1. Volume 139, Issue 2, Pages 609-619.e6 (August 2010)
Regulator of Calcineurin 1 Controls Growth Plasticity of Adult Pancreas 
Grzegorz T. Gurda, Stephen J. Crozier, Baoan Ji, Stephen A. Ernst, Craig D. Logsdon, Beverly A. Rothermel, John A. Williams 
Gastroenterology 
Volume 139, Issue 2, Pages e6 (August 2010)
DOI: /j.gastro
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2. Figure 1
Characterization of PI-induced and CN-dependent genes in pancreatic growth. (A) Top: experimental design of the microarray experiments. Arrows show biologically meaningful comparisons: genes altered with PI feeding (red) and inhibited by FK506 (blue). Bottom: PI-feeding significantly (>3-fold, q ≤ 0.08) increased the expression of 81 genes, whereas FK506 significantly (>70%, q ≤ 0.08) blocked 38 genes. (B) RNA samples were obtained from pancreases of 4 sets (n = 3–8/group) of mice: (1) control mice fasted overnight; (2) mice fasted overnight and refed PI-chow for 2 hours; (3) same as previous, except pretreated twice daily with 3 mg/kg of FK506; and (4) CCK-deficient mice fasted overnight and refed PI. Bars represent mean ± standard error of mean expression by qPCR relative to fasting controls; #P < .05 and ##P < .01 vs 2-hour PI (black bars). (C) Network association of molecular concepts for the 38 CN-dependent and 81 PI-induced genes, represented as 2 light green nodes. Each node in the network stands for a molecular concept or a set of biologically related genes, color coded in the legend. The size of the node is proportional to the number of genes, and connections signify statistically significant enrichment (P < .005). Links to concepts related to growth/differentiation are shown in blue.
Gastroenterology  , e6DOI: ( /j.gastro )
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3. Figure 2
Delineating NFAT-regulated genes. (A) Proximal promoters (MatInspector) or regulatory regions (rVISTA) for mouse, rat, and human orthologs of 38 CN-dependent genes were obtained and aligned. Of note, MatInspector did not automatically retrieve the proximal promoter of the exon4 transcript of RCAN1. With manual retrieval, RCAN1 was a high confidence identification. (B) The promoters of 21 experimentally established, NFAT-regulated genes (gold standard) and a set of 21 random genes analyzed by MatInspector were evaluated using the same methods and criteria as A. (C) Chromatin immunoprecipitation (ChIP) assay of A23187-stimulated binding of NFATc1 to promoters of 5 CN-dependent genes computationally predicted to be regulated by NFAT. Studies were carried out in cells. Pol II binding was used as a readout of transcriptionally active genes, and a primer set for EF1α was used as a control. (D) ChIP assay of CCK-stimulated binding of NFATc1 to the promoters of the same 5 CN-dependent genes as in C carried out in primary isolated mouse acinar cells.
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4. Figure 3
PI-containing chow increases RCAN1 expression. Mice were were fasted overnight, refed PI-containing chow for 1–8 hours, and killed. (A) Messenger RNA expression of RCAN1, but not RCAN2 or RCAN3, significantly (**P ≤ .01, n = 5 or 6) increased with PI feeding. 18S RNA was used as a control. (B) Exon4 form of RCAN1 was significantly induced (*P < .05 or **P < .01) by PI feeding with a peak at 2 hours. Representative Western blot for 26-kilodalton form of RCAN1 and cyclophilin A (CycloA) loading control are shown above a bar graph quantifying the results of 3 independent experiments from 5 or 6 mice per time point. (C) CCK dose dependently activates Rcan1-luciferase reporter in a CN-dependent fashion in NIH3T3 CCK-AR cells (*P ≤ .05, n = 5 or 6). (D) Representative Western blot for the 24-kilodalton RCAN1 form and cyclophilin A (loading control) of pancreases of wild-type mice fasted, free fed, fasted overnight and refed either standard or PI-containing diet; CCK-deficient mice were fasted and refed PI-containing diet.
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5. Figure 4
RCAN1 overexpression functionally inhibits CN-NFAT pathway. (A) Dose-response activation of NFAT-luciferase reporter by CCK in isolated acini incubated with either RCAN1 or β-gal/GFP adenovirus (n = 3, *P < .05 vs time zero; #P < .05 vs control virus). (B) Dose-response activation of NFAT-luciferase reporter in fresh isolated acini from either TAM-injected Ela-Cre(ER)/Rcan1 or wild-type littermates (n = 3, *P < .05 vs time zero; #P < .05 vs controls) (C) RNA samples from fasted controls as well as Ela-Cre(ER)/RCAN1 or wild-type littermates fasted and fed PI-containing chow for 2 hours were used for reverse-transcription quantitative polymerase chain reaction. All mice were injected with TAM. Shown are the mean ± standard error of mean induction relative to fasting controls for a set of 8 genes (n = 4–7; #P < .05 vs 2-hour PI wild type).
