Stimulation Of Melanocytes Research Articles

Growth stimulation of normal melanocytes and nevocellular nevus cells by gastrin releasing peptide (GRP)

In order to know the possible effects of gastrin releasing peptide (GRP) on nevus cells and melanocytes, we studied the effect of GRP on the proliferation of cultured human nevus cells and normal melanocytes. MTS assay showed that GRP stimulated the growth of viable melanocytes at 1000 ng/ml. GRP also stimulated the growth of nevus cells in a dose dependent manner and maximum stimulation was obtained at 100 ng/ml of GRP. GRP was less effective for growth stimulation of normal melanocytes than nevus cells. The cytoplasm of nevus cells were positively stained by polyclonal anti-GRP antibody. We also detected the expression of GRP and GRP receptor mRNAs in these cells by RT-PCR. These results suggest that GRP acts as an autocrine growth factor for nevus cells and normal melanocytes.

Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides.

The significance of melanotropic hormones as physiologic regulators of cutaneous pigmentation in humans is still controversial. Until recently, no direct effect for melanotropins could be demonstrated on human melanocytes. Here we present conclusive evidence that alpha-melanotropin (alpha-melanocyte-stimulating hormone, alpha-MSH) and the related hormone corticotropin (adrenocorticotropic hormone, ACTH) stimulate the proliferation and melanogenesis of human melanocytes maintained in culture in a growth medium lacking any AMP inducer. The minimal effective dose of either hormone is 0.1 nM. In time-course experiments, the increase in cell number and tyrosinase activity became evident after one treatment of the melanocytes with 100 nM alpha-MSH for 48 hr. The mitogenic effect gradually increased to 50-270% above control, depending on the individual melanocyte strain, with continuous treatment with 100 nM alpha-MSH for 8 days, whereas the melanogenic effect became maximal (70-450% increase above control) after 4 days of treatment. Western blot analysis of tyrosinase and the tyrosinase-related proteins TRP-1 and TRP-2 revealed that alpha-MSH increased the expression of those three melanogenic proteins. This was not accompanied by any change in their mRNA levels after brief (1.5-24 hr) or prolonged (6 days) treatment with 100 nM alpha-MSH, suggesting that the increased expression of these melanogenic proteins was due to posttranscriptional events. These results demonstrate both mitogenic and melanogenic effects of alpha-MSH and ACTH on human melanocytes. That both hormones are effective at subnanomolar concentrations, combined with the presence of melanotropin receptors on human melanocytes, strongly suggests that these melanotropins play a physiologic role in regulating human cutaneous pigmentation.

Photochemotherapy beyond psoriasis.

Photochemotherapy involves the therapeutic use of nonionizing radiation in combination with a photosensitizing chemical to trigger a photochemical reaction that mediates a beneficial effect. The successful introduction and widespread use of psoralen photochemotherapy (PUVA) in the management of psoriasis was the chief stimulus for recent interest in the therapeutic use of nonionizing radiation in various other dermatoses. This article discusses the expanding spectrum of diseases responding to PUVA therapy. More than 30 conditions such as atopic dermatitis, mycosis fungoides, vitiligo, the photodermatoses, chronic graft-versus-host disease, and granuloma annulare have been successfully treated with oral psoralen photochemotherapy. Various mechanisms of response to treatment are discussed including photoimmunologic effects, selective cytotoxicity, alterations of cell function, and stimulation of melanocytes. Finally, the limitations to the use of PUVA therapy are identified and its future use in other cutaneous and systemic diseases are discussed.

The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.

We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors.

Chronic growth stimulation of human adult melanocytes by inflammatory mediators in vitro: implications for nevus formation and initial steps in melanocyte oncogenesis.

In the human epidermis, melanocytes are distributed at a distance from each other. In contrast, melanocytes in nevi, which are considered benign neoplasms of melanocytes, are grouped in nests. Although still not well defined, environmental factors are thought to play an important role in the development of nevi. We found that chronic growth stimulation by leukotriene C4, a compound found in increased amounts in inflamed skin, induced pleiotropic modifications in the normal melanocyte phenotype. These changes include loss of contact inhibition and formation of structures resembling tumor spheroids. In parallel with these changes, there was a constitutive expression of Fos protein. Switching these cultures to medium supplemented with phorbol ester sustained growth with reversion of the altered phenotype. In contrast, a cAMP stimulator, cholera toxin, induced features of terminal differentiation. Our findings suggest a role for inflammatory mediators in human epidermal melanocytes. This observation provides insight into melanocyte growth alterations which may have relevance in early stages of melanocyte oncogenesis.

