Francois Rouzaud

Studies of mammalian pigmentation have identified many genes involved in the development, migration, and function of melanocytes. Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two signaling molecules, alpha-Melanocyte Stimulating Hormone (alphaMSH) and Agouti Signal Protein (ASP). AlphaMSH affects various aspects of melanocyte behavior, stimulating melanocyte dendricity, attachment to extracellular matrix proteins, but also up-regulating the expression of eumelanogenic genes. In response to ASP, melanocytes switch from producing eumelanin to pheomelanin concomitant with the down-regulation of melanogenic genes transcription. Since activation of signaling pathways leads to mRNA expression, microarray technology can provide a quantitative assessment of the consequences of this activation. Significant up/down regulation of all known melanogenic genes by alphaMSH/ASP in cultured melanocytes ...

Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma/KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an 8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells. cancer | melanosomes | skin | tumor therapy | multidrug resistance

ATP-binding cassette (ABC) transporters play a pivotal role in physiology and pathology. We identified and cloned two novel mRNA isoforms (ABCB 5α and ABCB 5β) of the ABC transporter ABCB 5 in human melanoma cells. The deduced ABCB 5α protein appears to be an altered splice variant containing only a putative ABC, whereas the ABCB 5β isoform shares approximately 70% similarity with ABCB1 (MDR1) and has a deduced topological arrangement similar to that of the whole carboxyl terminal half of the ABCB1 gene product, P-glycoprotein, including an intact ABC. Northern blot, real-time PCR, and conventional RT-PCR were used to verify the expression profiles of ABCB 5α/β. We found that the melanomas included among the NCI-60 panel of cell lines preferentially expressed both ABCB 5α and ABCB 5β. However, ABCB 5α/β expression was undetectable in two amelanotic melanomas (M14 and LOX-IMVI). The expression profile of ABCB 5α/β in all of the other melanomas of the panel was confirmed both by RT-PCR and by sequencing. Neither ABCB 5α nor ABCB 5β expression was found in normal tissues such as liver, spleen, thymus, kidney, lung, colon, small intestines or placenta. ABCB 5α/β mRNAs were also expressed in normal melanocytes and in retinal pigment epithelial cells, suggesting that ABCB 5α/β expression is pigment cell-specific and might be involved in melanogenesis. Our findings indicate that expression of ABCB 5α/β might possibly provide two novel molecular markers for differential diagnosis of melanomas and constitute potential molecular targets for therapy of melanomas.

ABSTRACT Mouse DOPAchrome tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn mice and were used in these studies. Dct activity is threefold lower in slaty cells compared with C57BL/6J black melanocytes whilst tyrosinase enzymatic activity is sevenfold higher. Homology modeling of the active site of Dct suggests that the slaty mutation (R194Q) is located in the first metal binding domain and may alter the ability of the enzyme to transform the substrate. Chemical analysis showed that eumelanin content is 25 times lower in slaty cells; however, the pheomelanin concentration was four times higher than in black melanocytes. In contrast, slaty light melanocytes have a surprisingly 28-fold lower Dct activity whilst tyrosinase activity was increased by fivefold. Slaty light melanocytes produce four times less eumelanin. Tyrosinase seems to be involved in the pheomelanin production as its intracellular concentration is increased 25-fold in slaty light cells compared with black melanocytes. Our results show that mutations in the Dct gene differentially affect not only the intracellular enzymatic activity of Dct as expected, but surprisingly also the tyrosinase activity and the pheomelanin production. Thus, Dct enzymatic activity may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis. Further characterization of melanocytes carrying mutations in the Dct gene will contribute to a better understanding of the importance of Dct expression for melanocyte function.

