Biomedical subjects

G Imokawa

Publications and source records attributed to G Imokawa.

At least 37 records · Page 2Linked to original sources

PubMed2000

High-expression of sphingomyelin deacylase is an important determinant of ceramide deficiency leading to barrier disruption in atopic dermatitis.

We have previously demonstrated that there is abnormal expression of sphingomyelin (SM) deacylase-like enzyme in the epidermis of patients with atopic dermatitis (AD), which results in decreased levels of ceramides in their involved and uninvolved stratum corneum. For quantitation of the expression of SM deacylase in AD, we synthesized 16-(9-anthroyloxy) hexadecanoylsphingosylphosphorylcholine or [palmitic acid-14C] SM and used them as substrates to directly measure the activity of SM deacylase by detecting the release of labeled free fatty acid. Direct enzymatic measurements demonstrated that stratum corneum from lesional forearm skin (volar side) of AD patients has an extremely high SM deacylase activity that is at least five times higher than in the stratum corneum from healthy controls. In stratum corneum from nonlesional skin of AD patients, SM deacylase activity is still at least three times higher than in healthy controls. In contrast, stratum corneum from contact dermatitis patients shows levels of SM deacylase similar to healthy controls. In extracts of whole epidermis biopsies from AD patients, SM deacylase activities are significantly (3-fold) increased over healthy controls in the particulate fraction, whereas there is no significant difference in the activity of sphingomyelinase between AD and healthy control. In peripheral blood lymphocytes of AD patients, there is no increase in activity compared with healthy controls, indicating a possibility that the high expression of SM deacylase is highly associated with the skin of AD patients. These findings suggest that, in contrast to changes in sphingolipid metabolism due to aging, the hitherto undiscovered enzyme SM deacylase, is highly expressed in the epidermis of AD patients, and competes with sphingomyelinase or beta-glucocerebrosidase for the common substrate SM or glucosylceramide, which leads to the ceramide deficiency of the stratum corneum in AD.

Adolescent↗

PubMed2000

Age-related alterations of echogenicity in Japanese skin.

BACKGROUND: Ultrasonic methods are commonly used for the noninvasive measurement of skin thickness. The effects of ageing on the skin have been reported to differ between Asians and Caucasians. Therefore, it is possible that the echogenicity of the skin on ultrasonographic images reflects differences in the skin architecture and properties that may vary among races and environments. OBJECTIVE AND METHODS: Since there have been a few reports on ageing-related changes in ultrasonic echogenicity in Caucasian skin, but not in Asian skin, we evaluated age-related changes in the echogenicity of ultrasonographic images by the ultrasonic B mode at 3 sites on the face (forehead, eye corners, cheeks) considered as sun-exposed areas and on the ventral forearms considered as weakly sun-exposed areas in 130 Japanese females aged from 18 to 83 years. The dermis was divided into 3 layers (upper, middle, lower), and echogenicity was evaluated in each area. RESULTS: No age-related change in echogenicity was observed in the entire dermis. When the dermis was divided into 3 layers (upper, middle and lower dermis), the echogenicity was decreased in the upper layer and increased in the lower layer with age in all skin areas tested. In contrast, there were no age-related changes of echogenicity in the middle dermis. CONCLUSION: These findings suggest that, as for Caucasians, echogenicity in the upper and lower dermis is a useful tool for evaluating skin ageing in the Japanese.

Adolescent↗

PubMed1999

Photohapten TCSA painting plus UVA irradiation of murine skin augments the expression of MHC class II molecules and CD86 on Langerhans cells.