Allergic Contact Dermatitis Releases Soluble Factors That Stimulate Melanogenesis Through Activation of Protein Kinase C-Related Signal Transduction Pathway

Phenylazo-naphthol (PAN) allergy induces visibly well-defined and late-appearing hyperpigmentation of brownish yellow guinea pig skin in clear contrast to dinitrochlorobenzene (DNCB) allergy, which has very low incidence of hyperpigmentation. Skin extract from PAN allergy at 20-29 d post-challenge exhibited marked melanogenic stimulatory effects (3H2O release and 14C-thiouracil incorporation) when added to cultured guinea pig melanocytes. The time course in the appearance of melanogenic factor was definitely consistent with the induction pattern of visible pigmentation. By contrast, the addition of DNCB-challenged skin extract demonstrated no significant stimulating effect on melanogenesis in either assay system on any of the post-challenge days tested. Assay of intracellular inositol 1,4,5-trisphosphate formed through incubation with the melanocytes demonstrated that the PAN-allergy skin extract at day 28, which contains definite melanogenic factors, stimulated the formation of inositol 1,4,5-trisphosphate that occurs around 50 seconds in contrast to no or little increase with extracts obtained at days 0 and 1 post-challenge. Gel chromatographic analysis revealed that the PAN-allergy skin extract at day 28 contained a newly generated melanogenic fraction with a molecular weight of approximately 9000 Da which was also capable of stimulating DNA synthesis and activating the signal-transduction process (inositol trisphosphate formation) when added to guinea pig melanocytes. Both stimulations of melanogenesis and DNA synthesis by the 9000 Da fraction were completely abolished by the prior and simultaneous addition of protein kinase C (PKC) inhibitor (H-7) or its down-regulatory agent, phorbol 12,13-dibutyrate (PdBu). Taken together, these results suggest that PAN allergy provides a new mechanism of hypermelanization in which endogenous factors synthesized within skin induce the activation of signal-transduction pathways such as phosphoinositide turnover through ligands-receptor binding, resulting in the stimulation of melanocytes possibly through the activation of PKC.

Smoker's melanosis may explain the lower hearing loss and lower frequency of Parkinson's disease found among tobacco smokers — A new hypothesis

A new hypothesis is presented explaining the preventive effect of tobacco smoking found on noise induced hearing loss and on the frequency of Parkinson's disease. The hypothesis is based on the finding of a melanocyte stimulation of tobacco smoking in the human oral mucosa, resulting in a higher melanin content in the epithelial cells, and a higher frequency of visible oral melanin pigmentation — smoker's melanosis. The preventive influence of smoking found in the cochlea and substantia nigra may also be due to a higher melanin content and to the ability of melanin to strongly bind specific chemical agents for a long time. Melanin may in this way act as a scavenger against cell toxic factors in these organs.

Design, synthesis, and biological activities of a potent and selective α‐melanotropin antagonist

Based on structure-activity relationships of the potent alpha-MSH agonist, Ac-Nle4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10-NH2, several analogs of the general formula Ac-Nle4-Asp5-Waa6-Xaa7-Yaa8-Zaa9-Lys10+ ++-NH2 were synthesized and tested on frog and lizard skin bioassays for their possible inhibitory actions against alpha-MSH on melanocyte stimulation. When Waa6 = Trp, Xaa7 = D-Phe, Yaa8 = Nle and Zaa = Trp, a highly potent alpha-MSH antagonist, Ac-Nle-Asp-Trp-D-Phe-Nle-Trp-Lys-NH2, with selectivity on the frog skin alpha-MSH receptor system (pA2 = 8.4) was obtained. However, several modifications in the amino acid sequence of the peptide resulted in a complete loss of antagonistic activity and a recovery of very weak agonistic action. The following changes in the amino acid sequence of the peptide were examined; His or D-Trp for Waa, L-Phe for Xaa, Arg, Ala or Pro for Yaa, and D-Trp for Zaa. All resulted in full agonists with no antagonistic activity. In addition, lactam cyclization between the Asp5 and Lys10 side chains in the antagonist gave a full agonist and a complete loss of antagonistic activity. Efforts to develop a rational approach for the design of selective alpha-MSH antagonists for the frog skin alpha-MSH receptor will be discussed.

