Journal of Dermatology 2014; 41: 716–723
doi: 10.1111/1346-8138.12570
ORIGINAL ARTICLE
Prevention of hair graying by factors that promote the growth and differentiation of melanocytes Mariko ENDOU,* Hitomi AOKI,* Tatsushi KOBAYASHI, Takahiro KUNISADA Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu, Japan
ABSTRACT Epidermal melanocyte precursors migrate into developing hair follicles to form the melanocyte stem cell system required to supply pigmented melanocytes necessary for hair pigmentation in repetitive hair cycles. Hair graying is caused by irreversible defects in the self-renewal and/or development of follicular melanocyte stem cells in the hair follicles. To investigate the mechanism(s) of hair graying during the normal aging process, we established a hair graying model in mice by repeatedly plucking or shaving trunk hairs. We repeatedly plucked or shaved trunk hairs to induce and accelerate the hair graying and counted the gray hairs. By using this functional model of hair graying in mice, we assessed the effects of genes known to affect melanocyte development, such as Kitl, hepatocyte growth factor (HGF) and endotheline 3 (ET3). After increasing the total numbers of cumulative hair cycles by plucking or shaving, we observed a significant increase in the gray hair of C57BL/6 mice. Kitl expression in the skin was the most effective for preventing hair graying and a significant effect was also confirmed for HGF and ET3 expression. The repeated hair plucking or shaving led to hair graying without any genetic lesion. Kitl is a more effective factor for prevention of hair graying than HGF or ET3. Our simple model of hair graying may provide a basic tool for screening the molecules or reagents preventing the progression of hair graying.
Key words:
endotheline 3, hair graying, hair plucking, Kit ligand, melanocyte stem cells.
INTRODUCTION Melanocytes develop from pluripotent neural crest cells that migrate along characteristic pathways to various destinations, such as the dermis and epidermis, the inner ear and the choroid of the eye.1–3 Once epidermal melanocyte precursors reside in the developing hair follicles, they form the melanocyte stem cell system that constantly supplies pigmented melanocytes necessary for hair pigmentation in the cycling hair follicles.4 While follicular melanocyte stem cells have the ability to self-renew during multiple hair cycles, the eventual loss of melanocyte stem cells is thought to cause hair graying.5–8 Taking advantage of the dispensable nature of the melanocyte cell lineage for the survival of individual organisms, the mechanisms controlling the self-renewal of follicular melanocyte stem cells manifested as hair graying have been studied.9–12 However, wild-type mice are almost free of hair graying during their natural aging when compared with human hairs that become totally gray or white by the seventh decade.13 To investigate the mechanism(s) of hair graying during the normal aging process, we first established a hair graying model without any genetic lesion by the repeated plucking or
shaving of trunk hairs. Since shaving and plucking are known to induce prompt entry into the anagen hair cycle stage,14 we repeatedly plucked or shaved the mice to increase the total number of cumulative hair cycles. After establishing a functional model of hair graying in mice by repeated plucking or shaving, we assessed the effects of genes known to stimulate melanocyte development, such as Kit ligand (Kitl), also known as stem cell factor (SCF), hepatocyte growth factor (HGF) and endothelin3 (ET3)15,16 for their effects on the progression of hair graying in this mouse model.
MATERIALS AND METHODS Animals All animal experiments were approved by the Animal Research Committee of the Graduate School of Medicine, Gifu University. C57BL/6 (B6) mice were obtained from Japan SLC (Shizuoka, Japan). The following transgenic mice were maintained in our animal facility: those generated with the human cytokeratin 14 promoter (hk14) driving cytokine/growth factor cDNAs [hk14-Kitl,17 hk14-HGF,18 hk14-TE3,19], and DCT-lacZ transgenic mice.20
Correspondence: Takahiro Kunisada, Ph.D., Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan. Email: [email protected] *These authors contributed equally to this work. Received 17 May 2014; accepted 12 June 2014.
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Mouse model for a prevention of hair graying
Mice were housed in standard animal rooms with food and water ad libitum under controlled humidity and temperature (22 2°C) conditions. The room was illuminated by fluorescent lights that were on from 08:00 to 20:00 h.
Hair plucking According to the telogen-hair plucking method,21 we plucked hairs from mice 3 to 4 weeks after birth. When the hair follicles reached their telogen stage, we started a second round of plucking and so on. Shaving of the hair was mostly performed simultaneously with the plucking using a razor blade taking special care not to cut the skin.
LacZ staining LacZ staining was performed as reported in detail previously.15
Skin melanocyte culture The cell preparation procedure was performed as described previously15,16 with minor modifications. Briefly, mice were sacrificed by decapitation, and their dorsal skins were quickly collected on ice. Specimens were treated overnight at 4°C or for 1 h at 37°C with 0.25% trypsin⁄ 1 mmol/L EDTA (Invitrogen, Carlsbad, CA, USA), 0.1% collagenase 1 (Sigma-Aldrich, St Louis, MO, USA) and 1X dispase (Roche, Basel, Switzerland). The epidermis was then peeled off the underlying dermis and dissected into very small pieces. These small pieces of epidermis were dissociated by gentle pipetting to obtain single cell suspensions which were then strained through 100-mesh nylon (Sansho, Tokyo, Japan). Fifty thousand cells prepared as described above were inoculated into 6-well plates previously seeded with ST2 cells and were then cultured in Alpha Minimum Essential Medium (Invitrogen) supplemented with 10% fetal calf serum (Equitech-Bio, Kerrville, TX, USA or Nichirei Bioscience, Tokyo, Japan), 40 pM basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA), 10 nmol/L dexamethasone (Sigma) and 10 pmol/L cholera toxin (Sigma).22 The medium was changed every 3 days. Cultures were maintained under 5% CO2 at 37°C.
