Kuninori Hirosaki

Sapporo Medical University, Sapporo, Hokkaidō, Japan

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Publications (12)41.59 Total impact

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    ABSTRACT: How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase-related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild-type (Rab7WT), constitutively active (Rab7Q67L) or dominant-negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio-distribution of melanosomal proteins between Rab7WT-expressing cells and mutant Rab7-expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.
    The Journal of Dermatology 09/2010; 38(5):432-41. · 1.77 Impact Factor
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    ABSTRACT: Melanosome biogenesis consists of multistep processes that involve synthesis of melanosomal protein, which is followed by vesicle transport/fusion and post-translational modifications such as glycosylation, proteolysis, and oligomerization. Because of its complexity, the details of the molecular mechanism of melanosome biogenesis are not yet fully understood. Here, we report that, in MMAc melanoma cells, wild-type (WT) Rab7 and its dominant-active mutant (Rab7-Q67L), but not its dominant-negative mutant (Rab7-T22N), were colocalized in the perinuclear region with granules containing Stage I melanosomes, where the full-length, immature gp100/Pmel17/Silv was present. It was also found that overexpression of Rab7-Q67L and, to a lesser extent, Rab7-WT increased the amount of proteolytically processed, mature gp100. However, Rab7-T22N did not show such an effect. Moreover, siRNA-mediated Rab7 knockdown considerably inhibited gp100 maturation. These results collectively suggest that the GTP-bound form of Rab7 promotes melanogenesis through the regulation of gp100 maturation in melanoma cells.
    Journal of Investigative Dermatology 02/2008; 128(1):143-50. · 6.19 Impact Factor
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    ABSTRACT: Psoralen plus ultraviolet A (PUVA) photochemotherapy is widely used for the therapy of mycosis fungoides (MF). Clinical progression of MF is often associated with an increase in the size of tumour cells known as transformation. We report two patients with CD30+ large cell transformation that appeared after low-dose PUVA therapy for MF. Clinical data, histopathology, immunohistopathology and T-cell receptor gene rearrangement were studied. Nodules consisted of atypical large cells that expressed CD30. Monoclonal rearrangement of T-cell receptors was observed in one case. Low-dose PUVA therapy may be associated with CD30+ large cell transformation in patients with MF.
    British Journal of Dermatology 02/2007; 156(1):148-51. · 3.76 Impact Factor
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    ABSTRACT: We have devised a bilobed skin flap for reconstruction after excision of small skin tumours. The sutured part serves as a zig-zag that leads to only slight postoperative contracture of the scar. The rotation centre of the flap is nearer to the affected area than other conventional bilobed flaps, resulting in less dog-ear deformity and distortion of tissue.
    Scandinavian Journal of Plastic and Reconstructive Surgery and Hand Surgery 02/2006; 40(1):32-40. · 0.94 Impact Factor
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    ABSTRACT: The synthesis of melanin intermediates through tyrosinase (TYR) involves the production of cytotoxic free radicals. By using recombinant adenoviruses that express TYR, tyrosinase-related protein 1 (TYRP1) or DOPAchrome tautomerase (DCT), we analyzed the biological function of these proteins with regard to melanin production and the growth of melanocytes, fibroblasts, melanoma cells and nonmelanoma cancer cells. High-level expression of TYR produced newly synthesized melanin and induced cell death in all of these cells. However, when TYRP1 or DCT was coexpressed with TYR in melanocytes and melanoma cells, TYR-mediated cell death was clearly decreased. This decrease was not observed in nonmelanocytic cells. Western blot analysis and measurement of enzyme activity revealed that the expression of TYRP1 or DCT had little effect on the amount or activity of cointroduced TYR in either the melanocytic or nonmelanocytic cells. In cells expressing both TYR and TYRP1 or TYR and DCT, the total amount of melanin and/or eumelanin increased substantially more than that in cells expressing TYR alone. On the other hand, the level of pheomelanin was similar in these three cell types. These findings suggest that TYRP1 and DCT play an important role in suppressing TYR-mediated cytotoxicity in melanocytic cells without decreasing TYR expression and/or activity. These biological activities of TYRP1 and DCT may work through the interaction with TYR in melanosomal compartment.
    Experimental Cell Research 09/2004; 298(2):317-28. · 3.56 Impact Factor
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    Experimental Cell Research - EXP CELL RES. 01/2004; 301(2):338-338.
  • International Journal of Dermatology 07/2003; 42(6):501-2. · 1.34 Impact Factor
