The Study Of TPA-Induced Melanocyte Stem Cells For Epidermal And Hair Follicular Repigmentation And Its Possible Mechanisms

BackgroundSelf-renewal and pluripotent stem cells hold great promise of tissue regeneration and organ repairation. A hair follicle is a unique and excellent model for stem cell study because it undergoes cyclical growth and regression. Melanocyte stem cells(Mc SCs) and hair follicle stem cells(HFSCs) reside in hair bulge-secondary hair germ(s HG). Moreover, Mc SCs are unique and excellent for stem cell study because they live among HFSCs, exist alone without interacting with their progenies, and live in the simplest micro-enviroment. And differenciated Mc SCs could produce melanin, which is easy to obersve.During the process of embryogenesis, melanoblasts origin from neural crest, then migrate into epidermis through dermis and finally reside in hair follicles. Melanoblasts differentiate into Mc SCs located in hair bulge which express DCT(Tyrosinase related proteins-2) and into mature melanocytes located in hair matrix which is DCT, TRP1(Tyrosinase related proteins-1), TYR(Tyrosinase) and MITF(Microphthalmia-associated transcription factor) positive. Mc SCs show cyclical activation accompanied with hair cycle. In the eraly anagen, dermal papilla(DP) signaling synergisticly activates Mc SCs and HFSCs to provide cell resources for pigmented hair regeneration. In catagen, due to plenty of apoptosis in outer root sheath, inner root sheath and hair bulb, hair follicles start to regress. And survived cells migrate back to stem cell niche and st ay in quiescent state. Once receiving DP signaling stimulation again, Mc SCs will be recuited for regenerated hair pigmentation. Functional lack of Mc SCs is associated with canities. Therefore, study on how to induce Mc SCs for melanocyte regeneration and pigmentation of hair follicles will provide scientific and useful therapic option for depigmentation disorders.TGF-β(Transformation growth factor-β), Wnt, Notch, and NFIB signaling play important roles in Mc SCs development, proliferation and differentiation. Wnt/β-catenin signaling has been demonstrated to initiate Mc SC proliferation and differentiation. Firstly, over-expression of Wnt1 or β-catenin could promote the proliferation and differentiation of neural crest-derived melanocytes. Secondly, blockade of Wnt1, Wnt3 a or β-catenin expression could inhibit neural crest cells to develop into melanocytes. Recently, rese arches have demonstrated that Wnt/β-catenin sinaling could synergisticly activate HFSCs and Mc SCs for pigmented hair follicle regeneration. Pigmented hair follicle regeneration is dependent on the coordinated activation during the hair growth. Therefore, it is important for the regeneration of pigmented hair follicles to investigate the signaling mechanism regulating the interaction between HFSCs and Mc SCs. Although ectogenous over-expression of Wnt3 a or Wnt10 b in the adult mouse dorsal skin could significa ntly induce melanogenesis in hair follicles, how to control Wnt/β-catenin signaling to promote Mc SCs and HFSCs for pigmented hair regeneration remains clusive.Melanocytes disappear in the adult mouse dorsal epidermis, while survive in human epidermis for epidermal pigmentation. Loss or functional loss of melanocytes in human epidermis could cause depigmentation-related disorders, such as vitiligo. Previous studies have found that both ultraviolet(UV) radiation and Chinese medicine treatment on vitiligo lesion area could induce pigmented spots around hair follicles, suggesting that hair follicular Mc SCs might contribute to epidermal pigmentation. Recent study has found that UV radiation or wound could recruit Mc SCs to migrate toward epidermis. However, t he detailed process of epidermal repigmentation and related signaling mechanism needs to be further investigated.SCF and b FGF(basic fibroblast growth factor) signaling have been reported to be crucial in epidermal melanocyte survival. SCF is encoded by steel locus and activates c-kit signaling by binding the c-kit receptor. In the early stage of melanocyte development, functional loss of kit receptor or SCF could block the melanocyte migration and cause melanocyte apoptosis. Null mutation of SCF or c-kit, or injection of anti-c-kit antibody(ACK2) could induce hair depigmentation. But conditional expression of SCF in mouse skin keratinocytes expressing Keratin 14 maintains melanocyte survival in epidermis. And in vitro experiment showed that SCF could also promote human melanocyte migration and proliferation. However, whether SCF/c-kit signaling is involved in regulating Mc SCs for epidermal pigmentation remains unknown.12-O-tetradecanoylphorbol-13-acetate(TPA) is the most commonly used phorbol ester. For the molecular structure of TPA is similar with diacylglycerol(DAG), it is usually used as PKC activator. TPA is able to regulate cell proliferation, differentiation and migration through affecting PKC, endothelin and mitogen-activated protein kinase(MAPK) sinaling. TPA is used in myeloid leukemia therapy, and