Regulation Of DHICA-mediated Antioxidation By Dopachrome Tautomerase

Human epidermal melanocytes function as a pivotal protective barrier against ultraviolet (UV) radiation and oxidative stress by generating the radical-scavenging pigment melanin within compartments called melanosomes. In melanosomes, which are the membrane-bound organelles wherein melanin is produced, tyrosinase, tyrosinase-related protein 1 (Tyrpl) and dopachrome tautomerase (Dct) form a complex. Dct is a member of the tyrosinase-related protein family, and the two other members of this family, tyrosinase and Tyrpl, are also melanogenic enzymes with distinct functions. Mutations in the genes encoding tyrosinase or Tyrpl also lead to hypopigmentation and, in humans, have been associated with two different pigmentary disease, OCA (oculocutaneous albinism) typesⅠandⅢrespectively. To date, Dct has not been associated with a human pigmentary disease. The gene encoding Dct maps to mouse chromosome 14, at the coat colour locus slaty. Mutant Dct produced by slaty mice has a single amino acid difference compared with wild-type Dct, namely an R194Q (Arg194→Gln) substitution in the first metal-binding domain. Dct activity in immortalized slaty melanocytes was reduced to 36% of the enzyme level in immortalized wild-type dermal melanocytes. The detailed melanosome biogenesis in slaty melanocytes is not still well studied. In this study, we attempted to define the effects of mutation in Dct on melanosome maturation in cultured slaty melanocytes.It has long been recognized that certain melanogenic intermediates, such as L-3,4-dihydroxyphenylalanine and 5,6-dihydroxyindole (DHI), exhibit intrinsic cytotoxic effects toward active melanocytes per se. However, no toxicity derived from such melanin precursors is normally discerned under physiological circumstances, which suggests that MCs possess a potent enzymatic and/or nonenzymatic defense mechanisms against oxidative stress in addition to the mechanical confinement of melanin-synthetic reactions within melanosomes to prevent toxic intermediates from potential leakage into the cytoplasm. To date, Dct has been well characterized as a specific enzyme that isomerizes the intermediate dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), aiming to detoxify its decarboxylated counterpart DHI, which is produced spontaneously in the absence or insufficiency of Dct enzyme activity. It is generally accepted that Dct controls the content of carboxylic groups in natural eumelanins owing to the incorporation of various proportions of DHICA monomers into the DHI polymer backbone in nascent eumelanin, but the physiologic significance of the Dct-regulated DHICA/DHI ratio has remained elusive. Recently, we successfully generated Dct knockout (Dct-/-) mice by removing exon 1 of the mouse Dct gene, and the homozygous Dct mutant mice completely lack Dct mRNA and protein expression. Unexpectedly, Dct-/- mice show only a mild dilution of coat color, which differs from mutations in tyrosinase and Tyrpl that cause significant oculocutaneous albinism hypopigmented phenotypes. We speculate that Dct-deficient MCs in the knockout mice lose Dct enzyme activity, which is probably compensated for by the up-regulation of tyrosinase and Tyrp1 activities in vivo because the three melanogenic proteins (Tyr, Tyrp1, and Dct) reside in the same multiple-enzyme complex inside melanosomes. In this study, we attempted to define the effects of Dct deficiency on the scavenging capacity of epidermal reactive oxygen species (ROS) and skin photoprotection against UVA radiation, measuring sunburn cell (SBC) formation, epidermal cell apoptosis, and melanin composition in the skin of Dct-/- mice compared with the skin of wild-type C57BL/6 mice after chronic UVA radiation. We also provide new evidence that the antioxidative properties of eumelanins are influenced by changing the proportions of DHICA/DHI monomers incorporated.Part 1 Effects of mutation in dopachrome tautomerase on melanosome maturation in cultured melanocytesObjective To investigate whether the mutation in Dct affects melanosome maturation in cultured melanocytes. Methods Slaty melanocytes and melan-a melanocytes were derived from the skins of neonatal Dctslt and C57 BL/6J mice, respectively. Their detailed ultrastructures of melanosome were examined by a transmission electron microscopy (TEM). And their eu-melanin granules were characterized by Fontana-Masson staining. Furthermore, the tyrosianse activity activity was measured with a spectrophotometery method. And the protein expression levels of tyrosinase, tyrosinase-related protein 1, and Dct were determined with western blot assay.Results Mature stage IV melanosomes were greatly decreased in slaty melanocytes under the transmission electron microscopy. The brownish granules stained with Fontana-Masson silver method were far less in slaty melanocytes than in melan-a melanocytes. The cell pellet of slaty melanocytes was white in color, but the similarities between slaty and melan-a were found in tyrosinase activity and their protein expression.Conclusions The mutation in Dct causes hypopigmented phenotype in cultured slaty melanocytes, and inhibits melanosome maturation.Part 2 Defficiency of dopachrome tautomerase relates to UVA-induced apoptosis in the epidermisObjective To investigate the impacts of UVA irradiation on apoptosis of epidermic cells in wild-type C57BL/6J mice and Dct -/- mice.Methods Dct-/- knockout mice and wild-type C57BL/6J mice were irradiated with 5 J/cm2 of UVA irradiation, three times a week for up to 4 weeks. Total UVA dose was 60 J/cm2. And then sunburn cell (SBC) was counted by light microscopy based on their characteristic morphology and apoptotic cells was identified by TUNEL staining on paraffin-embedded section. The level of homogenate reactive oxygen species (ROS) was determined by DCFH-DA labeling.Results The results showed that SBC number, TUNEL-positive cells, in the UVA-irradiated skins were much higher in Dct -/- mice than in wildtype mice (P<0.01). The numbers of SBC and TUNEL-positive cell is much higher in both mice exposed to chronic uv irradiation than that in unirradiated control (P<0.01).But no difference was found in both unirradiated mice (P>0.05). ROS level in the UVA-irradiated skins were much higher in Dct -/- mice than in wildtype mice.Conclusions The findings implicate the possible role of Dct in oxidative stress scavenging and skin UVA photoprotection, the intrinsic defect of Dct in vivo facilitates ROS production, may account for UVA-induced apoptosis seen in Dct -/- knockout mice.Part 3 Regulation of DHICA-mediated antioxidation by dopachrome tautomeraseObjective To investigate the impact of Dct on the melanin composition and the regulation of DHICA-mediated antioxidation.Methods Dct-/- knockout and wild-type C57BL/6J mice were irradiated with 5 J/cm2 of UVA irradiation, three times a week for up to 4 weeks. Total UVA dose was 60 J/cm2. And then eumelanin amount in epidermis of tail skin was visualized by Fontana-Masson staining. The eumelanin/pheomelanin content in the skin was analyzed using a chemical degradation protocol with HPLC. The effects of DHICA-melanin, DHI-melanin,and mixtures of both on hydroxyl radical generation in the Fenton reaction was determined by ESR.Results The prominent supranuclear melanin granules were more easily visualized in skins of C57BL/6J mice exposed to chronic UVA irradiation than Dct -/-mice. And eumelanin amount in the UVA-irradiated skins was much less in Dct -/- mice than in wild type mice. Twofold increase in the eumelanin/pheomelanin ratio in C57BL/6J mouse skin was observed after chronic UVA irradiation, but negligible increase is seen in Dct -/- mouse skin. DHICA-melanin exhibits a potent hydroxyl radical-scavenging activity, whereas DHI-melanin dose not.Conclusions Dct plays a role in the switch over to pheomelanin production. DHICA-melanin exhibits a potent hydroxyl radical-scavenging activity. Dct plays an important role in the regulation of DHICA-mediated antioxidation.