The Identification and Partial Cloning by PCR of the Gene for Tyrosinase-Related Protein-1 in the Mexican Axolotl

The tyrosinase gene family is currently composed of three members, tyrosinase and two tyrosinase-related proteins, TRP-1 and TRP-2. These three gene products have all been found to act in the synthesis of melanin pigments with the enzyme tyrosinase catalyzing the initial rate-limiting steps. Thus far these genes have primarily been analyzed in higher vertebrates. We have used degenerate PCR primers to isolate a large fragment of an axolotl tyrosinase-related protein. Sequence analysis of the entire 1,057-bp fragment isolated indicates a high degree of similarity to the mouse TRP-1, the product of the brown locus. Phylogenetic analysis supports the conclusion that the fragment isolated corresponds to the axolotl TRP-1 homolog. This is the first TRP-1 gene to be identified in an amphibian species.     

Composition of Mammalian Eumelanins: Analyses of DHICA-Derived Units in Pigments From Hair and Melanoma Cells

The proportions in which two eumelanin monomers, namely 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), compose the eumelanin polymer are believed to determine properties of the pigment including its color. These proportions are, however, not well elucidated for naturally occurring eumelanins, largely because of methodological difficulties. In this study we estimate the content of DHICA-derived units in mammalian eumelanins using a combination of two analytical techniques: 1) quantitation of DHICA-derived eumelanin by measuring the yield of pyrrole-2,3,5-tricarboxylic acid (PTCA index) and 2) spectrophotometrical quantitation of total (DHI + DHICA) eumelanin at 350 nm (A₃₅₀ index). The ratio of PTCA/A₃₅₀ measured for melanins synthesized from DHI and DHICA mixed in various molar proportions correlates well with the content of DHICA in synthetic polymers. Using this relationship as a standard curve we estimated the proportion of DHICA-derived units in mammalian eumelanins from hair and melanoma cells and found it to be much higher in rodent pigments (58.8%-98.3%; two species, mouse and hamster were examined) as compared to human eumelanins (19.2%-41.8%; one Caucasian and one Oriental individual were examined). No relationship between proportion of DHICA-derived units in eumelanin and hair color is found. The latter seems to be determined predominantly by the ratio of pheo- to eumelanin synthesis.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

Mutational Analysis of the Modulation of Tyrosinase by Tyrosinase‐Related Proteins 1 and 2 In Vitro

The albino (tyrosinase, Tyr^c), brown (tyrosinase‐related protein 1, Tyrp1^b) and slaty (tyrosinase‐related protein 2, tyrp2^slt) loci are all involved in the regulation of melanogenesis. Phenotypes of inbred mice mutant at two or more of these loci are not always explicable by simple summation of the established or suspected catalytic functions of the gene products. These phenotypes suggest that relationships among the proteins extend beyond the obvious fact that they catalyze different steps in the same melanogenic pathway, and that they may also interact intimately in such a way that a mutation in one impacts the function of the other(s). Previous studies have attributed catalytic activities to each member of this trio; however, it has been difficult to study the proteins individually, either in vivo or in tissues or cells. Therefore, we undertook to transfect the genes, in revealing combinations, into COS‐7 cells (which have no melanogenic apparatus of their own) to clarify the interacting functions of their encoded proteins. Specifically, we attempted to evaluate the effects of Tyrp1 and Tyrp2 proteins on tyrosinase protein. We report evidence that Tyrp1 stabilizes tyrosinase, confirming previous observations, and, in addition, demonstrate that Tyrp1 decreases tyrosinase activity. By contrast, Tyrp2 increases tyrosinase activity by stabilizing the protein. We conclude that both Tyrp1 and Tyrp2, in addition to other catalytic functions they may possess, act together to modulate tyrosinase activity.     

