Melanocyte homeostasis in vivo tolerates Rb1 loss in a developmentally independent fashion

There has been uncertainty regarding the precise role that the pocket protein Rb1 plays in murine melanocyte homeostasis. It has been reported that the TAT-Cre mediated loss of exon 19 from a floxed Rb1 allele causes melanocyte apoptosis in vivo and in vitro. This is at variance with other findings showing, either directly or indirectly, that Rb1 loss in melanocytes has no noticeable effect in vivo, but in vitro leads to a semi-transformed phenotype. In this study, we show that Rb1-null melanocytes lacking exon 19 do not undergo apoptosis and survive both in vitro and in vivo, irrespective of the developmental stage at which Cre-mediated ablation of the exon occurs. Further, Rb1 loss has no serious long-term ramifications on melanocyte homeostasis in vivo, with Rb1-null melanocytes being detected in the skin after numerous hair cycles, inferring that the melanocyte stem cell population carrying the Cre-mediated deletion is maintained. Consequently, whilst Rb1 loss in the melanocyte is able to alter cellular behaviour in vitro, it appears inconsequential with respect to melanocyte homeostasis in the mouse skin.

Ras controls melanocyte expansion during zebrafish fin stripe regeneration.

Regenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.

New prodrugs derived from 6-aminodopamine and 4-aminophenol as candidates for melanocyte-directed enzyme prodrug therapy (MDEPT)

Two novel tyrosinase mediated drug delivery pathways have been investigated for the selective delivery of cytotoxic units to melanocytes from urea and thiourea prodrugs. The synthesis of these prodrugs is reported, as well as oximetry data that illustrate that the targets are substrates for tyrosinase. The stability of each of the prodrugs in (i) phosphate buffer and (ii) bovine serum is discussed, and the urea prodrugs are identified as lead candidates for further studies. Finally, HPLC studies and preliminary cytotoxicity studies in a melanotic and an amelanotic cell line, that illustrate the feasibility of the approach, are presented.

MARCKS functions as a novel growth/tumour suppressor in cells of melanocyte origin

Protein kinase C (PKC) plays a pivotal role in modulating the growth of melanocytic cells in culture. We have shown previously that a major physiological substrate of PKC, the 80 kDa myristoylated alanine-rich C-kinase substrate (MARCKS), can be phosphorylated in quiescent, non-tumorigenic melanocytes exposed transiently to a biologically active phorbol ester, but cannot be phosphorylated in phorbol ester-treated, syngeneic malignant melanoma cells. Despite its ubiquitous distribution, the function of MARCKS in cell growth and transformation remains to be demonstrated clearly. We report here that MARCKS mRNA and protein levels are down-regulated significantly in the spontaneously derived murine B16 melanoma cell line compared with syngeneic normal Mel-ab melanocytes. In contrast, the tumourigenic v-Ha-ras-transfonned melan-ocytic line, LTR Ras 2, showed a high basal level of MARCKS phosphorylation which was not enhanced by treatment of cells with phorbol ester. Furthermore, protein levels of MARCKS in LTR Ras 2 cells were similar to those expressed in Mel-ab melanocytes. However, in four out of six murine tumour cell lines investigated, levels of MARCKS protein were barely detectable. Transfection of B16 cells with a plasmid containing the MARCKS cDNA in the sense orientation produced two neomycin-resistant clones displaying reduced proliferative capacity and decreased anchorage-independent growth compared with control cells. In contrast, transfection with the antisense MARCKS construct produced many colonies which displayed enhanced growth and transforming potential compared with control cells. Thus, MARCKS appears to act as a novel growth suppressor in the spontaneous transformation of cells of melanocyte origin and may play a more general role in the tumour progression of other carcinomas.

Protein kinase C down-regulation, and not transient activation, correlates with melanocyte growth

