HEDGEHOG PATHWAY ACTIVATION IN ACUTE INJURY AND REGENERATION IN ADULT MOUSE PANCREAS

Abstract

Background & Aims: The Hedgehog (Hh) signaling pathway specifies patterns of cell growth and differentiation during embryogenesis, and is also involved in mature tissue homeostasis, particularly in response to injury. Recently, the seminal role of this pathway in carcinogenesis, particularly in tumors of endodermal derivation such as pancreatic cancer, has been elucidated. Currently, little is known of the distribution of Hh-responsive cells in the mature exocrine pancreas, or the requirement for this pathway in epithelial regeneration. In this study, we examined the status of the Hh signaling pathway during pancreatic regeneration following exocrine injury, and also assessed the effect of Hh pathway inhibition on pancreatic regeneration, using both pharmacologic and genetic approaches. Methods: Acute exocrine pancreatic injury was induced by caerulein injection in C57BL/6J mice. To identify Hh-responding cells, we utilized a reporter mouse harboring a lacZ "knock-in" in one of the Ptch alleles (Ptch-lacZ mice). A second group of mice was additionally treated by, a specific inhibitor of the Hh-pathway. To genetically inhibit the Hh pathway in all pancreatic epithelial cell types, we used PdxI-Cre/smoflox/flox mice, while Ela-Cre-ERT2/smoflox/flox mice were used for acinar cell-specific Hh inactivation. Pancreatic tissue was harvested on days 1-7 following injury, and processed for Xgal staining (as a surrogate for Ptch activation), as well as for quantitative real-time PCR and immunohistochemical analysis. Results: We observed striking activation of the Hh pathway during acute pancreatic injury and regeneration, confirmed by marked Shh and Ptch-lacZ expression, as well as overexpression of Hh target genes, including Ptch and Gli1. Global blockade of pancreatic Hh signaling in either cyclopamine-treated or PdxI-Cre/smoflox/flox mice led to impaired regeneration of exocrine pancreas and the abnormal persistence of a novel cell population expressing both nestin and pdx1. Acinar cell-specific inhibition of Hh signaling in Ela-Cre-ERT2/smoflox/flox mice resulted in an identical phenotype. Conclusion: Our studies confirm that the Hh pathway is activated in acute pancreatic exocrine injury and repair. Inhibition of the Hh pathway prolonged the process of exocrine pancreatic regeneration. Because chronic injury in both pancreas and other solid organs is often associated with future cancer risk, activation of the Hh pathway during pancreatic injury may play a role in the initiation of pancreatic tumorigenesis.