Liu Y, Nusrat A, Schnell F J, Reaves T A, Walsh S, Pochet M, Parkos C A
Division of Gastrointestinal Pathology, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA.
J Cell Sci. 2000 Jul;113 ( Pt 13):2363-74. doi: 10.1242/jcs.113.13.2363.
Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35-39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.
上皮细胞形成一个高度选择性的屏障并覆盖许多器官。上皮屏障由包含紧密连接的紧密相邻的细胞间接触维持，其调节机制尚未完全了解。在此，我们报告了一种免疫球蛋白超家族成员的克隆、组织定位及其在上皮屏障调节中作用的证据，该成员可能代表小鼠连接粘附分子（JAM）的人类同源物。对从T84上皮细胞克隆的人类JAM一级结构的分析预测，它是一种跨膜蛋白，其胞外结构域包含两个IgV环。针对假定的胞外结构域产生的单克隆抗体与来自T84上皮细胞和人类中性粒细胞的35 - 39 kDa蛋白发生反应。通过免疫荧光，JAM单克隆抗体标记肠道、肺和肾脏的上皮细胞，在紧密连接区域（与闭合蛋白共定位）以及紧密连接下方的侧细胞膜上尤为明显。流式细胞术研究证实JAM在上皮细胞中主要表达，但也揭示其在所有谱系的内皮细胞和造血细胞上表达。功能研究表明，JAM特异性单克隆抗体在通过短暂钙耗竭破坏细胞间连接（包括紧密连接）后，显著抑制T84单层细胞的跨上皮电阻恢复。形态学分析显示，在细胞间连接解体后，抗JAM对屏障功能恢复的抑制与重新组装紧密连接结构时闭合蛋白和JAM的丢失相关，但与ZO - 1无关。主要粘附连接成分E - 钙粘蛋白的重新组装不受JAM特异性单克隆抗体的影响。我们的研究结果表明，JAM在上皮细胞紧密连接组装的调节中起重要作用。此外，这些JAM介导的效应可能通过与闭合蛋白的直接或间接相互作用发生。