Stereological Studies of the Effects of α-MSH and cAMP on Melanosomes in Melanoma Cells

The effects of α-MSH and cAMP on melanosomes in Cloudman S91 melanoma cells were investigated by modern stereological techniques. Cells were cultured for 4 days in medium containing α-MSH or cAMP harvested at 24 hour intervals; some were frozen for melanin assay and the reminder embedded in Epon for light and electron microscopy. Cellular and melanosomal parameters were estimated by new stereological probes. We found that both stimulators induced increases in nuclear volume, cell volume, and the volume fractions and volumes of premelanosomes (V_Vpm,cell’V_pm) and mature melanosomes (V_Vmm,cell’V_mm) and the number of mature melanosomes (N_mm). Both stimulators also caused declines in the volume of individual mature melanosomes (_Vimm) the melanin content per mature melanosome unit volume and the melanin content per individual mature melanosome. The increases in the volume of individual premelanosomes and the number of premelanosomes were only induced by cAME The effect cAMP on some parameters occurred 24 hours prior to α-MSH and was more marked. The response of premelanosomes to the stimulators was more sensitive than mature melanosomes. These results suggest that both stimulators enchanced melanogenesis by increasing the V_Vpm,cell’V_Vmm,cell’V_pm, V_mm and N_mm. The melanogenic level did not depend on the _Vimm and melanin concentration in melanosomes. The maturation of premelanosomes was involved in melanogenesis induced by both stimulators, but, de novo synthesis and enlargement of premelanosomes were only stimulated by cAME It imply that exogenous cAMP may affect melanosomes, and hence melanogenesis in quantitatively or qualitatively different ways to α-MSH.     

Tyrosinase Activity (TH,DO, PAGE-Defined Isozymes) and Melanin Production in Regenerating Hairbulb Melanocytes of Lethal Yellow (Ay/a), Black (a/a), Agouti (AwJ/AwJ) and Albino (a/a/c2J/c2J) Mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in participate and soluble fractions of hairbulb melanocytes of lethal yellow (A^y/a C/C), nonagouti black (a/a C/C), and albino (a/a c^2J/c^2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, A^y/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (A^wJ/A^wJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and A^y/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and A^y/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in A^y/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

STAP-2 Protein Expression in B16F10 Melanoma Cells Positively Regulates Protein Levels of Tyrosinase,Which Determines Organs to Infiltrate in the Body

Melanoma is the most serious type of skin cancer, with a highly metastatic phenotype. In this report, we show that signal transducing adaptor protein 2 (STAP-2) is involved in cell migration, proliferation, and melanogenesis as well as chemokine receptor expression and tumorigenesis in B16F10 melanoma cells. This was evident in mice injected with STAP-2 shRNA (shSTAP-2)-expressing B16F10 cells, which infiltrated organs in a completely different pattern from the original cells, showing massive colonization in the liver, kidney, and neck but not in the lung. The most important finding was that STAP-2 expression determined tyrosinase protein content. STAP-2 colocalized with tyrosinase in lysosomes and protected tyrosinase from protein degradation. It is noteworthy that B16F10 cells with knocked down tyrosinase showed similar cell characteristics as shSTAP-2 cells. These results indicated that tyrosinase contributed to some cellular events beyond melanogenesis. Taken together, one possibility is that STAP-2 positively regulates the protein levels of tyrosinase, which determines tumor invasion via controlling chemokine receptor expression.     

