1. Volume 122, Issue 7, Pages 1898-1912 (June 2002)
Transgenic overexpression of amphiregulin induces a mitogenic response selectively in pancreatic duct cells 
Martin Wagner, Christoph K. Weber, Frank Bressau, Florian R. Greten, Volker Stagge, Matthias Ebert, Steven D. Leach, Guido Adler, Roland M. Schmid 
Gastroenterology 
Volume 122, Issue 7, Pages (June 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Expression of AR in TGF-α transgenic mice. (A) RT-PCR analysis of TGF-α transgenic mice (TGF-α, 2 individual animals) at the ages of 28 and 180 days compared with littermate controls (WT, 2 individual animals) shows amplification of AR mRNA (125–base pair PCR product) after 40 cycles in the pancreas of 180-day-old TGF-α transgenic mice but not in littermate controls and 28-day-old TGF-α transgenic mice. DNA was visualized using ethidium bromide staining. Amplification of cyclophilin (101–base pair PCR product) serves as internal control. (B) Western blot analysis shows up-regulation of 50-, 43-, 28-, and 26-kilodalton bands of AR in whole pancreatic extracts in TGF-α transgenic mice (TGF-α) compared with littermate controls (WT). Immunohistochemical staining revealed nuclear AR expression in (C) tubular complexes of TGF-α transgenic mice and in (D) ductal cells of wild-type controls. Original magnification 100×, counterstaining with hematoxylin.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Generation of AR transgenic mice. (A) A 3.5-kb AflIII/PvuI fragment containing the rat elastase promoter (E0.2), the human AR cDNA (hAR), and a genomic fragment of human growth hormone (hGH/pA) was injected into Cl57/Bl6–DBA2 F2 zygotes. (B) Seven RT-PCR–positive founder animals (RT-PCR+) were tested for transgene expression compared with a RT-PCR–negative (RT-PCR-) transgenic animal (F41) and a littermate control (WT). Slot blot hybridization of 10 μg of total pancreatic RNA detects different levels of AR expression. The progeny of F30 (transgenic line AR-1) and of F59 (transgenic line AR-2) were further analyzed. (C) Western analysis indicates overexpression of various forms of AR in the pancreas of both transgenic lines (AR-1 and AR-2, 2 individual animals at the age of 360 days for each line). The 43-, 28-, and 26-kilodalton bands are predominately increased in both transgenic lines. An additional 16-kilodalton band was visible after long exposure (lower panel). Immunostaining with a goat anti-AR antibody (R&D Systems) confirmed expression of AR restricted to the acinar cells of transgenic animals (D, line AR-1, 360-day-old pancreas). (F) No immunoreactivity for endogenous AR was observed in littermate controls with this antiserum. Using a different antibody (rabbit anti-AR; NeoMarkers), AR expression was confirmed in (E) acinar cells of transgenic mice and in ductal cells both in (E) transgenic animals and (G) littermate controls. (H–K) The expression of the transgene restricted to the acinar cells was further confirmed by nonradioactive in situ hybridization. (H) Hybridization to the acinar cells of transgenic animals was evident with the human AR probe (line AR-2) but not on (I) an adjacent section with the sense probe nor in (J and K) littermate controls. Original magnification 50× (D–K).
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Different morphologic phenotypes of TGF-α and AR transgenic mice. (A) Tubular complex formation and a massive fibrosis are characteristic for the pancreas of adult TGF-α transgenic mice. AR transgenic animals show focal accumulations of enlarged ductal structures within the pancreas (arrow in B, line AR-1, 35 days old). These ductal structures differ from the regular pancreatic ducts (arrowhead). (C) After 120 days, these ductal structures are surrounded by normal acinar tissue (transgenic line AR-1) and (D) are strongly positive for PAS. (E) At the age of 320 days, progressive branching and papillary growth can be detected within the ductal clusters in the transgenic animals (line AR-1). Furthermore, the ductal clusters are surrounded by increased connective tissue. (F) Survival rates are comparable for line AR-1, AR-2, and littermate controls (■, ▴, and cirf;, respectively). Original magnification: B and E, 50×; A, C, and D, 100×.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Electronmicroscopy of duct cells in AR transgenic mice. (A) The intralobular ducts and centro-acinar cells in AR transgenic animals show normal morphology, although the number of cells is focally increased (open arrows, semithin section, AR-1, 60 days) compared with (B) the littermate control pancreas. Ductal cells share typical characteristics such as apical microvilli, basal indentations, and tight junctions in comparable-sized ducts in (C) AR transgenic mice and (D) littermate controls. The nuclei of small duct cells were irregularly shaped in (C) AR transgenic mice (asterisk) compared with (D) littermate controls. Original magnification: A and B, 400×; and C and D, 4000×.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Expression of ductal markers in AR transgenic mice. (A) Hematoxylin staining shows the morphologic structure of ductal and acinar cells on an adjacent frozen section. (B–F) Sections were stained with various antibodies as described in Materials and Methods (transgenic line AR-2, 180 days). Ductal structures in AR transgenic animals are positive for (B) cytokeratin 8, (C) 18, and (D) DUCT1. Double staining demonstrates the absence of (E) amylase (red fluorescence) and (F) ACINAR1 (red fluorescence) expression within ductal structures that are positive for cytokeratin 8 (E and F, green fluorescence). Original magnification 200×.