Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Gastroenterology. Author manuscript; available in PMC 2011 August 1.
Published in final edited form as:
PMCID: PMC2929702

Regulator of calcineurin 1 (Rcan1) controls growth plasticity of adult pancreas


Background & Aims

Growth of exocrine pancreas is regulated by GI hormones, notably cholecystokinin (CCK). CCK-driven pancreatic growth requires calcineurin (CN), which activates NFATs (Nuclear Factor of Activated T-cells), but the genetic underpinnings and feedback mechanisms that regulate this response are not known.


Pancreatic growth was stimulated by protease inhibitor (PI)-containing chow, which induces secretion of endogenous CCK. Expression profiling of PI-stimulation was performed on Affymetrix 430A chips and CN was inhibited via FK506. Exocrine pancreas-specific overexpression of CN inhibitor Rcan1 (Regulator of Calcineurin 1) was achieved by breeding elastase-Cre(ER) transgenics (TG) with ‘flox-on’ RCAN1 mice.


CN inhibitor FK506 blocked expression of 38 genes, as confirmed by qPCR. The CN-dependent genes were linked to growth-related processes, while their promoters were enriched in NFAT and NFAT/AP1 sites. Multiple NFAT targets, including Rcan1, Rgs2, HB-EGF, Lif and Gem, were validated by ChIP. One of these, a CN feedback inhibitor Rcan1, was induced >50 fold during 1–8h course of pancreatic growth and strongly inhibited (>99%) by FK506. To examine its role in pancreatic growth, we overexpressed Rcan1 in an inducible, acinar-specific fashion. Rcan1 overexpression inhibited CN-NFAT signaling, as shown using an NFAT-luciferase reporter and qPCR. Most importantly, the increase in exocrine pancreas size, protein/DNA content and acinar proliferation were all blocked in Rcan1 overexpressing mice.


We profile adaptive pancreatic growth, identify Rcan1 as an important new feedback regulator and firmly establish that CN-NFAT signaling is required for this response.

Keywords: Rcan1, NFAT, calcineurin, pancreas, growth


An adequate supply of pancreatic digestive enzymes is critical for digestion and absorption of food. To maintain its function in response to changes in diet or following injury1, the normally quiescent exocrine tissue can undergo a rapid expansion. The gastrointestinal hormone cholecystokinin (CCK) is a key regulator of adult pancreatic growth and is necessary and sufficient for this response2. Administration of exogenous CCK leads to both increased in pancreatic size in vivo and robust proliferation of acinar cells in culture. An alternative model used in this study involves increased endogenous secretion of CCK induced by feeding a chow containing a protease inhibitor (PI)3. CCK signals via its G-protein coupled receptor to activate MAPK, mTOR and calcineurin. How these early signaling events translate into growth, however, is largely unknown.

Calcium/calmodulin-dependent phosphatase calcineurin (CN or PP2B) has been shown to be an important mediator of adaptive responses such as T-cell proliferation, cardiac hypertrophy, osteogenesis and lung maturation4. CN activates several substrates, including a family of four Nuclear Factor of Activated T-cells (NFATc1-c4) transcription factors. In a basal state NFATs are heavily phosphorylated and sequestered in the cytoplasm, but upon stimulation rapidly shuttle into the nucleus due to CN-mediated dephosphorylation. The role of CN-NFAT axis in growth has thus far been addressed primarily in the heart. Expression of a constitutively active CN or NFATc4 is sufficient to produce cardiac hypertrophy5 and eventually leads to heart failure6. Using CN inhibitors CsA and FK506, our laboratory has shown that CN may also be important for CCK-mediated growth of the exocrine pancreas7. More recently, we have also shown that CCK activates NFATs both in vitro and in vivo, in a CN-dependent fashion8. Nonetheless, how activation of CN-NFAT leads to growth and the feedback regulation of this adaptive response are still poorly understood.

