Melanocytes and keratinocytes morphological changes in vitiligo patients. A histological, immunohistochemical and ultrastructural analysis.

Vitiligo is an idiopathic acquired chronic stigmatizing disease. It is a pigmentary disorder that affects the skin and the mucous membranes, and it is characterized by well-circumscribed, depigmented milky white macules and patches. It has an estimated prevalence of 0.5-2% of the population worldwide. In the previous studies, several mechanisms such as autoimmune, oxidative stress, genetic factors, melanocytorrhagy, and neural hypothesis have been suggested for vitiligo pathogenesis.We aimed to assess the morphological changes of epidermal melanocytes and keratinocytes in patients with vitiligo. This aim will be fulfilled by histological, ultrastructural, and immunohistochemical analysis of skin biopsies from lesioned and non-lesioned sites in vitiligo patients.The study was carried out on 15 selected patients with stable vitiligo vulgaris but not receiving treatment in the last year and they fulfilled our inclusion criteria.Biopsies were taken from lesioned and non-lesioned sites in the same vitiligo patients, and they are processed for examinations by LM (using Hx & E, and Masson Fontana stain), immunohistochemical analysis (using Melan-A, E-cadherin, and caspase-3), and TEM (to demonstrate the ultra-structures).By LM, staining with Hx & E, lesioned skin in vitiligo patients showed hyperkeratosis, basal vacuolization, acanthosis with an increase in the epidermal thickness, ballooning of keratinocytes, and spongiosis. Regarding melanocytes, we observed a few numbers of melanocytes, also we detected some basal epidermal cells contain brown melanin granules. Using Fontana-Masson stain, we found that the melanin pigment is present in both lesioned and non-lesioned skin of vitiligo patients. We confirmed the presence of melanocytes in the lesioned skin by the immunohistochemical staining with Melan-A. The epidermal cells in lesioned skin of vitiligo patients showed weak positive expression of E-cadherin between them and an increase in the number of apoptotic Caspase-3 positive cells. BY TEM, the lesioned skin in vitiligo patients showed that the keratinocytes and melanocytes had various degenerative changes, disturbance of desmosomes in between keratinocytes, and absence of melanosomes in the keratinocytes. The detected melanocytes were degenerated and contained some melanosomes, melanin granules, and autophagosomes.We concluded that vitiligo pathogenesis is a combination of several factors and cannot be explained by only one mechanism. The pathology in the lesioned vitiliginous skin is a combination of several degenerative changes in keratinocytes, and melanocytes.

In vitro disease modeling of oculocutaneous albinism type 1 and 2 using human induced pluripotent stem cell-derived retinal pigment epithelium.

Oculocutaneous albinism (OCA) encompasses a set of autosomal recessive genetic conditions that affect pigmentation in the eye, skin, and hair. OCA patients display reduced best-corrected visual acuity, reduced to absent ocular pigmentation, abnormalities in fovea development, and/or abnormal decussation of optic nerve fibers. It has been hypothesized that improving eye pigmentation could prevent or rescue some of the vision defects. The goal of the present study was to develop an in vitro model for studying pigmentation defects in human retinal pigment epithelium (RPE). We developed a "disease in a dish" model for OCA1A and OCA2 types using induced pluripotent stem cells to generate RPE. The RPE is a monolayer of cells that are pigmented, polarized, and polygonal in shape, located between the neural retina and choroid, with an important role in vision. Here we show that RPE tissue derived in vitro from OCA patients recapitulates the pigmentation defects seen in albinism, while retaining the apical-basal polarity and normal polygonal morphology of the constituent RPE cells.

Novel strategy for applying hierarchical density-based spatial clustering of applications with noise towards spectroscopic analysis and detection of melanocytic lesions.

