Mobilization of Melanocytes During NB-UVB Treatment of Vitiligo

Yue Li and Ru-Zhi Zhang∗

Department of Dermatology,The Third Affiliated Hospital of Soochow University,Changzhou,Jiangsu213003,China.

Abstract Vitiligo,a common skin depigmentation disorder,is the result of complex interactions of genetic,immunological,environmental,and biochemical events.Treatments for vitiligo include drugs,phototherapy,surgical transplantation,and so on.Among them,the efficacy of narrow band-ultraviolet B has been confirmed.By inducing keratinocytederived factors and signalings,narrow band-ultraviolet B can trigger and/or promote the mobilization of melanocytes which migrate to lesional epidermis ultimately,leads to the repigmentation of white patches.The mobilization of melanocytes includes stages of activation,migration,proliferation,and differentiation.Elucidating processes that enable the specific mobilization of melanocytes and the signaling pathways and factors involved,will help the development of new drugs and methods for the treatment of vitiligo.

Keywords:vitiligo,narrow band-ultraviolet B,melanocyte,repigmentation

Introduction

There are different patterns of repigmentation of vitiligo.1The perifollicular distribution is the most prevalent repigmentation pattern in vitiligo,suggesting that melanocyte stem cells(MSCs)in the hair follicles bulge region is the main reservoir that supplies melanocytes(MCs).The hair follicle is often unaffected by the T cell-mediated attack,because the hair follicle bulge is a site of immune privileged location.The marginal pattern,the secondary repigmentation pattern,is characterized by an increase in pigmentation at the edge of the lesion,which suggested that there may be a pool of MCs for repigmentation at the lesional borders.The diffuse pattern is described as generalized darkening across the white patches.The combined pattern contains more than one pattern or just not belong to any one of the above patterns.Recently,some researchers observed a new repigmentation pattern called medium spotted repigmentation in pediatric vitiligo patients,it occurs in the sites where contain few or no hair follicles,such as the wrist flexors and lips.2In addition,there are extrafollicular MSCs in the dermis of vitiligo lesions,which also have the ability to replace damaged MCs in the basal layer of the epidermis.3-4

Among the numerous methods currently used to treat vitiligo,narrow band-ultraviolet B(NB-UVB)is the most effective.Goldstein et al.5analyzed the distribution of MC precursors and differentiated MCs in the hair follicle bulge,the infundibulum,and the epidermis following NBUVB irradiation by monitoring the expression of four pigmentation-related genes(PAX3,TYR,DCT,and KIT),a proliferation marker(KI-67)and a migration marker(MCAM).Their study showed that after NB-UVB irradiation,MSCs(C-KIT-/DCT+/TYR–)were activated and gradually migrated upwards,which was accompanied by cell proliferation and MSCs differentiated into functional MCs in the epidermis.These events are parallel,closely related,and it must be emphasized that these processes are inseparable from well regulation of the signaling pathways,adhesion molecules,proteins,enzymes,and other factors.In this study,we made a searching in PubMed from1990to2019with strategies as follows:(‘vitiligo"and"repigmentation")OR("vitiligo"and"NB-UVB")OR("vitiligo"and"melanocytes")OR("melanocytes"and"activation"and"NB-UVB")OR("melanocytes"and"proliferation"and"NB-UVB")OR("melanocytes"and"differentiation"and"NB-UVB"),and based on the literature collected,we summarize current understanding of the specific repigmentation process,as well as the pathways and factors involved.

MC activation

MSCs and/or MC precursors in the hair follicle bulge region lack expression of melanogenesis-related enzymes,and their activation involves the process of initiating the synthesis of proteins and enzymes required for melanin production,6where the signalings and factors from keratinocytes(KCs)are essential.The activation of melanin production requires regulation of the Pax3-Mitf transcription network.Under normal conditions,TGF-β signaling expressed by KCs can inhibit the Pax3-Mitf network,thereby maintaining MCs in an undifferentiated and quiescent state.However,the activation of p53 induced by UVB can inhibit the expression of TGF-β by epidermal KCs,which allows MCs to respond to external melanogenic stimuli and begin to synthesize melanogenesis-related proteins and enzymes that initiates activation procedures.7In addition to its role in inhibiting TGF-β,p53also stimulates KCs to release paracrine/growth factors that activate melanogenesis,including α-melanocyte-stimulating hormone(α-MSH),which is a potent inducer of microphtalmia-associated transcription factor,and kit ligand.8P53and its downstream effectors including α-MSH act synergistically to activate MCs,thereby initiating the repigmentation process.