Hapten painting of skin is known to augment the expression of major histocompatibility complex (MHC) class II, CD54, and CD86 on Langerhans cells. We investigated whether painting with 3,3',4',5-tetrachlorosalicylanilide (TCSA), the representative photohapten, and subsequent irradiation with ultraviolet A (UVA) alter the expression of these surface molecules on epidermal Langerhans cells (LC). BALB/c mice were painted with 5 microl of 0.1% TCSA on the earlobes and irradiated with 16 J/cm2 (at 365 nm) of UVA. Epidermal cells were prepared from these earlobes 24 h later, and the levels of MHC class II, CD54, CD80, and CD86 on these cells were analyzed by flow cytometry. As compared with untreated earlobes, the levels of MHC class II and CD86 on LC were markedly augmented and those of CD54 and CD80 were slightly elevated in earlobes treated with TCSA/UVA. Since neither TCSA painting nor UVA exposure alone enhanced the expression, both treatments were essential for enhancement. A dot plot analysis showed the presence of subpopulations of LC expressing MHC class II and CD86 at high levels. The percentage of these highly expressing LC was increased with increasing concentrations of TCSA and doses of UVA up to 1% and 24 J/cm2, respectively. In addition, keratinocyte expression of CD54 was also augmented by TCSA plus UVA. These results suggest that photohaptens, with following UVA exposure, augment the expression of immunologically functional molecules on LC as do ordinary haptens, leading to effective sensitization and elicitation of contact photoallergy.

Animals↗

PubMed1999

All-trans retinoic acid promotes the repair of tortuosity of elastic fibres in rat skin.

To elucidate the repair effects of all-trans retinoic acid (t-RA) on ultraviolet (UV)-induced tortuosity of elastic fibres in rat skin, the hind limbs of Sprague-Dawley rats were irradiated at a suberythemal dose of UV (three times/week for 6 weeks) and 0.025% t-RA in ethanol was applied topically five times/week for 6 weeks. The three-dimensional arrangement of elastic fibres with special reference to their linearity was quantified by image analysis using a scanning electron microscope following a combination of intravascular resin injection and selective digestion using formic acid. When t-RA was topically applied for 6 weeks on wrinkles formed by 6 weeks' exposure of the skin of rat hind limbs to UV radiation, the wrinkles disappeared to control levels, concomitant with an improvement in skin elasticity. The linearity of the elastic fibres was significantly (P < 0.01) increased compared with age-matched UV-irradiated controls. These findings suggest that the degeneration and deposition of elastic fibres accompanied by an increase in their linearity is involved in the process of wrinkle repair by topical application of t-RA.

Administration, Topical↗

PubMed1999

Sphingosylphosphorylcholine is a potent inducer of intercellular adhesion molecule-1 expression in human keratinocytes.

We recently reported that the epidermis of patients with atopic dermatitis contains an abnormally expressed sphingomyelin deacylase that yields a large amount of sphingosylphosphorylcholine (SPC) rather than ceramide. In this study, we characterize inflammatory roles of newly discovered chemicals in the epidermis by elucidating biologic effects of SPC on intercellular adhesion molecules-1 (ICAM-I) expression by human keratinocytes in culture in comparison with other sphingolipids. Using fluorescence-activated cell sorter analysis, we found that SPC treatment at concentrations of 10-20 microM significantly enhanced the expression of ICAM-I by cultured human keratinocytes in a dose-dependent manner after incubation for 15-24 h, and, using northern blot analysis, that this was accompanied by increased expression of ICAM-1 mRNA within 4 h of incubation. Transforming necrosis factor-alpha (TNF-alpha) levels in the medium of keratinocytes treated at a 10 microM concentration of SPC were significantly increased by 200%. Furthermore, the SPC-induced ICAM-1 expression was partially abolished by the concomitant addition of anti-TNF-alpha, suggesting a partial autocrine involvement of TNF-alpha in ICAM-1 expression. Assay of mitogen-activated protein kinase revealed that 10 microM SPC induced a rapid activation of mitogen-activated protein kinase in human keratinocytes, including an increase in its phosphorylation within 5 min, which then declined to the baseline control level after 30 min. In contrast, sphingomyelin or sphingosine had no significant potential to activate mitogen-activated protein kinase at the same concentration. These findings suggest that SPC plays an important role in the inflammatory process of epidermis in skin diseases, such as atopic dermatitis, with high expression of sphingomyelin deacylase.

Calcium↗

PubMed1999

Beta-glucocerebrosidase activity in mammalian stratum corneum.

