Volume 124, Issue 7, Pages 1901-1914 (June 2003) Transgenic overexpression of the oncofetal RNA binding protein KOC leads to remodeling of the exocrine pancreas  Martin Wagner, Steffen Kunsch, Daniel Duerschmied, Michael Beil, Guido Adler, Friederike Mueller, Thomas M Gress  Gastroenterology  Volume 124, Issue 7, Pages 1901-1914 (June 2003) DOI: 10.1016/S0016-5085(03)00402-5

Figure 1 (A) Map of the construct used to generate KOC-transgenic mice. MT-1: mouse metallothionein I promoter; hGH: poly(A) region of the human growth hormone gene; 10 kb 5′ LCR and 7 kb 3′ LCR: 10 kb EcoRI fragment of the 5′ LCR and 7 kb EcoRI fragment of the 3′ LCR of the mouse MT I and II gene locus. (B) Tissue distribution of KOC transgene expression in line IX. Total RNA (30 μg) of tissues from 2-month-old transgenic mice and littermate controls maintained on drinking water containing 25 mmol/L ZnSO4 was hybridized with 32P-labeled human KOC cDNA (B). (C) Western blot analysis of human KOC expression in the pancreas. A polyclonal rabbit antibody directed against human KOC was generated and used to detect the 69-kilodalton human KOC protein in the pancreas of the 2 transgenic mouse, lines IX and XIX, used for further analysis. No human KOC protein was detected in littermate controls. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 2 Phenotype of the pancreas in KOC-transgenic mice of line IX and littermate controls. (A) H&E staining of the pancreas of a 3-month-old littermate control shows a homogeneous, regular pancreatic structure. (B) Incipient interstitial cells (arrowheads) and duct-like structures (arrows) evident in a 6-week-old transgenic mouse. (C) Fifty-week-old transgenic mouse exhibiting net loss of acinar tissue, increasing amounts of adipose tissue (asterisk), and a progressive increase of interstitial cells (arrowhead) and duct-like structures (arrow) throughout the pancreas. (D) Transitional phenotypes of acini composed of acinar cells, ductal cells, and an intermediate cell type displaying characteristics of both; acinar and ductal cells are indicated by arrowheads. (E) Overview of the pancreas of a 60-week-old transgenic mouse demonstrating the involvement of the complete pancreas displaying wide areas with adipose replacement (black asterisk) of pancreatic tissue, remnants of pancreatic tissue with multiple interspersed areas consisting of interstitial cells, and duct-like structures and individual peripancreatic lymph nodes (white asterisk). (F-H) The ultrastructure of ductal and acinar cells in the pancreas of KOC-transgenic mice is depicted. Two types of ducts were found in the transgenic pancreas. (F) First, we found an increased number of ducts showing all features of normal interlobular ducts such as apical microvilli and mucin granules, tight junctions, and basal indentations. (G) Second, small ducts reminiscent of the morphology of ducts during pancreatic development with nonlobulated nuclei and single nucleoli were found. (H) Acinar cells in unaffected pancreatic lobules of the transgenic pancreas were not different from the ones found in littermate controls. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 3 Expression of the KOC-transgene in the pancreas. Immunohistochemical analysis reveals the expression of the KOC protein in the pancreas of transgenic mice from line IX (A and B) but not in littermate controls (D and E). Arrows indicate acinar cells as the unique site of transgenic protein expression in A and B; littermate controls show no specific signal (D and E). Nonradioactive mRNA in situ hybridization with the antisense probe (C) confirms that expression of transgenic human KOC is restricted to acinar cells (white arrowhead). (F) Panel depicts a sense probe hybridization of a serial section of the transgenic pancreas shown in C. The asterisk in C highlights a typical area of interstitial cells and duct-like structures in the transgenic pancreas that shows no specific staining with the antisense probe. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 4 Amylase staining of the pancreas of KOC-transgenic mice and littermate controls. Amylase-staining (red signal in A-D) without (A and B) and with Yo-Pro counterstaining of the nuclei (C and D) of the pancreas of a 12-week-old KOC-transgenic mouse (B-D) and a littermate control (A). Arrowheads in B highlight the loss of amylase staining in areas consisting of interstitial cells and duct-like structures. (C) An area with interstitial cells and duct-like structures is shown surrounded by normal acini. Duct-like structures display luminal amylase staining (arrows). (D) The arrowheads highlight a transitional acinar unit composed of duct-like cells still displaying cytoplasmic amylase staining and acinar cells. Normal acini are indicated by asterisks in B and D. Biochemical analyses indicate decreased amylase and lipase concentration (E) and increased DNA content (F) in the pancreas of transgenic mice (TG) compared with littermate controls (WT). Protein levels are unchanged (F). Data shown represent mean values ± standard deviation (SD); the asterisks indicate significant differences (P < 0.05) as determined by the Mann-Whitney Wilcoxon test. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 5 Carbonic anhydrase expression and activity in the pancreas. In the pancreas of a 15-week-old littermate control mouse (A and B), carbonic anhydrase staining was detected in centroacinar cells (arrows in A) and duct cells (white asterisk in B). The black asterisk in B depicts a blood vessel showing no carbonic anhydrase activity. The pancreas of a 15-week-old line IX transgenic mouse is shown in C displaying a strong carbonic anhydrase staining in duct-like structures (arrowheads). (D) Panel displays an H&E-stained serial section to the area shown in C. (E) An RT-PCR analysis of the carbonic anhydrase gene expression in the pancreas of KOC-transgenic mice and littermate controls aged 7–12 weeks is shown. The lower panel shows the internal β-actin control PCR. (F) Panel shows the results of the biochemical analysis of the total carbonic anhydrase activity in the pancreas of transgenic mice (TG) and littermate control mice (WT). Mean values ± standard deviation are shown; the asterisk indicates significant differences (P < 0.05) as determined by the Mann-Whitney Wilcoxon test. