Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier:: role of myosin light-chain kinase protein expression
Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier:: role of myosin light-chain kinase protein expression
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DOI：
10.1152/ajpgi.00412.2004
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发表时间：
2005-03-01
影响因子：
4.5
通讯作者：
Said, HM
中科院分区：
文献类型：
--
作者：
Ma, TY;Boivin, MA;Said, HM
TNF-α在包括克罗恩病（CD）在内的各种炎性疾病的肠道炎症中起核心作用。TNF-α诱导的肠上皮紧密连接（TJ）通透性增加已被认为是导致肠道炎症的促炎机制之一。TNF-α诱导的肠TJ通透性增加的细胞内机制尚不清楚。本研究的目的是研究TNF-α诱导的肠上皮TJ通透性增加的可能性，通过肌球蛋白轻链激酶（MLCK）蛋白表达调节，使用体外肠上皮模型系统组成的过滤生长的Caco-2肠上皮单层。TNF-α（10 ng/ml）导致Caco-2 MLCK表达呈时间依赖性增加。MLCK蛋白表达的TNF-α增加抑制了Caco-2 TJ渗透性的增加，并且TNF-α诱导的MLCK表达的抑制（通过放线菌酮）阻止了Caco-2 TJ渗透性的增加，表明MLCK表达可能是Caco-2 TJ渗透性增加所必需的。MLCK蛋白表达的TNF-α增加之前是MLCK mRNA表达的增加，但MLCK蛋白降解没有改变。放线菌素D阻止了MLCK mRNA表达中TNF-α的增加以及MLCK蛋白表达和Caco-2 TJ渗透性的随后增加，表明MLCK mRNA转录的增加导致MLCK表达的增加。MLCK蛋白表达的TNF-α增加也与Caco-2 MLCK活性的增加相关。放线菌酮对MLCK蛋白表达的抑制阻止了TNF-α增加MLCK活性和Caco-2 TJ渗透性。此外，MLCK、Mg 2 +-肌球蛋白ATP酶和代谢能的抑制剂阻止了TNF-α增加Caco-2 TJ渗透性，表明MLCK活性的增加是TNF-α诱导的Caco-2 TJ屏障开放所必需的。总之，我们的结果首次表明：1）TNF-α增加Caco-2 TJ渗透性是由MLCK蛋白表达增加介导的，2）MLCK蛋白表达增加是由MLCK mRNA转录增加调节的，3）Caco-2 TJ渗透性增加需要MLCK活性的MLCK蛋白表达依赖性增加。
TNF-alpha plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-alpha-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-alpha-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-alpha (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-alpha increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-alpha-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-alpha increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-alpha increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-alpha increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-alpha increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg2+-myosin ATPase, and metabolic energy prevented the TNF-alpha increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-alpha-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-alpha increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.