Dissecting The Molecular Mechanisms Involved In Melanocyte Migration And Melanosome Degradation

Backgrounds and ObjectivesVitiligo is the most common depigmentary disorder of the skin,affecting 0.5-2%of the population worldwide.Vitiligo is predominantly characterized by white patches of the skin,due to the chronic and progressive loss of melanocytes from the epidermis and the follicular reservoir,which may cause significant social and psychological impacts on patients,especially teenagers.Current therapeutic modalities for vitiligo are aimed at arresting the progression of the disease and stimulating skin repigmentation(a process that recruits existing melanocytes and their precursors or stem cells back to the depigmented skin lesions).UVB-based phototherapy has been clinically proven to be efficient for inducing repigmentation.Different skin repigmentation patterns,such as perifollicular,marginal,diffuse and combined patterns,have been observed in vitiligo patients who have undergone UVB-based phototherapy,suggesting that the replenishment of melanocytes probably comes from diverse sources in the epidermis and/or in hair follicles.Over the past decades,efforts have been devoted to investigating whether dormant melanocyte stem cells that reside in the bulge region of hair follicles are awakened to participate in the perifollicular repigmentation of the skin.In contrast,much less is understood about the induction of marginal repigmentation of the skin,which possibly relies on the activation and migration of melanocytes existing at the border of the depigmented vitiligo macules.The migration of melanocytes released from the microenvironment of basal and suprabasal layers in the pigmented human epidermis in response to UVB phototherapy raises intriguing questions.The critical issues that remain to be resolved whether the migrating cells can secrete one or more proteolytic enzymes that enable melanocytes to move out by dissolving the adhesive structures between cells and basement menbrane.Several studies have demonstrated that the expression of matrix metalloproteases(MMPs)in melanocytes appears to be correlated with tissue remodeling or replasticity,little is known about whether MMP9 affects melanocyte migration in vitiligo repigmentation.This study attempts to explore the possible molecular mechanism of marginal repigmentation in vitiligo induced by UVB.In the acquired pigmentary disorders,the molecular mechanism of melanosome degradation and the changes in biological properties in keratinocytes remain elusive.In the physiological condition,melanosomes are synthesized by melanocytes located in the basal layer of the epidermis and transferred to neighboring keratinocytes for photoprotection.At last,melanosomes are delivered to the granular layer and completely degraded following by the keratinocytes terminal differentiation.Abnormal melanosome degradation can lead to the development of hyperpigmented diseases.Cathepsin L2(CTSL2)is a lysosomal cysteine protease and is expressed in macrophages,thymus,testes,corneal epithelium and epidermis.Some studies have demonstrated that melanosomes are completely degraded in corneocytes of lightly pigmented skin,while some melanosomes have not been degraded in the cornified layer of darkly pigmented skin.A further study has shown that the expression of CTSL2 m RNA in the epidermis of Caucasian was 7.5 times higher than the darkly pigmented skin.Therefore,we speculate that the lysosomal cysteine protease CTSL2may be involved in the degradation of melanosomes.This study includes three parts:1.(hypopigmented dermatosis)To explore the possible molecular mechanism involved in the pattern of marginal repigmentation in Vitiligo.We had treated melanocytes with single or repeated exposures to establish the UV irradiation model in vitro to detect the levels of p53-TRPM1/mi R-211-MMP9 gene and the ability of melanocyte migration.2.We developed the mi R-211 mimic and p53-lentiviral vector transfection model in vitro to examine the regulation of mi R-211 on MMP9 and the validity of p53-TRPM1/mi R-211-MMP9axis.3.(hyperpigmented dermatosis)To investigate the possible mechanism of melanosome degradation in hyperpigmented diseases and study whether CTSL2 was involved in the process of melanosomes degradation,Fontana-Masson and TEM were used to analysis the ultrastructural changes of melanin granules and melanosomes in pigmented skin tissues.Methods and Results1.The primary human epidermal melanocytes were isolated from healthy juvenile foreskin tissues.Cells were treated with single or repeated exposures to UVB to establish the cell model in vitro.After irradiation,the changes of the p53-TRPM1/mi R-211-MMP9 axis were measured by RT-q PCR and western blotting.Transwell was performed to detect the ability of migration.GM6001,a matrix metalloproteinase inhibitor,was added to the UVB-exposed group and UVB-unexposed group to measure the migration of melanocytes.The results revealed that the expression levels of p53 and MMP9 m RNAs and proteins were upregulated in a UVB dose-dependent manner(8.75-70 m J/cm~2).TRPM1 m RNA and protein levels were increased at lower doses(8.75-17.5 m J/cm~2)but were reduced at higher doses of UVB(35-70 m J/cm~2).To simulate clinical settings with multiple exposures to UVB,cells were treated with repeated small UVB exposures.The results showed that the upregulation of p53 and MMP9 m RNA and protein levels,whereas m RNA and protein levels of TRPM1 were significantly decreased in cells repeatedly exposed to UVB compared with the UVB-unexposed control.The Transwell assays found that the melanocyte migration was significantly increased in cells that exposed to UVB compared with the control group.Moreover,melanocytes treated with UVB irradiation induced a higher migration,which could be neutralized by GM6001treatment.2.The chemically synthesized mi RNA mimic and p53-GFP lentiviral vectors were transfected into melanocytes to determine the effects of mi R-211 and p53 on the p53-TRPM1/mi R-211-MMP9 axis.Western blotting and q PCR were performed to assess the expression of MMP9 and relevent genes.Transwell was performed to detect the ability of migration.These results found that the m RNA and protein levels of MMP9 were significantly suppressed after transfection with the mi R-211 mimic and the migration was significantly reduced in the mi R-211 mimic transfected cells;Melanocytes were transfected with p53-GFP lentiviral vectors.Western blotting and q PCR analysis demonstrated that the levels of p53 and MMP9 were significantly increased in cells transfected with the p53-GFP lentiviral vector compared with those levels in the GFP vector control group,which was concomitant with decreases in TRPM1 and mi R-211 levels.The Transwell assays showed that melanocyte migration was significantly increased in cells that had a lentiviral vector-mediated overexpression of p53 compared with the control group.Moreover,the overexpression of p53 induced a higher migration of melanocytes,which could be neutralized by GM6001 treatment.3.Fontana-Masson staining and TEM were performed to observe the pigmentary skin tissues,the data showed that a large number of melanin granules were deposited in the lesions,while the linear deposition of melanin granules was only seen in the basal layer of the normal skin.TEM showed that the percentage of damaged melanosomes was much higher in the normal skin than in the SK lesions.RT-PCR and enzyme activity revealed that the m RNA expression and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin.Conclusions1.Single and repeated exposures to UVB can induce an increase in endogenous p53,which may activate the p53-TRPM1/mi R-211-MMP9 axis to promote melanocyte migration by upregulating the expression of MMP9.2.mi R-211 exerts an anti-migration effect via the suppression of MMP9;3.The over-expression of exogenous p53 reconfirmed the validity of the p53-TRPM1/mi R-211-MMP9 axis by inhibiting the expression of TRPM1/mi R-211 and upregulating the expression of MMP9 to promote the migration of melanocytes,this process may be involved in vitiligo migration repigmentation.4.CTSL2 may participate in the degradation of melanosomes in keratinocytes.