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 6 Intermediate filament system in pancreatic cells. Results of the immunohistochemical analyses with antibodies for pancytokeratin (A and B) visualized by immunofluorescence and the duct-cell specific cytokeratin 19 (C-F) visualized by DAB staining in the pancreas of littermate controls (A and C) and transgenic mice (B and D-F). Intermediate filaments appear as cytoplasmic web in acini and as web with a more subcortical pattern in normal duct cells in the pancreas of littermate controls (A) when using the pancytokeratin antibody. (B) A transitional acinar unit in the pancreas of a transgenic mouse stained with the pancytokeratin antibody is shown. Note that the transitional acinar unit is composed of cells with a ductal (arrow) and an acinar (arrowhead) distribution pattern of intermediate filaments. In the pancreas of littermate control mice, only normal ducts stained positive for the duct cell-specific cytokeratin 19 (C). (D) An area is shown exhibiting cytokeratin 19 staining in duct-like structures (arrows) and in individual interstitial cells (arrowheads). (E) Panel displays transitional acinar units composed of acinar cells without (arrowheads) and of ductal cells with (arrows) the typical cytokeratin 19 staining. (F) Panel shows an area of the pancreas of a 50-week-old transgenic mouse composed of multiple cytokeratin 19-positive, fully differentiated duct-like structures. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 7 Immunohistochemical characterization of interstitial cells in the pancreas of transgenic mice. Immunostaining of serial sections from a 21-week-old KOC-transgenic mouse from line IX for endocrine lineage markers glucagon (A), insulin (B), Pax6 (C), and NkX 2.2 (D). Note staining of subpopulations of interstitial cells highlighted by arrows in each serial section, whereas no specific signal is detected in acinar cells. Inflammatory cells were only seen in older KOC-transgenic mice as exemplified for B lymphocytes stained with an anti-CD45 antibody (E) and for macrophages stained with an anti-Mac-3 antibody (F) in a 50-week-old mouse. (E) Arrows highlight CD45-positive B-cells, whereas arrowheads in F show Mac-3 positive cells. (G) Arrows denote an individual lobule staining positive for MMP-7 in the transgenic pancreas (100-day-old mouse), whereas MMP-7 immunoreactivity was homogeneously low in the exocrine pancreas of littermate controls (H). (I) Ductal clusters (arrows) in a 21-week-old transgenic mouse stained with an anti-TGF-α antibody. (J) Panel shows the littermate control pancreas stained for TGF-α with weak immunoreactivity in ductal cells. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 8 Development of mild fibrosis in the pancreas of KOC-transgenic mice. Collagen staining of pancreatic sections of a littermate control mouse (A) and a KOC-transgenic mouse (B), each 3 months of age. Asterisks indicate periacinar and interstitial collagen deposition in B. Fibronectin staining (C and D) of pancreatic sections of the same littermate control (C) and KOC-transgenic mouse (D) shown in A and B. Arrows highlight fibronectin deposition in D. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 9 Proliferation and apoptosis in the transgenic pancreas. PCNA immunostaining in the pancreas of a littermate control (A) and a KOC-transgenic mouse (B), each 13 weeks of age. The arrows indicate PCNA-positive acinar cell nuclei surrounding areas with interstitial cells and duct-like structures. Note the lack of PCNA-positive cell nuclei in interstitial cells and duct-like structures (asterisk). (C and D) Panels depict double immunohistochemical staining for Ki-67 (red nuclei, arrowheads in D) and KOC (brown, in D) of the transgenic (D) and control (C) pancreas. Proliferating acinar cells in the transgenic pancreas express the KOC protein (D). (E and F) Panels show the results of a TUNEL assay done on pancreatic sections of a KOC-transgenic mouse (F) and a littermate control (E). Arrows depict apoptotic cell bodies, which are predominantly found in interstitial cells. (G) Panel shows the results of a RT-PCR analysis for the cyclin D1 gene in the pancreas of KOC-transgenic mice and littermate controls between 10 and 20 weeks of age. Probes were size fractionated on a 1.5% agarose gel, blotted onto nylon membranes, and probed with a 32P labeled cyclin D1 probe. The lower panel shows the β-actin control PCR. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)

Figure 10 Expression of NGF-α and GEM in the pancreas of transgenic mice and littermate controls. (A) Reverse images of ethidium bromide stained agarose gels used to size fractionate RT-PCR reactions obtained with primers specific for the kir/GEM and NGF-α transcripts using cDNA from the pancreas of KOC-transgenic mice (line IX) and littermate controls each between 10 and 20 weeks of age as templates. The β-actin RT-PCR was done to control for the quantity of RNA used as template. (B and C) Panels depict immunostaining for NGF in littermate control (B) and transgenic pancreas (C). Arrowheads denote ductal clusters positive for NGF expression. (D and E) Panels show expression of TRK-A in the control (D) and the transgenic pancreas (E). (E) Arrowheads denote enhanced TRK-A immunoreactivity of ductal clusters, whereas asterisks are positioned over islet cells in D. Gastroenterology 2003 124, 1901-1914DOI: (10.1016/S0016-5085(03)00402-5)