Wnt/β-catenin is another major signaling pathway that activates the repigmentation process and is required for the development of MCs.Using in vitro experiments performed on the immortalized MC progenitor cell line iMC23and in vivo experiments performed in Dct-LacZ mice,researchers confirmed that Wnt/β-catenin signaling pathway activates and promotes the differentiation of MSCs.9UVB irradiation induces Wnt activation,resulting in stabilizing the β-catenin/Lymph enhancer complex and in transactivating downstream target genes(such as microphtalmia-associated transcription factor),ultimately promoting the fate determination and the differentiation of MCs.10

MC migration(decoupling-movementrecoupling)

The migration of MCs is different in perifollicular and marginal repigmentation patterns induced by NB-UVB.The perifollicular repigmentation pattern is the result of migration of MCs present in hair follicular bulge.However,the marginal repigmentation pattern seems to be the result of inward migration of MCs present in the nonlesional epidermis.11In this review,we only introduce the migration of MCs in the perifollicular repigmentation pattern since it’s the primary repigmentation pattern induced by NB-UVB.

After activation,MSCs and/or MC precursors located in the hair bulge region migrate upwards to the epidermis to replace the destroyed MCs,which accompanied by their proliferation and differentiation.However,MCs are tightly linked to adjacent KCs and basement membranes,so decoupling of MCs from the basement membrane and from KCs is essential for subsequent cell movement.The process of MC migration is accompanied by dynamic changes in factors that mediate cell-cell adhesion.

MC decoupling from KCs

The primary mediator of adhesion between MCs and KCs is E-cadherin.Researchers found that the mRNA levels of E-cadherin in the lesional specimens from non-segmental vitiligo patients were significantly decreased when compared with the perilesion and normal skin,12which suggested that decreased cell adhesion resulting from reduced E-cadherin may be a favorable condition for the loss of MCs in the lesional skin of vitiligo patients.In addition,they also showed that UVB exposure upregulated E-cadherin.12During wound healing,E-cadherin expressed in KCS of wound area can induce MCs to migrate from surrounding normal skin into the wound area.13Therefore,it is reasonable to speculate that increased expression of E-cadherin in KCs might induce migration of MCs to the lesion area during the irradiation of UVB.

MC decoupling from the basement membrane

In order to migrate,MCs must break through extracellular matrix barriers,and therefore,timely degradation of the extracellular matrix is an essential condition for cell migration.Integrins are a class of proteins known to mediate the attachment of cells to the extracellular matrix.Changes in the expression of integrin lead to decreased cell-matrix adhesion and thus facilitate cell movement.Studies have shown that MCs express β1,α2,α3,α5,α6,αvintegrin subunits,and αvβ3heterodimers.During the repigmentation process,changes in the expression of integrin subunits in MC precursors can reduce cell adhesion to the basement membrane and coordinate cell dissociation.14

Laminin is the major non-collagen component of the basement membrane and provides strong attachments between cells and basement membranes by interacting with cell surface receptors such as β1and β4integrin.MCs migrate along the basement membrane via the binding of integrin α6β1or α3β1to laminin.A recent study developed a mouse model for the KC-specific deletion of laminin γ1 chain(Lamc1EKO),which showed delayed skin pigmentation despite the fact that the number of MCs in the skin was normal.15This result indicates that deletion of the laminin γ1chain hinders the migration of MCs,suggesting that laminin is involved in decoupling and/or recoupling of MCs to the basement membrane.

MC movement

The migration of MCs may follow the four characteristic mechanisms of movement:(a)Chemotaxis,(b)Chemokinesis,(c)Haptotaxis(increased migration on a particular matrix molecule,increased in pace with higher concentration),(d)Random motion.In human MCs in culture,the baseline random movement is very slow.Four MC mitogens,leukotriene C4,ET-1,basic fibroblast growth factor(bFGF),and stem cell factor,stimulate the migration of MCs by chemokinesis or chemotaxis.

It has been speculated that one of the mechanisms that mediate the direction of migration of MCs may be changes in the expression of adhesion molecules in the vertical and horizontal directions of hair follicles.Kovacs et al.16used immunohistochemistry to evaluate E-cadherin levels in tissue samples collected from vitiligo patients after punch grafts.They found that levels of E-cadherin were strongly positive in all epidermal layers of the donor samples,while the overall expression of E-cadherin was reduced in the repigmented skin samples,suggesting that a gradual enlargement of the intercellular space caused by decreased E-cadherin reactivity may be one of the mechanisms that mediate MCs migration.