The nontumorigenic, immortal line of murine melanocytes, Mel-ab, requires the continual presence of biologically active phorbol esters for growth (R. E. Wilson et al., Cancer Res., 49: 711–716, 1989). Comparable treatments of B16 murine melanoma cells result in partial inhibition of cell proliferation. The role of protein kinase C (PKC) in the modulation of growth of cells from these two melanocytic cell lines has been investigated. Significant levels of PKC were present in quiescent Mel-ab cells as determined by Western blotting, whereas no immunoreactive protein was detected in cell extracts from either proliferating Mel-ab or B16.F1 cells. Phosphorylation of a Mr 80,000 protein, which by one- and two-dimensional gel analysis comigrated with the known Mr 80,000 protein substrate of PKC in fibroblasts, was induced in 12-O-tetradecanoylphorbol-13-acetate-stimulated quiescent Mel-ab cells but not in proliferating Mel-ab cells or B16.F1 melanoma cells. Direct measurement of PKC activity in these cells demonstrated a 10-fold greater level of activity in quiescent Mel-ab cells (262 ± 50 pmol/min/mg SD) compared with growing cells (22.8 ± 11.8 pmol/min/mg SD). An intermediate level of activity was detected in proliferating B16.F1 melanoma cells (148.5 ± 20.4 pmol/min/mg SD). The subcellular distribution of PKC was dependent upon the growth state of the cells such that quiescent Mel-ab cells displayed a higher level of activity in the cytosol, whereas growing Melab cells displayed greater activity in the particulate fraction. Like many other transformed lines, B16.F1 melanoma cells constitutively expressed the majority of enzyme activity in the particulate fraction. Measurement of [3H]phorbol ester binding in intact cells paralleled the PKC activation data such that quiescent Mel-ab cells displayed binding of 1612 ± 147 cpm/106 cells, whereas proliferating Mel-ab and B16.F1 melanoma cells displayed binding of 652 ± 28 and 947 ± 81 cpm/106 cells, respectively. Membrane-permeant diacylglycerol analogues, which activated but did not down-regulate PKC, were devoid of growth-stimulating effects on melanocytes, even in the presence of the specific diacylglycerol kinase inhibitor, R59022. Together, these data show that PKC down-regulation, and not activation, correlates with the growth of melanocytes in culture.

THE EFFECT OF TUBEROINFUNDIBULAR DOPAMINERGIC-NEURONS ON PROLACTIN AND ALPHA-MELANOCYTE STIMULATING HORMONE-RELEASE

The effect of tubero-infundibular dopaminergic neurons (TIDA) on the release of prolactin (PRL) and alpha-melanocyte stimulating hormone (alpha-MSH) was studied in median eminence-lesioned (MEL) male rats (N = 6-28). Plasma PRL and alpha-MSH levels were significantly elevated 2 (86.1 +/- 19.8 and 505.1 +/- 19.1 ng/ml), 4 (278.7 +/- 15.5 and 487.4 +/- 125.1 ng/ml), 7 (116.2 +/- 16.2 and 495.8 +/- 62.6 ng/ml) and 14 (247.3 +/- 26.1 and 448.4 +/- 63.8 ng/ml) days after MEL when compared to sham-operated control animals (55.5 +/- 13.4 and 56.2 +/- 6.1 ng/ml, respectively). MEL altered plasma PRL and alpha-MSH levels in a differential manner, with a 1.5-to 5.0-fold increase in PRL and an 8.0-to 9.0-fold increase in alpha-MSH. The increase of alpha-MSH levels occurred abruptly and remained constant from days 2 to 14. These observations indicate that TIDA plays an important role in the pituitary release of PRL and alpha-MSH and provide evidence that the release of the two hormones occurs in a differential manner.

Morphologic changes and the expression of alpha-melanocyte stimulating hormone and melanocortin-1 receptor in melasma lesions: A comparative study

Melasma is a common acquired symmetrical hypermelanosis characterized by irregular light- to dark-brown macules on sun-exposed skin areas. The literature shows few studies on its physiopathogeny. However, changes in α-melanocyte stimulating hormone (α-MSH) secretion and melanocortin-1 receptor (MC1-R) expression may play a role to trigger this condition. Biopsies were taken from both melasma skin and adjacent perilesional normal skin of 44 patients. The biopsies were submitted for hematoxylin and eosin and Fontana-Masson staining and immunohistochemistry with Melan-A, α-MSH, and MC1-R, and processed for transmission electron microscopy. In some cases, they were submitted to MC1-R gene expression analysis by real-time polymerase chain reaction. Increased lymphohistiocytic infiltrate and solar elastosis, higher epidermal melanin were observed in melasma skin. Electron microscopy revealed a greater number of mature melanosomes in keratinocytes and melanocytes, and more prominent cytoplasmic organelles in melasma skin. There was no difference in melanocyte number between areas. However, melanocytes were larger and more dendritic in melasma skin. Immunohistochemistry with α-MSH and MC1-R showed significant labeling in melasmic epidermis but MC1-R messenger ribonucleic acid (RNAm) did not show significant quantitative difference between melasma and normal skin. © 2010 by Lippincott Williams & Wilkins.

Visceral colouration of Eupemphix nattereri (Anura: Leiuperidae): effects of NDP-α-melanocyte stimulating hormone

Amphibians have melanin-containing cells in visceral organs that are similar to pigmentary cells from the epidermis. Both of them are derived from the ectodermal neural crest. Epidermal cells respond to α-melanocyte stimulating hormone (α-MSH), which is associated to the dispersion of melanin granules within melanocytes. Therefore, our aim was to test whether a non-degradable analogue of the α-MSH changes the superficial colouration of organs of Eupemphix nattereri. The hormone rapidly increases (within 12 hours) the colouration on the surface of the pericardium, heart, testes, nerves of the lumbar plexus, and lumbosacral parietal peritoneum. Colouration increased late (after 24 hours) in the kidneys and mesentery following hormone administration. However, this hormone did not change colouration of intestine, rectum and lungs. Our findings could be explained by the similarities between epidermal and visceral melanocytes, since both cells have a common embryonic origin. Furthermore, the increase in visceral colouration may be related to the dispersion of melanosomes within melanocytes, which causes the darkening of organs. Our results demonstrate for the first time that the visceral colouration is responsive, thereby altering the internal pattern of organs' colouration in anurans. © 2013 Copyright 2013 Unione Zoologica Italiana.

