Keratinocyte Extracellular Matrix-Mediated Regulation of Normal Human Melanocyte Functions

Active roles of cell-cell interaction between melanocytes and neighboring keratinocytes for the regulation of melanocyte functions in the skin have been suggested. We examined substantial regulatory mechanisms of keratinocyte extracellular matrix (kECMs) for normal human melanocyte functions without direct cell-cell contact. We specially devised kECMs from proliferating or differentiating keratinocytes and further treated them with environmental stimulus ultraviolet B (UVB) for skin pigmentary system. Normal human melanocytes (NHM) were cultured on the various keratinocyte ECMs and initially the effects of the kECMs upon melanocyte morphology (dendrite formation and extension), growth, melanin production and expressions of pigmentation-associated protein (MEL-5) and proliferation-associated protein (proliferating cell nuclear antigen; PCNA/cyclin) were studied. Then we compared the effects of these cell-matrix interactions with those of direct melanocyte-keratinocyte, cell-cell contact in co-culture on melanocyte functions. Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on the kECM prepared from UVB irradiated differentiating keratinocytes resulted in 219% increase in the number of dendrites. The growth of melanocytes on kECMs was also stimulated up to 280% of control. The kECM produced by proliferating keratinocytes had a more significant effect on the growth than kECM from differentiating keratinocytes. This melanocyte growth stimulating effect was decreased with kECM from UVB treated differentiating keratinocytes. The melanin content per melanocyte was constant on any of the kECMs. Expression of pigmentation-associated protein detected by monoclonal antibody, MEL-5, was not changed on the kECM, while it was increased in melanocytes in co-culture with keratinocytes. Expression of PCNA/cyclin in melanocytes cultured on kECMs was generally downregulated on kECM and in co-culture compared to that in a control culture. We demonstrated that the kECMs play important roles in the melanocyte morphology and proliferation. These observations suggest that environmental (UVB) and physiological (Ca⁺⁺) stimuli can regulate melanocyte functions through the keratinocyte extracellular matrix in vivo.     

Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.     

Sound needs sound melanocytes to be heard

Intermediate cells in the stria vascularis of the mammalian cochlea are melanocytes, which contain melanin pigments and are capable of synthesizing melanin. These melanocytes are required for normal development of the cochlea, as evidenced by studies of mutant mice with congenital melanocyte anomalies. Melanocytes are also needed for developed cochleae to function normally, as evidenced by studies of mutant mice with late-onset melanocyte anomaly and humans with acquired melanocyte anomaly. Melanin, per se, does not seem to be essential for normal hearing function, but it may protect against traumata to the cochlea, e.g., noise and ototoxic aminoglycosides. Recent electrophysiological studies have revealed that strial intermediate cells are provided with specific ionic channels, such as inwardly rectifying K+ channels (Kir4.1) and voltage-dependent outwardly rectifying K+ channels. These channels may play central roles in strial function and thus in normal hearing.     

lpa-miR-nov-66通过调控cAMP路径抑制α-MSH介导的羊驼黑色素生成

α-黑素细胞刺激素(α-MSH)和lpa-miR-nov-66在羊驼黑色素细胞产生黑色素过程中均起重要的调控作用,但二者之间的关系尚未报道.本研究在体外培养的羊驼黑色素细胞中通过转染lpamiR-nov-66和添加α-MSH处理,用实时定量PCR和Western印迹检测黑色素细胞内基因表达水平,ELISA法检测c AMP和cGMP的产量,RTCA实时无标记细胞功能分析黑色素细胞增殖以及紫外分光光度法检测黑色素产量,证实二者在调控羊驼黑色素细胞产生黑色素颗粒过程中的关系.结果显示,与单纯α-MSH处理相比,lpa-miR-nov-66转染结合α-MSH处理组中,小眼转录因子(MITF)和酪氨酸酶(TYR)在转录水平和翻译水平的表达均降低,而酪氨酸酶相关蛋白2(TYRP2)在转录和翻译水平的表达均升高;cGMP的产量升高,cAMP的产量下降;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量下降.与单纯转染lpa-miR-nov-66相比,lpa-miR-nov-66转染结合α-MSH处理组中,MITF、TYR和TYRP2在转录水平和翻译水平的表达均升高;cGMP的产量下降,cAMP的产量升高;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量升高.上述结果证明,lpa-miR-nov-66通过调控羊驼黑色素细胞中毛色形成的c AMP路径,抑制α-MSH对黑色素细胞产生黑色素的促进作用.     