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    ABSTRACT: We have recently identified the association of Rab7 in melanosome biogenesis and proposed that Rab7 is involved in the transport of tyrosinase-related protein 1 from the trans-Golgi network to melanosomes, possibly passing through late-endosome-delineated compartments. In order to further investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and tyrosinase-related protein by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells (SK-mel-24) in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection of the recombinant adenoviruses carrying tyrosinase (Ad-HT) and TRP-1 (Ad-TRP-1) resulted in the enhancement of tyrosinase activity and melanin production compared to a single infection of Ad-HT. In the Ad-HT-infected SK-mel-24 cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal lgp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization of tyrosinase and lgp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected SK-mel-24 cells, TRP-1, which is reported to be present exclusively in melanosomes, was detected throughout the cytoplasm, but not colocalized in prelysosomal (early endosomal) EEA-1 granules. In the presence of Rab7N125I, however, TRP-1 was retained in the EEA-1-positive granules. Our findings indicate that the dominant-negative mutant of Rab7 impairs vesicular transport of tyrosinase and TRP-1, suggesting that the transport of these melanogenic proteins from the trans-Golgi network to maturing melanosomes requires passage through endosome-delineated compartments.
    Journal of Investigative Dermatology 09/2002; 119(2):475-80. · 6.19 Impact Factor
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    ABSTRACT: The melanosome is a unique secretory granule of the melanocyte in which melanin pigments are synthesized by tyrosinase gene family glycoproteins. Melanogenesis is a highly regulated process because of its inherent toxicity. An understanding of the various regulatory mechanisms is important in delineating the pathophysiology involved in pigmentary disorders and melanoma. We have purified and analyzed the total melanosomal proteins from B16 mouse melanoma tumors in order to identify new proteins that may be involved in the control of the melanogenesis process. Melanosomal proteins were resolved by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, a predominant spot (27 kDa with isoelectric point 5.8-6.4) was excised and digested with cyanogen bromide, and the fragments were sequenced. Synthetic oligonucleotide primers were synthesized corresponding to the peptide sequences, and reverse transcriptase polymerase chain reaction amplification of total RNA from B16 cells was carried out. Sequencing of one of the polymerase-chain-reaction-mediated clones demonstrated 80%-97% sequence homology of 200 bp nucleotide with GTP-binding proteins at the 3'-untranslated region. GTP-binding assay on two-dimensional gels of melanosomal proteins showed the presence of several (five to six) small GTP-binding proteins, suggesting that small GTP-binding proteins are associated with the melanosome. Among the known GTP-binding proteins with similar molecular weight and isoelectric point ranges, rab3, rab7, and rab8 were found to be present in the melanosomal fraction by immunoblotting. Confocal immunofluorescence microscopy showed that rab7 is colocalized with the tyrosinase-related protein 1 around the perinuclear area as well as, in part, in the perikaryon, thereby suggesting that rab7 might be involved in the intracellular transport of tyrosinase-related protein 1. Tyrosinase-related protein 1 transport was blocked by the treatment of B16 cells with antisense oligonucleotide to rab7. We suggest (i) that rab7 is a melanosome-associated molecule, (ii) that tyrosinase-related protein 1 is present in late-endosome delineated granules, and (iii) that rab7 is involved in the transport of tyrosinase-related protein 1 from the late-endosome delineated granule to the melanosome.
    Journal of Investigative Dermatology 08/2001; · 6.19 Impact Factor
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    ABSTRACT: 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.
    Pigment Cell Research 09/2000; 13(4):222-9. · 4.29 Impact Factor
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    ABSTRACT: The intracellular vesicular trafficking in the melanosome biogenesis (melanogenesis) is reviewed with the incorporation of our own experimental findings. The melanosome biogenesis involves four stages of melanosome maturation, which reflect the transport of structural and enzymatic proteins from Golgi (trans-Golgi network: TGN) to the melanosomal compartment and their organization therein. The major melanosomal proteins include tyrosinase gene family protein (tyrosinase and tyrosinase-related protein; TRP), lysosome-associated membrane protein (Lamp) and gp100 (pmel 17). They are glycosylated in the endoplasmic reticulum, and transported by vesicles from the TGN to the melanosomal compartment. During the formation of transport vesicles, they assemble on the cytoplasmic face of the TGN to select cargo by interacting directly or indirectly with coat proteins. Tyrosinase and TRP-1 possess the dileucine motifs at the cytoplasmic domain, to which adapter protein-3 binds to transport them from the TGN to stage I melanosomes (related to late endosomes) and then to stage II melanosomes. A number of small guanosine triphosphate-binding proteins, including rab 7, appear to be involved in this vesicular transport. Phosphatidyl inositol 3 kinase also regulates this membrane trafficking of melanosomal glycoprotein. Eumelanogenesis is controlled by melanocyte-stimulating hormone, and all three tyrosinase gene family proteins are transported from the TGN to stage II melanosomes that are elliposoidal and contain the structural matrix of filaments/lamellae. In contrast, pheomelanogenesis is primarily regulated by agouti signal protein, and only tyrosinase is transported from stage I melanosomes to stage II melanosomes that are spherical and related to lysosomes. Because of the absence of TRP-1 and TRP-2 in pheomelanogenesis, it may be suggested that tyrosinase is involved in lysosomal degradation after forming dopaquinone, to which the cysteine present in the lysosomal granule binds to form cysteinyldopas that will then be auto-oxidized to become pheomelanin.
    Pigment Cell Research 02/2000; 13 Suppl 8:110-7. · 4.29 Impact Factor
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    ABSTRACT: To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.
    Journal of Biochemistry 02/1999; 125(1):82-9. · 3.07 Impact Factor