Tyrosinase mRNA Levels Research Articles

Base Substitution at Different Alternative Splice Donor Sites of the Tyrosinase Gene in Murine Albinism

Thec2jalbino mutation at the mouse tyrosinase locus arose spontaneously in the C57BL/6 inbred strain and causes complete absence of melanin synthesis, as does the “classical”cmutation of long-established albino inbred strains. Sequence analysis ofc2jcDNA reveals a G → T point mutation at nt 291, causing an arginine → leucine substitution in codon 77, where the arginine position has been conserved in vertebrate tyrosinases and tyrosinase-related proteins. Whilec2jdiffers fromc,in which there is a G → C mutation at nt 369 causing a cysteine → serine substitution, both mutations change the G1 position of alternative 5′ splice donor sites in exon 1. Bothc2jandcabolish the usage of the respective sites for alternative splicing of the tyrosinase pre-mRNA in skin melanocytes. Inc2j,there results an almost eightfold increase in activation of the 5′ splice site located 78 nt downstream, but incthere is no activation of the intact upstream splice site. Although the tyrosinase mRNA levels are similar inc2jand wildtype, the protein is virtually absent inc2j,as inc,possibly due to proteolytic degradation.

α-Melanocyte Stimulating Hormone-Induced Pigmentation Is Blocked by Depletion of Protein Kinase C

We have explored the role of protein kinase C (PKC) in pigmentation induced by α-melanocyte stimulating hormone (α-MSH). Using the well-studied S91 Cloudman mouse melanoma model system in which 10−7Mα-MSH is known to produce a time-dependent increase in pigmentation, we found an increase in the activity of tyrosinase, the key enzyme in pigmentation, between Days 2 and 6 accompanied by an increase in mRNA and protein levels of tyrosinase, as well as an increase in the level of specifically the β isoform of PKC. When S91 cells were treated with phorbol dibutyrate, 95% of PKC activity was lost within 48 h and the α-MSH-induced melanogenesis was completely blocked, as was the induction of tyrosinase mRNA and protein. Serially passaged S91 cells no longer capable of responding to α-MSH had an undetectable level of PKC-β, although the tyrosinase protein level was identical to that of α-MSH-responsive cells. Furthermore, in these S91 cells α-MSH also did not increase the level of tyrosinase mRNA. Thus, induction of murine melanogenesis by α-MSH involves up-regulation of tyrosinase mRNA and protein mediated in part by the PKC-dependent pathway, associated with an up-regulation of the β isoform previously demonstrated to specifically activate tyrosinase in human melanocytes.

Melanogenesis, biosynthetic phenotype of fibronectin and collagen, and migrating activity in cloned B16 mouse melanoma cells

Three clones, melanotic (M3), amelanotic (A4 and A7) cells were isolated from B16 F10 mouse melanoma cell line. These clones exhibited different biosynthetic activities of fibronectin and collagen: A4 clone showed relatively active synthesis of both collagen and fibronectin, and A7 clone exhibited most active fibronectin synthesis, whereas no significant synthesis of these molecules was observed in M3 clone. No significant difference in growth rate was observed in these three clones. Migrating activities measured by basement membrane matrix-coated dishes were greater in A7 clone than in A4 and M3 clones. Messenger RNA levels of collagen and fibronectin paralleled collagen and fibronectin synthesis in these clones whereas tyrosinase mRNA level was unaltered between melanotic (M3) and amelanotic (A4 and A7) cells. These results indicate that B16 melanoma cells have heterogeneous cell populations consisting of different biosynthetic and metastatic properties. These clones may provide good tools for studying the relationship between the phenotypes of melanogenesis biosynthesis of extracellular macromolecules and migrating activity of melanoma cells.