The Melanogenesis Alteration Effects of Achillea millefolium L. Essential Oil and Linalyl Acetate: Involvement of Oxidative Stress and the JNK and ERK Signaling Pathways in Melanoma Cells

The mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK)1/2 and p38 MAPK, is known to be activated by ultraviolet (UV) radiation in melanocytes to regulate melanin production. Reactive oxygen species (ROS) play important roles in the pathway of ERK and JNK activation. It has been established that the essential oil of Achillea millefolium L. (AM-EO) has activities that suppress the oxidative stress and inflammatory responses. Thus, we analyzed the effects of AM-EO on melanogenesis in melanocyte stimulating hormone (α-MSH) treated melanoma cells. The results demonstrated that AM-EO suppresses melanin production by decreasing tyrosinase activity through the regulation of the JNK and ERK signaling pathways. This effect might be associated with the AM-EO activity leading to the suppression of ROS, and linalyl acetate is its major functional component. Therefore, we propose that AM-EO has the potential to treat hyperpigmentation in the future.     

黑色素羽毛装饰反映了鸟类的抗氧化和免疫能力吗?

鸟类信号系统研究认为,不同于类胡萝卜素,基于黑色素的羽色装饰不需要昂贵的生理代价。然而,羽毛色素细胞的黑色素沉积与抗氧化能力和免疫系统有很强的联系,而抗氧化能力和免疫系统在提高有机体适合度方面具有重要的功能。黑色素细胞的生长对氧化环境压力十分敏感;并且,黑色素本身似乎就具有抗氧化剂的功能。相应地,把抗氧化剂用于以黑色素为基础羽色发育,还是用于其它方面例如免疫调节和免疫刺激等,个体也许必须对此做出权衡。组织中的抗氧化功能大多与代谢活动有关,也就是和自由基的最高水平有关。此外,人们发现,在哺乳动物中调节黑色素沉积的激素,即α黑素细胞刺激素,在鸟类上皮组织的色素沉积中也具有同样的功能。这种进化保守的激素是免疫和炎症反应的一个重要介体。它下调前炎性细胞因子、免疫介导细胞因子和协同刺激分子,以及主要组织相容性复合体Ⅰ类分子在单核细胞的表达以及抗体的产生,而上调抑制因子白介素。黑色素在羽毛上的大量沉积,也可能反映出免疫系统在局部免疫应答的负调控能力。这些将黑色素沉积和抗氧化剂以及免疫功能联系起来的机制,表明基于黑色素的羽色信号具有一定的生理代价,这些对以往关于黑色素的优势作用的假说提出质疑     

The ret oncogene can induce melanogenesis and melanocyte development in Wv/Wv mice.

We recently reported the establishment of transgenic mouse lines carrying the mouse metallothionein/ret fusion gene in which severe melanosis and melanocytic tumors developed. In the present study, we demonstrate that a significant number of pigmented hairs developed in Wv/Wv mice crossed to one of the transgenic mouse lines. The pigmented hair of Wv/Wv mice carrying the ret oncogene did not lose color during aging and reappeared after shaving, indicating that the melanocytes in the hair follicle function. The melanocytic tumors also developed in these mice, although the incidence was lower than that in the wild transgenic mice. Furthermore, the neutral tube culture of mouse embryos indicated that neural crest cells of the transgenic mice gave rise to a cell population that autonomously produced melanin even in the absence of melanocyte stimulating hormone. These results strongly suggested that the introduced ret oncogene could compensate for the defect of c-kit in Wv mice during both embryogenesis and postnatal life and induce a high level of melanin synthesis in the process of melanocyte development.     