初代培養ヒト色素細胞に対する炎症性化学伝達物質の影響

Skin darkening after cutaneous inflammation is a well known phenomenon but its mechanisms for this hyperpigmentation have not been clarified yet. Because in inflamed skin various mediators such as arachidonic acid metabolites and histamine are found in increased amounts, we have studied their effects on cultured normal human melanocytes. As reported previously we have found that prostaglandine E2 stimulated normal human melanocytes. In addition histamine, platelet activating factor, bradykinin and arachidonic acid metabolites such as leukotriene (LT) C4 and LTD4 also stimulated melanocytes.They were found to increase the total amounts of immunoreactive tyrosinase and tyrosinase related protein, the number of dendrites and the size of melanocytes. In these proinflammatory mediators, LTC4 and histamine showed far strong stimulatory effect. On the other hand, serotonin, heparin and other arachidonic acid metabolites such as PGE1 PGFs and 12-hydroxy eicosatetraenoic acid (12-HETE) did not show any significant stimulatory effect.Present studies suggest that various proinflammatory chemical mediators, especially LTC4 and histamine are involved in the stimulation of melanocytes to accelerate the production of melanine and its active transfer to neighboring keratinocytes, resulting into the formation of hyperpigmentation after skin inflammation.

Ultrastructural findings in oral hyperpigmentation of HIV-infected patients.

Oral hyperpigmentation has been observed in six HIV-infected patients, in two of whom systemic medication (ketokonazole, clofazimine) was supposed to be etiologically involved. Histologically, pigment was found in epithelial basal cells and particularly in subepithelial connective tissue. Ultrastructurally, the presence of premature melanosomes in subepithelial keratinocytes was of interest. Stimulation of melanocytes during HIV infection may occur in association with immunopathologic changes in the oral mucosa.

HMB-45 recognizes stimulated melanocytes.

A monoclonal antibody (HMB-45) was previously reported to bind to melanoma cells, and the junctional component of nevus cells, but not to normal adult melanocytes. We have tested HMB-45 binding in several conditions under which melanocyte stimulation might be expected in adults, i.e. 3 simple lentigines, 2 solar lentigines, 7 recent surgical scars (from re-excision of non-melanocytic tumors), 2 surgical scars from re-excisions of melanomas (after complete primary excisions), 9 hemangiomas from non-sun-exposed skin, 1 basal cell carcinoma, 1 acute ecchymosis, 1 keloid, and 1 dermatofibroma. Positive controls included 6 malignant melanomas and 1 fetal skin sample. Melanocytes were strongly positively stained overlying hemangiomas, within or near recent surgical scars of melanocytic and non-melanocytic tumor re-excisions, near basal cell carcinoma, and in fetal skin. Melanocytes either were not stained or were stained only focally for trace amounts in the normal skin near the new margins of the wide re-excision specimens for melanoma, i.e., at a distance from the scar, in the simple lentigines and in the fibrotic lesions. Thus, HMB-45 is staining an antigen which appears in adult melanocytes during stimulation and in fetal skin, as well as in melanomas. This stimulation is associated with conditions that would have increased vascularity, suggesting a melanocyte response to a plasma factor, or other endothelial cell derived factor. HMB-45 would not be a useful marker for residual melanoma cells in melanoma re-excision specimens.