Detection and quantitation of melanoma cells in the circulation of patients

A method for the quantitation of tyrosinase mRNA in the bloodstream of melanoma patients has been developed by using competitive polymerase chain reaction (PCR) with a heterologous DNA (PCR MIMIC) as an internal standard. The method was validated by demonstration of similar amplification efficiencies for both molecules and by accurate quantitation of an artificial fourfold difference in the level of tyrosinase mRNA. The ratio of amplified target to amplified standard (At/As ratio) was determined for a range of melanoma patients who had previously tested positive for tyrosinase. Sequential samples were also analysed to examine the level of tyrosinase in individual patients over time. When tyrosinase levels in melanoma cell lines were compared for a constant amount of total RNA, or for a constant number of cells, tyrosinase mRNA was found to vary more than a thousand-fold between cell lines. Because of this, the At/As ratio of patients was compared with the At/As ratios obtained when 10-fold serial dilutions of cells from a control melanoma cell line (MM200) were added to 2 ml of packed blood. An 'MM200 equivalence' value was thus calculated, giving the equivalent number of MM200 cells in the bloodstream of melanoma patients. Prolonged follow-up will be needed to determine the prognostic significance of the detection and levels of tyrosinase mRNA in the bloodstream of melanoma patients.

Correlation between the number of melanosomes, tyrosinase mrna levels, and tyrosinase activity in cultured murine melanoma cells in response to various melanogenesis regulatory agents

Tyrosinase is the rate limiting enzyme critically associated with melanin synthesis. The melanosomes are specialized membrane-bound organelles within melanocytic cells in which melanin polymers are ultimately deposited. To determine whether tyrosinase correlates with the number of melanosomes, we examined the relationship between tyrosinase activity, tyrosinase mRNA levels, and the number of melanosomes in B16 murine melanoma cells, using melanogenesis regulatory agents. 12-O-Tetradecanoylphorbol-13-acetate (TPA) or linoleic acid decreased tyrosinase activity, while dibutyryl cyclic adenosine monophosphate (dbcAMP) or palmitic acid increased it. The tyrosinase mRNA levels were not always correlated with tyrosinase activity, i.e., TPA down-regulated, dbcAMP upregulated, while linoleic acid or palmitic acid did not alter the message levels, indicating that fatty acid regulation of melanogenesis was due to post-transcriptional events. The number of melanosomes changed when agents which modulate the tyrosinase gene expression were added, since TPA decreased, dbcAMP increased, and linoleic acid or palmitic acid did not alter their number. These results suggest that the number of melanosomes changed in relation to tyrosinase mRNA level but not to tyrosinase activity in response to melanogenesis regulatory agents.

Alteration of tyrosinase activity in human melanocytes and melanoma cells by histamine H2 and H3 ligands

Nontoxic doses of the histamine H2 antagonists ranitidine, cimetidine, lamtidine and mifentidine rapidly and reversibly increased tyrosinase activity in an amelanotic human melanoma cell line (MM96L) with low constitutive activity. The H2 antagonists, famotidine and MGTI, and the imidazol(in)e receptor ligand clonidine had no effect either alone or in competition with ranitidine, whilst metiamide decreased tyrosinase activity. Lysosomotropic amines had a similar effect to ranitidine, except that induction reached a plateau at 6 h and was insensitive to amiloride. Human melanocytes and pigmented human melanoma cell lines exhibited minimal levels of tyrosinase induction, which was dependent on protein synthesis but not on RNA or DNA synthesis. Constitutive tyrosinase activity in MM96L cells was much less stable than in melanocytes and pigmented melanoma cells. No change was observed in expression of gp75, neural specific octamer binding proteins, or in mRNA levels of tyrosinase, Pmel-17 and gp75 (TRP-1). Tyrosinase was inhibited by the H3 agonist imetit but not by alpha-methylhistamine or the H3 antagonist thioperamide. Overall, this work showed that certain H2 antagonists activate an unstable form of tyrosinase in amelanotic melanoma cells by a post-transcriptional mechanism dependent on protein synthesis. An imidazoline/guanidinium receptor site rather than the H2 receptor appeared to be involved.

Regulation of Melanogenesis Induced by 5‐Methoxypsoralen Without Ultraviolet Light in Murine Melanoma Cells