内皮素-2促进体外培养的绵羊皮肤黑色素细胞增殖及黑色素生成

内皮素（endothelin,ET）及其受体在黑色素细胞成熟分化时起着有效的促进作用.然而,内皮素-2（ET-2）在黑色素生成的作用方面,还处于争论中或报道不一致.在此,我们研究ET-2对体外培养的绵羊皮肤黑色素细胞增殖和黑色素生成的影响.比较实验组ET-2（1,10,100 nmol/L）与空白对照组,通过MTT法和Ando等的方法检测出黑色素细胞的增殖率和黑色素含量显著增加.荧光定量PCR和Western 印迹分别检测出内皮素受体B(Bdnrb);酪氨酸激酶受体(Kit);酪氨酸酶(Tyr);酪氨酸相关蛋白-1(Tyrp-1)基因的mRNA水平及蛋白水平的相对表达量显著增加.这些数据表明,ET-2可能促进绵羊的皮肤黑色素细胞增殖和黑色素生成.     

α-MSH and Melanogenesis in Normal Human Adult Melanocytes

Normal human skin melanocytes do not pigment consistently to α-melanocyte stimulating hormone (α-MSH) in culture. The aim of this study was to establish media conditions in which to obtain a reproducible melanogenic response to α-MSH in these cells. Twenty-five media of varying mitogen composition were examined. As previously noted by other workers, melanocyte morphology and proliferation are greatly affected by media composition. However, under the majority of media conditions that supported melanocyte survival and proliferation, cells did not respond to α-MSH with any consistent increase in dopa oxidase activity or melanin content. In only one medium condition, where basic fibroblast growth factor (bFGF) was the sole mitogen present, α-MSH induced both an increase in dopa oxidase activity (at 48%) and in melanin content (of 283%).     

Enzymatic control of pigmentation in mammals.

Visible pigmentation in mammals results from the synthesis and distribution of melanin in the skin, hair bulbs, and eyes. The melanins are produced in melanocytes and can be of two basic types: eumelanins, which are brown or black, and phaseomelanins, which are red or yellow. In mammals typically there are mixtures of both types. The most essential enzyme in this melanin biosynthetic pathway is tyrosinase and it is the only enzyme absolutely required for melanin production. However, recent studies have shown that mammalian melanogenesis is not regulated solely by tyrosinase at the enzymatic level, and have identified additional melanogenic factors that can modulate pigmentation in either a positive or negative fashion. In addition, other pigment-specific genes that are related to tyrosinase have been cloned which encode proteins that apparently work together at the catalytic level to specify the quantity and quality of the melanins synthesized. Future research should provide a greater understanding of the enzymatic interactions, processing, and tissue specificity that are important to pigmentation in mammals.     

Melanocyte function and its control by melanocortin peptides.

Melanocytes are cells of neural crest origin. In the human epidermis, they form a close association with keratinocytes via their dendrites. Melanocytes are well known for their role in skin pigmentation, and their ability to produce and distribute melanin has been studied extensively. One of the factors that regulates melanocytes and skin pigmentation is the locally produced melanocortin peptide alpha-MSH. The effects of alpha-MSH on melanogenesis are mediated via the MC-1R and tyrosinase, the rate-limiting enzyme in the melanogenesis pathway. Binding of alpha-MSH to its receptor increases tyrosinase activity and eumelanin production, which accounts for the skin-darkening effect of alpha-MSH. Other alpha-MSH-related melanocortin peptides, such as ACTH1-17 and desacetylated alpha-MSH, are also agonists at the MC-1R and could regulate melanocyte function. Recent evidence shows that melanocytes have other functions in the skin in addition to their ability to produce melanin. They are able to secrete a wide range of signal molecules, including cytokines, POMC peptides, catecholamines, and NO in response to UV irradiation and other stimuli. Potential targets of these secretory products are keratinocytes, lymphocytes, fibroblasts, mast cells, and endothelial cells, all of which express receptors for these signal molecules. Melanocytes may therefore act as important local regulators of a range of skin cells. It has been shown that alpha-MSH regulates NO production from melanocytes, and it is possible that the melanocortins regulate the release of other signalling molecules from melanocytes. Therefore, the melanocortin signaling system is one of the important regulators of skin homeostasis.     