Although previous studies have demonstrated a crucial role for the enzyme beta-glucocerebrosidase (GlcCer'ase) in the final steps of membrane structural maturation in mammalian stratum cornuem (SC) and epidermal homeostasis, the precise in vivo localization of GlcCer'ase activity and protein is not known. Here, we developed a fluorogenic in situ assay on histologic sections (zymography) to elucidate the in vivo distribution of GlcCer'ase activity, and further characterized and localized the SC GlcCer'ase activity in vitro. The zymographic technique revealed higher GlcCer'ase activity in upper stratum granulosum and SC, both in murine and human SC; activity that was both inhibited by conduritol B epoxide, a specific GlcCer'ase inhibitor, and pH-dependent; i.e., present at pH 5.2, and absent or significantly reduced at neutral pH (7.4), consistent with the known pH optimum for epidermal GlcCer'ase in vitro. Immunohistochemical staining for GlcCer'ase protein showed enhanced fluorescent signal in the outer layers of human epidermis, concentrated at the apex and margins of stratum granulosum and lower SC. Moreover, in extracts from individual epidermal layers, GlcCer'ase activity was present throughout murine epidermis, with the highest activity in the SC, peaking in the lower-to-mid-SC. The SC activity was stimulated >10-fold by sodium taurocholate, and inhibited by bromoconduritol B epoxide. Finally, isolated membrane couplets, prepared from SC sheets, also demonstrated significant GlcCer'ase activity. These data localize GlcCer'ase activity to the outer epidermis by three different techniques, and support the role of this enzyme in extracellular processing of glucosylceramides to ceramides, required for permeability barrier maturation and function.

Animals↗

PubMed1998

Tyrosinase stabilization by Tyrp1 (the brown locus protein).

Mammalian melanogenesis is regulated directly or indirectly by over 85 distinct loci. The Tyr/albino locus, in which mutations cause a lack of pigmentation, encodes tyrosinase (Tyr), the critical and rate-limiting melanogenic enzyme. Other melanogenic enzymes include Tyrp1 (or TRP1) and 3,4-dihydroxyphenylalanine-chrome tautomerase (Dct or TRP2) encoded at the Tyrp1/brown and Dct/slaty loci, respectively. Murine Tyrp1 can oxidize 5, 6-dihydroxyindole-2-carboxylic acid (DHICA) produced by Dct, but mutations in Tyrp1 also affect the catalytic functions of Tyr. All three enzymes are membrane-bound melanosomal proteins with similar structural features and are thought to interact within and stabilize a melanogenic complex. We have now further investigated the effect of a Tyrp1(b) mutation on Tyr stability. Pulse/chase labeling experiments show that Tyr is degraded more quickly in Tyrp1(b) mutant melanocytes than in melanocytes wild type at that locus. This reduced stability of Tyr can be partly rescued by infection with the wild type Tyrp1 gene, and this is accompanied by phenotypic rescue of infected melanocytes. In sum, these results suggest that, in addition to its catalytic function in oxidizing DHICA, Tyrp1 may play an important role in stabilizing Tyr, a second potential role in the regulation of melanin formation.

3T3 Cells↗

PubMed1998

Purification, cDNA cloning, and expression of UDP-Gal: glucosylceramide beta-1,4-galactosyltransferase from rat brain.

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29. 7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N-glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn2+ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse beta-1, 4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to beta-1,4-GlcNAc in glycoproteins.

Amino Acid Sequence↗

PubMed1998

Biological characterization of human fibroblast-derived mitogenic factors for human melanocytes.

