Topical application of a novel, hydrophilic gamma-tocopherol derivative reduces photo-inflammation in mice skin.
Emiko Yoshida; Tatsuya Watanabe; Jiro Takata; Akihiko Yamazaki; Yoshiharu Karube; Shizuko Kobayashi
The Journal of investigative dermatology, Jul. 2006
We previously demonstrated that a novel hydrophilic gamma-tocopherol (gamma-Toc) derivative, gamma-tocopherol-N,N-dimethylglycinate hydrochloride (gamma-TDMG) converts to gamma-Toc in the mouse skin and has a higher bioavailability than gamma-Toc itself. In the present study, we determined whether gamma-TDMG could reduce photo-inflammation in mouse skin, and compared its effectiveness to that of alpha-Toc acetate (alpha-TA). Topical pre- or post-application of 5% gamma-TDMG significantly reduced the formation of edema and tempered the increase in cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin E2 (PGE2) that were induced by a single dose of UV irradiation of 2 kJ/m2 (290-380 nm, maximum 312 nm). The pre-treatment of mouse skin with 10% alpha-TA had the same anti-inflammatory effect as did gamma-TDMG. In spite of same having the ability to reduce PGE2 levels, the effect of gamma-TDMG pre-treatment on the inhibition of COX-2 mRNA/protein expression was less than that seen with 10% alpha-TA. In contrast, the increase in COX-2 activity seen after UV exposure was reduced more by gamma-TDMG than by alpha-TA, suggesting that the reduction in PGE2 levels might have been due to the direct inhibition of COX-2 activity by gamma-TDMG-derived gamma-Toc. Both Toc derivatives strongly suppressed inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide (NO) production, both of which play important roles in UV-induced inflammation. Both derivatives also significantly reduced lipid peroxidation in response to UV exposure, though gamma-TDMG's ability in this regard was less than that seen with alpha-TA, which correlated with their abilities to suppress COX-2 expression. Thus, the gamma-TDMG-derived gamma-Toc acts as an antioxidant, suppresses iNOS expression and directly inhibits COX-2 activity, all of which likely play a role in mediating its suppressive effects on photo-inflammation. Our data further suggest that the topical application of gamma-TDMG, a novel hydrophilic gamma-Toc derivative, may be efficacious in preventing and reducing UV-induced inflammation in humans.

Topical application of gamma-tocopherol derivative prevents UV-induced skin pigmentation.
Yuka Kuwabara; Tatsuya Watanabe; Shingo Yasuoka; Kohsuke Fukui; Jiro Takata; Yoshiharu Karube; Yuko Okamoto; Shin Asano; Eiko Katoh; Toshi Tsuzuki; Shizuko Kobayashi
Biological & pharmaceutical bulletin, Jun. 2006
We previously reported that a novel hydrophilic gamma-tocopherol (gamma-Toc) derivative, gamma-tocopheryl-N,N-dimethylglycinate hydrochloride (gamma-TDMG) gets converted to the antioxidant gamma-Toc in skin. We also found that this derivative displayed greater bioavailability than gamma-Toc itself. In the present study, we determined whether gamma-TDMG could reduce UV-induced skin pigmentation in brownish guinea pigs. gamma-TDMG (0.1 or 0.5%) was topically applied to the skin before and after it was exposed to UVB plus UVA (3 times/week for 1 week), and then 10 times/week for 4 weeks thereafter. Treatment with 0.5% gamma-TDMG resulted in significant skin lightening (70% of the pigmentation of irradiated controls). We also found that melanin synthesis was dose-dependently inhibited by gamma-TDMG in murine B16 melanoma cells. When gamma-TDMG or kojic acid (250 microM) were added to homogenates of B16 melanoma cells, their tyrosinase activity was significantly inhibited by approximately 40% and 75%, respectively. Mushroom tyrosinase activity was significantly inhibited by 200 microM gamma-Toc and kojic acid, but not gamma-TDMG. When B16 cells were incubated with 250 microM gamma-TDMG for 24 or 48 h, their intracellular gamma-Toc concentrations rose over 100 fold to 10.5 and 11.2 nmol/10(6) cells, respectively, suggesting that gamma-TDMG was rapidly converted to gamma-Toc in these cells and that their reduced melanin synthesis may have been due to the activity of gamma-Toc. Our data further suggest that the topical application of gamma-TDMG may be efficacious in preventing photo-induced skin pigmentation in humans.

