NO-induced apoptosis in human melanocytes from lightly and darkly pigmented skin.

Introduction: Nitric oxide (NO) is a potent modulator of programmed cell death, with the ability to both induce and prevent apoptosis. Some of the factors that are capable of triggering apoptosis of skin cells also cause NO overproduction in the epidermis. Unlike keratinocytes, melanin-producing melanocytes are highly resistant to apoptotic death. Aim: To investigate whether NO can induce apoptosis in normal human epidermal melanocytes and whether the pigmentation phenotype of the cells can affect their response to NO. Material and methods: Human epidermal melanocytes, derived from lightly and darkly pigmented neonatal foreskins, were cultured in the presence of various concentrations of SPER/NO. The effect of NO released from its donor on the cell morphology, viability, and proliferation was assessed. The ability of NO to induce cell apoptosis was evaluated by Hoechst 33342 staining, DNA fragmentation assay, flow cytometry with annexin V and propidium iodide staining, determination of caspase 3/7, 8, and 9 activities, and assessment of changes in the cell expression levels of BAX and BCL-2. Results: We have shown that NO is capable of inducing apoptosis in normal human epidermal melanocytes in vitro, with preferential activation of the intrinsic (mitochondrial) pathway. Melanocytes from darkly pigmented skin showed a strong increase in BCL-2 expression in response to NO and were significantly more resistant to apoptosis than those from lightly pigmented skin. Conclusions: The pigmentation phenotype may be an important factor modulating the response of human epidermal melanocytes to proapoptotic activity of extracellular NO.

The pigmentation phenotype of melanocytes affects their response to nitric oxide in vitro.

Introduction: It has been shown that nitric oxide (NO) can modulate the immune properties of epidermal melanocytes, and that overexpression of NO in the skin may contribute to inflammation-related pigmentary disorders. Little is known about whether constitutive cell pigmentation affects the sensitivity of melanocytes to NO. Aim: To compare the effect of NO on melanin synthesis and the expression of key melanogenesis-related genes in normal human melanocytes of various degrees of constitutive pigmentation. Material and methods: Human epidermal melanocytes derived from lightly and darkly pigmented skin (HEMn-LP and HEMn-DP, respectively) were cultured with or without a NO donor (SPER/NO). Then the total melanin content, the pheomelanin content, the activity and concentration of tyrosinase, and the expressions of TYR and DCT were assessed. Results: NO released from SPER/NO did not alter the total amount of melanin produced by cultured cells but increased the proportion of pheomelanin, especially in HEMn-DP. Transcriptional activity of the melanogenesis-related genes, in particular DCT, was downregulated in HEMn-DP and upregulated in HEMn-LP cultured with SPER/NO. Conclusions: NO can promote pheomelanogenesis in human epidermal melanocytes, and the cell response in this respect is associated with the pigmentation phenotype. During exposure to NO, melanocytes from dark skin produce much more pheomelanin than lightly pigmented cells. NO-induced overproduction of pheomelanin in the skin could be one of the factors responsible for the greater propensity to develop severe inflammatory dermatoses in dark-skinned individuals, or even melanoma de novo formation based on local chronic inflammation.

Differential expression of inflammatory cytokines and chemokines in lipopolysaccharide-stimulated melanocytes from lightly and darkly pigmented skin.

Increasing evidence suggests that human epidermal melanocytes play an important role in the skin immune system; however, a role of their pigmentation in immune and inflammatory responses is poorly examined. In the study, the expression of Toll-like receptor 4 (TLR4) and inflammatory cytokines and chemokines by cultured normal melanocytes derived from lightly and darkly pigmented skin was investigated after cell stimulation with lipopolysaccharide (LPS). The basal TLR4 mRNA level in heavily pigmented cells was higher as compared to their lightly pigmented counterparts. Melanocyte exposure to LPS upregulated the expression of TLR4 mRNA and enhanced the DNA-binding activity of NF-κB p50 and p65. We found substantial differences in the LPS-stimulated expression of numerous genes encoding inflammatory cytokines and chemokines between the cells with various melanin contents. In lightly pigmented melanocytes, the most significantly upregulated genes were nicotinamide phosphoribosyltransferase (NAMPT/visfatin), the chemokines CCL2 and CCL20, and IL6, while the genes for CXCL12, IL-16 and the chemokine receptor CCR4 were the most significantly upregulated in heavily pigmented cells. Moreover, the lightly pigmented melanocytes secreted much more NAMPT, CCL2 and IL-6. The results of our study suggest modulatory effect of melanogenesis on the immune properties of normal epidermal melanocytes.

