Anhui Medical University, Hefei, Anhui, China
Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
Gut. 2023 May;72(5):870-881. doi: 10.1136/gutjnl-2021-326534. Epub 2022 May 10.
Intestinal barrier loss is a Crohn's disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically.
Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects.
MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects.
Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.
肠屏障缺失是克罗恩病（CD）的一个风险因素。这可能与肌球蛋白轻链激酶 1（MLCK1）的表达和酶活性增加有关，它增加了肠细胞旁通透性，并与 CD 的严重程度相关。此外，临床前研究表明，MLCK1 募集到细胞连接对于 TNF 诱导的屏障缺失以及实验性炎症性肠病的进展是必要的。我们试图确定 MLCK1 募集的机制，并从药理学上靶向这一过程。
体外评估 FK506 结合蛋白 8（FKBP8）与 MLCK1 之间的蛋白相互作用。使用转基因和敲除肠上皮细胞系、人类肠类器官和小鼠作为临床前模型。在 CD 患者和对照患者的活检中验证了发现。
MLCK1 与 FKBP8 的他克莫司结合 PPI 结构域特异性相互作用。敲除或显性失活 FKBP8 表达可防止 TNF 诱导的 MLCK1 募集和体外屏障缺失。FKBP8 可阻断 MLCK1-FKBP8 结合，逆转 TNF 诱导的 MLCK1-FKBP8 相互作用、MLCK1 募集和体外及体内屏障缺失。与对照受试者相比，CD 患者的活检显示细胞间连接处 MLCK1-FKBP8 相互作用的数量增加。
与 FKBP8 的结合，FKBP8 可被他克莫司阻断，这是 MLCK1 募集到细胞连接和下游导致免疫介导的屏障缺失所必需的。在 CD 中观察到的 MLCK1 活性增加、MLCK1 在细胞连接处的定位以及周细胞 MLCK1-FKBP8 相互作用增加表明，靶向这一过程可能对人类疾病具有治疗作用。这些对与疾病相关的屏障缺失机制的新见解为 FKBP8-MLCK1 相互作用的治疗利用提供了重要基础。