melanocyte primary culture. TPA has also been demonstrated to induce neural crest cells to develop into mature melanocytes and promote melanocyte differentiation. Researches have found that TPA could reserve TGF-β-induced melanocyte growth inhibition through decreasing Smad activity. Additionally, previous study has also demonstrated that TPA could induce hair follicle regeneration. However, the effect of TPA on regulating Mc SCs for hair follicle and epidermis pigmentation remains unknown.Mc SCs and HFSCs reside in hair follicle bulge-s HG and share the same niche. In the early stage of anagen, HFSCs and Mc SCs are activated by DP signaling in a coordinated manner, and migrate downward along outer root sheath to hair matrix where they differentiate into pigmentated hairs. TPA or wound could recruit HFSCs to migrate toward to epidermis. And previous study has also demonstrated that TPA could induce HFSCs migrate downwards along outer root sheath to hair matrix for hair follicle regeneration. Thus, whether TPA could recruit Mc SCs to migrate to epidermis or hair follicle for epidermal or hair follicular pigmentation remains unknown. Additionally, TPA is able to enhance the c-kit expression in cultured embryonic avian peripheral nerves, and induces neural crest cells to differentiate into melanocytes. Now that both neuro and melanocytes origin from neural crest, and SCF/c-kit signaling plays an important role in melanocyte migration, proliferation and differentiation, we speculate that SCF/c-kit signaling might be required in TPA-induced Mc SCs for epidermal pigmentation. Moreover, β-catenin expression is significantly increased in TPA-treated GT1-1 hypothalamic neurons and mouse osteoblastic cells. Thus, we speculate that Wnt/β-catenin signaling is involved in TPA-induced Mc SCs for hair pigmentation.Objective1. To investigate whether TPA could promote Mc SCs to contribute to epidermal and hair follicular pigmentation.2. To investigate whether SCF/c-kit signaling is required in TPA-induced Mc SCs for epidermal pigmentation.3. To investigate whether Wnt/β-catenin signaling is involved in TPA-induced Mc SCs for hair pigmentation.Materials and methods1. Smear TPA or acetone on 7 week-old C57BL/6 female mouse dorsal skin. Use haematoxylin and eosin(H&E) staining and Masson Fontana staining to detect the mouse epidermal pigmentation after TPA or acetone treatment, and use L-DOPA staining to detect the TYR activity in TPA or acetone treated skin. Use double immunostaining label ing DCT and PCNA to detect the proliferation of Mc SCs and melanocyte precursors, and use double immunostaining labeling MITF and DCT to detect the distribution of melanocyte lineage in hair follicle and epidermis.2. Use wound healing and cell migration assay to detect the migration of melanoblast i MC23 treated by TPA at different concentration, and use Transwell technology to detect whether TPA-treated keratinocyte JB6 cl 30-7b could enhance the migration of i MC23 cell. Detect whether TPA could promote i MC23 melanogenesis and enhance the TYR activity. Use western blot and immunostaining to analyze the expression of DCT, TRP1 and TYR in TPA-treated i MC23 cells.3. Use immunostaining to detect the SCF and c-kit expression in TPA- or acetone-treated skin, and use quantitative reverse transcription-polymerase chain reaction(q RT-PCR) to detect the SCF m RNA expression in TPA-treated skin and JB6 cl 30-7b cells. Use western blot analysis to detect c-kit expression in TPA-treated i MC23 cells.4. Use H&E staining and Masson Fontana staining to detect the effect of intracutaneous injection of ACK2 on TPA-induced epidermal pigmentation, and TPA-enhanced TYR activity. And analyze the effect of ACK2 on the number of DCT, TYR, and MITF in TPA-treated skin.5. Use Transwell technology to detect whether ACK2 could inhibit the migration of i MC23 cells enhanced by TPA-treated JB6 cl 30-7b cells.6. Smear TPA or acetone on 7 week-old C57BL/6 female mouse dorsal skin to induce hair regeneration, and use H&E staining and Masson Fontana staining to compare the hair pigmentation difference among TPA-induced, acetone-treated and anagen VI hair follicles. Use immunostaining to further investigate the expression of DCR, TRP1, TYR and MITF in various groups.7. Use double immunostaining labeling DCT and 5-bromo-2-deoxyuridine(Brd U) to detect the proliferation of Mc SCs and melanocyte precursors in various groups, and labeling MITF and Brd U to further confirm the melanocyte precursor proliferation.8. Use immunostaining to detect β-catenin expression, a key indicator of Wnt/β-catenin signaling, in TPA-indcued, acetone-treated hair follicles and anagen VI hair follicles.9. Use immunostaining to examine the GFP expression in TPA-treated skin combined with DKK1 or N1 plasmid injection in order to show the transfection and expression of DKK1. Use immunostaing to detect nuclear β-catenin expression in various groups in order to show whether DKK1 inhibits TPA-activated Wnt/β-catenin signaling. Further analyze hair pigmentation in TPA-induced, acetone-treated hair