RESULTS Melanocyte stem cell numbers in B6 mice during aging In contrast to the increase in gray hair during human aging, there was no significant increase of gray hairs in aged (day 450 after birth) B6 mice (Fig. 1a). Dct-lacZ transgenic mice expressing the lacZ reporter gene in melanocyte lineage cells, including their precursor cells, were used to visualize melanocyte precursors (Fig. 1b). The number of precursor or stem cells in Dct-lacZ transgenic mice was counted (Fig. 1c) and a slight decrease of stem cell numbers from nearly 4 per hair follicle in young mice to around 2 per hair follicle in the aged mice was observed. To count the number of melanocyte stem cells using a functional method, we performed a colony formation assay from the dissociated skin cells containing melanocyte stem cells. In day 30 or older mice, melanocyte stem cells or precursors dis-
© 2014 Japanese Dermatological Association
appeared from the interfollicular skin and most melanocyte stem cells were restricted to hair follicles.23 Dissociated skin cells harvested from the skin formed pigmented colonies on the ST2 stromal cell layers, as shown in Figure 1d and each colony consisted of typical dendritic melanocytes (Fig. 1e). The total number of colonies derived from 1 cm2 skin was divided by the previously counted mean hair follicle numbers per 1 cm2 skin to estimate the number of melanocyte stem cells per hair follicle. The number of melanocyte stem cells counted by the colony formation assay changed from 1 per hair follicle in the young mice to around 2.5 per hair follicle in the aged mice (Fig. 1f). The discrepancy in stem cell numbers assessed by these two methods is likely due to the efficiency of recovery from the skin and the viability of the recovered melanocyte stem cells during the in vitro colony formation assay. In any case, we did not observe a significant loss of melanocyte stem cells in the aged mice, which is in accordance with the fact that normal B6 mice rarely produce gray hairs in aged individuals.
Induction of gray hairs by serial plucking or shaving To establish a hair graying mouse model, we plucked or shaved hairs of B6 mice continuously during their whole life. To pluck or shave them as often as possible, we repeatedly plucked or shaved the hairs when the regenerated hairs reached the telogen state. Figure 2a–h shows typical results of the plucking process; from the first to the third plucking within 90 days after birth did not affect the percentage of gray hair compared to normal littermates that were not plucked. After the third plucking, the hair regenerated as shown in Figure 2a (day 95 after birth) after which the mice were plucked as shown in Figure 2b. At this point, gray hair in the regenerated hair areas did not increase, indicated as P4 in Figure 2i. Regenerated hairs after the 4th plucking showed an increase of gray hairs, as clearly recognized in Figure 2c, i. After the photography, we performed the 5th plucking at day 246 and counted the gray hairs as represented in Figure 2f, which indicates that all hairs were evenly depigmented. The percentage of gray hairs after the 4th plucking was increased to 1.6%, indicated as P5 in Figure 2I. It should be noted that from 100 days after birth, plucked or shaved hairs took a longer time to fully regenerate over the plucked or shaved skin areas. The percentage of gray hairs continued to increase after the 5th plucking (P6) and the 6th plucking (P7). At P8, the percentage of gray hairs decreased, probably because the hair regeneration rate was reduced at this stage, observed clearly as sparsely distributed regenerated hair (Fig. 2d; just before the 8th plucking) and gray hairs seemed to be lost preferentially. We performed a forced hair regeneration also by shaving, indicated as S3 to S7 in Figure 2i. Although the observed percentages of gray hair were lower than those observed in the plucking experiment as shown in Figure 2g, pictured just before the 7th shaving, the percentages were constantly higher than the unshaved control (Fig. 2i). These data demonstrate that the continuous plucking or shaving of mouse skin represents an experimental gray hair induction model.
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Figure 1. Changes in the number of melanocyte stem cells in B6 mice during aging. (a) The coat color of young (30 days old) and aged (450 days old) B6 mice. Even at day 450, white hair is not apparent in these wild-type mice. (b) Dct-lacZ transgenic mouse skin containing hair follicles were stained to visualize melanocyte stem cells. Tissues were inspected using a high-magnification binocular and the number of lacZ-positive cells around the bulge region was carefully inspected and counted at various ages as summarized in (c). (d) Skins dissected from Dct-lacZ transgenic mice (1 cm2) were dissociated and cultured on ST2 cells to visualize individual colonies derived from follicular melanocyte stem cells. Each colony consisted of pigmented, dendritic melanocytes as shown in (e). (f) To calculate the number of stem cells per hair follicle, the number of hairs in age-matched mice skin per cm2 were counted and the total number of colonies derived from 1 cm2 skin were divided with these counted hair numbers.
Prevention of gray hair induction by the expression of melanocyte stimulation factors in the skin Kitl is known to stimulate the survival, proliferation, differentiation and migration of melanocyte precursors.17 Although Kitl is not indispensable for the self-renewal of follicular melanocyte stem cells of adult mice,23 the forced expression of Kitl in the basal layer of the skin sustained the stable maintenance of
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melanocytes and pigmented epidermis during the entire lifespan of the mouse. To test the protective effect of Kitl against hair graying, we used a transgenic mouse line expressing Kitl in the basal layer of the epidermis driven by the human keratin 14 promoter (hk14-Kitl). Continuous plucking was performed similarly as in B6 mice and virtually no increase of gray hair was observed, as summarized in Figure 3g (P3 to P8). The
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Age Figure 2. Changes in the number of gray hairs during repeated plucking or shaving of the trunk hairs of B6 mice. The dorsal skin of B6 mice was plucked repeatedly and until after the fourth plucking, no increase of gray hair was observed in the regenerated hair (a). The dorsal skin of each mouse was plucked as shown in (b), and after the fourth plucking a significant increase of gray hair was observed (c). After the seventh plucking, hair regeneration was perturbed and appeared as a hairless region in (d) and these regions did not produce hair later on. Age-matched unplucked 450 day old control mice appeared to have no gray hair (e). (f) Gray hairs regenerated in the plucked mice (m:monotrich; a:awl; z:zigzag). The regenerated coat hair after the sixth shaving is shown in (g) and a significant increase of gray hair was observed. (h) Normal control. (i) Quantitative analysis of the gray hair at various timings of plucking or shaving of B6 mice. P6 indicates the sixth plucked hair and S4 indicates the fourth shaved hair, for example.
