Campos-Blázquez Jessica P, Flores-Maldonado Catalina, Gallardo Juan M, Bonilla-Delgado José, Pedraza-Ramírez Alan A, López-Méndez Octavio, Cortés-Malagón Enoc M, Contreras Rubén G
Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the IPN (Cinvestav-IPN), Mexico City, Mexico.
Unidad de Investigación Médica en Enfermedades Nefrológicas, Hospital de Especialidades, Centro Médico Nacional "Siglo XXI" Instituto Mexicano del Seguro Social, Mexico City, Mexico.
Autophagy Rep. 2023 Oct 2;2(1):2256146. doi: 10.1080/27694127.2023.2256146. eCollection 2023.
Epithelial cells transport substances through the cellular and paracellular pathways. The last one depends on tight junctions, particularly on claudins, the family of integral membrane proteins responsible for the permeability and selectivity of these junctions. 300 nM ouabain (OUA) induces endocytosis and lysosomal degradation of claudin-2 and -4 in an Src and ERK1/2 kinases-dependent manner. Here we investigate whether OUA-induced lysosomal degradation of claudins implicates autophagy in renal epithelial Madin-Darby canine kidney cells. During autophagy, LC3 protein binds phosphatidylethanolamine and incorporates, together with protein p62, into the phagophore. Subsequently, the autolysosome degrades both LC3 and p62 proteins. OUA's occupancy of its site in the Na⁺/K⁺ATPase (300 nM, 10 h) increases autophagic flux because of degradation of LC3 and p62 and an increase in the number of autophagosomes, as detected by fluorescent LC3 and p62 and the rise in autolysosomes seen by the GFP-LC3-RFP probe. Finally, OUA increases the colocalisation of claudin-1, -2, or -4 with p62 in these . OUA induces autophagy increasing reactive oxygen species generation that activates AMP-activated protein kinase, phosphorylating ULK1 at S555. The autophagy inducer rapamycin causes a degradation of the studied claudins comparable to the one generated by OUA. Furthermore, the autophagy inhibitor dorsomorphin blocks OUA-induced autophagy and claudin-1, -2, and -4 degradation. These results demonstrated that OUA induces claudin-1, -2, and -4 autophagy through oxidative stress. AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATP: Adenosine triphosphate; DM: dorsomorphin; EGFR: epidermal growth factor receptor; ERK: extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LC3: microtubule-associated protein 1A/1B-light chain 3; MDCK: Madin-Darby canine kidney; mTOR: mammalian target of rapamycin; NAC: N-acetylcysteine; OUA: ouabain; PCC: Pearson's correlation coefficient; PE: phosphatidylethanolamine, Rapa: rapamycin; ROS: reactive oxygen species; SNK: Student-Newman-Keuls; TER: transepithelial electrical resistance; TJs: tight junctions; ULK1: Unc-51-like kinase 1.
上皮细胞通过细胞途径和细胞旁途径转运物质。后者依赖于紧密连接，特别是依赖于闭合蛋白，这是一类整合膜蛋白家族，负责这些连接的通透性和选择性。300 nM哇巴因（OUA）以Src和ERK1/2激酶依赖性方式诱导闭合蛋白-2和-4的内吞作用和溶酶体降解。在此，我们研究OUA诱导的闭合蛋白溶酶体降解是否涉及肾上皮性犬肾细胞中的自噬。在自噬过程中，LC3蛋白与磷脂酰乙醇胺结合，并与p62蛋白一起整合到吞噬体中。随后，自噬溶酶体降解LC3和p62蛋白。OUA占据其在Na⁺/K⁺ATP酶中的位点（300 nM，10小时）会增加自噬通量，这是因为LC3和p62的降解以及自噬体数量的增加，这通过荧光LC3和p62检测到，并且通过GFP-LC3-RFP探针观察到自噬溶酶体的增加。最后，OUA增加了这些细胞中闭合蛋白-1、-2或-4与p62的共定位。OUA诱导自噬，增加活性氧的产生，从而激活AMP活化蛋白激酶，使ULK1在S555位点磷酸化。自噬诱导剂雷帕霉素导致所研究的闭合蛋白降解，其程度与OUA产生的降解相当。此外，自噬抑制剂多柔比星可阻断OUA诱导的自噬以及闭合蛋白-1、-2和-4的降解。这些结果表明，OUA通过氧化应激诱导闭合蛋白-1、-2和-4的自噬。AMP：腺苷一磷酸；AMPK：AMP活化蛋白激酶；ATP：腺苷三磷酸；DM：多柔比星；EGFR：表皮生长因子受体；ERK：细胞外信号调节激酶；GAPDH：甘油醛-3-磷酸脱氢酶；LC3：微管相关蛋白1A/1B轻链3；MDCK：犬肾细胞；mTOR：雷帕霉素哺乳动物靶点；NAC：N-乙酰半胱氨酸；OUA：哇巴因；PCC：皮尔逊相关系数；PE：磷脂酰乙醇胺；Rapa：雷帕霉素；ROS：活性氧；SNK：学生-纽曼-库尔斯检验；TER：跨上皮电阻；TJs：紧密连接；ULK1：Unc-51样激酶1