follicles and anagen VI hair follicles using H&E staining and Masson Fontana staining, and the expression of melanocyte markers in various groups using immunostaining.10. Use double immunostaining labeling DCT and Brd U, and labeling MITF and Brd U to investigate the proliferation of Mc SCs and melanocyte precursors in various groups.Results1. TPA significantly increased epidermal thickness, and TPA treatment for 2 doses induced epidermal pigmentation and increased the TYR activity. Immunostaining showed that TPA treatment for 1 dose increased the number of DCT in hair bulge-s HG, while TPA treatment for 2 doses significantly increased all of DCT, TYR, and MITF expression in hair infundibulum and epidermis, suggesting that TPA could promote Mc SC proliferation and induce Mc SC migration towards epidermis. Double immunostaining showed that TPA treatment could obviously promote DCT positive Mc SC proliferation, significantly induce Mc SCs to directly exit hair bulge-s HG and migrate to epidermis for epidermal pigmentation.2. TPA could significantly promote i MC23 migration. Compared with alcohol –treated JB6 cl 30-7b cells, TPA-treated JB6 cl 30-7b cells could also enhance the i MC23 migration. Moreover, TPA increased i MC23 melanogenesis in a concentration-dependent manner, and the TYR activity of i MC23 in a concentration-dependent and time-dependent manner. TPA significantly increased the expression of DCT, TYR and TRP1 in i MC23 cells.3. TPA treatment for 1 dose significantly increased the SCF expression in epidermis, while c-kit was not expressed in hair follicular infundibulum and epidermis until TPA treatment for 2 doses. And the SCF m RNA expression was significantly increased in TPA-treated mouse dorsal skin and JB6 cl 30-7b cells. Additionally, TPA treatment also promoted c-kit expression in i MC23.4. ACK2 treatment did not decrease the TPA-increased epidermal thichness but inhibited TPA-induced epidermal pigmentation and TPA-enhanced TYR activity. ACK2 also inhibited the expression of DCT, TYR, and MITF in hair infundibulum and epidermis, suggesting that ACK2 blocked the migration of hair follicular melanocytes.5. The Transwell results showed that ACK2 significantly inhibited i MC23 cell migration induced both by TPA treatment and by TPA-treated JB6 cl 30-7b cells, indicating that inhibiting SCF/c-kit signaling could significantly decrease the Mc SC migration.6. TPA treatment for 4 weeks induced normally differentiated hair follicle regeneration. Compared with acetone-treated hair follicles and anagen VI hair follicles, TPA-induced hair follicles showed increased hair matrix pigmentation, and bigger size of hair bulb and shaft. Additionally, TPA also increased the expression of DCT, TRP1, TYR and MITF in hair bulb.7. Double immunostaining labeling DCT and Brd U, and labeling MITF and Brd U showed that TPA promoted proliferation of DCT positive Mc SCs in hair bulge and MITF positive melanocyte precursors in hair matrix.8. The number of nuclear β-catenin was significantly increased in TPA-induced hair follicles, compared to that in acetone-treated hair follicles and anagen VI hair follicles, suggesting that TPA significantly enhanced the activity of Wnt/β-catenin signaling in melanocytes.9. After DKK1 injection in the TPA-treated skin, GFP expression was obviously detected in hair follicles, dermis and subcutaneous tissue, suggesting that DKK1 was expressed in TPA-treated skin. DKK1 also significantly inhibited the number of nuclear β-catenin in TPA-induced hair follicles, suggesting that DKK1 significantly inhibited TPA-enhanced Wnt/β-catenin signaling. Moreover, DKK1 decreased the TPA-increased hair bulb and hair shaft size, and hair matrix pigmentation to a relatively normal level. DKK1 also inhibited the TPA-increased expression of DCT, TRP1, TYR and MITF expression in hair bulb. These data suggested that inhibition of Wnt/ β-catenin signaling could decrease the TPA-increased hair bulb and shaft size, hair matrix pigmentation and the number of melanocytes to a relatively normal level.10. Double immunostaining labeling DCT and Brd U showed that DKK1 significantly inhibited the TPA-increased the proliferation of Mc SCs in hair bulge and of melanocyte precursors in hair bulb. Double immunostaining labeling MITF and Brd U showed that DKK1 significantly inhibited the TPA-increased number of proliferating MITF positive melanocyte precursors in hair bulb.Conclusions1. TPA treatment promotes Mc SCs proliferation and direct migration from bulge towards epidermis for epidermal pigmentation, and SCF/c-kit signaling is required in TPA induced Mc SCs for epidermal pigmentation. Our data provides a potential therapeutic option for skin pigmentation disorders.2. Constant TPA treatment could activate Mc SCs for pigmentation of regenerated hair follicles, and Wnt/β-catenin signaling is involved in TPA-induced hair pigmentation, which might provide a useful experimental model for the study of signals controlling the activation of Mc SCs and a potential therapeutic option for hair pigmentation disorders.