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coat color of day 146 hk14-Kitl transgenic mice just before the 4th plucking showed a blackly pigmented phenotype (Fig. 3a) and dense epidermal pigmentation after the 4th plucking (shown in Fig. 3b) in contrast to B6 mice (Fig. 2b). At day 548, just before the 8th plucking, although repeated plucking reduced the density of regenerated hairs, gray hair was very rare (Fig. 3c) as in the unplucked control (Fig. 3d). White hairs were very rarely detected after the 4th plucking of hk14-Kitl transgenic mice (shown in Fig. 3e). Shaving also did not affect hair graying (S3 to S6 in Fig. 3g) and the coat color of just mice before the 6th shaving is shown in Figure 3f. HGF and ET3 are also known for their effects on stimulating the growth and differentiation of melanocyte precursors.15,18,22 We performed continuous plucking or shaving of hk14-HGF transgenic mice expressing human HGF in the basal layer of the epidermis. Repeated plucking did not affect the coat color of hk14-HGF transgenic mice, as shown in Figure 4a–d, and summarized in Figure 4i. Repeated shaving also did not affect hair graying in hk14-HGF transgenic mice (Fig. 4i). A slightly increased number of gray hairs was observed in hk14-ET3 transgenic mice as shown in Figure 4e–h and summarized in Figure 4j. White hairs appeared in the plucked area (enlarged in Fig. 4g), just before the 8th plucking. The number of gray hairs was also increased by repeated shaving, however, repeated plucking or shaving of hk14-ET3 transgenic mice decreased hair graying compared to the shaved B6 control (Fig. 4j). Thus, HGF and ET3 both reduced the number of gray hairs after repeated plucking or shaving.
DISCUSSION Hair graying in humans is not a disease but is a natural physiological trait that is closely related with aging. A special aspect of hair graying is the loss of stem cell self-renewal capacity with age which finally leads to the loss of all follicular melanocyte stem cells. This situation does not typically occur in other tissue stem cells which would readily lead to the death of the organism. This highlights the study of hair graying as an ideal system to specifically characterize the molecular basis for the self-renewal of stem cells. Here we first established a hair graying model in mice which recapitulates hair graying with age, just by repeatedly plucking or shaving coat hairs. Mice usually never show significant hair graying during their normal life span. Thus, the induced hair graying is not strictly due to natural aging, however, the health of repeatedly plucked or shaved mice seems to be normal and is not accompanied by an increased death rate. This suggests that the artificial stimulation of hair regeneration by plucking or shaving extends the limit of the number of possible hair cycles set by the normal life span of mice which is normally insufficient to elicit the loss of self-renewing melanocyte stem cells that would result in age-related hair graying in mice. Still, after the repeated plucking, only up to 4% of all hair follicles of the mice turned gray, showing a clear difference in that most humans over 70 years of age have mostly gray or white hairs.13 Interestingly, the numbers of melanocyte stem cells were rather constant in the two different measuring methods
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(Fig. 1), in contrast to those reported in hematopoietic stem cells in the bone marrow,24 in which a significant age-related increase in the number of hematopoietic stem cells was observed. It was also reported that hematopoietic stem cells present in aged bone marrow lose their self-renewing activity.24 However, we did not observe an apparent reduction in the size of individual colonies derived from aged skin, indicating that the self-renewal activity of aged follicular melanocyte stem cells is rather constant. The self-renewal potential of melanocyte stem cells during aging should be further tested since cellular aging is affected in several aspects including genetic variability.25,26 Using the mouse hair graying model, we demonstrate that factors which had been shown to sustain ectopic melanocyte stem cells in the interfollicular skin of mice, namely Kitl, HGF and ET3, are all effective in preventing the plucking- or shaving-induced hair graying. Although Kitl promotes the differentiation of melanocyte stem cells in the hair follicle, these melanocyte stem cells are maintained or survive in the absence of Kit signaling, as indicated previously.17,22,27 Recent analysis revealed that Wnt signaling is a possible selfrenewal promoting cue for adult follicular melanocytes.28 If that Kit signaling does not affect the self-renewal of follicular melanocyte stem cells, mechanisms underlying the life-time protective effect of Kitl against hair graying might be more complicated, since the loss or lack of the self-renewal capacity of melanocyte stem cells is a theoretical cause of the hair graying. Since melanocytes were maintained continuously in the interfollicular skin of hk14-Kitl transgenic mice,17 Kit signaling is thought to enable the maintenance of the melanocyte stem cell system in interfollicular skin, otherwise melanocytes would be eliminated from the region shortly after birth. It might be possible that Kit signaling promotes the asymmetric division of melanocyte stem cells into differentiated precursor and stem cells which may at least maintain the melanocyte stem cell population. Alternatively, Kit signaling may induce the de-differentiation of melanocyte stem cells from their descendant melanoblasts. It should be noted that if this is the case, the maintenance or survival of melanocyte stem cells should be controlled by other factors.28 The protective effect of HGF and ET3 may also be explained in the same manner and it should be noted that ET3 was recently suggested as a factor involved in the stimulated self-renewal of follicular melanocytes.28 It is also possible that Kitl promotes the selfrenewal of melanocyte stem cells in association with other factors, such as ET3, and in circumstances where Kitl is eliminated from the environment, available ET3 signaling solely and effectively maintains their self renewal.22 Whatever the molecular details are, the continuous expression of one or more of these factors may well stimulate the self-renewal of melanocyte stem cells revealed as a phenotype resistant to hair graying. It should be noted that the constitutive activation of Wnt signaling causes exhaustive proliferation and successive differentiation of melanocyte stem cells revealed as early hair graying.28 however, we never observed this kind of exhaustive hair graying in any of the Kitl, ET3 and HGF transgenic mice.
