Lahey Kelcie A, Ronaghan Natalie J, Shang Judie, Dion Sébastien P, Désilets Antoine, Leduc Richard, MacNaughton Wallace K
Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada.
Département de Pharmacologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.
PLoS One. 2017 Jul 3;12(7):e0180259. doi: 10.1371/journal.pone.0180259. eCollection 2017.
Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn's disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function.
胃肠道屏障功能的改变被认为与炎症性肠病（克罗恩病和溃疡性结肠炎）有关。我们实验室之前的研究表明，肠道上皮细胞系顶端暴露于丝氨酸蛋白酶会导致跨上皮电阻（TER）增加。然而，这种反应的潜在机制尚不清楚。我们旨在确定在使用犬肠道上皮细胞系（SCBN）进行蛋白酶处理后，启动和维持增强的屏障功能时，蛋白水解活性、表皮生长因子受体（EGFR）激活以及下游细胞内信号传导的必要性。我们还通过研究肌球蛋白调节轻链磷酸化在丝氨酸蛋白酶诱导的TER增加中的作用。结果发现，胰蛋白酶和matriptase的丝氨酸蛋白酶的蛋白水解活性是启动和维持蛋白酶介导的TER增加所必需的。我们还表明，不依赖基质金属蛋白酶（MMP）的EGFR激活对于蛋白酶反应的持续阶段至关重要，并且Src激酶可能介导EGFR的反式激活。PI3-K和ERK1/2信号传导在蛋白酶刺激后TER达到最大增加中很重要；然而，它们的上游激活剂尚未确定。CK2抑制可阻止丝氨酸蛋白酶诱导的TER增加。缓激肽B(2)受体不参与丝氨酸蛋白酶引起的TER变化，并且在胰蛋白酶或matriptase处理后未观察到MLC磷酸化的变化。综上所述，我们的数据表明，在蛋白酶介导的肠道上皮细胞屏障增强中，持续的蛋白水解活性、EGFR反式激活以及下游PI3-K、ERK1/2和CK2信号传导是必需的。蛋白酶介导增强屏障功能的途径可能是治疗以上皮屏障功能破坏为特征的肠道疾病的新靶点。