Tetraspanins CD9and CD151are involved in cell movement and intercellular adhesion of several types of cells including MCs and KCs.MCs express tetraspanins CD9and CD151at their intercellular contacts and at the tips of their dendrites.A study using blocking monoclonal antibodies against CD9and CD151showed no significant effect on cell–extracellular matrix adhesion,whereas the migration of MCs was significantly enhanced,demonstrating that tetraspanins affect the migration ability of MCs.17bFGF is secreted by KCs,and activation of the bFGF-induced extracellular regulated protein kinases(ERK)and PI3K/Akt-Rac1-FAK-JNK signaling pathways leads to cytoskeleton reorganization,thereby contributing to MC migration.18

Matrix metalloproteinases(MMPs)degrade matrix proteins and non-matrix proteins,and the main functions of MMP-2and MMP-9are to degrade proteins in the extracellular matrix.Significant decreases in the activities of MMP-2and/or MMP-9can reduce the migration of MC precursors from the outer root sheath or the migration of MCs from the edge of vitiligo lesions to the depigmented epidermis,thereby inhibiting the repigmentation process.19On the other hand,the increased expression and activity of MMPs promotes the migration of MCs.Focal adhesion kinase(FAK)is closely related to cell migration.Increases in MMP-2activity and in phosphorylated FAK(p125FAK)expression by MCs have been observed in supernatants of NB-UVB irradiated MCs.The NB-UVBirradiation-induced migration of MCs was significantly inhibited by the addition of a p125FAK inhibitor(herbimycin-A)or a MMP-2inhibitor(GM6001).20These results indicate that p125FAK and MMP-2play important roles in the NB-UVB-induced migration of MCs.

MCs recoupling and relocalization

Upon reaching their destination,MCs extend their dendrites and re-establish multiple contacts and adhesion relationships with KCs and the basement membrane,at which time the proliferation of MCs is reduced,indicating that KCs control the growth of MCs and the expression of cell surface receptors by direct cell-cell contacts or by paracrine factors.UV irradiation induces the expression of pro-inflammatory cytokines in KCs.Among them,IL-1β plays a role in up-regulating the secretion of the matricellular protein nephroblastoma overexpressed gene(NOV/CCN3).Studies performed on normal MCs have shown that CCN3ensures the adhesion of MCs to the epidermal microenvironment.21MCs increase CCN3 secretion after stimulation by interleukin(IL)-1β,and it continued for48h.Besides,the depletion of IL-1β by using neutralizing antibodies decreases CCN3in cocultures.Therefore,IL-1β secreted by KCs promotes the adhesion of MCs through CCN3.21

In human reconstituted skin,the increased adhesion to collagen IV found in the basement membrane is regulated by discoidin domain receptor1,a tyrosine kinase receptor for several collagens.22The knockdown of DDR1in MCs reduces their adhesion to collagen IV and promotes the relocalization of cells from the basement membrane to the upper layer of the epidermis,mimicking the phenotype of CCN3depletion.In UV-irradiated skin,DDR1-collagen IV binding may be critical for the localization of MCs.In general,the CCN3-DDR1-collagen axis is involved in the process of adhesion and localization of MCs in the vitiligo repigmentation process.

MC proliferation

NB-UVB irradiation induces an increase in the number of MSCs(c-KIT–/DCT+)and melanoblasts(c-KIT+/DCT+)located in the hair follicle bulge region,while different patterns of repigmentation have different characteristics,the follicular repigmentation pattern is associated with the proliferation of MCs denoted by an increase in c-KIT expression,and the diffusion repigmentation pattern is mainly characterized by the migration of MCs.23The marginal repigmentation pattern showed an increased expression of markers of MC proliferation and migration that rarely appeared in non-repigmented regions.