To clarify the paracrine linkage between human fibroblasts and melanocytes in cutaneous pigmentation, we studied the effects of human fibroblast-derived factors on the proliferation of human melanocytes. In medium conditioned for 4 days with human fibroblast culture, factors were produced that markedly stimulated DNA synthesis of human melanocytes. The stimulatory effect was higher in medium conditioned with fibroblasts from aged skin than in medium conditioned with fibroblasts from young skin, and was interrupted by inhibitors of tyrosine kinase, such as tyrphostin, genistein and herbimycin, but not by inhibitors of protein kinases C and A, such as H-7 and phloretin. The conditioned medium was also capable of activating mitogen-activated protein kinase of human melanocytes, with old fibroblasts being more effective than young ones. Analysis of factors released into the conditioned medium revealed that levels of hepatocyte growth factor (HGF) and stem cell factor (SCF) were increased in old-fibroblast-conditioned medium compared with young-fibroblast-conditioned medium. In contrast, levels of basic fibroblast growth factor (bFGF) were similar in both media. When the conditioned medium was treated with HGF antibody with or without SCF antibody, the increase in DNA synthesis by human melanocytes was decreased to 20% of the elevated level, whereas antibodies to bFGF had no effect. Analysis of the medium conditioned for 4 days after cytokine application demonstrated that, of the cytokines tested, interleukin 1alpha and tumour necrosis factor alpha are highly effective in stimulating HGF secretion by old fibroblasts. HGF and SCF, but not bFGF, were markedly increased in culture medium in the presence of IL-1alpha, and this stimulatory effect was confined to young human fibroblasts. These findings suggest that SCF and HGF derived from human fibroblasts may play a part in regulating cutaneous pigmentation during inflammation and aging.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Purification and characterization of an allergy-induced melanogenic stimulating factor in brownish guinea pig skin.

We have demonstrated recently that phenylazonaphthol (PAN) allergy-induced hyperpigmentation in brownish guinea pig skin is associated with the concomitant appearance of a melanogenic soluble factor(s) that activates the intracellular signal transduction system, including phosphatidylinositol turnover subsequent to ligand-receptor binding in cultured guinea pig melanocytes. In this study we have purified and characterized the PAN-induced melanogenic stimulating factor (PIMSF) that occurs in allergy-associated hyperpigmented skin. By successive column chromatography on TSK 2000SW, Mono Q, and octadecyl-NPR, the PIMSF was purified to homogeneity with a single band of apparent molecular mass of 7.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific bioactivity of PIMSF increased by 5,195-fold over the original skin homogenate. In cultured guinea pig melanocytes, this purified PIMSF had the potential of activating an intracellular signal transduction system such as inositol 1,4,5-trisphosphate formation and intracellular calcium levels through a pertussis toxin-sensitive G protein-coupled receptor. PIMSF consistently caused a rapid translocation of cytosolic protein kinase C (PKC) to membrane-bound PKC within 5 min of treatment with a return to the basal level after 120 min. The stimulating effects of PIMSF on proliferation and melanization of cultured guinea pig melanocytes were abolished completely by a PKC down-regulating agent (phorbol 12,13-dibutyrate). PIMSF was similar in molecular mass to rat growth-related oncogene alpha (GRO-alpha; molecular mass of 7.9 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had immunocross-reactivity with GRO-alpha upon Western immune blotting analysis. Further, the stimulatory effect of purified PIMSF on DNA synthesis of cultured guinea pig melanocytes was suppressed markedly by the addition of anti-rat GRO-alpha antibody, implying that the PIMSF is apparently identical to GRO-alpha. These findings suggest that PAN allergy provides a new mechanism of hyperpigmentation in which biological factors such as the GRO-alpha superfamily generated within allergy-induced skin stimulate melanocytes through activation of the PKC-related signal transduction pathway.

Allergens↗

PubMed1998

Percutaneous sensitization with allergens through barrier-disrupted skin elicits a Th2-dominant cytokine response.