Melanogenesis in melanoma cells can be enhanced by psoralens in the absence of UV light. Melanin biosynthesis is regulated by a number of melanocyte-specific proteins, including tyrosinase, DOPAchrome tautomerase (DCT), and tyrosinase-related protein-1 (TRP-1, gp75). To get more insight on the molecular mechanisms involved in psoralens-induced melanogenesis, we determined tyrosinase and DCT activities as well as mRNA and protein levels of tyrosinase, DCT, and TRP-1 in S91 mouse melanoma cells treated by 5-MOP. High concentration of 5-MOP (5 x 10(-5) M) induced a time-dependent increase of tyrosinase activity and melanin content, which was correlated to an increase of both mRNA and protein levels of tyrosinase. These results demonstrate that the 5-MOP stimulation of melanogenesis is related to increased tyrosinase synthesis. In addition, 5-MOP stimulated TRP-1 synthesis and induced a dose-dependent decrease of DCT activity without any modification in the expression of the protein. We explored then the signalling pathways involved in 5-MOP-induced melanogenesis and, particularly, the role of cyclic AMP and protein kinase C (PKC). A small stimulation of cyclic AMP production was observed in presence of 5-MOP. Furthermore, 1-oleoyl-2-acetylglycerol (OAG), a PKC activator, potentiated the 5-MOP stimulation of tyrosinase activity, while calphostin, a specific PKC inhibitor, inhibited the 5-MOP induction of tyrosinase activity. Phorbol-myristate acetate (PMA), described as a strong activator of PKC, inhibited also the effect of 5-MOP when used at long term. Taken together, these results demonstrate that in murine melanoma cells 5-MOP stimulates melanogenesis by increasing activity and synthesis of tyrosinase. Tyrosinase and TRP-1 expression are coordinately regulated by 5-MOP. Furthermore, a negative correlation between melanogenesis and DCT activity was observed under 5-MOP stimulation. At least, PKA and PKC systems appear to play an important role in the melanogenic effect of 5-MOP.

Eumelanin Biosynthesis Is Regulated by Coordinate Expression of Tyrosinase and Tyrosinase-Related Protein-1 Genes

Melanin is specifically produced in melanocytes. The pathway for melanin biosynthesis is regulated by a number of melanocyte-specific proteins, including tyrosinase and tyrosinase-related protein-1 (TRP-1, b locus protein). To understand the regulation of melanogenesis, we examined tyrosinase activities, mRNA levels of tyrosinase and TRP-1, and eumelanin and pheomelanin contents in mouse B16-F1 melanoma cells after they had been treated with some melanotropic reagents. Cholera toxin, α-melanocyte-stimulating hormone, and dibutyryl cyclic AMP increased tyrosinase activity and stimulated eumelanin biosynthesis. These reagents elevated intracellular cAMP levels. In contrast, 12-O-tetradecanoylphorbol 13-acetate reduced tyrosinase activity and eumelanin synthesis. In all cases, the mRNA levels of tyrosinase and TRP-1 changed in parallel with tyrosinase activity and eumelanin content. TRP-1 was induced simultaneously with tyrosinase, although its inducibility was lower than that of tyrosinase. These results suggest that the expressions of tyrosinase and TRP-1 genes are coordinately regulated by melanotropic reagents through cAMP-dependent protein kinase and protein kinase C in mouse B16-F1 cells, and that their coordinate expression causes eumelanin biosynthesis.

Role of Tyrosinase as the Determinant of Pigmentation in Cultured Human Melanocytes

Variations in human pigmentation among different racial groups are due to differences in the production and deposition of melanin in the skin. Although melanin synthesis is known to be controlled by the rate-limiting enzyme tyrosinase, the role of this enzyme as the principal determinant of skin pigmentation is unclear. Results from studies with human melanocyte cultures derived from different racial skin types reveal an excellent correlation between the melanin content of melanocyte cultures and the in situ activity of tyrosinase. Melanocytes derived from black skin have up to 10 times more tyrosinase activity and produce up to 10 times more melanin than melanocytes derived from white skin. However, the higher level of tyrosinase activity in melanocytes derived from black skin is not due to a greater abundance of tyrosinase. Results from immunotitration experiments and Western immunoblots reveal that the number of tyrosinase molecules present in white-skin melanocytes may equal the number found in highly pigmented black skin types. Moreover, approximately equivalent levels of tyrosinase mRNA are present in white and black skin cell strains. In contrast, melanocytes derived from red-haired neonates with low tyrosinase activity contain low numbers of tyrosinase molecules and low levels of tyrosinase mRNA. These results show that tyrosinase activity and melanin production in most light-skinned people is controlled primarily by a post-translational regulation of pre-existing enzyme and not by regulating tyrosinase gene activity. In contrast, melanocytes from red-haired (type I) people have low levels of tyrosinase protein and mRNA, suggesting that transcriptional activity of the tyrosinase gene is suppressed.

