Skin hyperpigmentation is characterized by increased melanin synthesis and deposition that can cause significant psychosocial and psychological distress. Although several cytokine-receptor signaling cascades contribute to the formation of ultraviolet B-induced cutaneous hyperpigmentation, their possible involvement in other types of skin hyperpigmentation has never been clearly addressed. Since our continuous studies using skin specimens from more than 30 subjects with ethnic skin diversity emphasized a consistent augmentation in the expression of endothelin-1 (ET-1) and its receptor (Endothelin B receptor, ET-B) in hyperpigmented lesions, including senile lentigos (SLs), the precise function of ET-1 signaling was investigated in the present study. In line with previous studies, ET-1 significantly induced melanogenesis followed by increases in melanosome transport in melanocytes and in its transfer to keratinocytes while inhibition of ET-B function substantially depressed melanogenic ability in tissue-cultured SLs. Additionally, in agreement with a previous report that the formation of autophagosomes rather than melanosomes is stimulated according to starvation or defective melanosome production, ET-1 was found to remarkably augment the expression of components necessary for early melanosome formation, indicating its counteraction against autophagy-targeting melanosome degradation in melanocytes. Despite the lack of substantial impact of ET-1 on keratinocyte melanogenic functions, the expression of ET-1 was enhanced following melanosome uptake by keratinocytes. Taken together, our data suggest that ET-1 plays a substantial role in the development and/or maintenance of skin hyperpigmentation in reciprocal cooperation with increased melanosome incorporation.
Intracellular organelles mediate complex cellular functions that often require ion transport across their membranes. Melanosomes are organelles responsible for the synthesis of the major mammalian pigment melanin. Defects in melanin synthesis result in pigmentation defects, visual deficits, and increased susceptibility to skin and eye cancers. Although genes encoding putative melanosomal ion transporters have been identified as key regulators of melanin synthesis, melanosome ion transport and its contribution to pigmentation remain poorly understood. Here we identify two-pore channel 2 (TPC2) as the first reported melanosomal cation conductance by directly patch-clamping skin and eye melanosomes. TPC2 has been implicated in human pigmentation and melanoma, but the molecular mechanism mediating this function was entirely unknown. We demonstrate that the vesicular signaling lipid phosphatidylinositol bisphosphate PI(3,5)P2 modulates TPC2 activity to control melanosomal membrane potential, pH, and regulate pigmentation.
As interest in skin beauty increases, the development of new skin whitening agents has attracted substantial attention; however, the action mechanism of the agents developed so far remains largely unknown. Tranexamic acid (TXA) is commonly being used to reduce melanin synthesis in patients with melasma and also used as a raw material for functional whitening cosmetics, although its action mechanism is poorly understood. Autophagy has been well known to be essential for tissue homeostasis, adaptation to starvation, and removal of dysfunctional organelles or pathogens. Recent studies have shown that autophagy regulators might have prominent roles in the initial formation stage of the melanosome, a lysosome-related organelle synthesizing melanin pigments. However, there is still no direct evidence showing a relationship between the activation of the autophagy system and the melanogenesis.
During skin pigmentation in amniotes, melanin synthesized in the melanocyte is transferred to keratinocytes by a particle called the melanosome. Previous studies, mostly using dissociated cultured cells, have proposed several different models that explain how the melanosome transfer is achieved. Here, using a technique that labels the plasma membrane of melanocytes within a three-dimensional system that mimics natural tissues, we have visualized the plasma membrane of melanocytes with EGFP in chicken embryonic skin. Confocal time-lapse microscopy reveals that the melanosome transfer is mediated, at least in part, by vesicles produced by plasma membrane. Unexpectedly, the vesicle release is accompanied by the membrane blebbing of melanocytes. Blebs that have encapsulated a melanosome are pinched off to become vesicles, and these melanosome-containing vesicles are finally engulfed by neighboring keratinocytes. For both the membrane blebbing and vesicle release, Rho small GTPase is essential. We further show that the membrane vesicle-mediated melanosome transfer plays a significant role in the skin pigmentation. Given that the skin pigmentation in inter-feather spaces in chickens is similar to that in inter-hair spaces of humans, our findings should have important consequences in cosmetic medicine.
