Hébert-Milette Isabelle, Lévesque Chloé, Paquette Jean, Rivard Marie-Ève, Villeneuve Louis, Boucher Gabrielle, Goyette Philippe, Charron Guy, Rioux John D
Montreal Heart Institute Research Centre, 5000 rue Bélanger, Montreal, Quebec, Canada.
Université de Montréal, Montreal, Quebec, Canada.
bioRxiv. 2025 Mar 15:2025.03.14.643205. doi: 10.1101/2025.03.14.643205.
C1ORF106 has previously been associated with inflammatory bowel diseases (IBD) via large-scale genetic studies. Increased intestinal permeability is a hallmark of IBD and is observed in at-risk individuals prior to the appearance of clinical symptoms. C1ORF106 was previously shown to regulate intestinal barrier permeability through the regulation of adherens junction stability and through the formation of tight junctions, which impacted actin assembly. However, the downstream impact and molecular mechanisms involved in actin regulation by C1ORF106 haven't been explored. Our study aimed at identifying which pathways involved in intestinal epithelial barrier regulation and F-actin regulation are impacted by C1ORF106 and its IBD-associated variant.
We knocked down (KD) the expression of in human colonic epithelial cells and characterized the function of the 333F variant in intestinal epithelial spheroid cultures obtained from patient-derived human induced pluripotent stem cell (hiPSC). We measured barrier permeability and characterized spheroid formation, actin regulation and cell migration though immunofluorescence, western blots and permeability assays.
C1ORF106 KD leads to impaired cortical actin belt dynamics and regulation of stress fiber formation, resulting in increased cell constriction, impaired barrier permeability, cell polarity and cell migration. Moreover, we demonstrated that an inhibition of ROCK rescues the actin belt and cell polarity phenotypes in C1ORF106 KD cells, demonstrating that C1ORF106 regulates these phenotypes through a ROCK-dependent mechanism. We also observed an altered nmMYO2-P localization in C1ORF106 KD cells associated with the formation of Vacuolar Apical Compartments (VACs), which are important for 3D epithelial spheroid formation. We observed a similar impact on cell polarity in intestinal epithelial spheroids obtained from hiPSC carrying the 333F variant, providing additional support that this pathway is involved in disease development.
We provide insights into the molecular mechanisms by which C1ORF106 controls actin dynamics to regulate intestinal epithelial integrity.
通过大规模基因研究，C1ORF106先前已被发现与炎症性肠病（IBD）相关。肠道通透性增加是IBD的一个标志，并且在临床症状出现之前在高危个体中就可观察到。先前的研究表明，C1ORF106通过调节黏附连接稳定性和紧密连接的形成来调节肠道屏障通透性，而这会影响肌动蛋白组装。然而，C1ORF106对肌动蛋白调节的下游影响和分子机制尚未得到探索。我们的研究旨在确定C1ORF106及其IBD相关变体影响了肠道上皮屏障调节和F - 肌动蛋白调节中的哪些途径。
我们在人结肠上皮细胞中敲低C1ORF106的表达，并在从患者来源的人诱导多能干细胞（hiPSC）获得的肠道上皮球状体培养物中表征333F变体的功能。我们通过免疫荧光、蛋白质免疫印迹和通透性测定来测量屏障通透性，并表征球状体形成、肌动蛋白调节和细胞迁移。
C1ORF106基因敲低导致皮质肌动蛋白带动力学受损和应力纤维形成调节异常，从而导致细胞收缩增加、屏障通透性受损、细胞极性和细胞迁移受损。此外，我们证明抑制ROCK可挽救C1ORF106基因敲低细胞中的肌动蛋白带和细胞极性表型，表明C1ORF106通过ROCK依赖性机制调节这些表型。我们还观察到C1ORF106基因敲低细胞中nmMYO2 - P定位改变，这与液泡顶端隔室（VACs）的形成有关，而VACs对三维上皮球状体形成很重要。我们在携带333F变体的hiPSC获得的肠道上皮球状体中观察到对细胞极性的类似影响，这为该途径参与疾病发展提供了额外支持。
我们深入了解了C1ORF106控制肌动蛋白动力学以调节肠道上皮完整性的分子机制。