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Age Figure 3. Prevention of the increase of hair graying in ectopically activated Kit signaling in hk14-Kitl transgenic mice. The dorsal skin of hk14-Kitl mice was plucked or shaved in the same manner as described for Figure 2. Regenerated hairs after the third plucking (a) were plucked as in (b) and so on. Even after the seventh plucking, no significant increase of gray hair was observed (c), as compared with the age-matched control of unplucked hk14-Kitl transgenic mice (d). Gray hairs were rarely found in the plucked mice (e) and the regenerated coat hairs after the sixth shaving (f). (g) Quantitative analysis of gray hair at various timings of plucking or shaving of hk14-Kitl transgenic mice. For comparison, the results of B6 mice (presented in Figure 2i) are shown (black line: plucked skin; gray line: shaved skin). Abbreviations used are the same as in Figure 2.
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Figure 4. Prevention of increased hair graying in ectopically activated HGF and ET3 signaling in hk14-HGF and hk14-ET3 transgenic mice. The dorsal skins of hk14-HGF mice just before (a) and after (b) the sixth plucking and those after the ninth plucking (c) and age-matched unplucked hk14-HGF control mice (d). hk14-ET3 hairs regenerated after the fourth plucking (e) were shaved (f) and after the eighth plucking patchy areas containing the gray hairs (g, enlarged underneath) were found compared with unplucked control mice (h). Please note that hairless skin areas were increased in hk14-ET3 mice after the repeated plucking (g). Quantitative analysis of gray hair at various timings of plucking or shaving of hk14-HGF (i) and hk14-ET3 (j) transgenic mice. The results of B6 mice (presented in Figure 2i) are shown for comparison (black line: plucked skin; gray line: shaved skin). Abbreviations used are the same as in Figure 2.
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This new animal model of hair graying in mice is simple and reproducible and can be easily combined with drug screening or testing to develop effective ways to prevent hair graying. Based on the protective effects of Kitl and also HGF and ET3 revealed in this study, these signaling molecules are promising targets for the prevention of hair graying.
ACKNOWLEDGMENTS: We thank Drs. Emi K. Nishimura, Masatake Osawa, Hisahiro Yoshida and Tomohisa Hirobe for their thoughtful advice. This study was supported in part by grants from the Japan Science and Technology Agency, by Grants-in-Aid for Scientific Research of JSPS and MEXT, and a grant supported by Gifu University KASSEIKA-KEIHI. CONFLICT OF INTEREST:
The authors declare no conflict-
ing interests.
REFERENCES 1 Hall BK. The Neural Crest and Neural Crest Cells in Vertebrate Development and Evolution. Springer, New York 1999. 2 Bronner ME, Le Douarin NM. Evolution and development of the neural crest: an overview. Dev Biol 2012; 366: 2–9. 3 Yajima I, Larue L. The location of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color. Pigment Cell Melanoma Res 2008; 21: 471–476. 4 Lamoreux ML, Delmas V, Larue L, Bennett D. The Colors of Mice. Wiley-Blackwell, Hoboken, NJ 2010. 5 Tobin DJ, Paus R. Graying: gerontobiology of the hair follicle pigmentary unit. Exp Gerontol 2001; 36: 29–54. 6 Freedberg IM, Eisen AZ, Wolff K et al., eds. Fitzpatricks Dermatology in General Medicine, 6th edn. Mcgraw-Hill, New York, 2003. 7 Steingrimsson E, Copeland NG, Jenkins NA. Melanocyte stem cell maintenance and hair graying. Cell 2005; 121: 9–12. €b RM. Pharmacologic interventions in aging hair. Clin Interv 8 True Aging 2006; 1: 121–129. 9 Nishimura EK, Granter SR, Fisher DE. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche. Science 2005; 307: 720–724. 10 Osawa M. Melanocyte stem cells. Mol Vis 2008; 14: 1064–1070. 11 Nishimura EK, Suzuki M, Igras V et al. Key roles for transforming growth factor beta in melanocyte stem cell maintenance. Cell Stem Cell 2010; 5: 130–140. 12 Nishikawa-Torikai S, Osawa M, Nishikawa S. Functional characterization of melanocyte stem cells in hair follicles. J Invest Dermatol 2011; 131: 2358–2367.