NFATs are well-characterized transcriptional regulators of inflammation. CN-NFAT pathway is also involved in a range of other responses, but relatively few of these NFAT-regulated genes have been identified. In the initial part of the study we carried out a genome-wide transcriptional profiling of a 2h point of pancreatic growth. We identified and analyzed the transcriptional regulation of 38 genes both increased in response to PI-feeding and blocked by the CN inhibitor FK506. Our efforts then focused on one of the 38 target genes, Regulator of Calcineurin 1 (Rcan1/Dscr1/Mcip1), an endogenous inhibitor of CN activity with several alternative transcripts. In the heart, exon4 transcript is controlled by CN-NFAT whereas exon1 transcript is expressed independent of this pathway9. Here, we tested the role of Rcan1 as a feedback inhibitor of the adaptive, and in contrast to muscle largely hyperplastic, growth of exocrine pancreas. We demonstrate a sustained, CN-dependent increase in Rcan1 throughout early course of pancreatic growth. We show that Rcan1 inhibits CCK-stimulated CN-NFAT signaling. Most significantly, we note that with tissue specific, inducible expression of Rcan1, CCK-induced acinar cell proliferation and pancreatic growth are nearly abolished. Our work shows that Rcan1 is an important new regulator of pancreatic growth and CN-NFAT signaling is required for this response. These results represent the first molecularly-defined feedback mechanism that delimits hormonally-mediated growth of the exocrine pancreas.



The protease inhibitor (PI) camostat was provided by Ono Pharmaceuticals (Osaka, Japan). TaqMan reverse transcription and Expand PCR kits were purchased from Roche (Basel, Switzerland). Antibodies and all other reagents are listed in supplementary materials.

Animals and Treatment

Animal experiments, including PI-feeding and FK506 injections, are outlined in supplementary materials or performed as previously described8. To overexpress Rcan1 in pancreatic acini, we bred FLAG-Rcan1 [Rcan1] transgenic mice10 with Elastase(ER)-Cre [Ela-Cre] mice11, which were generated as previously described. Rosa26-LacZ reporter mice were from Jackson Laboratories. All strains of mice were genotyped by PCR with primers in supplementary Table II (C). β-gal expression was examined as previously described12.

Cell Culture

NIH 3T3 cells stably transfected with CCK-AR were described previously13, HEK-293 and 266-6 cells were obtained from ATCC (Rockville, MD), and all cells cultured as instructed. Pancreatic acini were isolated from 6–8 week old male mice as previously described8.

RNA isolation, bioinformatics and microarray analysis

RNA isolation, cDNA synthesis and PCR followed previously described procedures14. cDNA synthesis, hybridization and fluorescence signal optimization on Affymetrix 430A gene chips was performed at University of Michigan microarray core according to manufacturer’s protocols. Detailed methods and computational analysis are outlined in supplementary materials.

Quantitative, real-time RT-PCR and Western blotting

1 μg of total RNA was reverse transcribed using TaqMAN reagents. Real-time PCR was performed using Bio-Rad I-Cycler IQ, as previously described14. The primers were designed with Primer315 based on gene sequences retrieved from GenBank (supplementary Table IIA). Western blots were performed as previously described 8.


ChIP in 266-6 cells and isolated pancreatic acini was carried out using ChIP-IT express (Active Motif). The cells were left untreated or stimulated with CCK or A23187 and fixed in either 1% (266-6 cells) or 0.75% formaldehyde (isolated acini) for 10 min at room temperature. Glycine solution was applied, cells were harvested, lysted and sonicated. 25μL of each chromatin sample was precleared, blocked with protein G agarose beads and incubated with appropriate antibody. The complex was washed, eluted and cross-links reversed by 4h incubation at 65°C. The samples were then treated with proteinase K for 2h, DNA was purifed and used for PCR. The primers used in PCR amplification are in Supplementary Table IIB.

Quantification of Pancreatic Growth

Determination of body weight, protein, DNA concentration and BrdU-based acinar cell proliferation were performed as previously described16. DAPI-stained nuclei were quantified using CellProfiler software per authors’ instructions17, whereas BrdU-positive cells were counted by hand. The percentage of BrdU labeled cells was a ratio of BrdU-positive/DAPI-stained nuclei, averaged for 3–5 fields per animal.

