Identification and action of N-myc downstream regulated gene 4 A2 in rat pancreas Jia Fang Wang 1 and David J Hill 1,2,3,4 1 Lawson Health Research Institute, St Joseph’s Health Care, 268 Grosvenor Street, Room H404, London, Ontario, Canada N6A 4V2 Departments of 2 Medicine, 3 Physiology and Pharmacology and 4 Paediatrics, University of Western Ontario, London, Ontario, Canada N6A 4V2 (Correspondence should be addressed to D J Hill; Email: dhill@lri.sjhc.london.on.ca) Abstract Pancreatic islets and acinar tissue develop from duct epithelium and share expression of several transcription factors and other molecular markers also involved with the development of neural tissues. We examined rat pancreatic tissue from fetal life until adulthood for the expression of N-myc downstream regulated gene 4 (Ndrg4), a gene shown to be expressed during neuronal cell differentiation. Isolated pancreatic ducts from neonatal rats were maintained in culture and gave rise to clusters of cells expressing nestin (NES) and PDX-1, which subsequently contained immunoreactive glucagon. Using reverse transcription PCR (RT-PCR), we identified mRNA expression and immunoreactive protein presence for NDRG4 in cultured duct-derived cells, and brain of neonatal rats. By PCR cloning of the ductal cell- derived DNA the molecular form of NDRG4 expressed in pancreatic ducts and ARIP rat pancreatic cells was identified as NDRG4A2, and its presence in intact pancreas of fetal and neonatal rats was demonstrated by immunohistochemistry. Incubation of ARIP cells with glucagon-like polypeptide-1 (GLP-1), increased the expression of NDRG4A2 and PDX-1, while decreasing DNA synthesis and promoting the appear- ance of glucagon-positive cells. This inhibitory effect of GLP-1 on DNA synthesis and the stimulatory effect on endocrine differentiation were reversed when the translation of NDRG4A2 was prevented using siRNA. These findings indicate that NDRG4A2 is expressed in pancreatic duct cells under GLP-1 control and may be related to a reduction in proliferation and the onset of the pancreas cell differentiation. Journal of Endocrinology (2009) 201, 15–25 Introduction Both islet endocrine cells and acinar tissue develop from pancreatic epithelium cells during the fetal and neonatal development of the rat, and in the human fetus (Cerf 2006, Murtaugh 2007). The initial development of both lineages depends on the expression of key transcription factors such as PDX-1 and PTF1a within the ductal cells (Murtaugh 2007). PDX-1 is also required in the mature b-cell where it trans- activates the insulin and GLUT2 gene promoters. Other transcription factors including neurogenin3, NEUROG 3, (previously known as NEUROD) BETA2, PAX-4 and -6, and NKX2.2 that are necessary to complete the differentiation of individual endocrine cell lineages, and many of these have a determining role in the differentiation of both neural cells and pancreatic cell types (Habener et al. 2005). Pancreatic ductal cells can be manipulated in vitro giving rise to pseudo-islet structures with multiple endocrine cell types (Ramiya et al. 2000, Soria 2001, Bonner-Weir et al. 2004). Typically, this trans-differentiation process involves the expression of PDX-1. However, there is a paucity of specific markers of pancreatic progenitor cells prior to their expression of endocrine hormones. One such marker was thought to be the intermediate filament protein, the nestin that is also expressed in developing neural tissues (Lendhal et al. 1990). While nestin positive cells were described in isolated adult human islets and identified as a sub-fraction expressing the ATP- binding cassette transporter (ABCG2; Lechner et al. 2002), the corresponding cells in vivo are usually mesenchymal in nature and have been linked to the development of new endothelial cells within the supporting vasculature (Treutelaar et al. 2003). This does not preclude nestin expression also being associated within the lineage development of some pancreatic endocrine or acinar precursor cells (Delacour et al. 2004, Joanette et al. 2004). We therefore hypothesized that there might be other genes co-expressed in developing pancreas and brain that might be involved in pancreatic cell differentiation from ductal cells. One such candidate is the N-myc downstream regulated gene 4 (Ndrg4)/Bdm1 family. These genes are expressed through- out regions of the brain in the neonatal rat and are required for neurite outgrowth (Yamauchi et al. 1999, Ohki et al. 2002). A mutant Ndrg1 gene is the cause of hereditary motor and sensory neuropathy-Lom, marked by progressive axonal loss on the peripheral nervous system. All Ndrg proteins contain an a/b hydrolase structure common to hydrolytic Journal of Endocrinology (2009) 201, 15–25 DOI: 10.1677/JOE-08-0296 0022–0795/09/0201–015 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org 15