhancing the Caspase3 expression as well as the apoptosis induction.

Gsdma3 gene is needed for the induction of apoptosis-driven catagen during mouse hair follicle cycle

Gsdm is a newly found gene family, which is restricted in its expression to the gastrointestinal tract and the skin epithelium. As a main member of the Gsdma subfamily, Gsdma3 is expressed specifically in the hair follicle of mouse skin, but its function remains largely unclear. By hematoxylin and eosin staining, we showed that Gsdma3 gene mutation caused an abnormal catagen phase with unshortened length and unshrunk structure of the hair follicle, in which the development of catagen phase was inhibited. TUNEL staining further revealed that the apoptosis of the hair follicle was obviously decreased in mutant mice. Caspase-3 downregulation was also detected by immunofluorescence, Western blot and RT-PCR in the hair follicle of the mutant mice. After intradermal injection of Gsdma3 gene expression plasmid, apoptosis as well as Caspase-3 expression in the hair follicle of mutant mice was enhanced, and so the catagen retardation of Gsdma3-mutant mice was rescued. Our results confirmed that Gsdma3 gene mutation interfered with catagen formation during mouse hair follicle cycle and, by upregulation of Caspase-3 expression and promotion of apoptosis, Gsdma3 gene could play an essential role in normal catagen induction.

Gsdma3 is required for hair follicle differentiation in mice.

Hair follicle differentiation is regulated by multiple signaling pathways. However, the known cellular and molecular mechanisms are limited. Gsdma3 is a novel murine gene and considered to be a mutation hotspot. Six mutants have been reported in Gsdma3 and all these mutants exhibit hair loss and hyperkeratosis phenotypes. In order to verify how the lack of Gsdma3 affects the hair defects, we use alopecia and excoriation mice, a new mouse mutation in this gene, as our research model. This mutation exhibits progressive hair loss, from head to the whole back, and followed by hair regrowth. We test that Gsdma3 is expressed in matrix, inner root sheath, and hair shaft. Ultrastructural and histological analyses show abnormal hair structures and reduced hair keratins in AE mice. The loss of interlocking structures and abnormal constitutive protein indicate defects in anchoring hair shaft in the hair follicle and resisting external forces. Molecular analysis of Gsdma3 deficiency and overexpression shows an Msx2/Foxn1/acidic hair keratin genetic pathway is involved. Thus, Gsdma3 is necessary for normal hair follicle differentiation.

Immunolocalization of Wnt5a during the hair cycle and its role in hair shaft growth in mice.

Previous studies have shown that the Wnt signaling pathway plays an important role in the growth and development of hair follicles. It has been generally accepted that Wnt5a, a non-canonical Wnt gene, inhibits the Wnt/β-catenin signaling pathway. Several reports have addressed its mRNA expression in embryonic and postnatal hair follicles, but its exact role in the growth of hair follicles is currently unknown. In this study, we investigated the immunolocalization of Wnt5a protein in pelages of the dorsal skin and whisker follicles of mice. We found that in the anagen phase, dermal papilla cells showed the highest staining levels of Wnt5a protein, while in the catagen and the telogen phases the staining levels were lower. During the growth stage, Wnt5a protein was prominently located in the matrix and precortex cells in addition to the inner root sheath, outer root sheath and the dermal papilla. As the hair cycle progresses, the immunostaining of Wnt5a was gradually decreased in the catagen phase and was located in the bulge and secondary hair germ in the telogen phase. This Wnt5a immunostaining profile was consistent between dorsal skin pelages and whisker follicles. Furthermore, in an in vitro study using whisker follicle organ culture, we demonstrated that the growth of the hair shaft was significantly inhibited by adenovirus Wnt5a. Our findings suggest that Wnt5a is a dynamic factor in the hair cycle and it is important for the regulation of hair shaft growth.

Wnt3a inhibits proliferation but promotes melanogenesis of melan-a cells

Melanocytes are pigment-producing cells responsible for coloration of skin and hair. Although the importance of Wnt3a in melanocyte development has been well recognized, the role of Wnt3a in mature melanocytes has not been elucidated. This study was conducted to further explore the effects of Wnt3a on melanocyte proliferation and melanogenesis, and to elucidate the possible mechanisms involved. We infected melan-a cells with AdWnt3a to serve as the production source of the Wnt3a protein. MTT assay, 5-bromodeoxyuridine incorporation assay and flow cytometric analysis showed that Wnt3a inhibited the proliferation of melan-a cells and this was associated with decrease of cells in the S phase and increase of cells in the G(1) phase. Melanin content and tyrosinase activity assay revealed that Wnt3a significantly promoted melanogenesis of melan-a cells. Furthermore, western blot analysis showed that Wnt3a upregulated the expression of microphthalmia-associated transcription factor and its downstream target genes, tyrosinase and tyrosinase-related protein 1 in melan-a cells. Collectively, our results suggest that Wnt3a plays an important role in melanocyte homeostasis.