Luciferase Reporter Assay

NFAT-luciferase adenovirus6 was a gift from Dr. JMolkentin (University of Cincinnati). High concentrations (≥1011 PFU) of NFAT-luciferase, AdRcan1 and control β-gal/EGFP adenovirus were produced as described8. For in vitro Rcan1 expression, isolated acini were pre-incubated with AdRcan1, then with 5×106 PFU of NFAT-luc adenovirus for 1h and finally with CCK for 5½h. For in vivo Rcan1 overexpression, isolated acini from TAM-injected Ela-Cre(ER)/Rcan1 or littermate controls were incubated with 5×106 PFU of NFAT-luc for 30min and then treated for 5½h. Rcan1-luciferase construct was a gift of Dr. CGlembotski (San Diego State) and transfected into AR42J NIH 3T3 cells with Superfect reagent (Invitrogen). For all luciferase assays, cells were washed with PBS, lysed in reporter lysis buffer (Promega, Madison, WI) and measured in a Berthold luminometer. Each condition was assayed based on >3 separate isolations with 3 replicates per preparation, normalized to protein concentration in the lysate.


Comparisons of multiple data sets were analyzed by one-way ANOVA followed by Dunnett’s or Bonferroni’s post-test carried out on Graphpad Prism and expressed as mean ± SE. Pair-wise analysis was performed using a two-tailed Student’s t-test. Large microarray data sets were initially compared by ANOVA with p-values of the groups subject to F-test. False discovery rate and associated q-values were then derived as described18.


RNA microarray analysis of PI-induced and CN-dependent genes

We performed genome-wide expression profiling of the 2h time point of CCK-driven pancreatic growth, focused on CN-dependent genes in particular. Pancreatic RNA was harvested from mice fasted overnight or fasted and re-fed PI-containing chow for 2h and injected with either 3mg/kg of FK506 or vehicle, then hybridized with Affymetrix 430A genechips (4 mice/group, 16 chips total), as outlined in Figure 1A, top. PI-feeding led to a significant (>3 fold, q≤0.08) increase in expression of 81 genes and decrease in expression of 2 genes, whereas FK506 significantly (>70%, q≤0.08) blocked PI-induced expression of 38 genes. The top 12 of the 38 genes, which we termed calcineurin (CN)-dependent, were inhibited >90% by FK506 and are listed in Table I, whereas the remaining 26 and the entire microarray are available in supplementary materials. Underlining the importance of CN for pancreatic growth, CN-dependent genes comprised a third of those induced by PI-feeding (Figure 1A, bottom). A set of six genes induced by PI-feeding and strongly inhibited by FK506 were independently examined by qPCR (Figure 1B). The results closely correlated with changes seen by microarray. As an additional control, we examined CCK-deficient mice. These animals do not exhibit significant pancreatic growth in response to PI and their qPCR expression profile was nearly identical to FK506-treated mice (Figure 1B). Most CN-dependent genes appeared to function in growth, cell-cell communication, transcriptional regulation or negative feedback (Suppl. Table I); statistical analysis of gene ontology (GEO) annotations confirmed these observations (Suppl. Fig. 1). A network association analysis of the 38 CN-dependent and 81 PI-induced genes, showed significant overlap with concepts related to growth, differentiation and stress response (Figure 1C, thick lines). Interestingly, NFAT and AP-1, two transcription factors previously shown to be activated by CCK, were also linked to our two sets of genes.

Figure 1
Characterization of PI-induced and CN-dependent genes in pancreatic growth
Table I
FK506 inhibits numerous genes induced by PI-feeding

NFAT regulation of CN-dependent genes

Based above findings, we next assessed the potential role of NFATs as transcriptional regulators of CN-dependent genes in pancreatic growth. We retrieved non-coding regulatory sequences of these 38 genes along with their rat and human orthologs, performed exon-anchored alignments and searched for conserved NFAT binding sites. Any gene with at least one NFAT site conserved across three species was termed as ‘NFAT-regulated’ and a subset within this group with several closely-spaced sites as ‘high-confidence’. Genes which did not contain any sites with 3-way conservation or those that could not be aligned were termed ‘unlikely’ or ‘undetermined’, respectively. Independent analysis using two computational tools, MatInspector and rVISTA, produced largely overlapping results (Figure 2A). Subject to the same analysis, nearly all of experimentally established, immune system-related ‘gold standard’ genes were found to be NFAT-regulated, whereas only a few and no high confidence identifications were found among genes selected at random (Figure 2B). Like many other transcription factors, NFATs are known to control gene expression as parts of larger regulatory ‘modules.’ Using the frameworker tool in Genomatix software, we found several statistically overrepresented modules connected to CN or CN-NFAT signaling (Suppl. Figure 2A). The well-known NFAT/AP1 transcription factor complex was then analyzed analogously to NFATs, identifying several CN-dependent genes that contained this conserved promoter module (Suppl. Figure 2B and C). Computational analysis is a good point of departure, though results may not always reflect the biology. To experimentally test our predictions, we examined several high confidence NFAT-regulated genes with previously known functions relevant to growth via chromatin immunoprecipitation (ChIP) assay. Treatment of the mouse pancreatic 266-6 acinar cells with calcium ionophore A23187 and primary isolated pancreatic acini with CCK both stimulated NFATc1 binding to promoter regions of five CN-dependent genes (Figure 2C and D).