We investigated whether percutaneous sensitization with different allergens through barrier-disrupted skin regulates the balance of Th1/Th2 cytokine expression. When mice were sensitized with the typical hapten picryl chloride (PiCl) by a single topical application to intact skin, there was an up-regulation in the lymph nodes (LN) of mRNA expression for the Th1 cytokines IL-2 or IFN-gamma, and for the Th2 cytokine IL-4. In contrast, sensitization with PiCl after barrier disruption of the skin down-regulated the expression of mRNA for IFN-gamma in a tape-stripping number-dependent manner without changing the expression of mRNA for IL-4. When mice were sensitized with house dust mite antigens (MA) by a single topical application to barrier-disrupted abdominal skin, there was a tape-stripping number-dependent up-regulation in the LN of mRNA expression for IL-4 but not for IL-2 or IFN-gamma. In the LN, mRNA for the IL-4-inducible immunoglobulins IgE and IgG1, but not for the IFN-gamma-inducible IgG2a, were up-regulated after sensitization with MA, while all three immunoglobulin mRNA were augmented after PiCl sensitization through intact skin. Antigenic elicitation by a topical application of PiCl in aural skin of mice sensitized through intact skin consistently increased the expression of mRNA for all three cytokines in the challenged skin, whereas elicitation in mice sensitized through barrier-disrupted skin decreased the expression of mRNA for IL-2 and IFN-gamma, but not for IL-4. Antigenic elicitation by subcutaneous injection of MA in aural skin consistently increased the expression of mRNA for IL-4, but not for IL-2 or IFN-gamma in the challenged skin. Infiltration of eosinophils in the dermis was more prominent following elicitation with MA in mice sensitized through barrier disruption than with PiCl in mice sensitized through intact skin. These findings suggest that the percutaneous entry of environmental allergens through barrier-disrupted skin is strongly associated with the induction of Th2-dominant immunological responses, as is seen in atopic dermatitis.

Allergens↗

PubMed1998

The effects of UVA and UVB irradiation on the viscoelastic properties of hairless mouse skin in vivo.

BACKGROUND: Age-related changes in the elasticity of the human skin have been demonstrated as well as those due to proto ageing. AIMS AND METHODS: To investigate if UV irradiation causes similar changes in the elastic properties of skin, hairless mice (HR/ICR) were irradiated chronically with suberythemal doses of UVA or UVB. Using a recently designed, commercially available noninvasive in vivo instrument, we compared skin thickness and elasticity after 8 weeks of UV exposure with age-matched control mice. RESULTS: Skin thickness increased significantly after UVB irradiation. Skin elasticity (Ur/Uf) decreased significantly after UVA and UVB irradiation, and the ratio of viscosity to elasticity (Uv/Ue) increased significantly after UVA irradiation. Intrinsic immediate strain (Ue*) and intrinsic delayed strain (Uv*) decreased significantly after UVB and UVA exposure. These changes were similar to those reported previously in an in vivo study on human face skin using the same instrument. CONCLUSION: This finding suggests that the Ue* and Uv* changes observed in human facial skin are associated with actinic ageing caused by chronic UV exposure.

Aging↗

PubMed1997

Modulation of murine melanocyte function in vitro by agouti signal protein.

Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two intercellular signaling molecules, alpha-melanocyte-stimulating hormone (MSH) and agouti signal protein (ASP). In this study, we have characterized the physiological effects of ASP on eumelanogenic melanocytes in culture. Following exposure of black melan-a murine melanocytes to purified recombinant ASP in vitro, pigmentation was markedly inhibited and the production of eumelanosomes was decreased significantly. Melanosomes that were produced became pheomelanosome-like in structure, and chemical analysis showed that eumelanin production was significantly decreased. Melanocytes treated with ASP also exhibited time- and dose-dependent decreases in melanogenic gene expression, including those encoding tyrosinase and tyrosinase-related proteins 1 and 2. Conversely, melanocytes exposed to MSH exhibited an increase in tyrosinase gene expression and function. Simultaneous addition of ASP and MSH at approximately equimolar concentrations produced responses similar to those elicited by the hormone alone. These results demonstrate that eumelanogenic melanocytes can be induced in culture by ASP to exhibit features characteristic of pheomelanogenesis in vivo. Our data are consistent with the hypothesis that the effects of ASP on melanocytes are not mediated solely by inhibition of MSH binding to its receptor, and provide a cell culture model to identify novel factors whose presence is required for pheomelanogenesis.

Agouti Signaling Protein↗

PubMed1997

Altered permeability and disordered cutaneous immunoregulatory function in mice with acute barrier disruption.

