Volume 138, Issue 4, Pages 1585-1594.e3 (April 2010) Betacellulin Protects From Pancreatitis by Activating Stress-Activated Protein Kinase  Maik Dahlhoff, Hana Algül, Jens T. Siveke, Marina Lesina, Rüdiger Wanke, Thomas Wartmann, Walter Halangk, Roland M. Schmid, Eckhard Wolf, Marlon R. Schneider  Gastroenterology  Volume 138, Issue 4, Pages 1585-1594.e3 (April 2010) DOI: 10.1053/j.gastro.2009.12.045 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Expression and effects of BTC in the pancreas. (A) Islet cells in the pancreas of a nontransgenic animal show immunoreactivity for BTC, whereas exocrine cells are not stained. In the pancreas of BTC-tg mice both islets and exocrine cells show intense immunostaining for BTC. Absolute (B) and relative (C) pancreas weights of male (n = 14) and female (n = 16) BTC-tg mice are significantly reduced as compared to control littermates (n = 14 males and 16 females). Quantitative reverse transcriptase polymerase chain reaction revealed unchanged expression of amylase (D) and cytokeratin 19 (E) in BTC-tg mice. All animals were sacrificed at the age of 9 weeks. Scale bars in (A) represent 50 μm. **P < .01; ***P < .001. Gastroenterology 2010 138, 1585-1594.e3DOI: (10.1053/j.gastro.2009.12.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Cell proliferation and apoptosis in the exocrine pancreas (n = 4 males/group; age: 4 months). The proliferation index (number of BrdU-positive cell nuclei/105 cell nuclei), (A) and the apoptosis index (number of activated caspase-3−positive cells/105 cell nuclei), (B) are significantly increased in exocrine cells of transgenic pancreas. *P < .05; ** P < .01. BTC-tg, betacellulin transgenic. Gastroenterology 2010 138, 1585-1594.e3DOI: (10.1053/j.gastro.2009.12.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Localization of the BTC receptors EGFR and ERBB4 in the pancreas. (A) Immunohistochemical staining for EGFR (ERBB1) is predominantly localized to islet cells of the pancreas of a nontransgenic animal. (B) Immunostaining for ERBB4 is predominantly localized to exocrine cells of the pancreas of a nontransgenic animal. The reduction in absolute (C) and relative (D) pancreas weight of BTC-tg mice is maintained after introduction into the Wa5 background with deficient EGFR signaling, showing that BTC effects in the pancreas are EGFR-independent. Scale bars in (A,B) represent 50 μm. **P < .01; ***P < .001. BTC-tg, betacellulin transgenic. Gastroenterology 2010 138, 1585-1594.e3DOI: (10.1053/j.gastro.2009.12.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Response of BTC-tg mice and control littermates to caerulein. Serum amylase (A) and lipase (B) levels, indistinguishable in both groups in the absence of caerulein, are significantly reduced or reduced with borderline significance (B, 3 hours; P = .08) in BTC-tg mice as compared to control animals. Representative histological sections of mouse pancreas stained with hematoxylin and eosin or immunostained against cleaved caspase-3 (bottom panels, positive cells are indicated by arrowheads) from caerulein-treated nontransgenic (left column) and BTC-tg mice (right column) before and after 3 or 24 hours of caerulein injection. (D) BTC-tg animals show significantly less severe necrosis, edema, and inflammation histological scores 24 hours after caerulein injection as compared to control mice. (E) Quantitative evaluation revealed a significantly higher number of cleaved caspase-3−positive cells in the pancreas of BTC-tg mice 3 hours and 24 hours after caerulein injection. Scale bars in (C) represent 200 μm (for 0 and 3 hours) or 100 μm (for 24 hours). *P < .05; **P < .01; ***P < .01. Gastroenterology 2010 138, 1585-1594.e3DOI: (10.1053/j.gastro.2009.12.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 The protective effect of BTC is also effective in the l-arginine pancreatitis model. The increase in serum amylase levels in BTC-tg mice at 24, 48, and 72 hours was significantly less pronounced as compared to nontransgenic control mice (A). Hematoxylin and eosin−stained histological sections revealed less-pronounced tissue necrosis, edema, and infiltration in the pancreas of BTC-tg mice as compared to control animals (B). Histological scoring revealed that the increase in acinar necrosis (C) and the severity of inflammatory cell infiltration (D) was significantly less prominent in the pancreas of BTC-tg mice as compared to control animals. (E) Quantitative evaluation of cleaved caspase-3−positive cells in the pancreas of BTC-tg mice. Data in (B)−(E) are at 72 hours after l-arginine administration. Gastroenterology 2010 138, 1585-1594.e3DOI: (10.1053/j.gastro.2009.12.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 The protective effect of BTC against AP is SAPK−dependent. (A) Western blot analysis revealed that basal pSAPK levels were practically undetectable in the pancreas of BTC-tg mice and their control littermates. However, 3 hours after induction of AP, pSAPK signals became evident in both groups. Densitometric analysis revealed significantly stronger pSAPK activation in the pancreas of BTC-tg mice as compared to control mice (B). Treatment of BTC-tg mice with the pSAPK inhibitor SP600125 did not alter the levels of SAPK, but resulted in much weaker pSAPK signals (C). Densitometric evaluation confirmed a significant reduction in the levels of activated pSAPK (D). Mice treated with SP600125 showed a significant reduction in the number of cleaved caspase-3−positive cells (E) and significantly higher amylase levels 3 hours after induction of AP (F). Gastroenterology 2010 138, 1585-1594.e3DOI: (10.1053/j.gastro.2009.12.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Treatment of nontransgenic mice with recombinant BTC protects from caerulein-induced AP. Serum levels of amylase 3 hours after induction of AP were significantly lower in BTC-treated mice as compared to the control (vehicle-treated) group (A). The protective effect observed in BTC-treated mice was associated with a significant increase in apoptotic cell number as compared to control animals (B). Gastroenterology 2010 138, 1585-1594.e3DOI: (10.1053/j.gastro.2009.12.045) Copyright © 2010 AGA Institute Terms and Conditions