Differential Regulation of Tyrosinase Activity in Skin of White and Black Individuals In Vivo by Topical Retinoic Acid

Tyrosinase activity is a key determinant of melanin production in skin. Because retinoic acid regulates tyrosinase activity in melanoma cells, we analyzed modulation of pigmentation in vivo by retinoic acid. Black and white subjects were either not treated, or treated topically for 4 d under occlusion with vehicle, retinoic acid (0.1%), or the irritant sodium lauryl sulfate (2%). In untreated skin, tyrosinase activity and melanin content were significantly greater (2.3 times, and 3.2 times, respectively) in blacks versus whites. Four days of treatment with topical retinoic acid did not alter tyrosinase activity or melanin content in black skin. In contrast, retinoic acid treatment significantly induced (2.7 times, n = 8) tyrosinase activity, compared to vehicle treatment, in white skin. Melanin content, however, remained unchanged at 4 d. In separate experiments, tyrosinase activity in white subjects (n = 25) was increased 16% (p = 0.01) in sodium lauryl sulfate-treated skin, and 77% (p = 0.0005) in retinoic acid-treated skin, compared to vehicle-treated skin. The effect of retinoic acid on tyrosinase activity could be differentiated from non-specific irritation, because tyrosinase activity in retinoic acid-treated skin was significantly greater (52%, p = 0.004) than sodium lauryl sulfate-treated skin. Similar results were obtained with the dihydroxyphenylalanine reaction done on vehicle, sodium lauryl sulfate-, and retinoic acid-treated white skin. Northern analysis (n = 6) and semi-quantitative polymerase chain reaction (n = 6) demonstrated that retinoic acid treatment did not alter tyrosinase mRNA levels in white skin. Western analysis revealed that induction of tyrosinase activity by retinoic acid also was not associated with increased tyrosinase protein content (n = 9), indicating that regulation of tyrosinase activity by retinoic acid occurs through a post-translational mechanism. These data demonstrate that low tyrosinase activity in white skin in vivo is retinoic acid inducible and high tyrosinase activity in black skin in vivo is neither further induced nor reduced by retinoic acid.

Effects of melanocyte-stimulating hormone on tyrosinase expression and melanin synthesis in hair follicular melanocytes of the mouse.

Tyrosinase mRNA, synthesis and activity were measured in the skin during the first 2 weeks of life of C3H-HeAvy mice. Tyrosinase mRNA levels were found to peak on days 3-4 and were followed by increases in tyrosinase synthesis and activity which peaked on days 6-7 and 7-8 respectively. These changes in tyrosinase expression were presumably associated with the growth of the first coat of hair that in neonatal C3H-HeAvy mice is yellow in colour as a result of the increased proportion of phaeomelanin. By the time hair growth had ceased there was no expression of tyrosinase at both mRNA and protein levels. Daily administration of alpha-melanocyte stimulating hormone (alpha-MSH) enhanced the expression of tyrosinase mRNA transcripts, tyrosinase synthesis and activity. The increase in tyrosinase activity paralleled the change in the amount of tyrosinase, suggesting that the primary action of alpha-MSH is to stimulate new synthesis of the enzyme. This induction of tyrosinase was associated with the growth of hair that was darker in colour than that of the controls and contained an increased proportion of eumelanin. This increase in eumelanin reflected a decrease in phaeomelanin content. It was concluded that, through its actions on the enzyme tyrosinase, alpha-MSH is able to switch the synthesis of phaeomelanin to that of eumelanin in hair follicular melanocytes.

Suppression of tyrosinase gene expression by bromodeoxyuridine in Syrian hamster melanoma cells is not due to its incorporation into upstream or coding sequences of the tyrosinase gene.