ABSTRACT Melanin synthesized by epidermal melanocytes protects the skin against UV induced damage. Exposure to UVR or stimulation by MSH increases melanogenesis through mechanisms involving direct effects on melanocytes which increase the synthesis of eumelanin upon activation of the melanocortin 1 receptor (MC1R), usually reported as a 317 amino acid (aa) protein. We now characterize the structure of MC1R350, a novel 350 aa human MC1R isoform bearing 5 Cys residues in an additional 33 aa C-ter sequence due to an alternative splicing. We examined its expression in cultured primary melanocytes, in paraffin embedded sections of human skin, and in melanocytes derived from melanotic, amelanotic or metastatic malignant melanomas. We used PEP19 (which detects both MC1R isoforms) and a novel polyclonal antibody specific to MC1R350 (PEP20) for Western blotting and immunofluorescence in addition to real time RT-PCR, to study the expression of both isoforms at the protein and mRNA levels. In normal melanocytes, the melanin content correlates well with the total amount of MC1R (r2 = 0.6) and even better with the levels of MC1R317 (r2 = 0.7) but only poorly with the amount of MC1R350. MC1R317 increased its expression upon MSH treatment whilst MC1R350 levels were reduced in some cases. Immunohistochemistry with PEP20 and N2-28 antibodies revealed similar low levels of MC1R350 in Asians and Caucasians and a significant increase in African Americans. Upon 1 MED UV, African Americans skins showed a peak of immunoreactivity for both MC1R isoforms 1 d after irradiation. Further studies will fully analyze the expression of the MC1R isoforms to better understand the role of MC1R350 in pigmentation and melanoma susceptibility and its interactions with MC1R317.

Melanosomes provide an intriguing model for study at many levels. In part this is due to their unique structure and function, but also in part to their involvement in pigmentary diseases and as a model to study basic cellular mechanisms of organelle biogenesis. Recent studies have elucidated the full proteome of the melanosome and the metabolic and molecular lesions involved in a number of pigmentary diseases have been resolved. This paper summarizes recent advances in the field in these areas.

The effect of androgens on human melanocytes has not been well clarified. We studied the effects of androgens on normal human melanocytes in the presence or absence of sex-hormone-binding globulin (SHBG), which complexes with those hormones. Immunohistochemically, testosterone and SHBG co-localized on the cell membrane. Androgens such as testosterone, 5α-dihydrotestosterone, and methyltrienolone (R1881, a potent synthetic androgen), reduced intracellular cAMP levels after treatment with SHBG, but hydrocortisone had no effect. We also found that testosterone and R1881 slightly suppressed tyrosinase activity in melanocytes when treated with SHBG, although they had no effect on the expression of tyrosinase at the transcriptional or translational level, as measured by semi-quantitative reverse transcriptase-polymerase chain reaction and by Western blot analysis, respectively. Our results suggest that androgens may modulate tyrosinase activity at the posttranslational level through the cell membrane signaling pathway.

Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form mature, fibrillar, and pigmented organelles. Recently, we reported that the less glycosylated form of Pmel17 (termed iPmel17) is sorted via the plasma membrane in a manner distinct from mature Pmel17 (termed mPmel17), which is sorted directly to melanosomes. To clarify the mechanism(s) underlying the distinct processing and sorting of Pmel17, we generated a highly specific antibody (termed alphaPEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation. alphaPEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with alphaPEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using alphaPEP25h demonstrate that iPmel17 differs from mPmel17 not only in its sensitivity to endoglycosidase H, but also in the content of core 1 O-glycans modified with sialic acid. This evidence reveals that iPmel17 is glycosylated differently in the Golgi and that it is sorted through the secretory pathway. Analysis of Pmel17 processing in glycosylation-deficient mutant cells reveals that Pmel17 lacking the correct addition of sialic acid and galactose loses the ability to form fibrils. Furthermore, we show that addition of sialic acid affects the stability and sorting of Pmel17 and reduces pigmentation. Alterations in sialyltransferase activity and substrates differ between normal and transformed melanocytes and may represent a critical change during malignant transformation.

Holstein (A), Limousine (B) and Charolaise (C) bovines showing the characteristic pigmentation pattern. See The Untranslated Side of Hair and Skin Mammalian Pigmentation: Beyond Coding Sequences by Francois Rouzaud, Ahmad Oulmouden, and Lidia Kos, pp. 340–346.