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13 Nordlund J, Boissy RE, Hearing VJ et al., eds. The Pigmentary System. Wiley-Blackwell, Hoboken, NJ 2006. 14 Hattori M, Ogawa H. Biochemical analysis of hair growth from the aspects of aging and enzyme activities. J Dermatol 1983; 10: 45–54. 15 Aoki H, Yamada Y, Hara A, Kunisada T. Two distinct types of mouse melanocyte: differential signaling requirement for the maintenance of non-cutaneous and dermal versus epidermal melanocytes. Development 2009; 136: 2511–2521. 16 Aoki H, Hara A, Motohashi T, Kunisada T. Protective effect of Kit signaling for melanocyte stem cells against radiation-induced genotoxic stress. J Invest Dermatol 2011; 131: 1906–1915. 17 Kunisada T, Yoshida H, Yamazaki H et al. Transgene expression of steel factor in the basal layer of epidermis promotes survival, proliferation, differentiation and migration of melanocyte precursors. Development 1998; 125: 2915–2923. 18 Kunisada T, Yamazaki H, Hirobe T et al. Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis. Mech Dev 2000; 94: 67–78. 19 Yamazaki H, Sakata E, Yamane T et al. Presence and distribution of neural crest-derived cells in the murine developing thymus and their potential for differentiation. Int Immunol 2005; 17: 549–558. 20 Mackenzie MA, Jordan SA, Budd PS, Jackson IJ. Activation of the receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the mouse embryo. Dev Biol 1997; 192: 99–107. 21 Potten CS. Radiation depigmentation of mouse hair: effect of the hair growth cycle on the sensitivity. J Invest Dermatol 1970; 55: 410–418. 22 Aoki H, Motohashi T, Yoshimura N et al. Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development. Dev Dyn 2005; 233: 407–417. 23 Hirobe T. Histochemical survey of the distribution of the epidermal melanoblasts and melanocytes in the mouse during fetal and postnatal periods. Anat Rec 1984; 208: 589–594. 24 Haan G, Nijhof W, Zant GV. Mouse strain-dependent changes in frequency and proliferation of hematopoietic stem cells during aging: correlation between lifespan and cycling activity. Blood 1997; 89: 1543–1550. 25 Haan G, Zant GV. Dynamic changes in mouse hematopoietic stem cell numbers during aging. Blood 1999; 93: 3294–3301. 26 Rubin H. Cell aging in vivo and in vitro. Mech Ageing Dev 1997; 98: 1–35. 27 Botchkareva NV, Khlgatian M, Longley BJ et al. SCF/c-kit signaling is required for cyclic regeneration of the hair pigmentation unit. FASEB J 2001; 15: 645–658. 28 Rabbani P, Takeo M, Chou W et al. Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration. Cell 2011; 145: 941–955.
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doi: 10.1111/1346-8138.12570
ORIGINAL ARTICLE
Prevention of hair graying by factors that promote the growth and differentiation of melanocytes Mariko ENDOU,* Hitomi AOKI,* Tatsushi KOBAYASHI, Takahiro KUNISADA Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu, Japan
ABSTRACT Epidermal melanocyte precursors migrate into developing hair follicles to form the melanocyte stem cell system required to supply pigmented melanocytes necessary for hair pigmentation in repetitive hair cycles. Hair graying is caused by irreversible defects in the self-renewal and/or development of follicular melanocyte stem cells in the hair follicles. To investigate the mechanism(s) of hair graying during the normal aging process, we established a hair graying model in mice by repeatedly plucking or shaving trunk hairs. We repeatedly plucked or shaved trunk hairs to induce and accelerate the hair graying and counted the gray hairs. By using this functional model of hair graying in mice, we assessed the effects of genes known to affect melanocyte development, such as Kitl, hepatocyte growth factor (HGF) and endotheline 3 (ET3). After increasing the total numbers of cumulative hair cycles by plucking or shaving, we observed a significant increase in the gray hair of C57BL/6 mice. Kitl expression in the skin was the most effective for preventing hair graying and a significant effect was also confirmed for HGF and ET3 expression. The repeated hair plucking or shaving led to hair graying without any genetic lesion. Kitl is a more effective factor for prevention of hair graying than HGF or ET3. Our simple model of hair graying may provide a basic tool for screening the molecules or reagents preventing the progression of hair graying.
Key words:
endotheline 3, hair graying, hair plucking, Kit ligand, melanocyte stem cells.
INTRODUCTION Melanocytes develop from pluripotent neural crest cells that migrate along characteristic pathways to various destinations, such as the dermis and epidermis, the inner ear and the choroid of the eye.1–3 Once epidermal melanocyte precursors reside in the developing hair follicles, they form the melanocyte stem cell system that constantly supplies pigmented melanocytes necessary for hair pigmentation in the cycling hair follicles.4 While follicular melanocyte stem cells have the ability to self-renew during multiple hair cycles, the eventual loss of melanocyte stem cells is thought to cause hair graying.5–8 Taking advantage of the dispensable nature of the melanocyte cell lineage for the survival of individual organisms, the mechanisms controlling the self-renewal of follicular melanocyte stem cells manifested as hair graying have been studied.9–12 However, wild-type mice are almost free of hair graying during their natural aging when compared with human hairs that become totally gray or white by the seventh decade.13 To investigate the mechanism(s) of hair graying during the normal aging process, we first established a hair graying model without any genetic lesion by the repeated plucking or
shaving of trunk hairs. Since shaving and plucking are known to induce prompt entry into the anagen hair cycle stage,14 we repeatedly plucked or shaved the mice to increase the total number of cumulative hair cycles. After establishing a functional model of hair graying in mice by repeated plucking or shaving, we assessed the effects of genes known to stimulate melanocyte development, such as Kit ligand (Kitl), also known as stem cell factor (SCF), hepatocyte growth factor (HGF) and endothelin3 (ET3)15,16 for their effects on the progression of hair graying in this mouse model.
