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The hepatic barrier is indispensable for the physiological functions of the liver and is impaired under various pathological conditions. Tight junctions reportedly play a central role in hepatic barrier regulation; however, there is limited direct evidence supporting this observation, with few in vivo models or confirmations of the implicated molecular mechanisms presented to date. We inactivated the tight junction component gene, Tjp2/ZO-2, and the related molecule, Tjp1/ZO-1, in mouse livers. In humans, TJP2/ZO-2 mutations have been implicated in the development of human progressive familial intrahepatic cholestasis 4 (PFIC4). The mice deficient in either ZO-1 or ZO-2 in the liver did not exhibit major abnormalities. However, the ablation of both molecules impaired the molecular architecture as well as the structure and function of hepatocyte tight junctions, which disrupted the hepatic barrier and was lethal to the mice by 6 weeks of age. In mutant mice, bile canaliculus formation and cellular polarity were compromised; also, transporter expression and localization were deregulated. Moreover, typical hepatic zonation and bile duct formation were inhibited, and sinusoidal vessels were disorganized. These findings clarify the role of tight junctions and polarity in the hepatic barrier as well as the effect that their disruption has on liver tissue. The observations also suggest that liver-specific ZO-1 -/- and ZO-2 -/- mice could be used as models for PFIC4, and this will provide new insights into liver pathophysiology and clinical applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Occludin and claudins are tetraspan-transmembrane proteins in tight junctions. Maculae occludentes, which are less-developed tight junctions, occur in the granular cell layer of the epidermis. The periderm, which overlies the developing epidermis and functions as a protective layer for the embryo, carries developed tight junctions as observed in simple epithelia. In both periderm and epidermis, occludin is expressed at the cell-cell border. To determine the difference between tight junctions of periderm and epidermis, claudin-6 expression was examined in periderm and epidermis. Immunofluorescence staining showed claudin-6 expression at the cell-cell border of the periderm, but not in the epidermis. Reverse transcription-polymerase chain reaction confirmed that claudin-6 was not expressed in mouse adult skin, whereas immunoelectron microscopy revealed that claudin-6 was localized at tight junctions of the periderm. Furthermore, L fibroblasts with stable expression of exogenous claudin-6 formed developed tight junctions at cell-cell borders. These findings indicate that molecular architecture of tight junctions of the periderm is different from that of the maculae occludentes of the epidermis, and that claudin-6 is important in the formation of tight junctions of the periderm.

Tight junctions (TJs) have been suggested to act both as barriers and fences, but lack of information on the constituents of TJ strands has hampered the direct assessment of these functions. Over the past year, our understanding of the molecular architecture of TJ strands has increased markedly and we are ready to experimentally examine how TJs are involved in their dual functions.

Occludin is an integral membrane protein that is concentrated at tight junctions (zonulae occludentes) in simple epithelial cells. ZO-1 and ZO-2 are peripheral membrane proteins that are localized at tight junctions in simple epithelial cells and at cadherin-based adherens junctions in nonepithelial cells. In this study, we investigated the expression and subcellular distribution of occludin, ZO-1, and ZO-2 in rodent skin. Immunoblotting detected all of these molecules in isolated epidermis, but the occludin/ZO-1 (or occludin/ZO-2) ratio was significantly lower than that in cultured simple epithelial cells. In the epidermis of adult skin, occludin was concentrated at cell-cell borders only in the most superficial zone of the granular cell layer, whereas ZO-1 and ZO-2 were distributed in a much broader zone from the spinous to the granular layers. During mouse skin development, this peculiar distribution of occludin in the epidermis appeared when the periderm, a simple epithelium bearing typical occludin-based tight junctions, was sloughed off at embryonic day 16.5 of gestation. Freeze-fracture electron microscopy identified the so-called focal strands or maculae occludentes, i.e., spot tight junction-like structures, between adjacent granular cells, and anti-occludin monoclonal antibody exclusively labeled these focal strands. In hair follicles, occludin and ZO-1 were colocalized at cell-cell borders in Henle's layer and the cornifying cuticle of the inner root sheath. In addition, ZO-1 but not occludin were localized weakly at the outer root sheath and intensely at the hair cortex/matrix.

Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions, and the cDNA encoding its mammalian homologue was identified very recently (Ando-Akatsuka, Y., M. Saitou, T. Hirase, M. Kishi, A. Sakakibara, M. Itoh, S. Yonemura, M. Furuse, Sh. Tsukita, J. Cell Biol. 133, 43-47 (1996)). Here we describe the basic properties of mammalian occludin in epithelial cells at the DNA, RNA, and protein levels. The human occludin gene was mapped to chromosome band 5q13.1 by fluorescent in situ hybridization. Northern blotting identified several occludin mRNA bands, suggesting the possible expression of several alternatively spliced products. Occludin mRNA was detected in cultured epithelial cells, but not in cultured fibroblasts. The mRNA level was high in the testis, kidney, liver, lung, and brain, which reportedly bear well-developed tight junctions. We then produced monoclonal and polyclonal antibodies using recombinant mouse occludin as the antigen, which reacted not only with mouse, but also human, dog and pig occludin. These antibodies recognized several bands around 60 kDa in epithelial cells but not in fibroblasts. Immunofluorescence microscopy of various tissues revealed that the staining intensity of occludin correlated well with the number of tight junction strands in epithelial cells. By contrast, the staining of ZO-1, a well-characterized tight junction-associated protein, was not specific for tight junctions. Furthermore, the exclusive concentration of occludin at tight junctions in epithelial cells was confirmed by immunoreplica electron microscopy.