Figure 2
Delineating NFAT-regulated genes

Rcan1 is strongly induced in the course of pancreatic growth

As the gene most sensitive to FK506 and among the top three induced by feeding PI-containing chow (Table I), Rcan1 expression appeared to be tightly linked to pancreatic growth. To examine changes in Rcan1 expression throughout the early time course of pancreatic growth, we obtained RNA and protein from mice either fasted overnight or fasted and refed PI-containing chow for 1–8h. PI-feeding produced a sustained, >50 fold increase in Rcan1 mRNA (Figure 3A), while the expression of Rcan2 and Rcan3, members of the same family independent of CN-NFAT pathway19, was not altered (Figure 3A). Parallel changes took place for the 26kDa exon4 RCAN1 protein (Figure 3B), whereas levels of thelarger exon1 form were not altered (data not shown). CCK also dose-dependently activated Rcan1 promoter-driven luciferase reporter, and this effect was shown to be CN-dependent as it was blocked by FK506 (Figure 3C). As an additional control, we examined the effect of feeding. In fasted mice circulating CCK is typically at 0.5–1 pM, in animals fed standard diet it increases to 3–5 pM, whereas in those fed PI-containing chow to ~15 pM16. No appreciable increase in Rcan1 expression was noted among feeding controls or CCK-deficient mice (Figure 3D).

Fiqure 3
PI-containing chow increases RCAN1 expression

Inducible, pancreatic acinar-cell specific Rcan1 overexpression in vivo

The sustained, CCK-dependent increase in expression of Rcan1 prompted us to test if this endogenous regulator of CN can function as a feedback inhibitor of CCK-driven pancreatic growth. To express Rcan1 in an inducible, acinar cell-specific fashion, we crossed mice expressing tamoxifen (TAM)-inducible, elastase promoter-driven Cre recombinase [Ela-Cre(ER)] with ‘flox-ON’ FLAG-Rcan1 mice. We examined Cre protein using immunohistochemistry and western blots. Cre was expressed in the exocrine pancreas of TAM-injected Ela-Cre(ER) mice, but not in islets or ducts (Suppl Figure 3A) and in the pancreas but not the salivary glands or liver (Suppl Figure 3B). To verify tissue specific activation of Ela-Cre(ER), we crossed this strain with Rosa26-LacZ reporter mice. Immunohistochemistry for β-gal showed Cre recombinase was activated in >80% of pancreatic acini, but not in islets, blood vessels or ducts (Suppl Figure 3C) of TAM-injected Ela-Cre(ER)/LacZ mice. No β-gal staining was noted in salivary acinar cells of double transgenic or the pancreas and salivary gland of single transgenic controls (data not shown). To assess acinar-specific expression of Rcan1 transgene, we prepared RNA from acini of TAM-injected Ela-Cre(ER)/Rcan1 mice or single transgenics. FLAG-Rcan1 transgene was expressed in acini of double transgenic mice independent of CCK, but not in single transgenic littermates (Suppl Figure 3D). By contrast, acini from uninjected animals showed equal, CCK-inducible expression of endogenous Rcan1 message for all three groups. RCAN1 protein was also strongly expressed in TAM-injected Ela-Cre(ER)/Rcan1 mice (Suppl Figure 3E, lane 2), but not in TAM-injected wild type animals and weakly for vehicle-injected double transgenics; no transgene expression was seen in the liver (Suppl Figure 3E, lanes 4–6). Lastly, to test dose-dependent effects of Rcan1 overexpression, we utilized a second, independently generated line of ‘Flox-ON’ Rcan1 animals10, called ‘Rcan1(a)’ mice. The double transgenic offspring of the cross between Ela-Cre(ER) and Rcan1(a) mice showed ~8 fold lower transgene expression (data not shown).