Melanocytes of the hair follicle produce melanin and are essential in determining the differences in hair color. Pigment cell-specific MELanocyte Protein (PMEL17) plays a crucial role in melanogenesis. One of the critical steps is the amyloid-like functional oligomerization of PMEL17. Beta Site APP Cleaving Enzyme-2 (BACE2) and γ-secretase have been shown to be key players in generating the proteolytic fragments of PMEL17. The β-secretase (BACE1) is responsible for the generation of amyloid-β (Aβ) fragments in the brain and is therefore proposed as a therapeutic target for Alzheimer’s disease (AD). Currently BACE1 inhibitors, most of which lack selectivity over BACE2, have demonstrated efficacious reduction of amyloid-β peptides in animals and the CSF of humans. BACE2 knock-out mice have a deficiency in PMEL17 proteolytic processing leading to impaired melanin storage and hair depigmentation. Here, we confirm BACE2-mediated inhibition of PMEL17 proteolytic processing in vitro in mouse and human melanocytes. Furthermore, we show that wildtype as well as bace2(+/-) and bace2(-/-) mice treated with a potent dual BACE1/BACE2 inhibitor NB-360 display dose-dependent appearance of irreversibly depigmented hair. Retinal pigmented epithelium showed no morphological changes. Our data demonstrates that BACE2 as well as additional BACE1 inhibition affects melanosome maturation and induces hair depigmentation in mice.
Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.
Southern right whales (SRWs, Eubalena australis) are polymorphic for an X-linked pigmentation pattern known as grey morphism. Most SRWs have completely black skin with white patches on their bellies and occasionally on their backs; these patches remain white as the whale ages. Grey morphs (previously referred to as partial albinos) appear mostly white at birth, with a splattering of rounded black marks; but as the whales age, the white skin gradually changes to a brownish grey color. The cellular and developmental bases of grey morphism are not understood. Here we describe cellular and ultrastructural features of grey-morph skin in relation to that of normal, wild-type skin. Melanocytes were identified histologically and counted, and melanosomes were measured using transmission electron microscopy. Grey-morph skin had fewer melanocytes when compared to wild-type skin, suggesting reduced melanocyte survival, migration, or proliferation in these whales. Grey-morph melanocytes had smaller melanosomes relative to wild-type skin, normal transport of melanosomes to surrounding keratinocytes, and normal localization of melanin granules above the keratinocyte nuclei. These findings indicate that SRW grey-morph pigmentation patterns are caused by reduced numbers of melanocytes in the skin, as well as by reduced amounts of melanin production and/or reduced sizes of mature melanosomes. Grey morphism is distinct from piebaldism and albinism found in other species, which are genetic pigmentation conditions resulting from the local absence of melanocytes, or the inability to synthesize melanin, respectively.
Analysis of melanosome biogenesis in the retinal pigment epithelium (RPE) is challenging because it occurs predominantly in a short embryonic time window. Here we show that the zebrafish provides an ideal model system for studying this process because in the RPE the timing of melanosome biogenesis facilitates molecular manipulation using morpholinos. Morpholino-mediated knockdown of OA1, mutations in which cause the most common form of human ocular albinism, induces a major reduction in melanosome number, recapitulating a key feature of the mammalian disease where reduced melanosome numbers precede macromelanosome formation. We further show that PMEL, a key component of mammalian melanosome biogenesis, is required for generation of cylindrical melanosomes in zebrafish, in turn required for melanosome movement into the apical processes and maintenance of photoreceptor integrity. Spherical and cylindrical melanosomes containing similar melanin volumes co-exist in the cell body but only cylindrical melanosomes enter the apical processes. Taken together our findings indicate that melanosome number and shape are independently regulated and that melanosome shape controls a function in the RPE that depends on localization in the apical processes.
Direct reprogramming provides a fundamentally new approach for the generation of patient-specific cells. Here, by screening a pool of candidate transcription factors, we identify that a combination of the three factors, MITF, SOX10 and PAX3, directly converts mouse and human fibroblasts to functional melanocytes. Induced melanocytes (iMels) activate melanocyte-specific networks, express components of pigment production and delivery system and produce melanosomes. Human iMels properly integrate into the dermal-epidermal junction and produce and deliver melanin pigment to surrounding keratinocytes in a 3D organotypic skin reconstruct. Human iMels generate pigmented epidermis and hair follicles in skin reconstitution assays in vivo. The generation of iMels has important implications for studies of melanocyte lineage commitment, pigmentation disorders and cell replacement therapies.
Skin pigmentation is a powerful defense against ultraviolet irradiation. Particularly in humans, the body surface needs to be widely covered by protective pigmentation, and melanocytes, a major lineage of neural crest derivatives, have evolved several maneuvers to transfer melanin pigment to the skin. Recent studies with embryonic melanocytes of chickens and mice have revealed sequential events mediated by melanocytes to maximize the skin coverage by pigmentation. These processes include the migration of melanocyte precursors in the embryo, the microscopic uniform spacing of individual melanocytes, and melanosome transfer from melanocytes to keratinocytes. In particular,in vivo/ex vivolive-imaging techniques of melanosome transfer and a quantitative method to evaluate the distribution patterns of melanocytes have greatly advanced our understanding of how a limited number of cells can implement a maximal coverage of the large surface area of a developing body.