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6. Figure 5
RCAN1 transgene blocks acinar cell proliferation in vivo. (A) TAM-injected wild-type and Ela-Cre(ER)/RCAN1 mice were fasted and refed standard or PI-containing chow for 2 days, treated with BrdU, and killed 2 hours later. Representative slides of BrdU immunostaining in red, 4′,6-diamidino-2-phenylindole (DAPI) in blue, and overlay in pink (white arrows). The results from experiments on 3–5 mice are quantified as percentage of cells with BrdU staining over total DAPI-staining. **P < .01 compared with standard chow; #P < .05 vs wild-type fed PI-containing chow. (B) BrdU (pink nuclei/white arrows) incorporates into morphologically mature, amylase expressing (green) acinar cells.
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7. Figure 6
Acinar-specific overexpression of RCAN1 blocks pancreatic growth. (A) TAM-injected wild-type and Ela-Cre(ER)/RCAN1 mice were fed standard or PI-containing chow for 10 days. Representative photographs of the pancreas for age and body weight (BW)-matched mice. Squares are 6 × 6 mm. (B) Relative pancreatic weights of wild-type and Ela-Cre(ER)/RCAN1 mice fed a standard or PI-containing chow for 0–10 days. All animals were preinjected with TAM; n = 3–5, **P < .01 vs day zero; ##P < .01 vs same day wild-type fed PI-containing chow. (C, D, and E) Changes in relative pancreatic weight (C), protein (D), and DNA content (E) at 7 days; TAM-injected wild-type, Ela-Cre(ER)/RCAN1 and single transgenic littermates (n = 4–6) were fed with standard or PI-containing chow (**P < .01 vs wild type; ##P < .01 vs wild-type PI-containing chow).
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8. Supplementary Figure 1
Broad functional gene ontology characterization of 38 CN-sensitive and 81 PI-induced genes. (A) GOTree machine software-generated table of statistically overrepresented gene ontology categories among CN-sensitive genes, grouped into Biological Process and Molecular Function. Growth, cell-cell communication, and negative feedback regulation were among the most notable overrepresented categories. (B) A diacryllic diagram of the hierarchical parent-child relationship of statistically overrepresented GO categories. Functional categories enriched among PI-induced genes are outlined in red, whereas categories enriched among FK560-sensitive (CN-dependent) genes are outlined in blue.
Gastroenterology  , e6DOI: ( /j.gastro )
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9. Supplementary Figure 2
Computational analysis of NFAT/AP1 regulation, carried out as described in Figure 2. (A) 1300bp promoters (MatInspector) or regulatory regions (rVISTA) for mouse, rat, and human orthologs of 38 CN-dependent genes were obtained from sequence databases and aligned, as described in methods. Genes with at least one NFAT/AP1 sites (spaced within 50bp) conserved across three species were termed NFAT/AP1-regulated. This group included a subset of genes with several conserved NFAT/AP1 modules, which we termed as higher confidence identifications. Genes which did not contain any sites with 3-way conservation or could not be aligned, were termed unlikely and undetermined, respectively. (B) The promoters of 21 genes known to be biologically regulated by NFATs (gold standard these are the same genes as Figure 2; they are largely inflammatory and the majority are known to be regulated not only by NFAT but also NFAT/AP1 complex) and a set of 21 random genes were analyzed by MatInspector using the same methods and criteria as in Figure 1. CN-dependent (FK506 inhibited) genes had fewer conserved NFAT/AP1 modules than the gold standard set, but considerably more than the random set.
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10. Supplementary Figure 3
(A) Immunohistochemistry for Cre (red) showing positive staining in exocrine tissues, but not islets of Elastase-Cre(ER) transgenics treated with TAM. (B) Representative Western blot for Cre of liver, pancreas, and salivary glands of elastase-Cre(ER) transgenic mice treated with TAM. (C) Immunohistochemistry for β-gal (red) showing positive staining in exocrine tissues, but not islets of double transgenic elastase-Cre(ER) × Rosa26-LacZ mice, treated with TAM (D) PCR carried out using mRNA obtained from pancreases of TAM-treated elastate-Cre. (ER)/Rcan1 double transgenic mice as well as single transgenic elastate-Cre (ER) only or Rcan1 only controls, utilizing primers for FLAG-Rcan1 transgene, endogenous Rcan1 and Cyclophilin A (CycloA) as reaction control. (E) Representative Western blot for Rcan1 using whole tissue lysates of pancreas and liver in vehicle (–) or TAM (+) treated, fasted, elastase-Cre(ER)/Rcan1 double transgenic mice (TG) or nontransgenic, wild type (wt) littermates.
Gastroenterology  , e6DOI: ( /j.gastro )
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11. Supplementary Figure 4
Isolated acini treated with adenovirus(es) and CCK as indicated in caption. All treatments were carried out for 30 minutes with near-maximal result at 5 minutes shown.
Gastroenterology  , e6DOI: ( /j.gastro )
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