In vivo and in vitro T-cell-activating ability of murine epidermal cells (EC) was investigated in acutely barrier-disrupted skin by extraction of epidermal lipids with acetone or removal of corneocytes by tape stripping. Contact sensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB) and picryl chloride (PCl) and contact photosensitivity (CPS) to tetrachlorosalicylanilide (TCSA) were significantly augmented when challenged or sensitized at sites treated with acetone 24 h before, compared with the intact skin. CS to DNFB was also enhanced by tape stripping, but not by water rubbing, suggesting that physical stress or a toxic effect of acetone was not responsible for the augmentation. Semi-quantification of TCSA-EC photoadducts showed markedly increased permeability of hapten in the epidermis 24 h after acetone treatment. Bioactive IL-1alpha was more pronounced in barrier-disrupted than in intact skin. Lymph node T cells from PCl-sensitized mice proliferated significantly more in a hapten-specific and co-stimulatory molecule-dependent manner in response to trinitrophenylated (TNP) EC from acetone-treated skin than to those from untreated skin. Immunofluorescence staining of epidermal sheets and flow cytometric analysis of dispersed EC showed that subpopulations of Langerhans cells (LC) in acetone-rubbed or tape-stripped skin expressed major histocompatibility complex class II CD54 and CD86 molecules at levels higher than the rest of LC and LC from water-treated or untreated epidermis. Therefore, not only increased permeability of hapten through the epidermis but also altered immune functions of EC potentiate T-cell activation in acute barrier disruption. Such augmentation of immune reactivity may be critical to elimination of environmental noxious agents that penetrate easily into the barrier-disrupted epidermis.

Acetone↗

PubMed1997

The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis.

The paracrine linkage of endothelins (ET) between keratinocytes and melanocytes suggested that ETs are intrinsic mediators for human melanocytes in UVB-induced pigmentation. In this study, the role of ET-1 in the epidermal hyperpigmentation was investigated in vivo and in vitro. The addition of 10 nM ET-1 induced a H-7 (10 microM) suppressible-increase in tyrosinase activity in cultured human melanocytes and was accompanied by elevated levels of tyrosinase and tyrosinase-related protein-1 mRNA expression as shown by Northern blotting. Analysis of signaling mechanisms leading to tyrosinase activation demonstrated the involvements of quick translocation of PKC, the H-7 (10 microM) suppressible-phosphorylation of the threonine residue of several proteins, and highly elevated level of cyclic AMP (4-fold over control). Reverse transcription polymerase chain reaction (RT-PCR) of RNA isolated from the epidermis of human skin exposed to UVB revealed that UVB irradiation with a dose of 2 MED caused a significant increase in the expressions of ET-1, IL-1 alpha, and tyrosinase mRNA signals 5 days after irradiation. The involvement of ET-1 in UVB-pigmentation was also corroborated by the experiments that the extracts of M. Chamomilla, which can act as an antagonist for ET-receptor binding-mediated signaling but has no inhibitory effect on tyrosinase activity in culture, had a significant inhibitory effect on UVB-induced pigmentation in vivo when daily applied immediately after UVB exposure to human skin. These findings suggest that ET-1 is an important mediator in the epidermis for UVB-induced pigmentation in vivo.

Cell Division↗

PubMed1996

Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes.

To understand the signalling mechanisms involved in the dual stimulatory effects of endothelin-1 (ET-1) on DNA synthesis and melanization in cultured human melanocytes, we analysed the biological profile of ET-1 receptor and determined the effects of ET-1 on the protein kinase C, cyclic AMP system and mitogen-activated protein kinase (MAP kinase) in comparison with their relevant stimulants. The photoaffinity labelling of ET-1 receptors with Denny-Jaff reagents revealed an ET-1 receptor with a molecular mass of 51 kDa in human melanocytes. The ET(A) receptor subtype-sensitive antagonist BQ123(50 nM) or pertussis toxin (100 ng/ml) significantly suppressed the ET-1-induced intracellular calcium mobilization, indicating the presence of pertussis toxin-sensitive G-protein-coupled ET(A) receptors. An assay of protein kinase C activity revealed that 10nM ET-1 translocated cytosolic protein kinase C to membrane-bound protein kinase C within 5 min of the start of incubation. In contrast, receptor-mediated melanocyte activation by ET-1 was accompanied by an elevated level of cyclic AMP (4-fold over control) after 10-60 min of incubation, whereas 60 min of incubation of human melanocytes with c-Kit or c-Met ligands such as stem cell factor (10 nM) or basic fibroblast growth factor (10 nM) did not elevate the cyclic AMP level. We have also demonstrated that a specific tyrosine kinase inhibitor, tyrphostin B-42 (10 microM), inhibited the ET-1-induced growth stimulation, suggesting the involvement of the tyrosine kinase pathway in growth stimulation. Consistently, an assay of MAP kinase revealed that ET-1 caused a 10-fold activation of MAP kinase after 5 min of incubation with human melanocytes in a similar way to tyrosine kinase ligands such as stem cell factor and hepatocyte growth factor. Further, the DNA synthesis stimulated by the c-Kit ligand stem cell factor at a concentration of 1 nM was synergistically enhanced by 5 nM ET-1. These results suggest that ET-induced dual cellular events in human melanocytes are closely associated with cross-talk between the protein kinase C and A and tyrosine kinase pathways.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Granulocyte/macrophage colony-stimulating factor is an intrinsic keratinocyte-derived growth factor for human melanocytes in UVA-induced melanosis.