5-Bromodeoxyuridine (BrdU), a thymidine analog, suppresses melanogenesis in Syrian hamster melanoma cells. Tyrosinase, which is the key enzyme for the synthesis of melanin, is suppressed by exposure to BrdU, and the drop in enzyme activity is correlated with a drop in tyrosinase mRNA level. In order to investigate whether suppression of tyrosinase mRNA by BrdU is due to BrdU substitution into coding sequences or upstream sequences of the tyrosinase gene, we carried out stable and transient transfection assays with constructs containing either the human tyrosinase cDNA sequence under the control of a nontyrosinase promoter or a chloramphenicol acetyltransferase (CAT) reporter gene under the control of 5' flanking sequences of the mouse tyrosinase gene. When the plasmid containing the tyrosinase cDNA was stably transfected into mouse fibroblasts, tyrosinase activity in the transfectants was not suppressed by BrdU. Since BrdU would be incorporated into the tyrosinase cDNA integrated in these transfectants, the results suggest that BrdU suppression of tyrosinase gene expression is not due to its incorporation into coding sequences of the tyrosinase gene. When plasmids with tyrosinase regulatory sequences were transfected into melanoma cells for transient expression assays, CAT gene expression was suppressed by BrdU. Because the CAT plasmids do not contain a mammalian origin of replication and should not replicate under the conditions of transient transfection, BrdU would not be incorporated into the DNA of those plasmids. Therefore, these results suggest that the suppression of tyrosinase gene expression by BrdU also is not due to the incorporation of BrdU into upstream sequences of the tyrosinase gene.

Tyrosinase Gene Transcription and Its Control by Melanogenic Inhibitors

The levels of tyrosinase mRNA and tyrosinase activity were analyzed in two amelanotic melanoma cell lines, D1(178) (hamster) and G-361 (human). Neither tyrosinase mRNA nor tyrosinase activity were detected in D1(178) cells. On the other hand, both tyrosinase mRNA and weak tyrosinase activity were detected in G-361 cells. Assuming that the different types of melanogenic inhibitors affected melanogenesis in these two amelanotic melanoma cells in different manners, we performed a screening of melanogenic inhibitors in these two cell lines. As an isolated tyrosinase suppressive melanogenic inhibitor, ascorbic acid and glutathione were identified from D1(178) cells and G-361 cells, respectively. Furthermore, lactic acid was identified from D1(178) cells as an isolated tyrosinase non-suppressive melanogenic inhibitor. B-16 mouse melanotic melanoma cells were depigmented by treatment with lactic acid. The melanogenesis suppression by lactic acid in B-16 cells was found to be due to inhibition of tyrosinase gene expression.

リノール酸のメラノジェネシスに対する抑制効果

Linoleic acid has an inhibitory effect on melanogenesis in B16 mouse melanoma cells. Linoleic acid did not affect the expression level of tyrosinase mRNA and the pattern of tyrosinase glycosylation. The production of premelanosomes and the transfer of tyrosinase into them were also not affected by linoleic acid. The decrease of tyrosinase activity in golgi apparatus and the inhibition of melanin polymer formation within premelanosomes were found in linoleic acid-treated B16 cells.Using cultured normal human melanocytes, it was shown that linoleic acid inhibited melanogenesis as well as using B16 cells.We also report here the evidence that linoleic acid can diminish UVB-induced pigmentation. Linoleic acid was topically applied daily for one month after UVB-induced pigmentation was fully developed on the back of brownish guinea pigs, and resulted to marked depigmentation.

Glucocorticoid Stimulates Melanogenesis and Tyrosinase Gene Expression in B16 Melanoma Cells

Effects of dexamethasone on melanogenesis and tyrosinase mRNA levels were determined in B16/F10 melanoma cells. Melanin content of B16 cells increased in a dose-dependent manner by the addition of dexamethasone to the culture medium. After 72 hr exposure, dexamethasone (10(-6) M) produced a 2.4-fold increase in melanin content. Northern blot analysis revealed that tyrosinase mRNA level also increased by the addition of dexamethasone to the culture medium. After 24 hr exposure, dexamethasone (10(-6) M) caused a 1.8-fold increase in tyrosinase mRNA levels. A tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) decreased tyrosinase mRNA level at 30 nM concentration. Dexamethasone antagonized this TPA-mediated decrease in tyrosinase mRNA. It is suggested that glucocorticoids are involved in the regulation of tyrosinase activity at the transcriptional level.