MATERIALS AND METHODS Animals All animal experiments were approved by the Animal Research Committee of the Graduate School of Medicine, Gifu University. C57BL/6 (B6) mice were obtained from Japan SLC (Shizuoka, Japan). The following transgenic mice were maintained in our animal facility: those generated with the human cytokeratin 14 promoter (hk14) driving cytokine/growth factor cDNAs [hk14-Kitl,17 hk14-HGF,18 hk14-TE3,19], and DCT-lacZ transgenic mice.20
Correspondence: Takahiro Kunisada, Ph.D., Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan. Email: [email protected] *These authors contributed equally to this work. Received 17 May 2014; accepted 12 June 2014.
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Mouse model for a prevention of hair graying
Mice were housed in standard animal rooms with food and water ad libitum under controlled humidity and temperature (22 2°C) conditions. The room was illuminated by fluorescent lights that were on from 08:00 to 20:00 h.
Hair plucking According to the telogen-hair plucking method,21 we plucked hairs from mice 3 to 4 weeks after birth. When the hair follicles reached their telogen stage, we started a second round of plucking and so on. Shaving of the hair was mostly performed simultaneously with the plucking using a razor blade taking special care not to cut the skin.
LacZ staining LacZ staining was performed as reported in detail previously.15
Skin melanocyte culture The cell preparation procedure was performed as described previously15,16 with minor modifications. Briefly, mice were sacrificed by decapitation, and their dorsal skins were quickly collected on ice. Specimens were treated overnight at 4°C or for 1 h at 37°C with 0.25% trypsin⁄ 1 mmol/L EDTA (Invitrogen, Carlsbad, CA, USA), 0.1% collagenase 1 (Sigma-Aldrich, St Louis, MO, USA) and 1X dispase (Roche, Basel, Switzerland). The epidermis was then peeled off the underlying dermis and dissected into very small pieces. These small pieces of epidermis were dissociated by gentle pipetting to obtain single cell suspensions which were then strained through 100-mesh nylon (Sansho, Tokyo, Japan). Fifty thousand cells prepared as described above were inoculated into 6-well plates previously seeded with ST2 cells and were then cultured in Alpha Minimum Essential Medium (Invitrogen) supplemented with 10% fetal calf serum (Equitech-Bio, Kerrville, TX, USA or Nichirei Bioscience, Tokyo, Japan), 40 pM basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA), 10 nmol/L dexamethasone (Sigma) and 10 pmol/L cholera toxin (Sigma).22 The medium was changed every 3 days. Cultures were maintained under 5% CO2 at 37°C.
RESULTS Melanocyte stem cell numbers in B6 mice during aging In contrast to the increase in gray hair during human aging, there was no significant increase of gray hairs in aged (day 450 after birth) B6 mice (Fig. 1a). Dct-lacZ transgenic mice expressing the lacZ reporter gene in melanocyte lineage cells, including their precursor cells, were used to visualize melanocyte precursors (Fig. 1b). The number of precursor or stem cells in Dct-lacZ transgenic mice was counted (Fig. 1c) and a slight decrease of stem cell numbers from nearly 4 per hair follicle in young mice to around 2 per hair follicle in the aged mice was observed. To count the number of melanocyte stem cells using a functional method, we performed a colony formation assay from the dissociated skin cells containing melanocyte stem cells. In day 30 or older mice, melanocyte stem cells or precursors dis-
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appeared from the interfollicular skin and most melanocyte stem cells were restricted to hair follicles.23 Dissociated skin cells harvested from the skin formed pigmented colonies on the ST2 stromal cell layers, as shown in Figure 1d and each colony consisted of typical dendritic melanocytes (Fig. 1e). The total number of colonies derived from 1 cm2 skin was divided by the previously counted mean hair follicle numbers per 1 cm2 skin to estimate the number of melanocyte stem cells per hair follicle. The number of melanocyte stem cells counted by the colony formation assay changed from 1 per hair follicle in the young mice to around 2.5 per hair follicle in the aged mice (Fig. 1f). The discrepancy in stem cell numbers assessed by these two methods is likely due to the efficiency of recovery from the skin and the viability of the recovered melanocyte stem cells during the in vitro colony formation assay. In any case, we did not observe a significant loss of melanocyte stem cells in the aged mice, which is in accordance with the fact that normal B6 mice rarely produce gray hairs in aged individuals.
Induction of gray hairs by serial plucking or shaving To establish a hair graying mouse model, we plucked or shaved hairs of B6 mice continuously during their whole life. To pluck or shave them as often as possible, we repeatedly plucked or shaved the hairs when the regenerated hairs reached the telogen state. Figure 2a–h shows typical results of the plucking process; from the first to the third plucking within 90 days after birth did not affect the percentage of gray hair compared to normal littermates that were not plucked. After the third plucking, the hair regenerated as shown in Figure 2a (day 95 after birth) after which the mice were plucked as shown in Figure 2b. At this point, gray hair in the regenerated hair areas did not increase, indicated as P4 in Figure 2i. Regenerated hairs after the 4th plucking showed an increase of gray hairs, as clearly recognized in Figure 2c, i. After the photography, we performed the 5th plucking at day 246 and counted the gray hairs as represented in Figure 2f, which indicates that all hairs were evenly depigmented. The percentage of gray hairs after the 4th plucking was increased to 1.6%, indicated as P5 in Figure 2I. It should be noted that from 100 days after birth, plucked or shaved hairs took a longer time to fully regenerate over the plucked or shaved skin areas. The percentage of gray hairs continued to increase after the 5th plucking (P6) and the 6th plucking (P7). At P8, the percentage of gray hairs decreased, probably because the hair regeneration rate was reduced at this stage, observed clearly as sparsely distributed regenerated hair (Fig. 2d; just before the 8th plucking) and gray hairs seemed to be lost preferentially. We performed a forced hair regeneration also by shaving, indicated as S3 to S7 in Figure 2i. Although the observed percentages of gray hair were lower than those observed in the plucking experiment as shown in Figure 2g, pictured just before the 7th shaving, the percentages were constantly higher than the unshaved control (Fig. 2i). These data demonstrate that the continuous plucking or shaving of mouse skin represents an experimental gray hair induction model.
