| Co-Investigator(Kenkyū-buntansha) |
FURUSE Mikio Kyoto University, Faculty of Medicine, Associate Prof., 医学研究科, 助教授 (90281089)
永渕 昭良 熊本大学, 発生医学研究センター, 教授 (80218023)
伊藤 雅彦 京都大学, 医学研究科, 助手 (70270486)
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| Budget Amount *help |
¥37,690,000 (Direct Cost: ¥36,400,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2001: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2000: ¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1999: ¥25,000,000 (Direct Cost: ¥25,000,000)
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| Research Abstract |
(1) The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia including the epidermis. In this study, we generated claudin-1-deficient mice and found that the animals died within one day of birth with wrinkled skin. Dehydration assay as well as transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to re-examine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an anti-occludin mAb, a Tjspecific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. In claudin-1-deficient mice, claudin-1 appeared to have simply been removed from these TJs leaving occlu
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din(and also claudin-4)-positive TJs. Interestingly, in the wild-type epidermis, these occludin-positive TJs efficiently prevented the diffusion of subcutaneously-injected tracer (〜600 dalton) toward the skin surface, whereas in the claudin-1-deficient epidermis, the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis, and that these TJs are crusial for the barrier function of the mammalian skin.
(2) The tight junction (TJ) stand is linear polymer, typical of proteinaceous polymers within plasma membranes, the physicochemical nature of which remains elusive. Recently, claudins were identified as major constituents of TJ strands, and they were shown to reconstitute TJ strands at cell-cell borders when exogenously expressed in mouse L fibroblasts. In this study, we show that the GFP (green fluorescent protein) fusion protein with claudin-1 can also reconstitute TJ strands in L fibroblasts, allowing us to directly observe individual TJ strands in live cells. Less
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