Paracrine Stimulation of Melanocytes by Keratinocytes through Basic Fibroblast Growth Factora

Melanocytes cultured in the presence of keratinocytes survive for weeks without added basic fibroblast growth factor (bFGF) and cyclic-adenosine-monophosphate (cAMP), the two factors needed for their proliferation in vitro. We show here that the growth factor for melanocytes produced by human keratinocytes is bFGF because its activity can be abolished by neutralizing antibodies to bFGF and by a bFGF synthetic peptide that inhibits the binding of the growth factor to its receptor. The melanocyte mitogen in keratinocytes is cell-associated and increases after irradiation with ultraviolet B (UVB). Northern blots reveal bFGF gene transcripts in keratinocytes but not melanocytes. These studies demonstrate that bFGF elaborated by keratinocytes in vitro sustains melanocyte growth and survival, and they suggest that keratinocyte-derived bFGF is the natural growth factor for normal human melanocytes in vivo.

Stimulation of Human Melanocytes by Vitamin D3 Possibly Mediates Skin Pigmentation After Sun Exposure

We found an increased amount of immunoreactive tyrosinase in human melanocytes after 6-d culturing with vitamin D3 (cholecalciferol). Most of these melanocytes became more dendritic and swollen in a fashion similar to that noted in the skin after ultraviolet irradiation. However, 7-dehydrocholesterol (pro-vitamin D3) or 1 alpha,25-dihydroxy-vitamin D3 (activated vitamin D3) were found to have little effect on the same system. Because vitamin D3 is known to be photochemically converted from pro-vitamin D3 in the skin by ultraviolet irradiation, the mechanism of human skin pigmentation after ultraviolet irradiation, thus far unknown, may be at least partly explained by this stimulating effect of vitamin D3 on melanocytes.

Growth stimulation of human melanocytes: Identification and characterization of melanoma-derived melanocyte growth factor (M-McGF)

The melanocyte growth stimulating factor from human melanoma cells grown in vitro [Eisinger, M. et al, (1985) Science 229 ,984–986] has been purified and identified as a 14kDa peptide. The activity was shown to be sensitive to acid and heat treatments but resistent to reduction. The factor has strong affinity for heparin-Sepharose and was separated into two fractions by ion-exchange chromatography. Based on its properties, it is grouped in the family of heparin-binding growth factors. Identification of this growth factor will provide a tool for studies of growth regulation of normal and transformed human melanocytes.

Hyperpigmentation and hypopigmentation of the skin after long term PUVA therapy. Light and electron microscopic observations on three patients.

An electron microscopic study was performed to demonstrate the pathological changes induced by long term PUVA treatment in recalcitrant psoriasis. Three patients developed mottling (hyperpigmentation and hypopigmentation) during two to three years of treatment. Three different types of morphological changes were found: disarrangement of keratinocytes, clustering and stimulation of melanocytes and homogenization of papillary dermis. Furthermore, the superficial blood vessels were loaded with the same type of amorphous granular substance. These changes might be specific to PUVA treatment or they might occur only in patients with previous treatment with, e.g., arsenic, methotrexate, anthralin + UVB or a combination of these.

5-S-Cysteinyldopa and dopa in serum during treatment with 8-methoxypsoralen and UVA light

5-S-Cysteinyldopa and dopa concentrations in serum were studied by high-performance liquid chromatography (HPLC) in patients with psoriasis treated by 8-methoxypsoralen and UVA light. A marked increase in 5-S-Cysteinyldopa was found after 3 days´ treatment, although no increase in pigmentation could yet be observed. The highest concentrations of 5-S-Cysteinyldopa in serum were noted after 1 or 2 weeks´ treatment. In one patient who showed no increase in serum 5-S-Cysteinyldopa treatment was unsuccessful. During PUVA treatment of 3 patients with psoriasis confined to the palms and/or soles a delayed increase in serum 5-S-Cysteinyldopa was noted after 5 weeks. This delayed response after treatment of a small area of the body is remarkable, and may indicate the formation of a systemic melanocyte stimulation factor. There was no increase in the serum dopa concentrations during PUVA treatment, and dopa analysis was of no value in assessing the activity of the melanocytes. Some unexpectedly high dopa values recorded before and during PUVA treatment may be explained by the fact that dopa originates in the nervous system or the adrenals.