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Activation of the Ras-Erk1/2 pathway in the pancreas of AR transgenic mice. (A) Ras affinity precipitation assay in pancreatic lysates of AR transgenic mice (AR-2 and AR-1, 240 days after birth) compared with littermate controls (WT). (A) An increase of guanosine triphosphate–loaded Ras was evident in both transgenic lines (upper panel; asterisk depicts an unspecific band), whereas the amount of Ras protein in the pancreatic lysate was comparable (lower panel). Each lane depicts protein preparations of individual animals. (B) Immunohistochemistry shows increased staining for phosphorylated Erk1/2 in intralobular ducts (AR-1, 60 days after birth) and in centro-acinar cells (insert, 60 days after birth). (C) Accumulations of duct cells in older animals are strongly positive for phosphorylated Erk1/2 (AR-1, 120 days after birth). (D) Nuclear staining for phosphorylated Erk1/2 is visible only in single duct cells in littermate controls. Original magnification, 50×. Scale bar, 100 μm.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Increased activity of cyclin-D1/CDK4 and cyclin-E/CDK2 complexes in the pancreas of AR transgenic mice. (A) Immunocomplex kinase assay for cyclin-D1/CDK4 activity in pancreatic lysates of AR transgenic mice (AR-2 and AR-1) and littermate controls (WT) using GST-Rb ( ) as substrate. Increased phosphorylation of GST-Rb is evident when pancreatic extracts of both transgenic lines were used compared with littermate controls (upper panel). Western blot for CDK4 (lower panel) shows the amount of immunoprecipitated CDK4 protein used in the kinase assay. Each lane depicts protein preparations of individual animals. Nuclear immunostaining of cyclin D is increased in ductal structures in (B) transgenic animals (AR-1, 120 days after birth) compared with (C) littermate controls. The insert in B shows cyclin D staining of centro-acinar cells (AR-1, 60 days after birth). (D) Immune complex kinase assay for cyclin-E/CDK2 activity of pancreatic lysates of AR transgenic mice (AR-2 and AR-1) shows increased phosphorylation of histon 1 compared with littermate controls (WT). The lower panel shows the amount of CDK2 immunoprecipitated protein used in the assay. Each lane depicts protein preparations of individual animals. (E) Immunostaining for cyclin-E (AR-1, 120 days after birth) is increased in the pancreas of AR transgenic mice compared with (F) littermate controls. The insert in E depicts cyclin E staining of centro-acinar cells. Original magnification: B, C, E, and F, 50×. Scale bar, 100 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
EGFR and ErbB2 are differentially expressed in the pancreas of AR and TGF-α transgenic mice. Quantitative mRNA analysis indicates up-regulation of EGFR in TGF-α transgenic mice (TGF-α, 180 days) compared with littermate controls (WT) and AR transgenic animals (AR-1, 180 days). AR transgenic mice show induction of ErbB2 mRNA (AR-1) that is absent in TGF-α transgenics (TGF-α) compared with control animals (WT). Expression of TGF-α is unchanged in the AR transgenic animals (AR-1, 180 days) compared with littermate controls (WT). (B) Western blot analysis shows increased levels of EGFR protein in the pancreas of TGF-α transgenic animals (TGF-α, 180 days, upper panel) compared with littermate controls (WT) and both AR transgenic mouse lines (AR-1 and AR-2, 180 days). ErbB2 is slightly induced in AR transgenic animals (AR-1 and AR-2, 180 days, lower panel) in comparison to the littermate control (WT) and TGF-α transgenic mice (TGF-α). Immunofluorescence staining for (C) EGFR (AR-2, 180 days, red fluorescence) and (D) ErbB2 (AR-2, 180 days, red fluorescence) demonstrates expression of both receptors restricted to duct cells. Sections were counterstained with the nuclear dye Yo-Pro-1 (green fluorescence). Original magnification: C and D, 100×.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Collagen gel culture of isolated acini confirms different biological effects of TGF-α and AR. (A) Phase contrast microscopy of collagen gel cultures shows different morphology of isolated acini after 5 days of culture. Acini maintain a solid-shaped appearance in the presence of 10% fetal calf serum and in medium supplemented with 500 ng/mL recombinant AR, whereas the addition of 100 ng/mL TGF-α induced the formation of tubular structures. The combination of TGF-α (100 ng/mL) and AR (0.5 ng/mL) enhanced branching of these structures. (B) Histologic examination shows tube-shaped structures in the presence of TGF-α but not in control cultures nor in the presence of 500 ng/mL AR. (C and D) Double immunofluorescence staining for amylase (green fluorescence) and cytokeratin 19 (red fluorescence) demonstrates strong amylase expression under 10% serum condition, whereas in the presence of TGF-α only single cells maintain amylase expression. Cytokeratin 19 expression is enhanced in the ductal structures. (E and F) Hoechst dye counterstained nuclei. (G) Quantitative analysis demonstrates a dose-dependent increase of ductal structures in the presence of TGF-α (0.1, 1.0, 10, and 100 ng/mL TGF-α, respectively) but not in the presence of AR (0.5, 5.0, 50, and 500 ng AR, respectively). The combination of TGF-α (100 ng/mL) with AR (0.5 ng/mL) further enhanced the formation of ductal structures.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions