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      Claudin-based tight junctions are crucial for the mammalian epidermal barrier : a lesson from claudin-1–deficient mice

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          Abstract

          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. Here we generated claudin-1–deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and 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 reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1–deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (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 D) 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 crucial for the barrier function of the mammalian skin.

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          Most cited references70

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            RADIOAUTOGRAPHIC STUDIES OF CHOLINE INCORPORATION INTO PERIPHERAL NERVE MYELIN

            This radioautographic study was designed to localize the cytological sites involved in the incorporation of a lipid precursor into the myelin and the myelin-related cell of the peripheral nervous system. Both myelinating and fully myelinated cultures of rat dorsal root ganglia were exposed to a 30-min pulse of tritiated choline and either fixed immediately or allowed 6 or 48 hr of chase incubation before fixation. After Epon embedding, light and electron microscopic radioautograms were prepared with Ilford L-4 emulsion. Analysis of the pattern of choline incorporation into myelinating cultures indicated that radioactivity appeared all along the length of the internode, without there being a preferential site of initial incorporation. Light microscopic radioautograms of cultures at varying states of maturity were compared in order to determine the relative degree of myelin labeling. This analysis indicated that the myelin-Schwann cell unit in the fully myelinated cultures incorporated choline as actively as did this unit in the myelinating cultures. Because of technical difficulties, it was not possible to determine the precise localization of the incorporated radioactivity within the compact myelin. These data are related to recent biochemical studies indicating that the mature myelin of the central nervous system does incorporate a significant amount of lipid precursor under the appropriate experimental conditions. These observations support the concept that a significant amount of myelin-related metabolic activity occurs in mature tissue; this activity is considered part of an essential and continuous process of myelin maintenance and repair.
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              Claudin-1 and -2: Novel Integral Membrane Proteins Localizing at Tight Junctions with No Sequence Similarity to Occludin

              Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band ∼22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as “claudin-1” and “claudin-2”, respectively. Although the precise structure/function relationship of the claudins to TJ still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.
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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                18 March 2002
                : 156
                : 6
                : 1099-1111
                Affiliations
                [1 ]Department of Cell Biology, Kyoto University Faculty of Medicine, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan
                [2 ]KAN Research Institute, Inc., Kyoto Research Park, Chudoji, Shimogyo-ku, Kyoto 600-8317, Japan
                [3 ]Department of Dermatology, Kyoto University Faculty of Medicine, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
                [4 ]Basic Research Laboratory, Kanebo, Ltd., Kotobuki-cho, Odawara, Kanagawa 250-0002, Japan
                [5 ]Department of Cell Biology, Japanese Foundation for Cancer Research-Cancer Institute, Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan
                [6 ]Department of Molecular Genetics, Tohoku University School of Medicine, Seryo-cho, Aoba-ku, Sendai 980-8575, Japan
                Author notes

                Address correspondence to Shoichiro Tsukita, Dept. of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-753-4372. Fax: 81-75-753-4660. E-mail: htsukita@ 123456mfour.med.kyoto-u.ac.jp

                Article
                0110122
                10.1083/jcb.200110122
                2173463
                11889141
                830e7a9d-f8f3-4377-a72c-ab608d748289
                Copyright © 2002, The Rockefeller University Press
                History
                : 25 October 2001
                : 30 January 2002
                : 31 January 2002
                Categories
                Article

                Cell biology
                claudin-1; tight junction; skin; epidermis; barrier
                Cell biology
                claudin-1; tight junction; skin; epidermis; barrier

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