Testosterone and UVL‐B stimulation of epidermal melanocytes in rat scrotal skin

The effects of UVL-B and/or testosterone replacemnt therapy are compared in normal and castrated rats in order to determine whether testosterone is required for UVL-B (290-315 nm) stimulation of melanogenesis in the testosterone-dependent epidermal melanocyte system of the scrotal skin of black Long Evans rats. Testosterone is not a prerequisite for UVL-B stimulation of melanocytes as in both castrates and normal animals the melanocytes respond to UVL-B by increases in size, length and number of dendrites (dendriticness), and tyrosinase activity (intensity of Dopa reaction). Addition of testosterone to castrates does enhance the effects of UVL-B. However, UVL-B with or without testosterone cannot maintain normal melanogenesis in rats irradiated immediately after castration nor can it restore normal melanogenesis following long term castration. Bth the amount of UVL nergy/exposure and the number of exposures are important variables in stimulation of the epidermal melanocytes. Administration of a dose of UVL-B to castrates in a single exposure is ineffective, while the same overall dose spread over several exposures increases the size and dendriticness of melanocytes. Testosterone and UVL-B act synergistically in affecting melanogenesis although neither singly nor in combination are they able to fully restore normal melanogenesis.

Organ culture of mammalian skin and the effects of ultraviolet light and testosterone on melanocyte morphology and function.

Scrotal skin of black Long-Evans rats and human thigh skin were maintained in vitro as organ cultures for as long as 14 days, and examined histologically using the combined skin splitting and Dopa techniques. Selected rat skin cultures received testosterone in the culture medium and/or were irradiated with ultraviolet light (290-320 nm UVL). With increased time in culture, scrotal melanocytes round up and there is an increase in epidermal pigmentation. Human skin behaves similarly; after eight days in vitro human melanocytes also become rounded, but remain strongly Dopa-positive. Addition of exogenous testosterone to cultured rat skin maintains dendritic morphology of melanocytes, but cell body size is still reduced. UVL irradiation stimulates melanocytes in rat skin cultures, maintaining their dendritic morphology and increasing epidermal and dermal pigmentation. Cultured skin receiving both UVL and testosterone illustrates a synergistic effect. Electron microscopic examination of cultured rat skin shows the presence of large melanosome complexes in keratinocytes, much larger than those found in vivo. Melanocytes appear to be active as they contain an extensive Golgi zone, rough endoplasmic reticulum, and melanosomes in various stages of formation. Dermis contained many dermal melanocytes and macrophages laden with melanosomes, correlating with the increased visible dermal pigmentation in vitro. This UVL stimulation of melanocytes in our skin organ cultures contrasts with the lack of melanogenic stimulation found in melanoma cell cultures. Our findings suggest that the intact epidermal melanin unit may be necessary for UVL stimulation of melanocytes.

The ``Half-and-Half Nail"

A 55-year-old patient had chronic renal failure and pigmentary changes. The distal appeared dark brown while the proximal was white. This pattern has been previously called the ``half-and-half nail and is believed to be a pattern uniquely found in certain patients with chronic renal failure. A biopsy showed the pigment to be melanin. Stimulation of matrix melanocytes during sudden renal decompensation is proposed as the cause of this finding.

The isolation and structure of β-melanocyte-stimulating hormone from horse pituitary glands

The complete amino acid sequence in equine β-melanocyte-stimulating hormone has been determined on highly purified material isolated from the neurointermediate lobes of horse pituitaries. In the final purification step (countercurrent distribution in 0.1% acid- n-butanol-pyridine, 11:5:3 v v ), as well as in zone electrophoresis at pH 4.9 on starch, this material behaved like bovine β-melanocyte-stimulating hormone; however, complete amino acid analysis of the hormone, as well as the elucidation of the structure of the peptide fragments isolated from chymotryptic and tryptic digests of the equine β-melanocyte-stimulating hormone, revealed its unique structure to be H-Asp.Glu.Gly.Pro.Tyr.Lys.Met.Glu.His.Phe.Arg.Try.Gly. Ser.Pro.Arg.Lys.Asp-OH. Although the sequence differs somewhat from that found in any other naturally occurring melanocyte-stimulating pituitary hormone (see Fig. 3), it still contains the nonvarying internal heptapeptide sequence… Met.Glu.His.Phe.Arg.Try.Gly… found in each of these hormones. On the basis of results of bioassay, it was concluded that the nature of the NH 2-terminal sequence in β-melanotropins is far more important for the in vivo melanocyte stimulation than is the amino acid composition near the COOH terminus.