Production of the long and short forms of MFG-E8 by epidermal keratinocytes.
Tatsuya Watanabe; Rakuno Totsuka; Seiji Miyatani; Shun-ichi Kurata; Shingo Sato; Iyoko Katoh; Shizuko Kobayashi; Yoji Ikawa
Cell and tissue research, Aug. 2005
Mouse milk fat globule-EGF factor 8, MFG-E8, is the ortholog to the human mammary tumor marker, lactadherin, and comprises two spliced variants, the L and S forms. Recent studies have suggested that MFG-E8-L produced by macrophages and Langerhans cells in the skin serves as a linker between phagocytic cells and apoptotic cells, and that MFG-E8-S, also termed SED1, facilitates sperm-egg interaction for fertilization. However, Mfge8 gene expression occurs in various tissues apparently unrelated to these critical events. Our in situ hybridization study has revealed that Mfge8 is expressed in the periderm (the premature epidermis) on embryonic day-14, well before Langerhans cells begin to grow in the prenatal phase. Mfge8 transcript is detectable in the basal and spinous layers throughout skin development, whereas immunostaining has revealed MFG-E8 protein accumulation in the spinous layer. Cultured keratinocyte stem cells consistently express Mfge8-L and -S mRNAs and produce the L protein, which is primarily detectable in the culture supernatant, and the S protein, which is mostly associated with the cells. Upon Ca(2+)-stimulated differentiation, which is detected by a decrease in keratinocyte stem cell marker p63(p51) and the induction of keratin1, we have observed suppression of Mfge8, and the protein becomes localized to the cell-cell borders. Papillomas and carcinomas caused by chronic UV-B irradiation produce MFG-E8 as determined by immunostaining. Thus, undifferentiated and poorly differentiated keratinocytes produce the L and S forms of MFG-E8 during normal and pathological tissue development, probably to support an as yet unidentified membrane function.

p51/p63 Controls subunit alpha3 of the major epidermis integrin anchoring the stem cells to the niche.
Shun-Ichi Kurata; Takeshi Okuyama; Motonobu Osada; Tatsuya Watanabe; Yoshiya Tomimori; Shingo Sato; Aki Iwai; Tsutomu Tsuji; Yoji Ikawa; Iyoko Katoh
The Journal of biological chemistry, 26 Nov. 2004
p51/p63, a member of the tumor suppressor p53 gene family, is crucial for skin development. We describe here identification of ITGA3 encoding integrin alpha(3) as a target of its trans-activating function, proposing that p51/p63 allows epidermal stem cells to express laminin receptor alpha(3)beta(1) for anchorage to the basement membrane. When activated by genotoxic stress or overexpressed ectopically in non-adherent cells, p51/p63 transduced a phenotype to attach to extracellular matrices, which was accompanied by expression of ITGA3. Motifs matching the p53-binding consensus sequence were located in a scattered form in intron 1 of human ITGA3, and served as p51/p63-responsive elements in reporter assays. In addition to the trans-activating ability of the TA isoform, we detected a positive effect of the DeltaN isoform on ITGA3. The high level alpha(3) production in human keratinocyte stem cells diminished upon elimination of p51/p63 by small interfering RNA or by Ca(2+)-induced differentiation. Furthermore, a chromatin immunoprecipitation experiment indicated a physical interaction of p51/p63 with intron 1 of ITGA3. This study provides a molecular basis for the standing hypothesis that p51/p63 is essential for epidermal-mesenchymal interactions.

Expression of a novel secretory form (Crb1s) of mouse Crumbs homologue Crb1 in skin development.
Tatsuya Watanabe; Seiji Miyatani; Iyoko Katoh; Shizuko Kobayashi; Yoji Ikawa
Biochemical and biophysical research communications, 09 Jan. 2004
Drosophila Crumbs and the mammalian homologues encoded by the Crb genes are transmembrane proteins required for determination of retinal cell polarity. We cloned a novel variant of mouse Crb1 and termed it Crb1s. Since the 3'-end of exon 6 remained unspliced, Crb1s coded for a short secretory protein lacking the transmembrane and cytoplasmic domains required for the function of Crb1. The Crb1 expression was confined to brain and eye, whereas Crb1s was detectable in various tissues including skin, lung, and kidney in adult mice. Active expression of Crb1s, but not Crb1, was observed during the skin development, in which localization of the Crb1s protein was altered from the basal layer to the upper layers. Cultured mouse keratinocytes synthesized the Crb1s protein and secreted a 80 kDa processed form to the supernatant. After Ca(2+)-induced differentiation, Crb1s became associated with focal adhesions and cell-cell contacts. Crb1s may play a role distinct from that of Crb1 in epidermal tissue morphogenesis.

リトドリン塩酸塩の経口投与が胎動に及ぼす影響について Fetal movement acceleration measurement recorder(FMAM recorder)を用いて
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日本薬学会