Kennedy Kanak V, Wang Joshua X, McMillan Emily, Zhou Yusen, Teranishi Ryugo, Semeao Ann, Mirchandani Leena, Umeweni Chizoba N, Dhakal Diya, Baccarella Alyssa, Ishikawa Satoshi, Sasaki Masaru, Itami Takefumi, Harman Adele C, Joannas Leonel, Karakasheva Tatiana A, Nakagawa Hiroshi, Muir Amanda B
Division of Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA 94304, USA.
Biomolecules. 2024 Dec 7;14(12):1560. doi: 10.3390/biom14121560.
In homeostatic conditions, the basal progenitor cells of the esophagus differentiate into a stratified squamous epithelium. However, in the setting of acid exposure or inflammation, there is a marked failure of basal cell differentiation, leading to basal cell hyperplasia. We have previously shown that lysyl oxidase (LOX), a collagen crosslinking enzyme, is upregulated in the setting of allergic inflammation of the esophagus; however, its role beyond collagen crosslinking is unknown. Herein, we propose a non-canonical epithelial-specific role of LOX in the maintenance of epithelial homeostasis using 3D organoid and murine models. We performed quantitative reverse transcriptase PCR, Western blot, histologic analysis, and RNA sequencing on immortalized non-transformed human esophageal epithelial cells (EPC2-hTERT) with short-hairpin RNA (shRNA) targeting mRNA in both monolayer and 3D organoid culture. A novel murine model with a tamoxifen-induced Lox knockout specific to the stratified epithelium (; was utilized to further define the role of epithelial LOX in vivo. We found that LOX knockdown decreased the proliferative capacity of the esophageal epithelial cells in monolayer culture, and dramatically reduced the organoid formation rate (OFR) in the shLOX organoids. LOX knockdown was associated with decreased expression of the differentiation markers filaggrin, loricrin, and involucrin, with RNA sequencing analysis revealing 1224 differentially expressed genes demonstrating downregulation of pathways involved in cell differentiation and epithelial development. Mice with Lox knockout in their stratified epithelium demonstrated increased basaloid content of their esophageal epithelium and decreased Ki-67 staining compared to the vehicle-treated mice, suggesting reduced differentiation and proliferation in the Lox-deficient epithelium in vivo. Our results demonstrate, both in vivo and in vitro, that LOX may regulate epithelial homeostasis in the esophagus through the modulation of epithelial proliferation and differentiation. Understanding the mechanisms of perturbation in epithelial proliferation and differentiation in an inflamed esophagus could lead to the development of novel treatments that could promote epithelial healing and restore homeostasis.
在稳态条件下，食管的基底祖细胞分化为复层鳞状上皮。然而，在酸暴露或炎症环境中，基底细胞分化明显失败，导致基底细胞增生。我们之前已经表明，赖氨酰氧化酶（LOX），一种胶原蛋白交联酶，在食管过敏性炎症环境中上调；然而，其在胶原蛋白交联之外的作用尚不清楚。在此，我们使用三维类器官和小鼠模型提出了LOX在维持上皮稳态中的一种非经典上皮特异性作用。我们在单层和三维类器官培养中，对靶向mRNA的短发夹RNA（shRNA）处理的永生化非转化人食管上皮细胞（EPC2-hTERT）进行了定量逆转录聚合酶链反应、蛋白质免疫印迹、组织学分析和RNA测序。利用一种新型小鼠模型，该模型中他莫昔芬诱导的Lox基因敲除特异性针对复层上皮（；），以进一步确定上皮LOX在体内的作用。我们发现，LOX基因敲低降低了单层培养的食管上皮细胞的增殖能力，并显著降低了shLOX类器官中的类器官形成率（OFR）。LOX基因敲低与分化标志物丝聚蛋白、兜甲蛋白和内披蛋白的表达降低有关，RNA测序分析显示1224个差异表达基因，表明参与细胞分化和上皮发育的通路下调。与载体处理的小鼠相比，复层上皮中Lox基因敲除的小鼠食管上皮的基底样细胞含量增加，Ki-67染色减少，这表明体内Lox缺陷上皮中的分化和增殖减少。我们的结果在体内和体外均表明，LOX可能通过调节上皮增殖和分化来调节食管上皮的稳态。了解炎症食管上皮增殖和分化的扰动机制可能会导致开发出能够促进上皮愈合和恢复稳态的新疗法。
