Ets Transcription Factor Etv5 Regulates Ductal Morphogenesis And Differentiation In Association With Sox9 In Vitro And Increases Susceptibility And Delays Recovery From Pancreatitis In Vivo

Introduction: The exocrine pancreas comprises a branched network of ducts that are connected to acini and lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesenchyme. The formation and maturation of pancreatic ductal cells during branching morphogenesis is controlled by a complex hierarchy of transcription factors, which is not fully understood. The Ets-transcription factor Etv5 has been reported to play an important role during the development of organs that undergo branching morphogenesis such as mammary and salivary glands. We therefore aimed to characterize the functional role of Etv5 in pancreatic ductal development and differentiation. Methods: Cells were isolated from wild-type mouse pancreata and lentiviral transduction was used to induce Etv5 overexpression. Cells were grown in 3D organotypic culture and analyzed with time lapsed microscopy for morphology and dynamics in the formation of duct-like cystic structures. Differentiation status of cells grown in 3D was analyzed by immunofluorescence-staining (IF) and qPCR. To asses the role of Etv5 in the normal mouse pancreas and its role in inflammation and regeneration in vivo , Pdx1cre;Etv5 -/- ; Rosa YFP mice were generated and aged up to six months and subjected to a cerulein induced acute pancreatitis protocol. Results: Normal pancreatic ductal cells grown in 3D-organotypic culture form spheroid cysts that resemble pancreatic ductal structures. Etv5 overexpression leads to early and exuberant formation of spheroid cysts within 2 days after seeding. Time-lapse microscopy demonstrated a significantly increased movement of cellular structures along the cyst as well as fusion of cysts into larger tubular structures. mRNA analysis of cysts harvested at day 7 displayed a strong upregulation of Sox9 and Foxa2, important regulators of ductal differentiation. Concurrently, E-cadherin was upregulated significantly whereas N-cadherin was downregulated indicating the terminal differentiation of these cells. IF-staining revealed co-localization of Etv5 and Sox9 in spheroid cysts of Etv5-overexpressing cells. Knockdown of Sox9 in Etv5-overexpressing cells with siRNA partially abrogated the formation of tubular structures and disrupted cyst architecture. Etv5 knockout mice ( Pdx1Cre;Etv5 -/- ) were generated and we performed a cerulein-induced acute pancreatitis model. We found significantly elevated levels of serum amylase in Etv5 -/- mice on Days 1 and 3 of the protocol. In a semiquantitative, blinded review of the histology, there was significantly more intense edema, inflammation, vacuolization and necrosis in Etv5 -/- mice in all time points, which was also confirmed by quantitative amylase area scoring. Conclusion: Our data suggest a novel role for Etv5 in pancreatic ductal morphogenesis and lumen formation that is at least in part mediated by Sox9. In addition, our data suggests that the loss of Etv5 expression increases susceptibility to pancreatitis and results in persistent pancreatitis with delayed regeneration. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A11.