Secretion of proinflammatory cytokines by normal human melanocytes in response to lipopolysaccharide.

A large body of evidence suggests that epidermal melanocytes are an integral part of the skin immune system and can be considered immunocompetent cells. Recently, it has been reported that human melanocytes constitutively express Toll-like receptors and may be involved in the induction of several inflammatory cytokines. In the study the secretion of IL-1ß, IL-6 and TNF-α by cultured normal melanocytes was investigated after stimulation with lipopolysaccharide. LPS increased the secretion of IL-1ß in a dose-dependent manner. IL-1ß stimulated release of IL-6 and TNF-α by melanocytes, whereas LPS activated production of TNF-α, but not of IL-6. These observations indicate that LPS can participate in the regulation of cytokine activity in normal human melanocytes and suggest that cytokines released by melanocytes could affect melanocytes themselves or/and other cells of the epidermis.

Melanin from epidermal human melanocytes: study by pyrolytic GC/MS.

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation.

[The role of neuromelanin in Parkinson's disease--new concepts]. / Rola neuromelaniny w chorobie Parkinsona--nowe koncepcje.

Progressive degeneration of dopaminergic neurons of the substantia nigra and the resulting dopamine deficiency in the striatum are neuropathological basis of the movement disorders in Parkinson's disease (PD). Neuromelanin-containing neurons are particularly susceptible to degeneration and their depigmentation is the hallmark of the advanced disease. The proposed mechanisms underlaying the pathogenesis and progression of neurodegeneration in the substantia nigra include iron-catalyzed oxidative stress, mitochondrial dysfunctions, inflammation and disturbances of protein metabolism. This review presents some new concepts concerning important but ambiguous role of neuromelanin in the above mentioned processes. It seems that the imbalance between cytoprotective and cytotoxic action of the pigment may cause neuronal death via mitochondrial oxidative stress, inhibition of ubiquitine-proteasome system and alpha-synuclein accumulation. Extraneuronal melanin may contribute to chronic inflammation by excessive secretion of cytokines and nitric oxide due to prolonged microglia activation. Recent reports suggest a possible role of the lipid component of neuromelanin in pigment-dependent cytotoxicity.

Alteration of growth and metabolic activity of cells in the presence of propranolol and metoprolol.

Mechanisms of action at the cellular level of a variety of drugs and xenobiotics may be assessed using Chlorella vulgaris cells. Synchronous culture, which consists of cells at the same phase of development, provides the most convenient model for studying processes at the cellular level. Stability of metabolic activity of synchronously growing cells is achieved by conducting cell culturing under strictly controlled conditions. The aim of the present study was to determine to what extent propranolol and metoprolol alter the Chlorella vulgaris metabolic activity, expressed by the number of progeny cells, the culture absorbance at lambda = 680 nm and the amount of selected photosynthetic pigments (chlorophyll a, chlorophyll b, antheraxanthin, lutein, violaxanthin and beta-carotene). Three different concentrations (10(-4), 10(-5) and 10(-6) M) of propranolol and metoprolol were administered to the Chlorella vulgaris cultures. It has been demonstrated that the higher the propranolol and metoprolol concentrations (from 10(-6) M to 10(-4) M) the lower the number of progeny cells in the cultures, expressed by the lower values of division coefficient. Both the propranolol and metoprolol caused a decrease in the photosynthetic pigments production in the mother cells. This effect was more important in the propranolol-treated cultures. The higher values of photosynthetic pigments concentrations in the progeny cells grown under the presence of a drug indicate that both the drugs tested influence mainly the cell growth and in a lower manner--their metabolic activity, expressed by the production of photosynthetic pigments.