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Dahlhoff, Maik (2009): Actions of betacellulin in the gastrointestinal tract: studies in transgenic mouse lines. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

This work employed a transgenic mouse model to investigate the functions and effects elicited by the peptide growth factor betacellulin (BTC), an EGFR ligand, on the physiology and pathology of the gastrointestinal tract. BTC, a 32-kDa glycosylated protein, was initially purified from a mouse pancreatic β-cell carcinoma cell line. In addition to the EGFR, BTC is also able to directly bind and activate the related ERBB4 receptor. BTC is expressed in a wide variety of tissues, particularly in the pancreatic β-cells, stomach, small intestine, lung, kidney and uterus. Three different transgenic mouse lines (L2, L4, and L5) overexpressing BTC under the control of a ubiquitous promoter were established by pronuclear DNA microinjection. Southern blot analysis revealed different integration sites for each line. RT-PCR, Northern and Western blot analysis revealed strong expression of the transgene in the lung, heart, brain and pancreas, with detectable levels in other tissues like stomach, intestine, muscle, bones, liver, thymus, spleen, kidneys, adrenal glands, ovaries, brain and eyes of transgenic mice. BTC-transgenic mice showed stunted growth and significantly reduced relative pancreas and carcass weights. In contrast, the absolute and relative weights of eyes, lung, stomach, intestine and spleen were significantly increased. BTC-transgenic mice exhibit a remarkable, age-dependent hyperplasia of the gastric epithelium. The lesions were characterized by a tumor-like hyperplasia of foveolar epithelium with large cystic formations and a severe depletion of the preexisting body of the mucosa, thus resembling some aspects of human gastric tumors found in patients with Ménétrier disease where TGFA, another ligand of the EGFR, plays an important role. BTC-transgenic animals showed hypoalbuminemia (due to protein loss across the mucosa) and an increase in the gastric pH (as a consequence of parietal cell depletion), while gastrin levels were not altered. The hyperplastic lesions originated exclusively in the lesser curvature of the stomach and were histologically detectable for the first time at the age of 4 weeks. Another important aspect of the gastric lesions was the pronounced infiltration with mononuclear cells. Gastric inflammation is regularly observed during infection with Helicobacter pylori in humans, which can lead to development of stomach cancer or Ménétrier disease. In conclusion, our study is the first to reveal an association between gastric hyperplastic lesions and overabundance of an EGFR ligand but TGFA. BTC-transgenic mice showed a significant reduction in the length of small and large intestine while the weight of theses tissues was significant increased. The villi were narrower and longer as compared with control littermates. No pathological alterations such as fibrosis, metaplastic or neoplastic lesions could be found in the intestinal mucosa of transgenic animals, but the cell proliferation index was increased. It was further demonstrated that the BTC effects in the intestine are EGFR-dependent and that BTC overproduction increases the multiplicity of intestinal adenomas in Apc+/Min mice. A distinct expression pattern of ERBB1 and ERBB4 could be responsible for the significantly reduced pancreas weight in BTC-transgenic mice: ERBB1 is expressed predominantly in the islets, while ERBB4 expression is nearly absent in the endocrine tissue but is high in the exocrine part. It can be postulated that the reduced exocrine pancreas is mediated by the ERBB4 receptor and not by the EGFR. This concept is supported by the maintenance of the pancreas weight reduction in BTC-transgenic mice in an EGFR-deficient background. To evaluate the functional consequence of BTC excess in the pancreatic tissue, we submitted transgenic mice to caerulein-induced acute pancreatitis. The serum markers indicative of pancreatitis were significant lower in the transgenic mice as compared to their control littermates, an indication for a weaker pancreatitis in this group. Further, BTC-transgenic animals developed less tissue inflammation, pancreatic edema and acinar necrosis. Interestingly, BTC-transgenic mice showed significantly higher levels of apoptosis during pancreatitis as compared to their littermates. Further studies will be necessary to investigate whether a shift in death response from necrosis to apoptosis is the mechanism resulting in a protection against caerulein-induced pancreatitis. If confirmed, this finding may have important therapeutic implications.