Current challenges in understanding melanogenesis: bridging chemistry,biological control,morphology, and function

Melanin is a natural pigment produced within organelles, melanosomes, located in melanocytes. Biological functions of melanosomes are often attributed to the unique chemical properties of the melanins they contain; however, the molecular structure of melanins, the mechanism by which the pigment is produced, and how the pigment is organized within the melanosome remains to be fully understood. In this review, we examine the current understanding of the initial chemical steps in the melanogenesis. Most natural melanins are mixtures of eumelanin and pheomelanin, and so after presenting the current understanding of the individual pigments, we focus on the mixed melanin systems, with a critical eye towards understanding how studies on individual melanin do and do not provide insight in the molecular aspects of their structures. We conclude the review with a discussion of important issues that must be addressed in future research efforts to more fully understand the relationship between molecular and functional properties of this important class of natural pigments.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

How are proliferation and differentiation of melanocytes regulated?

Coat colors are determined by melanin (eumelanin and pheomelanin). Melanin is synthesized in melanocytes and accumulates in special organelles, melanosomes, which upon maturation are transferred to keratinocytes. Melanocytes differentiate from undifferentiated precursors, called melanoblasts, which are derived from neural crest cells. Melanoblast/melanocyte proliferation and differentiation are regulated by the tissue environment, especially by keratinocytes, which synthesize endothelins, steel factor, hepatocyte growth factor, leukemia inhibitory factor and granulocyte-macrophage colony-stimulating factor. Melanocyte differentiation is also stimulated by alpha-melanocyte stimulating hormone; in the mouse, however, this hormone is likely carried through the bloodstream and not produced locally in the skin. Melanoblast migration, proliferation and differentiation are also regulated by many coat color genes otherwise known for their ability to regulate melanosome formation and maturation, pigment type switching and melanosome distribution and transfer. Thus, melanocyte proliferation and differentiation are not only regulated by genes encoding typical growth factors and their receptors but also by genes classically known for their role in pigment formation.     

Characterization of the bioactive motif of neuregulin-1, a fibroblast-derived paracrine factor that regulates the constitutive color and the function of melanocytes in human skin

Interactions between melanocytes and neighboring cells in the skin (keratinocytes and fibroblasts) play important roles in regulating human skin color. We recently reported that neuregulin-1 (NRG1) is highly expressed in fibroblasts from Fitzpatrick type VI skin (the darkest) and at least in part determines the constitutive color of human skin. We have now characterized the bioactive motif of NRG1 that is involved in modulating melanin production in human melanocytes. We found that 8-mer motifs (PSRYLCKC and LCKCPNEF) increased melanin production but did not increase the proliferation of melanocytes; the minimum fragment that could elicit that effect was the tetrapeptide LCKC. This smaller bioactive peptide might have an advantage in clinical applications in which it modulates only pigmentation and does not stimulate melanocyte proliferation.     

灌喂氧化鱼油后黄颡鱼胃肠道黏膜黑色素细胞分化和黑色素合成途径基因的差异表达

以黄颡鱼(Pelteobagrus fulvidraco)为实验对象, 灌喂氧化鱼油、鱼油7d后, 提取胃肠道黏膜总RNA, 采用RNA-seq测序并做转录组分析, 分析了黑色素生物合成途径关键酶(酪氨酸酶)及其相关蛋白基因、黑素体运动的3个蛋白基因、α黑素细胞刺激激素途径和WNT/β-catenin、EDN3和EDNRB、KIT及其配体KITL3个信号通路的主要蛋白基因的差异表达活性。结果显示, 黄颡鱼胃肠道黏膜中存在黑色素细胞分化和发育过程、黑色素合成及其调控途径的代谢网络, 通过绘制代谢网络得到了关键性酶或蛋白质的基因信息。在灌喂氧化鱼油后, 控制黑色素合成途径主要基因的表达活性显著下调, 可能导致黑色素合成量的不足; α-MSH激素途径主要基因差异表达上调, 具备促进黑色素细胞分化和发育的调控基础; 而调控黑色素细胞分化和发育的3个信号通路主要基因也有差异表达。因此, 黄颡鱼受灌喂氧化鱼油的影响, 黑色素细胞分化和发育过程受到较大影响, 会影响到鱼体成熟的黑色素细胞的数量, 同时, 黑色素的生物合成量不足将导致引起黄颡鱼体色的变化。