Advancements in dermoscopy techniques have elucidated identifiable characteristics of melanoma which revolve around the asymmetrical constitution of melanocytic lesions consequent of unfettered proliferative growth as a malignant lesion. This study explores the applications of hierarchical density-based spatial clustering of applications with noise (HDBSCAN) in terms of the direct diagnostic implications of applying agglomerative clustering in the spectroscopic analysis of malignant melanocytic lesions and benign dermatologic spots. 100 images of benign (n = 50) and malignant moles (n = 50) were sampled from the International Skin Imaging Collaboration Archive and processed through two separate Python algorithms. The first of which deconvolutes the three-digit tupled integer identifiers of pixel color in image composition into three separate matrices corresponding to the red, green and blue color channel. Statistical characterization of integer variance was utilized to determine the optimal channel for comparative analysis between malignant and benign image groups. The second applies HDBSCAN to the matrices, identifying agglomerative clustering in the dataset. The results indicate the potential diagnostic applications of HDBSCAN analysis in fast-processing dermoscopy, as optimization of clustering parameters according to a binary search strategy produced an accuracy of 85% in the classification of malignant and benign melanocytic lesions.

Melanocyte progenitor cells reside in human subcutaneous adipose tissue.

Based on the assumption that some progenitor cells in an organ might reside in neighboring adipose tissue, we investigated whether melanocyte progenitor cells reside in human subcutaneous adipose tissue. First, we examined the expression of human melanoma black 45 (HMB45) and microphthalmia-associated transcription factor (MITF) in undifferentiated adipose-derived stem cells (ADSCs) by immunostaining, RT-PCR, and western blotting. These two markers were detected in undifferentiated ADSCs, and their expression levels were increased in differentiated ADSCs in melanocyte-specific culture medium. Other melanocytic markers (Melan A, MATP, Mel2, Mel EM, tyrosinase, KIT, and PAX3) were also detected at variable levels in undifferentiated ADSCs, and the expression of some markers was increased during differentiation into the melanocyte lineage. We further showed that ADSCs differentiated in melanocyte-specific culture medium localized in the basal layer and expressed tyrosinase and HMB45 in a 3D epidermal culture system. Melanin deposits were also induced by ultraviolet-light-B (UVB) irradiation. These results demonstrate that melanocyte progenitor cells reside in human subcutaneous adipose tissue and that these cells might have the potential to differentiate into mature melanocytes. Melanocyte and keratinocyte progenitors residing in human subcutaneous tissue can be used for the treatment of skin diseases and skin rejuvenation in the future.

PINK1 in normal human melanocytes: first identification and its effects on H2 O2 -induced oxidative damage.

BACKGROUND: Oxidative stress plays an important role in initiating the destruction of melanocytes, which could be one possible mechanism of vitiligo. PINK1 is an outer membrane protein of mitochondria, which protects many cells from oxidative stress through regulating mitochondrial function. However, the role of PINK1 and its effects on oxidative damage in melanocytes have not been elucidated. AIM: To investigate the expression and effects of PINK1 on oxidative stress in human melanocytes. METHODS: Quantitative reverse transcription-PCR and western blot analysis were used to analyse the expression of PINK1 in PIG1 melanocyte and gene downregulation models. Levels of cell viability, cell apoptosis and intracellular reactive oxygen species (ROS), mitochondrial morphology, mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (MPTP) opening were measured in PIG1 models transfected with PINK1 small interfering RNA with or without hydrogen peroxide (H2 O2 ). RESULTS: We first observed the expression of PINK1 in human PIG1 melanocytes and found that downregulation of PINK1 made melanocytes more sensitive to oxidative stress induced by H2 O2 , with more cell apoptosis and increased intracellular ROS. Meanwhile, downregulation of PINK1 caused morphological changes in mitochondria, decreased the MMP and increased MPTP opening. CONCLUSIONS: Our study found PINK1 plays an essential role in protecting human melanocytes from oxidative stress.

Three-dimensional structure analysis of melanocytes and keratinocytes in senile lentigo.

