Leaphart Cynthia L, Dai Shipan, Gribar Steven C, Richardson Ward, Ozolek John, Shi Xia-hua, Bruns Jennifer R, Branca Maria, Li Jun, Weisz Ora A, Sodhi Chhinder, Hackam David J
Division of Pediatric Surgery, Rm. 4A-486 DeSoto Wing, Children's Hospital of Pittsburgh, and Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Am J Physiol Gastrointest Liver Physiol. 2008 Sep;295(3):G559-69. doi: 10.1152/ajpgi.90320.2008. Epub 2008 Jul 17.
Necrotizing enterocolitis (NEC) is associated with the release of interferon-gamma (IFN) by enterocytes and delayed intestinal restitution. Our laboratory has recently demonstrated that IFN inhibits enterocyte migration by impairing enterocyte gap junctions, intercellular channels that are composed of connexin43 (Cx43) monomers and that are required for enterocyte migration to occur. The mechanisms by which IFN inhibits gap junctions are incompletely understood. Lipid rafts are cholesterol-sphingolipid-rich microdomains of the plasma membrane that play a central role in the trafficking and signaling of various proteins. We now hypothesize that Cx43 is present on enterocyte lipid rafts and that IFN inhibits enterocyte migration by displacing Cx43 from lipid rafts in enterocytes. We now confirm our previous observations that intestinal restitution is impaired in NEC and demonstrate that Cx43 is present on lipid rafts in IEC-6 enterocytes. We show that lipid rafts are required for enterocyte migration, that IFN displaces Cx43 from lipid rafts, and that the phorbol ester phorbol 12-myristate 13-acetate (PMA) restores Cx43 to lipid rafts after treatment with IFN in a protein kinase C-dependent manner. IFN also reversibly decreased the phosphorylation of Cx43 on lipid rafts, which was restored by PMA. Strikingly, restoration of Cx43 to lipid rafts by PMA or by transfection of enterocytes with adenoviruses expressing wild-type Cx43 but not mutant Cx43 is associated with the restoration of enterocyte migration after IFN treatment. Taken together, these findings suggest an important role for lipid raft-Cx43 interactions in the regulation of enterocyte migration during exposure to IFN, such as NEC.
坏死性小肠结肠炎（NEC）与肠上皮细胞释放γ-干扰素（IFN）以及肠道修复延迟有关。我们实验室最近证明，IFN通过损害肠上皮细胞间隙连接来抑制肠上皮细胞迁移，间隙连接是由连接蛋白43（Cx43）单体组成的细胞间通道，是肠上皮细胞迁移所必需的。IFN抑制间隙连接的机制尚不完全清楚。脂筏是富含胆固醇-鞘脂的质膜微区，在各种蛋白质的运输和信号传导中起核心作用。我们现在假设Cx43存在于肠上皮细胞脂筏上，并且IFN通过将Cx43从肠上皮细胞脂筏上置换下来来抑制肠上皮细胞迁移。我们现在证实了我们之前的观察结果，即NEC中肠道修复受损，并证明Cx43存在于IEC-6肠上皮细胞的脂筏上。我们表明脂筏是肠上皮细胞迁移所必需的，IFN将Cx43从脂筏上置换下来，并且佛波酯佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）在IFN处理后以蛋白激酶C依赖的方式将Cx43恢复到脂筏上。IFN还可逆地降低了脂筏上Cx43的磷酸化，这可被PMA恢复。引人注目的是，PMA或用表达野生型Cx43而非突变型Cx43的腺病毒转染肠上皮细胞使Cx43恢复到脂筏上，与IFN处理后肠上皮细胞迁移的恢复有关。综上所述，这些发现表明脂筏-Cx43相互作用在暴露于IFN（如NEC）期间调节肠上皮细胞迁移中起重要作用。