Recently we demonstrated that endothelins secreted from human keratinocytes act as intrinsic mitogens and melanogens for human melanocytes in UVB-induced melanosis. We show here that UVA-induced melanosis is associated with other keratinocyte-derived growth factors, secretion of which is specifically stimulated after exposure of human keratinocytes to UVA. Medium conditioned by UVA-exposed human keratinocytes elicited a significant increase in DNA synthesis by cultured human melanocytes in a UVA dose-dependent manner. Analysis of endothelin-1 and interleukin (IL)-1 alpha in the conditioned medium by ELISA, both of which are major keratinocyte-derived cytokines involved in UVB-associated melanocyte activation, revealed that UVA exposure did not cause human keratinocytes to stimulate the secretion of the two cytokines. In contrast, the levels of several other cytokines such as IL-6, IL-8 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were significantly increased in the conditioned medium of human keratinocytes after exposure to UVA at a dose of 1.0 J/cm2. The gel chromatographic profile of UVA-exposed keratinocyte-conditioned medium demonstrated that there were two factors (P-1 and P-2) with molecular masses of approx. 20 and 1 kDa respectively that stimulate DNA synthesis in human melanocytes, and the larger species (P-1) also increased melanization as assessed by [14C]thiouracil incorporation. Quantitative analysis of cytokines in chromatographic fractions by ELISA revealed the P-1 fraction to be consistent with the molecular mass profile of GM-CSF. Furthermore the stimulatory effect of the P-1 fraction on DNA synthesis in human melanocytes was neutralized by antibodies to GM-CSF, but not to basic fibroblast growth factor or stem cell factor. Binding and proliferation assays with recombinant GM-CSF demonstrated that human melanocytes possess specific binding sites for GM-CSF(Kd 2.11 nM; binding sites, 2.5-3.5 x 10(4) per cell), and recombinant GM-CSF at concentrations of more than 10 nM significantly stimulated DNA synthesis and melanization. These findings suggest that GM-CSF secreted by keratinocytes plays an essential role in the maintenance of melanocyte proliferation and UVA-induced pigmentation in the epidermis.

Cells, Cultured↗

PubMed1996

Role of endothelin-1 in hyperpigmentation in seborrhoeic keratosis.

Seborrhoeic keratosis (SK) is a benign epidermal tumour with a varying degree of pigmentation. We have recently demonstrated that endothelin-1 (ET-1) is a strong keratinocyte-derived mitogen and melanogen for human melanocytes in UVB-induced melanosis. To clarify the role of ET-1 in hyperpigmentation in SK, we have used immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to see whether the production of ET-1 is accentuated in SK. Immunohistochemical analysis in SK (n = 7; acanthotic and deeply pigmented types) revealed marked immunostaining with anti-ET-1 in almost all basaloid and basal cells as compared with definite staining confined to basal cells in the perilesional normal controls. In parallel, RT-PCR of ET-1 mRNA demonstrated accentuated expression of ET-1 transcript in SK (n = 4) in comparison with that in the perilesional normal controls, accompanied by a similarly accentuated expression of tyrosinase mRNA. These findings suggest that ET-1 plays a part in the hyperpigmentation seen in SK.

Adult↗