Senile lentigo or age spots are hyperpigmented macules of skin that commonly develop following long-term exposure to ultraviolet radiation. This condition is caused by accumulation of large numbers of melanosomes (melanin granules) produced by melanocytes within neighboring keratinocytes. However, there is still no consensus regarding the melanosome transfer mechanism in senile lentigo. To date, most pathohistological studies of skin have been two-dimensional and do not provide detailed data on the complex interactions of the melanocyte-keratinocyte network involved in melanosome transfer. We performed a three-dimensional reconstruction of the epidermal microstructure in senile lentigo using three different microscopic modalities to visualize the topological melanocyte-keratinocyte relationship and melanosome distribution. Confocal laser microscopy images showed that melanocyte dendritic processes are more frequently branched and elongated in senile lentigo skin than in normal skin. Serial transmission electron micrographs showed that dendritic processes extend into intercellular spaces between keratinocytes. Focused ion beam-scanning electron micrographs showed that dendritic processes in senile lentigo encircle adjacent keratinocytes and accumulate large numbers of melanosomes. Moreover, melanosomes transferred to keratinocytes are present not only in the supranuclear area but throughout the perinuclear area except on the basal side. The use of these different microscopic methods helped to elucidate the three-dimensional morphology and topology of melanocytes and keratinocytes in senile lentigo. We show that the localization of melanosomes in dendritic processes to the region encircling recipient keratinocytes contributes to efficient melanosome transfer in senile lentigo.

Dermal Fibroblasts Internalize Phosphatidylserine-Exposed Secretory Melanosome Clusters and Apoptotic Melanocytes.

Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis.

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime.

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.

H2 O2 -induced oxidative stress disrupts mitochondrial functions and impairs migratory potential of human epidermal melanocytes.

Reactive oxygen species (ROS) have already been demonstrated to impede the migratory ability in non-melanocytic cell lines by depleting mitochondrial ATP production. Therefore, understanding the mitochondrial metabolic response to migration in the presence of ROS should be a key to understanding repigmentation in vitiligo. This study aimed to investigate the energy mechanism associated with the ROS-mediated attenuation of melanocyte migration. After melanocytes were pretreated with H2 O2 , their ATP production, migratory ability, ultrastructural changes and Mitochondrial Permeability Potential were analysed. The results showed that, in parallel with the decreased ATP production, the migratory ability of melanocytes was significantly inhibited by oxidative stress. Supplementation with exogenous ATP reversed the suppressed ATP-dependent migration of melanocytes. Melanocytes were then stressed with H2 O2 and Agilent Whole Human Genome microarray analysis identified 763 up-regulated mRNAs and 1117 down-regulated mRNAs. Among them, 11 of the encoded proteins were involved in mitochondrial ATP production and their expression levels were verified. The decreased expression of NADH dehydrogenase 2(ND2) , cytochrome c oxidase 1(COX1) and cytochrome c oxidase 3(COX3) was shown to be involved in the depletion of mitochondrial ATP production, which was coupled with the impaired migratory potential. These results indicate that the migration of melanocytes relies heavily on an inexhaustible supply of ATP from mitochondria.

Aberrant ETS-1 signalling impedes the expression of cell adhesion molecules and matrix metalloproteinases in non-segmental vitiligo.

Cell adhesion is a complex process that involves multiple molecules on the cell surface (ie cell adhesion molecules [CAMs]), surrounding cells and extracellular matrix (ECM). Repigmentation in vitiligo is dependent on the ECM remodelling and cellular migration, primarily attributed to the transcriptional activation of matrix metalloproteinases (MMPs). In this study, we aimed to demonstrate the role of ETS-1 signalling in the regulation of MMPs and CAMs. Therefore, we studied the expression of ETS-1, MMPs (MMP-2, MMP-9) and CAMs including E-cadherin, ITGA-1 and ICAM-1 in vitiligo (both active and stable) ex vivo. Further, we compared melanocyte morphology and their adhesion towards collagen IV and laminin between control and vitiligo groups in vitro. Also, we silenced ETS-1 in melanocytes cultured from control skin and observed post-silencing effect on above-mentioned MMPs and CAMs. We perceived absent ETS-1 and significantly reduced CAMs and MMPs in vitiligo compared with normal skin. Scanning electron microscopy (SEM) revealed a translucent material surrounding individual melanocytes in stable vitiligo and controls, whereas active vitiligo melanocytes demonstrated loss of this extracellular substance. Adhesion assays revealed significantly decreased binding of cultured melanocytes to collagen IV and laminin V plates in both stable and active vitiligo. Importantly, ETS-1 silencing resulted in significantly reduced expression of CAMs and MMPs. In conclusion, absent ETS-1 expression in both stable and active non-segmental vitiligo seems to impede the expression of CAMs, apart from MMPs, probably leading to progressive depigmentation in active disease and absence of spontaneous repigmentation in stable disease.

