Department of Animal Sciences, Purdue University, West Lafayette, IN, USA.
FASEB J. 2021 Feb;35(2):e21356. doi: 10.1096/fj.202002098R.
Intestinal epithelial cells are tightly bound by tight junction proteins (TJP) which are dynamic and sensitive to environmental stress. However, the role of the endocytic pathway in the regulation of TJP abundance and tight junction integrity during nutrient stress is poorly understood. Therefore, this study was conducted to investigate the regulation of TJP abundance during nutrient starvation and the role of the endocytic mechanism in this process. IPEC-J2 cells were subjected to nutrient starvation in Krebs-Ringer bicarbonate buffer (KRB) and abundance of TJP, an indication of tight junction remodeling, was characterized with RT-PCR, western blotting and immunofluorescence. Abundance of TJP was dynamically regulated by nutrient starvation. The protein levels of claudin-1, 3, and 4 were initially downregulated within the first 6 hours of starvation, and then, increased thereafter (P < .01). However, there was no change in occludin and ZO-1. Lysosome and proteasome inhibitors were used to determine the contribution of these protein degradation pathways to the TJP remodeling. Short-term starvation-induced degradation of claudin-1, 3, and 4 was found to be lysosome dependent. Specifically, the downregulation of claudin-3 and 4 was via a dynamin-dependent, but clathrin and caveolae independent, endocytic pathway and this downregulation was partly reversed by amino acids supplementation. Interestingly, the re-synthesis of TJP with prolonged starvation partly depended on proteasome function. Collectively, this study, for the first time, elucidated a major role for dynamin-dependent endocytosis of claudin-3 and 4 during nutrient stress in intestinal epithelial cells. Therefore, transient endocytosis inhibition may be a potential mechanism for preserving tight junction integrity and function in metabolic or pathological states such as inflammatory bowel disease that involves destruction of intestinal epithelial TJP.
肠上皮细胞被紧密连接蛋白 (TJP) 紧密结合，这些蛋白是动态的，对外界环境压力敏感。然而，在营养应激下，内吞途径在调节 TJP 丰度和紧密连接完整性方面的作用知之甚少。因此，本研究旨在探讨营养饥饿时 TJP 丰度的调节以及内吞机制在这一过程中的作用。将 IPEC-J2 细胞置于 Krebs-Ringer 碳酸氢盐缓冲液 (KRB) 中进行营养饥饿，并用 RT-PCR、western blot 和免疫荧光法研究 TJP 的丰度，这是紧密连接重塑的一个指标。TJP 的丰度受营养饥饿的动态调节。在饥饿的前 6 小时内，claudin-1、3 和 4 的蛋白水平最初下调，然后增加（P <.01）。然而，occludin 和 ZO-1 没有变化。溶酶体和蛋白酶体抑制剂用于确定这些蛋白降解途径对 TJP 重塑的贡献。发现短期饥饿诱导的 claudin-1、3 和 4 降解依赖于溶酶体。具体来说，claudin-3 和 4 的下调是通过依赖于 dynamin 的，但不依赖于网格蛋白和小窝的内吞途径，并且氨基酸补充部分逆转了这种下调。有趣的是，延长饥饿后的 TJP 重新合成部分依赖于蛋白酶体功能。总的来说，这项研究首次阐明了 dynamin 依赖性内吞在肠上皮细胞营养应激下 claudin-3 和 4 中的重要作用。因此，短暂的内吞抑制可能是在涉及肠道上皮细胞 TJP 破坏的代谢或病理状态（如炎症性肠病）中维持紧密连接完整性和功能的潜在机制。
