Pancreatic gastrin stimulates islet differentiation of transforming growth factor alpha-induced ductular precursor cells. T C Wang, … , E V Schmidt, S J Brand J Clin Invest. 1993;92(3):1349-1356. https://doi.org/10.1172/JCI116708. Research Article Find the latest version: https://jci.me/116708/pdf Pancreatic Gastrin Stimulates Islet Differentiation of Transforming Growth Factor a-induced Ductular Precursor Cells Timothy C. Wang, * Susan Bonner-Weir, Phillip S. Oates, * MaryBeth Chulak, * Babette Simon, * Glenn T. Merlino,I Emmett V. Schmidt,11 and Stephen J. Brand* *Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114; *Joslin Research Laboratory, Joslin Diabetes Center, Boston, Massachusetts 02215; ILaboratory ofMolecular Biology, National Cancer Institute, National Institutes ofHealth, Bethesda, Maryland 20892; and 'IMassachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129 Abstract mouse gestation, the protodifferentiated pancreas begins a phase of rapid growth and differentiation characterized by cy- Gastrin is transiently expressed in fetal islets during a critical todifferentiation of islet cells and a several hundredfold in- period of their development from protodifferentiated islet pre- crease in islet specific gene expression. Histologically, islet for- cursors in fetal pancreatic ducts. To examine the possible role mation (neogenesis) becomes apparent as proliferating islets of gastrin as an islet cell growth factor, postnatal islet growth budding from the pancreatic ducts. In the weeks after birth the was studied in transgenic mice which overexpress gastrin and rate of islet growth slows, with islet neogenesis becoming much TGFa in their pancreas. Overexpression of a TGFa transgene less apparent. The islets also attain a fully differentiated state causes metaplastic ductules containing numerous insulin ex- with maximal levels of insulin gene expression. Similar to pressing cells that resemble protodifferentiated precursors of many organs, the differentiated adult pancreas has reduced re- the fetal pancreas. However, islet mass of the TGFa transgenic generative potential and low levels of cell turnover, in contrast mice was not increased. Pancreatic overexpression of gastrin to the rapid proliferation seen in fetal development. from a chimeric insulin/ gastrin transgene transcribed from the As in many tissues, synergistic interactions between combi- insulin promoter markedly decreased the TGFa-stimulated in- nations of growth factors probably regulate pancreatic growth crease in pancreatic duct mass. Furthermore, pancreatic coex- and development (3). Although studies of fetal explants have pression of both gastrin and TGFa significantly increased islet identified important mesenchymal factors which stimulated mass in mice expressing both transgenes. These findings indi- pancreatic differentiation, these growth factors have not been cate that TGFa and gastrin can act synergistically to stimulate purified or structurally characterized (4). Some known regula- islet growth, although neither peptide alone is sufficient. Islet tory peptides have also been shown to stimulate pancreatic growth may possibly be stimulated through gastrin promoting growth and differentiation. EGF is a potent stimulus of ductu- the differentiation of insulin-positive cells in the TGFa-induced lar cell proliferation ( 5 ). Furthermore, both EGF and its homo- metaplastic ducts. This transgenic study suggests that islet logue TGFa are expressed in pancreatic duct and acinar cells, neogenesis can be reactivated in the ductular epithelium of the and EGF receptors are found on acinar cells and the apical adult pancreas by local expression of two growth factors, gas- surface of duct cells (6). Overexpression of TGFa, EGF, and trin and TGFa. (J. Clin. Invest. 1993. 92:1349-1356.) Key EGF receptor levels have been demonstrated in pancreatic words: pancreatic ducts * islets * gastrin * transforming growth duct neoplasms, supporting their role in stimulating pancreatic factor a proliferation. Transgenic overexpression of TGFa has distinc- Introduction tive effects on the pancreas which differ from other digestive organs (7, 8). Overexpression of TGFa in the liver and intes- The pancreatic islets develop from endodermal stem cells that tines causes hyperplasia. By contrast, TGFa overexpression in lie in the fetal ductular pancreatic epithelia, which also con- the mouse pancreas results in ductular hyperplasia and pro- tains stem cells that develop into the exocrine pancreas ( 1, 2). nounced interstitial fibrosis, without increasing the acinar cell Islet development proceeds through discrete developmental mass (9). TGFa overexpression also induces metaplastic states during fetal gestation, punctuated