Characterization of Induced Pluripotent Stem Cells from Human Epidermal Melanocytes by Transduction with Two Combinations of Transcription Factors.

OBJECTIVE: In order to generate induced Pluripotent Stem Cells (iPSCs) more efficiently, it is crucial to identify somatic cells that are easily accessible and possibly require fewer factors for conversion into iPSCs. METHODS: Human epidermal melanocytes were transduced with lentiviral vectors carrying 3 transcription factors (OCT-4, KLF-4 and c-MYC, 3F) or 4 transcription factors (OCT-4, KLF-4, c-MYC and SOX-2, 4F). Once the clones had formed, assays related to stem cell pluripotency, including alkaline phosphatase staining, DNA methylation levels, expression of stem cell markers and ultrastructure analysis were carried out. The iPSCs obtained were then induced to differentiate into the cells representing the three embryonic layers in vitro. RESULTS: Seven days after the transduction of epidermal melanocytes with 3F or 4F, clones were formed that were positive for alkaline phosphatase staining. Fluorescent staining with antibodies against OCT-4 and SOX-2 was strongly positive, and the cells showed a high nucleus-cytoplasm ratio and active karyokinesis. No melanosomes were found in the cytoplasm by ultrastructural analysis. There were obvious differences in DNA methylation levels between the cloned cells and their parental cells. However, there was not a significant difference between 3F or 4F transfected clonal cells. Meanwhile, the iPSCs successfully differentiated into the three germ layer cells in vitro. CONCLUSION: Human epidermal melanocytes do not require ectopic SOX-2 expression for conversion into iPSCs, and may serve as an alternative source for deriving patient-specific iPSCs with fewer genetic elements.

BAP1-inactivated melanocytic tumor with preserved BAP1 expression? Morphology to the rescue!

BAP1-inactivated melanocytic tumors typically present with distinctive histopathological changes and loss of nuclear BAP1 protein expression. Rare cases exhibit the typical morphology but with preserved expression of BAP1. In the current issue of Journal of Cutaneous Pathology, Linos et al. describe such a case and provide a comprehensive molecular-genetic exploration to explain such a phenomenon.

Integrin-linked kinase regulates melanosome trafficking and melanin transfer in melanocytes.

Melanosomes are melanin-containing organelles that provide pigmentation and protection from solar UV radiation to the skin. In melanocytes, melanosomes mature and traffic to dendritic tips, where they are transferred to adjacent epidermal keratinocytes through pathways that involve microtubule networks and the actin cytoskeleton. However, the role of scaffold proteins in these processes is poorly understood. Integrin-linked kinase (ILK) is a scaffold protein that regulates microtubule stability and F-actin dynamics. Here we show that ILK is necessary for normal trafficking of melanosomes along microtubule tracks. In the absence of ILK, immature melanosomes are not retained in perinuclear regions, and mature melanosome trafficking along microtubule tracks is impaired. These deficits can be attenuated by microtubule stabilization. Microtubules are also necessary for the formation of dendrites in melanocytes, and Ilk inactivation reduces melanocyte dendricity. Activation of glycogen synthase kinase-3 (GSK-3) interferes with microtubule assembly. Significantly, inhibition of GSK-3 activity or exogenous expression of the GSK-3 substrate collapsin response mediator protein 2 (CRMP2) in ILK-deficient melanocytes restored dendricity. ILK is also required for normal melanin transfer, and GSK-3 inhibition in melanocytes partially restored melanin transfer to neighboring keratinocytes. Thus, our work shows that ILK is a central modulator of melanosome movements in primary epidermal melanocytes and identifies ILK and GSK-3 as important modulators of melanin transfer to keratinocytes, a key process for epidermal UV photoprotection.

