Polyamines are necessary for synthesis and stability of occludin protein in intestinal epithelial cells
Polyamines are necessary for synthesis and stability of occludin protein in intestinal epithelial cells
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DOI：
10.1152/ajpgi.00407.2004
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发表时间：
2005-06-01
影响因子：
4.5
通讯作者：
Wang, JY
中科院分区：
文献类型：
--
作者：
Guo, X;Rao, JN;Wang, JY
Occludin是一种完整的膜蛋白，形成紧密连接的封闭元件，对上皮屏障功能至关重要。多胺参与多种信号通路，驱动肠上皮细胞(IEC)的不同生物学功能。本研究确定了多胺是否参与了occludin的表达，并在肠上皮屏障功能中发挥作用。研究是在稳定的Cdx2转基因IEC-6细胞(IEC-Cdx2L1)中进行的，该细胞具有高度分化的表型。用α-二氟甲基鸟氨酸(DFMO)耗竭多胺可降低occludin蛋白水平，但不影响其mRNA的表达。在多胺缺乏的细胞中，其他紧密连接蛋白ZO-1、ZO-2、Claudin-2和Claudin-3也减少。DFMO处理的细胞中紧密连接蛋白水平的降低与上皮屏障的功能障碍有关，而这一障碍可被外源性多胺亚精胺克服。在多胺缺乏的细胞中，封闭蛋白水平的降低并不是由于细胞内游离钙浓度([Ca~(2+)](Cyt))的降低，因为在存在或不存在多胺的情况下，[Ca~(2+)](Cyt)的增加或降低都不会改变封闭蛋白的水平。在多胺缺乏后，新合成的封闭蛋白水平下降了近70%，而其蛋白半衰期从对照细胞的接近120min缩短到多胺缺乏细胞的类似于75min。这些发现表明，多胺对闭锁蛋白的合成和稳定性是必需的，多胺的缺失至少部分地通过减少闭锁蛋白来破坏上皮屏障功能。
Occludin is an integral membrane protein that forms the sealing element of tight junctions and is critical for epithelial barrier function. Polyamines are implicated in multiple signaling pathways driving different biological functions of intestinal epithelial cells (IEC). The present study determined whether polyamines are involved in expression of occludin and play a role in intestinal epithelial barrier function. Studies were conducted in stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) associated with a highly differentiated phenotype. Polyamine depletion by alpha- difluoromethylornithine (DFMO) decreased levels of occludin protein but failed to affect expression of its mRNA. Other tight junction proteins, zonula occludens (ZO)-1, ZO-2, claudin-2, and claudin-3, were also decreased in polyamine-deficient cells. Decreased levels of tight junction proteins in DFMO-treated cells were associated with dysfunction of the epithelial barrier, which was overcome by exogenous polyamine spermidine. Decreased levels of occludin in polyamine- deficient cells was not due to the reduction of intracellular-free Ca2+ concentration ([Ca2+](cyt)), because either increased or decreased [Ca2+](cyt) did not alter levels of occludin in the presence or absence of polyamines. The level of newly synthesized occludin protein was decreased by similar to 70% following polyamine depletion, whereas its protein half- life was reduced from similar to 120 min in control cells to similar to 75 min in polyamine- deficient cells. These findings indicate that polyamines are necessary for the synthesis and stability of occludin protein and that polyamine depletion disrupts the epithelial barrier function, at least partially, by decreasing occludin.