Responses of melanocytes and melanomacrophages of Eupemphix nattereri (Anura: Leiuperidae) to Nle4, D-Phe7-α-melanocyte stimulating hormone and lipopolysaccharides

Melanocytes are found in various organs of ectothermic animals, playing a protective role against bacteria and free radicals. It is known that pigment cells from hematopoietic organs have immune functions. However, the role of visceral melanocytes is not well understood. Cutaneous melanocytes are responsive to α-melanocyte stimulating hormone (α-MSH), which is associated with the dispersion of melanin granules within melanocytes. α-MSH has also been reported to inhibit most forms of inflammatory responses by decreasing the pro-inflammatory cytokines and neutrophil migration. The present study evaluated the influence of an α-MSH analog (Nle4, D-Phe7-α-MSH) and lipopolysaccharides (LPS) from Escherichia coli on the liver and testicular tissues of the anuran Eupemphix nattereri. The tested hypotheses were: (i) the pigmented area will increase following hormone and LPS administration, (ii) pre-treatment with α-MSH will decrease the number of mast cells, and (iii) the hormone will have protective effects against LPS-induced responses. We found that hormone administration did not change hepatic pigmentation, but increased testicular pigmentation. Testicular pigmentation quickly increased after LPS administration, whereas there was a late response in the liver. The response of enhanced pigmentation was delayed and the number of mast cells decreased in animals previously treated with the α-MSH analog when compared to the LPS group. Hemosiderin and lipofuscin were found in melanomacrophages, but not in testicular melanocytes. Although both the liver and the testes of E. nattereri have pigmented cells, these are distinct in morphology, embryonic origin, and pigmentary substances. These differences may be responsible for the different responses of these cells to the α-MSH analog and LPS administration. © 2013 Elsevier GmbH.

PReS-FINAL-2357: Effects of anti-melanocyte stimulating hormone in murine pristine-induced lupus

Introduction Alfa-melanocyte stimulating hormone (α-MSH) has a variety of biological functions such as downregulation of pro-inflammatory pathways, reduction of skin delayed-type hypersensitivity and blockage of leukocyte migration. Inhibition of experimental disease models development including inflammatory bowel disease and rheumatoid arthritis has been shown, however the immunomodulatory and anti-inflammatory effects of α-MSH on murine lupus remain undetermined. Objectives To evaluate the effect of α-MSH analogue (NDP α-MSH) on pristane-induced murine lupus. Methods Thirty-five BALB/c mice were injected with 0.5 ml intraperitoneal (IP) pristane for lupus-like model induction and 5 age/gender matched control mice were given saline. Pristane-induced lupus animals received daily IP saline (n = 5) or treatments with 3.1 mg/kg/d chloroquine (n = 10), 1.25 mg/kg/d NDP α-MSH (n = 10) or 2.5 mg/kg/d NDP α-MSH (n = 10). Prior and 180 days after induction, clinical and laboratorial lupus-like parameters were examined. Sera ANA was tested by IF using Hep2 cells. Statistical analysis was performed by Mann-Whitney and Fisher test and P < 0,05 considered significant. Results Arthritis in both hind legs and large amounts of lipogranulomas in peritoneal cavity were observed in all lupus-like animals in contrast to all controls. By visual observation, all lupus animals treated with both doses of α-MSH had significant less amount and lower size lipogranulomas. Mean arthritis score in 5 untreated mice, 9 animals treated with chloroquine and 8 with α-MSH 2.5 mg/kg/d was 5.2, 3.33 and 3.1 respectively. Remarkably, mean arthritis score of animals treated with α-MSH 1.25 mg/kg/d was 1.6, significantly lower than untreated mice (1.6 vs 5.2, p = 0.0291). ANAs were negative in sera from all 40 animals before pristane lupus injection; 180 days after induction, ANAs remained negative in normal mice but became positive in all 5 (100%) untreated lupus animals, 7 (77%), 4 (50%) and 3 (35%) lupus models treated with chloroquine, α-MSH 2.5 mg/kg/d and α-MSH 1.25 mg/kg/d (100% vs 35%, p = 0,0256), respectively. Before the end of the experiment, by day 150, 3 animals died: 1 treated with chloroquine and 2 with higher doses of α-MSH. Conclusion NDP α-MSH promoted improvement of clinical and serological parameters in pristane-induced murine lupus suggesting a potential role for this drug in human SLE.