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Figure 1. Changes in the number of melanocyte stem cells in B6 mice during aging. (a) The coat color of young (30 days old) and aged (450 days old) B6 mice. Even at day 450, white hair is not apparent in these wild-type mice. (b) Dct-lacZ transgenic mouse skin containing hair follicles were stained to visualize melanocyte stem cells. Tissues were inspected using a high-magnification binocular and the number of lacZ-positive cells around the bulge region was carefully inspected and counted at various ages as summarized in (c). (d) Skins dissected from Dct-lacZ transgenic mice (1 cm2) were dissociated and cultured on ST2 cells to visualize individual colonies derived from follicular melanocyte stem cells. Each colony consisted of pigmented, dendritic melanocytes as shown in (e). (f) To calculate the number of stem cells per hair follicle, the number of hairs in age-matched mice skin per cm2 were counted and the total number of colonies derived from 1 cm2 skin were divided with these counted hair numbers.
Prevention of gray hair induction by the expression of melanocyte stimulation factors in the skin Kitl is known to stimulate the survival, proliferation, differentiation and migration of melanocyte precursors.17 Although Kitl is not indispensable for the self-renewal of follicular melanocyte stem cells of adult mice,23 the forced expression of Kitl in the basal layer of the skin sustained the stable maintenance of
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melanocytes and pigmented epidermis during the entire lifespan of the mouse. To test the protective effect of Kitl against hair graying, we used a transgenic mouse line expressing Kitl in the basal layer of the epidermis driven by the human keratin 14 promoter (hk14-Kitl). Continuous plucking was performed similarly as in B6 mice and virtually no increase of gray hair was observed, as summarized in Figure 3g (P3 to P8). The
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Age Figure 2. Changes in the number of gray hairs during repeated plucking or shaving of the trunk hairs of B6 mice. The dorsal skin of B6 mice was plucked repeatedly and until after the fourth plucking, no increase of gray hair was observed in the regenerated hair (a). The dorsal skin of each mouse was plucked as shown in (b), and after the fourth plucking a significant increase of gray hair was observed (c). After the seventh plucking, hair regeneration was perturbed and appeared as a hairless region in (d) and these regions did not produce hair later on. Age-matched unplucked 450 day old control mice appeared to have no gray hair (e). (f) Gray hairs regenerated in the plucked mice (m:monotrich; a:awl; z:zigzag). The regenerated coat hair after the sixth shaving is shown in (g) and a significant increase of gray hair was observed. (h) Normal control. (i) Quantitative analysis of the gray hair at various timings of plucking or shaving of B6 mice. P6 indicates the sixth plucked hair and S4 indicates the fourth shaved hair, for example.
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coat color of day 146 hk14-Kitl transgenic mice just before the 4th plucking showed a blackly pigmented phenotype (Fig. 3a) and dense epidermal pigmentation after the 4th plucking (shown in Fig. 3b) in contrast to B6 mice (Fig. 2b). At day 548, just before the 8th plucking, although repeated plucking reduced the density of regenerated hairs, gray hair was very rare (Fig. 3c) as in the unplucked control (Fig. 3d). White hairs were very rarely detected after the 4th plucking of hk14-Kitl transgenic mice (shown in Fig. 3e). Shaving also did not affect hair graying (S3 to S6 in Fig. 3g) and the coat color of just mice before the 6th shaving is shown in Figure 3f. HGF and ET3 are also known for their effects on stimulating the growth and differentiation of melanocyte precursors.15,18,22 We performed continuous plucking or shaving of hk14-HGF transgenic mice expressing human HGF in the basal layer of the epidermis. Repeated plucking did not affect the coat color of hk14-HGF transgenic mice, as shown in Figure 4a–d, and summarized in Figure 4i. Repeated shaving also did not affect hair graying in hk14-HGF transgenic mice (Fig. 4i). A slightly increased number of gray hairs was observed in hk14-ET3 transgenic mice as shown in Figure 4e–h and summarized in Figure 4j. White hairs appeared in the plucked area (enlarged in Fig. 4g), just before the 8th plucking. The number of gray hairs was also increased by repeated shaving, however, repeated plucking or shaving of hk14-ET3 transgenic mice decreased hair graying compared to the shaved B6 control (Fig. 4j). Thus, HGF and ET3 both reduced the number of gray hairs after repeated plucking or shaving.