The secretion of ET-1by KCs stimulates the synthesis of DNA(ie,proliferation)in MCs.Supernatants of NB-UVBirradiated KCs significantly increased the number of MCs and an obvious increase in ET-1was also detected in those supernatants.Treatment with an endothelin-B receptor antagonist(BQ788)remarkably reduced the proliferation of MCs induced by NB-UVB,suggesting an important role for ET-1in regulating MC proliferation.20

In addition,many KC-derived factors have also been shown to promote the proliferation of MCs,including α-MSH,leukemia inhibitory factor,stem cell factor,granulocyte-macrophage colony-stimulating factor,bFGF,HGF,and so on.These factors,produced and released by KCs,are involved in regulating the proliferation and differentiation of epidermal MCs via receptormediated signaling pathways.However,several studies have shown that MCs migrate and/or differentiate rather than proliferate during skin repigmentation,and even the proliferation of MCs is inhibited by UV irradiation.24-25

GLI1,an effector of the Sonic Hedgehog pathway,is required for MC proliferation and for melanoma growth and metastasis.Goldstein et al.26showed that the GLI1 was upregulated in both MCs and KCs of NB-UVB-treated and untreated vitiligo bulge,indicating that this may be an important bulge-associated gene involved in the response to the NB-UVB.Interestingly,the Sonic Hedgehog and Wnt/β-catenin pathways can interact through GLI1’s regulation of both nuclear localization and transcriptional activity of β-catenin.This suggests that in vitiligo patients,NB-UVB can activate different signaling pathways which interact with each other to induce the mobilization of MCs.

MC differentiation

Melanogenesis,melanosome transfer,dendrite formation,and morphological changes are all indicators of MC differentiation and functional maturation.It was observed that MCs gradually mature in function and morphology during their upward migration to the epidermis,and these events represent the differentiation process of MCs.At the beginning of repigmentation,MCs already exist in hair follicles but they have little or no ability to produce melanin and have almost no dendrites.When those MCs enter the epidermis,their ability to produce melanin becomes stronger and they produce longer dendrites,indicating that their melanogenic activity is enhanced and they gradually differentiate into functional MCs during their migration to the epidermis.The newly emerging MCs in repigmented skin regions have different morphological characteristics–some have large cell bodies and long branches,some have small cell bodies and shorter,finer dendrites,and some have flat or high cell bodies.27

The upper infundibulum of the hair follicle appears to be an important area of differentiation,where TYR+MCs(a marker of differentiated MCs)were found,while there was no TYR expression in the bulge region or lower infundibulum,suggesting that MC precursors have a differentiation ability in the upper infundibulum.These MCs and MC precursors continue to migrate to the epidermis.After reaching the epidermis,they continue to proliferate,migrate,and differentiate into mature MCs,eventually repigmenting the lesions.6

A part of NB-UVB-induced differentiation of MCs may be explained by NB-UVB-activated vitamin D3synthesis.28The photochemical effect of NB-UVB makes7-dehydrocholestero converted to pre-vitamin D3in the stratum spinosum and stratum basale,resulting in the synthesis of Vitamin D3which induces immature MCs in the bulge region of hair follicles to produce melanin by stimulating their differentiation and expression of endothelin B receptor.28

Yamada et al.10found that UVB irradiation induces a strong expression of Wnt7a(a classical Wnt signaling ligand)in hair follicle stem cells,in KCs in the outer root sheath and in KCs in epidermis,but not in MSCs.They also showed that treatment of the dorsal skin of mice with IWR-1(Wnt inhibitor1)and a Wnt7a siRNA inhibited the nuclear translocation of β-catenin,reduced the number of epidermal MCs,and decreased the mRNA expression level of melanogenic enzymes after UVB irradiation.These results suggest that Wnt7a secreted by neighboring cells can trigger the differentiation of MSCs during UVBinduced epidermal pigmentation by activating the Wnt/β-catenin signaling pathway.10

Conclusions

This review has two limitations:(1)it focuses on the activation,migration,proliferation and differentiation of MCs,however,due to the lack of relevant research,the content of NB-UVB involved in some parts is relatively small.(2)The content of this review is not comprehensive enough,and the references need to be expanded.The overall innovation of this paper needs to be improved.NBUVB irradiation induces various growth factors and cytokines,adhesion molecules signaling pathways that regulate MCs activation,migration,proliferation,differentiation,and then finally repigment vitiligo skin lesions.Certainly,P53and Wnt signaling pathways mediate activation,proliferation,and differentiation of MCs,and E-cadherin,integrin,laminin,tetraspanins,MMP,CCN3,and DDR1mediate the migration of MCs.However,it is still unclear how these factors and signaling pathways coordinately control MCs and how they change at different stages of repigmentation.

Acknowledgements

The authors are very grateful to Professor V.J.Hearing for help with the English-language editing.The work was supported by the National Nature Science Foundation of China(No.81673078).