Rcan1 is an inhibitor of CCK-driven pancreatic CN-NFAT signaling

Next, we tested if Rcan1 overexpression inhibits CN-NFAT signaling in CCK-mediated pancreatic growth. As a readout of CN-NFAT activation we used a previously characterized NFAT-luciferase (NFAT-luc) reporter8. CCK induced a robust, dose-dependent activation of this reporter in primary isolated pancreatic acini incubated with a control β-gal/GFP adenovirus, but not those incubated with Rcan1 virus (Figure 4A). Expression of Rcan1 also blocked the translocation of NFATc1GFP to the nucleus (Suppl Figure 4). Likewise, CCK-induced, dose-dependent response for NFAT-luc reporter in isolated acini of wild type mice was largely abrogated in acini from TAM-injected Ela-Cre(ER)/Rcan1 littermates (Figure 4B). Thus, in vivo expression of the Rcan1 led to functional inhibition of CN-NFAT signaling on par with that of adenovirally-mediated overexpression. Acinar-specific overexpression of Rcan1 in vivo, however, did not alter CCK-dependent phosphorylation of ribosomal protein S6 and 4E-BP1, two well-known downstream targets of mTOR, a key signal in PI-induced pancreatic growth (results not shown). Interestingly, Rcan1 also inhibited the PI-induced activation of many, but not all of the 38 CN-dependent, FK506 inhibited genes (Figure 4C, compare with Table I and Figure 1B). Upon closer examination, the genes significantly inhibited by Rcan1 in PI-feeding aligned with our computational predictions of NFAT-regulated genes (Figure 2A), implying NFAT-specific skewing of the inhibitory effects of Rcan1.

Figure 4
RCAN1 overexpression functionally inhibits CN-NFAT pathway

Rcan1 overexpression blocks pancreatic CCK-driven acinar proliferation

Rcan1 transgene clearly inhibits CN-NFAT signaling hence we asked if acinar cell-specific Rcan1 overexpression was sufficient to inhibit CCK-driven proliferation of pancreatic acinar cells. TAM-injected wild type and Ela-Cre(ER)/Rcan1 mice were fasted and refed with either control or PI-containing chow for 2 days and treated with BrdU. In both Ela-Cre(ER)/Rcan1 and controls fed normal diet, less than 0.5% of cells incorporated BrdU in their nuclei (Figure 5A). In wild-type mice fed a PI-containing diet there was a significant increase in BrdU incorporation and this increase was largely blocked in Ela-Cre(ER)/Rcan1 mice (Figure 6A). In addition, BrdU incorporated into morphologically identifiable, amylase-staining acinar cells (Figure 5B).

Figure 5
RCAN1 transgene blocks acinar cell proliferation in vivo. (A)
Figure 6
Acinar-specific overexpression of RCAN1 blocks pancreatic growth

Rcan1 overexpression blocks CCK-mediated pancreatic growth

Lastly, we tested if acinar cell-specific overexpression of Rcan1 was also sufficient to inhibit pancreatic growth in vivo. We compared TAM-injected Ela-Cre(ER)/Rcan1 and controls fed either standard or PI-containing chow for up to 10 days. In wild type animals, feeding PI-containing diet led to a large increase in the gross size of the pancreas compared to littermates on a standard chow (Figure 6A). Pancreatic weight relative to BW more than doubled, with an increase that peaked at 7 days (Figure 6B). CCK-driven pancreatic growth induced by PI, however, was completely abolished in Ela-Cre(ER)/Rcan1 mice throughout the timecourse (Figure 6). A detailed study of 4–6 mice/group at the 7 day point confirmed these findings, with doubling of relative pancreatic weight in TAM-injected wild type and single transgenics fed PI-containing chow, but almost no growth in Ela-Cre(ER)/Rcan1 littermates (Figure 6C). Interestingly, the Ela-Cre(ER)/Rcan1(a) line which expresses lower levels of Rcan1 showed an intermediate phenotype; increased growth compared to control mice fed standard chow, but a decrease compared to controls fed PI-containing chow (Figure 6C). Pancreatic protein and DNA content measured after 7 days of standard or PI-containing diet showed changes that paralleled growth (Figure 6C and D). These last sets of data point to the hyperplastic nature of pancreatic growth and agrees both with our BrdU incorporation data and previous work7.


