The interaction of stem cell factor with its receptor, c-kit, is well known to be critical to the survival of melanocytes. Little is known about the role(s) of the stem cell factor/c-kit interaction in epidermal pigmentation, however. To clarify whether the stem cell factor/c-kit signaling has a paracrine role in ultraviolet-B-induced pigmentation, we determined whether the exposure of human keratinocytes, melanocytes, and the epidermis to ultraviolet B light stimulates the expression of stem cell factor or c-kit at the gene and/or protein levels. We further examined whether interrupting the binding of stem cell factor to c-kit by subepidermal injection of a monoclonal antibody to c-kit affects ultraviolet-B-induced pigmentation in brownish guinea pig skin. When human keratinocytes and melanocytes in culture were exposed to ultraviolet B light, transcripts of stem cell factor and c-kit (as assessed by reverse transcription polymerase chain reaction) and expression of those proteins (by enzyme-linked immunosorbent assay and western blotting) increased significantly and peaked at a dose of 20-40 mJ per cm2. In ultraviolet-B-exposed human epidermis, stem cell factor transcripts and protein expression were also markedly enhanced compared with the nonexposed epidermis. Immunohistochemistry with antibodies to stem cell factor revealed an increased staining in the ultraviolet-B-exposed epidermis, which was accompanied by a slight epidermal hyperplasia. In the course of ultraviolet-B-induced pigmentation of brownish guinea pig skin, the subepidermal injection of c-kit inhibitory antibodies completely abolished the induction of pigmentation in the ultraviolet-B-exposed area, and there was no increase in the number of dihydroxyphenylalanine-positive melanocytes. These findings indicate that the stem cell factor/c-kit signaling is critically involved in the biologic mechanism of ultraviolet-B-induced pigmentation.

Stem cell factor (SCF) and endothelin-1 (ET-1) have been reported to be up-regulated at the protein and gene levels in human epidermis after ultraviolet B (UVB) irradiation and to play central roles in UVBinduced pigmentation. However, little is known about the time sequence of SCF and ET-1 expression in UVB-exposed human epidermis and the coordination of their roles during epidermal pigmentation. To clarify such parameters in UVB-exposed human skin, we measured the expression patterns of SCF and ET-1 (as well as of their corresponding receptors) at the gene level at various times during UVB-induced human pigmentation. When human forearm skin was exposed to UVB radiation at two minimal erythemal doses, the expression of SCF mRNA transcripts was significantly enhanced at 3 days after irradiation with an early decrease and subsequently constant expression of SCF receptor (c-KIT) mRNA transcripts. In contrast, up-regulation of ET-1 and endothelin B receptor (ET B R) mRNA expression was synchronized at 5 to 10 days after irradiation in concert with an increased expression of tyrosinase mRNA transcripts and the increase in pigmentation. In parallel the expression of tyrosinase and ET B R proteins as well as ET-1 was up-regulated at 7 to 10 days after irradiation, whereas KIT protein decreased at 3 days after irradiation and returned to the nonirradiated control level at 5 days after irradiation. When cultured human melanocytes were treated with human recombinant SCF, ET B R protein expression and the binding of 125 I-labeled ET-1 to the ET B R were significantly increased, further suggesting the preferential and coordinated role of early expression of SCF in UVB-induced melanogenesis. These findings suggest that SCF/KIT signaling is predominantly involved in the early phase of UVB-induced human pigmentation during which it stimulates the ET-1/ET B R linkage that is associated with the later phase of UVB-induced melanogenesis.

When the skin is irradiated with UVB light, various cytokines are released, act on normal human melanocytes (NHMC), and induce them to synthesize melanin pigment, to proliferate and to differentiate, which leads to increased pigmentation.1-3) Endothelin-1 (ET-1), 2,4) basic fibroblast growth factor (b-FGF), 5) and a-melanocyte-stimulating hormone, [6][7][8] all of which are produced by normal human keratinocytes (NHKC) and are up-regulated following UVB irradiation, act as mitogens for NHMC. Furthermore, an increase in ET-1 expression 3) in human skin after UVB irradiation has also been reported, and it has been suggested that ET-1 plays an important role in UVB-induced pigmentation. Therefore, we have investigated various botanical extracts that inhibit the ET-1-induced activation of NHMC. Since intracellular calcium mobilization is induced when ET-1 acts on NHMC, 1) we have continued to search for new agents which can block this calcium mobilization and we have found that an extract of Althaea officinalis L. has a potent inhibitory effect. In this study, we have clarified the inhibitory mechanism of the extract of A. officinalis on ET-1-induced activation of NHMC. MATERIALS AND METHODS Extract of A. officinalis L.A. officinalis is a large perennial plant belonging to the genus hollyhock in the mallow family. Pale pink hollyhock, velvet hollyhock, and marshmallow also belong to this family, and A. officinalis is cultivated for gardening, medication, and food in Europe. The genus name 'Althaea' is derived from 'althaino' in Greek, meaning therapy. The species name 'officinalis' means 'use for drug', and the plant is often prescribed for pain relief and treatment of renal function, inflammation, and irritation syndrome. The portions of the plant used as a drug are the roots and leaves that contain abundant mucus. Marshmallow sweet was originally sipped to treat sore throats, and can be jelled by soaking the root powder with sugar in water. We extracted A. officinalis roots with a 45% 1,3-butylene glycol solution and obtained a brown transparent extract, which was used in this evaluation.Materials NHMC were obtained from KURABO Industries, Ltd. (Osaka, Japan) and NHKC were obtained from Sanko Junyaku Co., Ltd. (Tokyo, Japan). Serum-free NHMC medium (MGM) was purchased from Sanko Pure Chemicals (Tokyo, Japan) and serum-free NHKC medium (SFM) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco Laboratories (Grand Island, NY, U.S.A.). ET derivatives were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Other chemicals were of reagent grade.Cell Culture NHMC were maintained in MGM supplemented with 1 ng/ml recombinant b-FGF, 0.5 mg/ml hydrocortisone, 5 mg/ml insulin, 10 ng/ml phorbol 12-myristate 13-acetate, 3 mg/ml heparin, antibiotics (50 mg/ml streptomycin), and 0.2% bovine pituitary extract (BPE). NHKC were maintained in modified SFM supplemented with 5 ng/ml epidermal growth factor and 50 mg/ml BPE. B16 F10 melanoma cells are a subline of the pigmented B16 melanoma (C57BL/6N)...