Extracellular vesicles released by melanocytes after UVA irradiation promote intercellular signaling via miR21.

Skin pigmentation is controlled by complex crosstalk between melanocytes and keratinocytes and is primarily induced by exposure to ultraviolet (UV) irradiation. Several aspects of UVA-induced signaling remain to be explored. In skin cells, UVA induces plasma membrane damage, which is repaired by lysosomal exocytosis followed by instant shedding of extracellular vesicles (EVs) from the plasma membrane. The released EVs are taken up by neighboring cells. To elucidate the intercellular crosstalk induced by UVA irradiation, EVs were purified from UVA-exposed melanocytes and added to keratinocytes. Transcriptome analysis of the keratinocytes revealed the activation of TGF-ß and IL-6/STAT3 signaling pathways and subsequent upregulation of microRNA (miR)21. EVs induced phosphorylation of ERK and JNK, reduced protein levels of PDCD4 and PTEN, and augment antiapoptotic signaling. Consequently, keratinocyte proliferation and migration were stimulated and UV-induced apoptosis was significantly reduced. Interestingly, melanoma cells and melanoma spheroids also generate increased amounts of EVs with capacity to stimulate proliferation and migration upon UVA. In conclusion, we present a novel intercellular crosstalk mediated by UVA-induced lysosome-derived EVs leading to the activation of proliferation and antiapoptotic signaling via miR21.

Autophagy induction can regulate skin pigmentation by causing melanosome degradation in keratinocytes and melanocytes.

Autophagy regulates cellular turnover by disassembling unnecessary or dysfunctional constituents. Recent studies demonstrated that autophagy and its regulators play a wide variety of roles in melanocyte biology. Activation of autophagy is known to induce melanogenesis and regulate melanosome biogenesis in melanocytes. Also, autophagy induction was reported to regulate physiologic skin color via melanosome degradation, although the downstream effectors are not yet clarified. To determine the role of autophagy as a melanosome degradation machinery, we administered several autophagy inducers in human keratinocytes and melanocytes. Our results showed that the synthetic autophagy inducer PTPD-12 stimulated autophagic flux in human melanocytes and in keratinocytes containing transferred melanosomes. Increased autophagic flux led to melanosome degradation without affecting the expression of MITF. Furthermore, the color of cell pellets of both melanocytes and keratinocytes was visibly lightened. Inhibition of autophagic flux by chloroquine resulted in marked attenuation of PTPD-12-induced melanosome degradation, whereas the expression of melanogenesis pathway genes and proteins remained unaffected. Taken together, our results suggest that the modulation of autophagy can contribute to the regulation of melanocyte biology and skin pigmentation.

Clinical & ultrastructural evaluation of the effect of fractional CO2 laser on facial melasma.

Melasma represents the most obvious and disfiguring change of the face leading to psychological problems especially in females. Ablative lasers have also been used by many professionals to treat melasma, although there is few scientific data supporting this indication. The exact mechanism of action of ablative lasers in melasma is not yet clear enough. We aimed to evaluate the ultrastructural effect of fractional ablative CO2 (FrCo2) laser on facial melasma. Eleven melasma patients evaluated clinically by clinical modified area and severity index (MASI) score, treated by two sessions of fractional CO2 laser one month a part. Two punch biopsies of 2 mm diameter were obtained from all subjects one before and the other 3 months after treatment. All biopsies were analyzed by light and electron microscopy. Clinically, significant improvement of pigmentation and 48% reduction of (MASI) score were observed after two sessions of laser treatments. Light microscopic analysis of specimens revealed significant decrease in melanocyte count after treatment. Electron microscopic analysis of specimens after treatment revealed significant decrease in the number and size of melanocytes and significant decrease or complete absence of melanin granules in the surrounding keratinocytes compared to pre-treatment specimens. No scarring or post inflammatory hyper or hypopigmentation. We concluded that repeated application of Fractional CO2 laser on melasma skin may result in long lasting improvement due to its destructive effect on melanocytes.

