Lysyl Oxidase Mediates Proliferation and Differentiation in the Esophageal Epithelium
Kanak V. Kennedy,
Joshua X. Wang,
Emily McMillan,
Yusen Zhou,
Ryugo Teranishi,
Ann Semeao,
Leena Mirchandani,
Chizoba N. Umeweni,
Diya Dhakal,
Alyssa Baccarella,
Satoshi Ishikawa,
Masaru Sasaki,
Takefumi Itami,
Adele C. Harman,
Leonel Joannas,
Tatiana A. Karakasheva,
Hiroshi Nakagawa,
Amanda B. Muir
Affiliations
Kanak V. Kennedy
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Joshua X. Wang
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Emily McMillan
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Yusen Zhou
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Ryugo Teranishi
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Ann Semeao
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Leena Mirchandani
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Chizoba N. Umeweni
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Diya Dhakal
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Alyssa Baccarella
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Satoshi Ishikawa
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Masaru Sasaki
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Takefumi Itami
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Adele C. Harman
Transgenic Core, CHOP Research Institute, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Leonel Joannas
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Tatiana A. Karakasheva
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Hiroshi Nakagawa
Division of Digestive and Liver Diseases, Department of Medicine and Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY 10032, USA
Amanda B. Muir
Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
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 LOX mRNA in both monolayer and 3D organoid culture. A novel murine model with a tamoxifen-induced Lox knockout specific to the stratified epithelium (K5CreER; Loxfl/fl) 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.