DISCUSSION Hair graying in humans is not a disease but is a natural physiological trait that is closely related with aging. A special aspect of hair graying is the loss of stem cell self-renewal capacity with age which finally leads to the loss of all follicular melanocyte stem cells. This situation does not typically occur in other tissue stem cells which would readily lead to the death of the organism. This highlights the study of hair graying as an ideal system to specifically characterize the molecular basis for the self-renewal of stem cells. Here we first established a hair graying model in mice which recapitulates hair graying with age, just by repeatedly plucking or shaving coat hairs. Mice usually never show significant hair graying during their normal life span. Thus, the induced hair graying is not strictly due to natural aging, however, the health of repeatedly plucked or shaved mice seems to be normal and is not accompanied by an increased death rate. This suggests that the artificial stimulation of hair regeneration by plucking or shaving extends the limit of the number of possible hair cycles set by the normal life span of mice which is normally insufficient to elicit the loss of self-renewing melanocyte stem cells that would result in age-related hair graying in mice. Still, after the repeated plucking, only up to 4% of all hair follicles of the mice turned gray, showing a clear difference in that most humans over 70 years of age have mostly gray or white hairs.13 Interestingly, the numbers of melanocyte stem cells were rather constant in the two different measuring methods
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(Fig. 1), in contrast to those reported in hematopoietic stem cells in the bone marrow,24 in which a significant age-related increase in the number of hematopoietic stem cells was observed. It was also reported that hematopoietic stem cells present in aged bone marrow lose their self-renewing activity.24 However, we did not observe an apparent reduction in the size of individual colonies derived from aged skin, indicating that the self-renewal activity of aged follicular melanocyte stem cells is rather constant. The self-renewal potential of melanocyte stem cells during aging should be further tested since cellular aging is affected in several aspects including genetic variability.25,26 Using the mouse hair graying model, we demonstrate that factors which had been shown to sustain ectopic melanocyte stem cells in the interfollicular skin of mice, namely Kitl, HGF and ET3, are all effective in preventing the plucking- or shaving-induced hair graying. Although Kitl promotes the differentiation of melanocyte stem cells in the hair follicle, these melanocyte stem cells are maintained or survive in the absence of Kit signaling, as indicated previously.17,22,27 Recent analysis revealed that Wnt signaling is a possible selfrenewal promoting cue for adult follicular melanocytes.28 If that Kit signaling does not affect the self-renewal of follicular melanocyte stem cells, mechanisms underlying the life-time protective effect of Kitl against hair graying might be more complicated, since the loss or lack of the self-renewal capacity of melanocyte stem cells is a theoretical cause of the hair graying. Since melanocytes were maintained continuously in the interfollicular skin of hk14-Kitl transgenic mice,17 Kit signaling is thought to enable the maintenance of the melanocyte stem cell system in interfollicular skin, otherwise melanocytes would be eliminated from the region shortly after birth. It might be possible that Kit signaling promotes the asymmetric division of melanocyte stem cells into differentiated precursor and stem cells which may at least maintain the melanocyte stem cell population. Alternatively, Kit signaling may induce the de-differentiation of melanocyte stem cells from their descendant melanoblasts. It should be noted that if this is the case, the maintenance or survival of melanocyte stem cells should be controlled by other factors.28 The protective effect of HGF and ET3 may also be explained in the same manner and it should be noted that ET3 was recently suggested as a factor involved in the stimulated self-renewal of follicular melanocytes.28 It is also possible that Kitl promotes the selfrenewal of melanocyte stem cells in association with other factors, such as ET3, and in circumstances where Kitl is eliminated from the environment, available ET3 signaling solely and effectively maintains their self renewal.22 Whatever the molecular details are, the continuous expression of one or more of these factors may well stimulate the self-renewal of melanocyte stem cells revealed as a phenotype resistant to hair graying. It should be noted that the constitutive activation of Wnt signaling causes exhaustive proliferation and successive differentiation of melanocyte stem cells revealed as early hair graying.28 however, we never observed this kind of exhaustive hair graying in any of the Kitl, ET3 and HGF transgenic mice.
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Age Figure 3. Prevention of the increase of hair graying in ectopically activated Kit signaling in hk14-Kitl transgenic mice. The dorsal skin of hk14-Kitl mice was plucked or shaved in the same manner as described for Figure 2. Regenerated hairs after the third plucking (a) were plucked as in (b) and so on. Even after the seventh plucking, no significant increase of gray hair was observed (c), as compared with the age-matched control of unplucked hk14-Kitl transgenic mice (d). Gray hairs were rarely found in the plucked mice (e) and the regenerated coat hairs after the sixth shaving (f). (g) Quantitative analysis of gray hair at various timings of plucking or shaving of hk14-Kitl transgenic mice. For comparison, the results of B6 mice (presented in Figure 2i) are shown (black line: plucked skin; gray line: shaved skin). Abbreviations used are the same as in Figure 2.
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Figure 4. Prevention of increased hair graying in ectopically activated HGF and ET3 signaling in hk14-HGF and hk14-ET3 transgenic mice. The dorsal skins of hk14-HGF mice just before (a) and after (b) the sixth plucking and those after the ninth plucking (c) and age-matched unplucked hk14-HGF control mice (d). hk14-ET3 hairs regenerated after the fourth plucking (e) were shaved (f) and after the eighth plucking patchy areas containing the gray hairs (g, enlarged underneath) were found compared with unplucked control mice (h). Please note that hairless skin areas were increased in hk14-ET3 mice after the repeated plucking (g). Quantitative analysis of gray hair at various timings of plucking or shaving of hk14-HGF (i) and hk14-ET3 (j) transgenic mice. The results of B6 mice (presented in Figure 2i) are shown for comparison (black line: plucked skin; gray line: shaved skin). Abbreviations used are the same as in Figure 2.
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This new animal model of hair graying in mice is simple and reproducible and can be easily combined with drug screening or testing to develop effective ways to prevent hair graying. Based on the protective effects of Kitl and also HGF and ET3 revealed in this study, these signaling molecules are promising targets for the prevention of hair graying.
ACKNOWLEDGMENTS: We thank Drs. Emi K. Nishimura, Masatake Osawa, Hisahiro Yoshida and Tomohisa Hirobe for their thoughtful advice. This study was supported in part by grants from the Japan Science and Technology Agency, by Grants-in-Aid for Scientific Research of JSPS and MEXT, and a grant supported by Gifu University KASSEIKA-KEIHI. CONFLICT OF INTEREST:
The authors declare no conflict-
ing interests.
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