Melanocyte spheroids are formed by repetitive long-term trypsinization.

BACKGROUND: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. OBJECTIVE: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. METHODS: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. RESULTS: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. CONCLUSION: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

The distribution of melanocytes and the degradation of melanosomes in fetal hair follicles.

There have been many studies about the formation, storage, transport and degradation of melanosomes in epidermal melanocytes but studies of melanocytes and melanosomes in fetal hair follicles (HFs) have been limited and ambiguous. The goal of this study was to investigate the distribution of melanocytes and the degradation of melanosomes in fetal HFs. After obtaining approval and informed consent for the study, a scalp specimen from a 5 month gestational age fetus was obtained and was divided into two parts. One part was subjected to immunohistochemical staining with the melanocyte-specific marker HMB-45 and was then observed by light microscopy to detect the distribution of melanocytes in HFs. The other part underwent conventional processing for transmission electron microcopy (TEM). Subsequently, the morphology of melanosomes in HF melanocytes and their degradation in cortical keratinocytes were observed. Immunohistochemically, scattered round melanocytes lacking dendrites were mainly observed along the outer root sheath of the lower part of the HF. A few fusiform or tri-dendritic melanocytes were located at the bottom of the hair bulbs. Significantly melanized melanocytes with multiple dendrites were concentrated in the pigmented area in the center of the hair bulbs, only above the dermal papilla. Analysis by TEM revealed melanocytes containing melanosomes at all stages of development. Autophagosomes containing stage mature IV melanosomes were observed in some melanocytes. Many phagolysosomes containing numerous melanosomes were observed in the cortical keratinocytes. Some phagolysosomes were concentrically surrounded by 3-5 layers of endoplasmic reticulum. Melanosomes that had been degraded or were being degraded in phagolysosomes in keratinocytes had lost their integrity and had become an ill-defined melanosomal dust that were arranged irregularly. Partial melanin particles were released into the cytosol. Melanocytes in different regions of fetal HFs had different morphologies and were at various stages of differentiation. Fetal HF melanocytes contained not only melanosomes at different developmental stages, but autophagosomes were seen occasionally. Melanosomes were degraded into irregular pigment particles in the phagolysosomes of cortical keratinocytes. These results provide important clues to elucidate the mechanism of melanosome biodegradation.

The physiology of melanin deposition in health and disease.

Eumelanin is the major pigment responsible for human skin color. This black/brown pigment is localized in membrane-bound organelles (melanosomes) found in specialized cells (melanocytes) in the basal layer of the epidermis. This review highlights the steps involved in melanogenesis in the epidermis and the disorders in skin pigmentation that occur when specific steps critical for this process are defective. Melanosomes, which contain tyrosinase, a major enzyme involved in melanin synthesis, develop through a series of steps in the melanocyte. They are donated from the melanocyte dendrites to the surrounding keratinocytes in the epidermis. In the keratinocytes, the melanosomes are found singly or packaged into groups, and as the keratinocytes move upward in the epidermis, the melanosomes start to degrade. This sequence of events is critical for melanin pigmentation in the skin and can be influenced by genetic, hormonal, and environmental factors, which all play a role in levels of melanization of the epidermis. The effects these factors have on skin pigmentation can be due to different underlying mechanisms involved in the melanization process leading to either hypo- or hyperpigmentary disorders. These disorders highlight the importance of mechanistic studies on the specific steps involved in the melanization process.

Effect of urea-extracted sericin on melanogenesis: potential applications in post-inflammatory hyperpigmentation.

BACKGROUND: Hyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented. METHODS: In this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed. RESULTS: The results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-ß were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components. CONCLUSION: In summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.