Leaphart Cynthia L, Qureshi Faisal, Cetin Selma, Li Jun, Dubowski Theresa, Baty Catherine, Beer-Stolz Donna, Guo Fengli, Murray Sandra A, Hackam David J
Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Gastroenterology. 2007 Jun;132(7):2395-411. doi: 10.1053/j.gastro.2007.03.029. Epub 2007 Mar 21.
BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is characterized by interferon-gamma (IFN-gamma) release and inadequate intestinal restitution. Because enterocytes migrate together, mucosal healing may require interenterocyte communication via connexin 43-mediated gap junctions. We hypothesize that enterocyte migration requires interenterocyte communication, that IFN impairs migration by impairing connexin 43, and that impaired healing during NEC is associated with reduced gap junctions.
NEC was induced in Swiss-Webster or IFN(-/-) mice, and restitution was determined in the presence of the gap junction inhibitor oleamide, or via time-lapse microscopy of IEC-6 cells. Connexin 43 expression, trafficking, and localization were detected in cultured or primary enterocytes or mouse or human intestine by confocal microscopy and (35)S-labeling, and gap junction communication was assessed using live microscopy with oleamide or connexin 43 siRNA.
Enterocytes expressed connexin 43 in vitro and in vivo, and exchanged fluorescent dye via gap junctions. Gap junction inhibition significantly reduced enterocyte migration in vitro and in vivo. NEC was associated with IFN release and loss of enterocyte connexin 43 expression. IFN inhibited enterocyte migration by reducing gap junction communication through the dephosphorylation and internalization of connexin 43. Gap junction inhibition significantly increased NEC severity, whereas reversal of the inhibitory effects of IFN on gap junction communication restored enterocyte migration after IFN exposure. Strikingly, IFN(-/-) mice were protected from the development of NEC, and showed restored connexin 43 expression and intestinal restitution.
IFN inhibits enterocyte migration by preventing interenterocyte gap junction communication. Connexin 43 loss may provide insights into the development of NEC, in which restitution is impaired.
坏死性小肠结肠炎（NEC）的特征是γ干扰素（IFN-γ）释放以及肠道修复不足。由于肠上皮细胞一起迁移，黏膜愈合可能需要通过连接蛋白43介导的缝隙连接进行肠上皮细胞间通讯。我们推测肠上皮细胞迁移需要肠上皮细胞间通讯，IFN通过损害连接蛋白43来损害迁移，并且NEC期间愈合受损与缝隙连接减少有关。
在瑞士韦伯斯特小鼠或IFN基因敲除（IFN-/-）小鼠中诱导NEC，并在存在缝隙连接抑制剂油酰胺的情况下或通过对IEC-6细胞进行延时显微镜观察来测定修复情况。通过共聚焦显微镜和35S标记在培养的或原代肠上皮细胞、小鼠或人类肠道中检测连接蛋白43的表达、运输和定位，并使用油酰胺或连接蛋白43小干扰RNA通过实时显微镜观察评估缝隙连接通讯。
肠上皮细胞在体外和体内均表达连接蛋白43，并通过缝隙连接交换荧光染料。缝隙连接抑制在体外和体内均显著降低了肠上皮细胞迁移。NEC与IFN释放以及肠上皮细胞连接蛋白43表达缺失有关。IFN通过使连接蛋白43去磷酸化和内化来减少缝隙连接通讯，从而抑制肠上皮细胞迁移。缝隙连接抑制显著增加了NEC的严重程度，而IFN对缝隙连接通讯的抑制作用的逆转恢复了IFN暴露后肠上皮细胞的迁移。引人注目的是，IFN-/-小鼠可免受NEC的发展，并显示出连接蛋白43表达和肠道修复的恢复。
IFN通过阻止肠上皮细胞间缝隙连接通讯来抑制肠上皮细胞迁移。连接蛋白43的缺失可能为NEC的发展提供见解，其中修复受损。