by dramatic transi- changes in the ductules manifest by the expression of amylase, tions. The initial period is a protodifferentiated state which is an acinar cell-specific product (8). Numerous ducts cells have characterized by the commitment of these pluripotent stem mixture of zymogen and mucin granules, suggesting that cells to the islet cell lineage, manifested by the expression of TGFa expression induces metaplasia of acinar cells into duct insulin and glucagon. These protodifferentiated cells comprise like cells (9). Thus, TGFa overexpression alters pancreatic dif- a population of committed islet precursor cells which express ferentiation by disturbing the normal balance between TGFa only low levels of islet specific gene products and lack the cyto- and other growth factors (7, 8). differentiation of mature islet cells ( 1). Around day 16 in Another peptide known to regulate pancreatic growth is cholecystokinin (CCK),' a gastrointestinal peptide hormone. Address correspondence to Stephen J. Brand, M. D., Ph. D., Gastroin- CCK stimulates proliferation of pancreatic acinar cell in vivo testinal Unit, Department of Medicine, Massachusetts General Hospi- and in vitro ( 10-12). Nevertheless, it is unlikely that CCK tal, Fruit Street, Boston, MA 02114. regulates pancreatic growth during fetal development since the Received for publication 4 January 1993 and in revised form 27 levels 'f CCK peptide expressed in fetal life are negligible ( 13 ). April 1993. The Journal of Clinical Investigation, Inc. 1. Abbreviations used in this paper: CCK, cholecystokinin; INSGAS, Volume 92, September 1993, 1349-1356 insulin/gastrin. Pancreatic Gastrin Stimulates Islet Growth 1349 However, gastrin, the homologue of CCK, is transiently ex- Northern blot hybridization assay pressed in the islets of the developing pancreas ( 13). In the For Northern analysis, total RNA was extracted from tissues by the adult, gastrin acts as a gastric hormone regulating acid secre- method of Cathala et al. (21). Samples of 20 msg of total RNA were tion, and consequently the major site of gastrin expression is resolved on a 1% agarose denaturing gel and transferred to nitrocellu- the stomach. In the fetus, however, the main site of gastrin lose. RNA blots were hybridized with 32P-labeled TGFa riboprobes or expression is the pancreatic islets ( 13). Although the role of exon II of human gastrin that did not cross-hybridize with endogenous pancreatic gastrin in islet development is undefined, some clin- mouse gastrin mRNA. ical observations suggest gastrin may influence islet growth. Peptide radioimmunoassays Hypergastrinemia, caused by gastrin-expressing islet cell tu- Gastrin. Tissues were extracted and assayed for gastrin immunoreactiv- mors and atrophic gastritis, is associated with nesidioblastosis, ity by radioimmunoassay as described previously using antibody 2604 which is characterized histologically by islet cells budding from which is specific for biologically active C terminally amidated gastrin, pancreatic ducts similar to that seen during in fetal islet devel- in a gastrin radioimmunoassay as previously described (22). Tyrosine opment (14, 15). An abnormal persistence of pancreatic gas- monoiodinated human gastrin 17 tracer was used in all assays and trin has also been documented in a case of infantile nesidioblas- synthetic human gastrin 17 was used as a standard. tosis ( 16). However, in neither circumstance was a causal rela- TGFa. Tissues were frozen in liquid nitrogen, ground to powder tionship established between the nesidioblastosis and gastrin with mortar and pestle, and subjected to acid-ethanol extraction as stimulation. Therefore, to elucidate the role of pancreatic gas- described (23). Extracts were reconstituted with water, and protein in we studies on the concentrations determined with a Coomassie blue dye binding assay trin expression islet development, report (Bio-Rad Laboratories, Richmond, CA). Aliquots from the pancreata transgenic overexpression of TGFa and gastrin in the pancreas, were tested in duplicate in a TGFa radioimmunoassay, which mea- which suggest that TGFa and gastrin regulate sequential stages sured competition with 1251 TGFa for binding to a solid-phase rabbit of islet development. antibody raised against the COOH terminus of rat TGFa (kit from BioTope, Seattle, WA). Methods Histological analysis and immunohistochemistry The pancreas was removed, weighed, similarly oriented in cassettes, Animals fixed in Bouin's solution, and embedded in paraffin. Routine 5-Mum FVB/N mice were obtained from Taconic Farms Inc. (Germantown, paraffin sections were stained with hematoxylin and eosin according to NY), and CD I mice were obtained from Charles River Laboratories, standard methods.