Endothelin-1 (ET-1), which was initially isolated and sequenced from cultured pig endothelial cells, is a potent vasoactive peptide consisting of 21 amino acid residues with two disulfide bridges.1) Vascular endothelial cells locally regulate the function of underlining smooth muscle cells by producing ET-1 as well as nitric oxide in response to various stimuli.2) ET-1 also plays an important role in regulating cutaneous melanogenesis, especially in ultraviolet B (UVB)-induced pigmentation, since ET-1 production by human keratinocytes and by the epidermis increased after irradiation with UVB in a dose-dependent manner, and this was accompanied by the significant secretion of interleukin 1a.3,4) It was demonstrated that proliferation and differentiation of human melanocytes, and tyrosinase expression, are stimulated by ET derivatives via a receptor-mediated signal transduction pathway. 3-7)In the search for whitening agents that can suppress such ET-1 functions, we have found that extracts of Matricaria chamomilla L. and Althaea officinalis L. inhibit ET-1-induced intracellular calcium mobilization and proliferation of human melanocytes. When an extract of M. chamomilla was applied to human skin after UVB irradiation, pigmentation was significantly diminished.8) This extract has been actually used as a whitening agent in our products. To improve the inhibitory effects of our whitening products on ET-1 function and melanin synthesis, we have continued to look for new agents which inhibit ET-1 synthesis and/or inhibit ET-1 function.It is well known that a membrane-bound neutral metalloprotease, termed endothelin-converting enzyme (ECE), catalyzes the specific cleavage of the inactive precursor Big endothelins (Big ETs) at Trp-21-Val-/Ile-22 to produce the 21-residue mature active ETs in mammalian endothelial cells. 9)Human endothelial cells express predominantly ECE-1a. 10)We have recently revealed that ECE-1a plays a pivotal role in the secretion of ETs by human keratinocytes using an ECE-1a-specific immunoprecipitating antibody (Hachiya et al., manuscript in preparation).Therefore, inhibition of ECE activity in human keratinocytes would be expected to suppress UVB-induced pigmentation. It was reported that ECE activity is inhibited by the metalloprotease inhibitor phosphoramidon, but not by thiorphan, a specific inhibitor of neutral endopeptidase 24.11 (NEP).11) However, phosphoramidon is not suitable for use as a cosmetic agent because of its low penetration of the skin and its expense. Therefore, we have tried to find an ECE inhibitor which is suitable for use as a cosmetic agent.We have found that an extract of Sanguisorba officinalis L. inhibits ECE activity in human endothelial cells. S. officinalis is found in China, Korea, Japan, Siberia and Europe, and extracts of S. officinalis have been used as fungicides, astringents and hemostatics. In this report, to clarify the suitability of this botanical extract as a whitening agent, we examined whether it inhibits UVB-induced pigmentation in vivo. MATERIALS AND...

Objective We aimed to investigate the association between the digit symbol test (DST) and clinical characteristics, including the nutritional status of liver cirrhosis patients. Methods Fifty-nine cirrhotic patients without a history of overt hepatic encephalopathy were retrospectively evaluated. We examined neuropsychological abnormalities (NPAs) using the DST. We also estimated the detailed nutritional status using the Food Frequency Questionnaire (FFQ). The patients were divided into two groups according to their DST status: patients with normal DST scores (DST-Nor group, n=45) and those with abnormal DST scores (DST-Abn group, n=14). The clinical and nutritional findings of the two groups were compared. Results Overall, 14 (23.7%) patients had a DST abnormality. There were significant differences between the two groups in serum albumin (Alb; p=0.0043), valine (Val; p=0.0016), leucine (Leu; p=0.0078), isoleucine (Ile; p=0.0022), the molar ratio of total branched-chain amino acids to tyrosine (BTR; p=0.00025), total-bilirubin (T-Bil; p=0.0071), prothrombin time(%) (PT; p=0.028), and serum sodium (Na; p=0.035). A multivariate analysis found the BTR to be the only independent predictor of a DST abnormality (hazard ratio, 9.24; p＜0.031). An FFQ analysis, revealed that the nutritional findings of patients with and without a DST abnormality, were similar. Conclusion The BTR was useful for predicting the risk of NPAs, as defined by a DST abnormality. The risk of NPAs may be estimated by monitoring the BTR.