Expression of DP2 (CRTh2), a Prostaglandin D2 Receptor,in Human Mast Cells

PGD₂ has long been implicated in allergic diseases. Recent cloning of a second PGD₂ receptor, DP2 (also known as CRTh2), led to a greater understanding of the physiological and pathophysiological implications of PGD₂. PGD₂ signaling through DP1 and DP2 mediates different and often opposite effects in many cell types of the immune system. Although mast cells (MC) are the largest source of PGD₂ in the body, there is little information about their potential expression of DP2 and its functional significance. In this study, we show that tissue MC in human nasal polyps express DP2 protein, and that human MC lines and primary cultured human MC express mRNA as well as protein of DP2. By immunohistochemistry, we detected that 34% of MC in human nasal polyps expressed DP2. In addition, flow cytometry showed that 87% of the LAD2 human MC line and 98% of primary cultured human MC contained intracellular DP2. However, we could not detect surface expression of DP2 on human MC by single cell analysis using imaging flow cytometry. Blocking of endogenous PGD₂ production with aspirin did not induce surface expression of DP2 in human MC. Two DP2 selective agonists, DK-PGD₂ and 15R-15-methyl PGD₂ induced dose-dependent intracellular calcium mobilization that was abrogated by pertussis toxin, but not by three DP2 selective antagonists. MC mediator release including degranulation was not affected by DP2 selective agonists. Thus, human MC express DP2 intracellularly rather than on their surface, and the function of DP2 in human MC is different than in other immune cells such as Th2 cells, eosinophils and basophils where it is expressed on the cell surface and induces Th2 cytokine and/or granule associated mediator release. Further studies to elucidate the role of intracellular DP2 in human MC may expand our understanding of this molecule and provide novel therapeutic opportunities.     

Expression of ODC Antizyme Inhibitor 2 (AZIN2) in Human Secretory Cells and Tissues

Ornithine decarboxylase (ODC) antizyme inhibitor 2 (AZIN2), originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s) of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3) to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.     

Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cells by Calcium lonophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. lonophore at a concentration of 10^–6 g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. lonophore A23187 also inhibits the PGEi mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10^–4M). lonophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGEi, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. lonophore causes a rapid and marked (> 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

Expression of Calmodulin-Dependent Phosphodiesterase, Calmodulin-Dependent Protein Phosphatase, and Other Calmodulin-Binding Proteins in Human SMS-KCNR Neuroblastoma Cells

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.     

Assembly,Target‐Signaling and Intracellular Transport of Tyrosinase Gene Family Proteins in the Initial Stage of Melanosome Biogenesis

Assembly, target‐signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein‐3 (AP‐3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di‐leucine motif in their cytoplasmic tail, to which AP‐3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Inhibitory Effects of Melanin Monomers,Dihydroxyindole-2-Carboxylic Acid (DHICA) and Dihydroxyindole (DHI) on Mammalian Tyrosinase,With a Special Reference to the Role of DHICA/DHI Ratio in Melanogenesis

DOPAchrome tautomerase (DCT) is known to control the ratio of DHICA/DHI formed within the melanocyte, but physiologic significance of this activity is not yet fully elucidated. In this study the two melanin monomers are shown to inhibit with different efficacy the initial, tyrosinase-controlled, melanogenic reaction, namely conversion of L-tyrosine to DOPAchrome (2-carboxy-2,3-dihydroindole-5,6-quinone). This is demonstrated in the test tube assay system whereby formation of DOPAchrome is catalyzed by i) isolated premelanosomes (PMS), ii) tyrosinase-rich PMS glycoproteins, or iii) tyrosinase purified from fibroblasts transfected with human tyrosinase gene. Both DHI and DHICA suppress the conversion of L-tyrosine to DOPAchrome when added to reaction mixture but the inhibitory effect is far more strongly pronounced by DHI. DHI inhibits both activities of tyrosinase—tyrosine-hydroxylation and DOPA-oxidation—more strongly than DHICA. The different extent of inhibition is shown to reflect i) the ability of the two monomers to compete with tyrosinase substrates for the enzyme's active center and ii) the rate of interaction between melanin monomers and DOPAquinone. Consequently, we demonstrate that the tyrosinase-catalyzed DOPAchrome formation can be modulated by the ratio of DHICA/DHI among melanin monomers with the increased proportion of DHICA resulting in more efficient DOPAchrome formation. These results raise the possibility that DOPAchrome tautomerase plays a role in positive control of the tyrosinase-catalyzed early phase of melanogenesis.     

Structural Insights into the Interaction between a Potent Anti-inflammatory Protein,Viral CC Chemokine Inhibitor (vCCI), and the Human CC Chemokine,Eotaxin-1

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.