Pigment content of cultured human melanocytes does not correlate with tyrosinase message level

Tyrosinase is considered to be the rate-limiting enzyme for the biosynthesis of melanin in epidermal melanocytes, and thus tyrosinase activity is thought to be a major regulatory step in melanogenesis. To determine whether the rate of pigment production was controlled at the level of tyrosinase gene expression, we developed a culture system capable of generating large populations of pure human melanocytes and then measured both melanin content as determined spectrophotometrically by absorption at 475 nm and mRNA levels as detected by hybridization with cloned cDNA Pmel 34, encoding human tyrosinase. We examined the relationship between pigment content and tyrosinase mRNA levels among human melanoma and melanocyte lines with very different levels of basal pigmentation; between two clones of a single human melanoma line, one pigmented and one amelanotic; and sequentially in melanocytes before and after simulation with isobutylmethylxanthine to increase melanin content per cell. Using Northern blot analysis and in-situ hybridization we found no correlation between tyrosinase message levels and melanin content, suggesting that posttranscriptional regulation of tyrosinase and/or other events determine the rate of pigment synthesis in human melanocytes.

L-tyrosine induces tyrosinase expression via a posttranscriptional mechanism

Exposure of hamster amelanotic melanoma cells to L-tyrosine caused a time-dependent increase of tyrosinase protein concentrations, tyrosinase activity and level of cell pigmentation. In contrast, Northern blot analysis using mouse tyrosinase cDNA showed a steady level of tyrosinase mRNA. Thus in hamster melanoma cells the stimulation of intracellular tyrosinase concentration by L-tyrosine is mediated mainly via a posttranscriptional mechanism.

Differential Expression and Activity of Melanogenesis-Related Proteins During Induced Hair Growth in Mice

In C57 Bl-6 mice, melanogenesis is strictly coupled to the growth phase of the hair cycle (anagen). To further study this phenomenon of concerted developmental and pigmentary activity, we followed the sequence of tyrosinase (key enzyme of melanogenesis) expression and activity and the presence of the melanosomal protein gp 75 during the development of traumatically induced anagen follicles (days 0 = telogen, and days 1-12, after anagen induction studied). In addition to performing Northern and Western blots for tyrosinase, tyrosine hydroxylase activity (THA) and dopa oxidase activity (DOA) were measured. On day 0, DOA was undetectable, and THA was very low. On days 1 and 2, both activities were undetectable; starting from day 3, they increased rapidly, reaching a plateau on days 8 and 12. DO-positive proteins had apparent molecular weights (MW) of 66-68 kD (days 3-12), 72-74 kD (days 5-12), and 130 kD (days 8 and 12). Western blotting emphasized proteins of MW 66-68 kD (tyrosinase), and 73-75 kD (gp 75); tyrosinase was undetectable on day 0, but already present on days 1 and 2; it increased by day 5 and had reached a plateau on days 8 and 12; gp 75 was undetectable on days 0-2; it was present on day 3, increased by day 5, and reached a plateau on days 8 and 12. Northern blot analysis revealed high levels of tyrosinase mRNA on days 5 and 8, low levels on days 1-3, and none on day 0. These data suggest a highly regulated, time frame-restricted, differential pattern of tyrosinase transcription, translation, and enzyme activity during the different stages of the developing murine anagen follicle, possibly as a result of complex interactions between follicular melanocytes and their environment.

Tyrosinase Expression and Melanogenesis in Melanotic and Amelanotic B16 Mouse Melanoma Cells

Tyrosinase activity, abundance, and mRNA transcription were examined in three sublines of the B16 mouse melanoma. Tyrosinase activity and melanin content were highest in the B16-F1 cells, slightly less in the B16-F10, and markedly lower in the B16-F10-DD cells. No differences in the level of tyrosinase mRNA or protein were found in the three different sublines. Thus, the differences in tyrosinase expression arise from the post-translational modification of the enzyme causing its activation or inhibition.

Molecular mechanism of tyrosinase regulation by L-DOPA in hamster melanoma cells

Exposure of hamster amelanotic melanoma cells to L-dihydroxyphenylalanine (L-DOPA) resulted in a time dependent increase of cell pigmentation, tyrosinase concentration and activity with peak after 24 hours. Northern blot analysis showed a small but reproducible increase of tyrosinase mRNA after 3 hours and a decrease below the control level after 9 hours. After 24 and 48 hours tyrosinase mRNA was undetectable. It is suggested that L-DOPA or its oxidation products can stimulate intracellular tyrosinase concentration and regulate tyrosinase mRNA level both in positive and negative fashion.