Physiological growth of the pancreas takes place in response to high protein diet, hyperphagia, pregnancy and lactation20. The molecular mechanisms that govern this adaptive response, however, are poorly understood. Here, we examined the expression profile of CCK-mediated pancreatic growth. We focused on CN-NFAT signaling axis, identifying several novel growth-related, NFAT-regulated genes. We also demonstrate that one of these genes, Rcan1, functions as a CN/NFAT-dependent feedback inhibitor that controls growth plasticity of adult pancreas.

The results from our expression profile both validate and extend past work. Sets of genes significantly altered in our expression profile significantly overlap with molecular concepts linked to pathways previously shown to be activated CCK, including MAPK-AP114, Rho-SRF21 and NF-κB22. Aside from CN-NFAT-Rcan1, our work also points to JAK-STAT and its inhibitors Socs2 and Socs3 as yet another signal regulating this response (Suppl. Table I and unpublished data). Several of our 38 CN-dependent genes have been tied to regulation of growth, differentiation and metabolism. FGF21 has been shown to be important for hepatic regeneration and β-cell survival23, HB-EGF in β-cell transdifferentiation and pancreatic cancer24, whereas Socs3 for proliferation and angiogenisis in the liver25. We posit that these and other membrane-bound paracrine/autocrine mediators alter local microenvironment, drive hyperplasia or differentiation and thereby direct pancreatic growth. Analogous mechanisms have already been shown in the liver following a partial resection26 and the pancreas following pancreatitis27. Broadly, our results argue that CCK-mediated pancreatic growth has much in common with these regenerative responses.

NFATs are best known as transcriptional mediators of inflammation. Their role as regulators of growth or tissue homeostasis in general, however, is not as clear. Our work is one of the most detailed analyses of genome-wide expression that underlies an adaptive response linked to CN-NFAT signals. We predict a sizable new set of NFAT-regulated genes related to pancreatic growth and show that NFATc1 binds to promoters of selected high quality identifications. The time frame of increased NFATc1 binding by ChIP corresponds to CCK-stimulated nuclear shuttling of NFATc1-GFP in acini8 and the kinetics of NFAT translocation in other cell types. Lastly, NFATs have intrinsically low affinity for DNA and therefore function within complexes or modules. Known partners of NFATs include GATA-4 in the heart, C/EBP in lung, MEF2 in muscle and AP-1/Foxp3 in immune cells28. Complex-dependent effects dictate timing, affinity and outcome of NFAT binding in a tissue or stimulus-dependent manner. Here, we identify several enriched transcriptional modules among CN-dependent genes, including the well-known synergistic NFAT/AP-1 complex29. As a site of convergence, transcriptional complexes may explain why inhibition of a single pathway can be sufficient to disrupt an entire physiological response.

Rcan1 expression increased throughout the early course of pancreatic growth and endogenous Rcan1 strongly inhibited CCK-induced CN-NFAT signaling. This led us to formulate the central paradigm of this study: CCK-mediated activation of CN leads to nuclear translocation and activation of NFATs, NFAT-dependent induction of Rcan1 and finally RCAN1-mediated inhibition of CN. The exact means whereby CN-NFAT-RCAN1 axis constrains growth, however, remains unknown. One rationale is that RCAN1 blocks proliferation; an alternative may be that RCAN1 limits angiogenesis. NFATc1 has been shown to regulate vascular endothelial growth factor (VEGF) expression30 and mice that overexpress Rcan1 showed decreased tumor formation due attenuated angiogenesis31. Rcan1 is also expressed at a higher level in Down Syndrome (Rcan1 is also known as Down Syndrome Critical Region 1 or DSCR1) and may decrease susceptibility to solid tumors in these individuals. CCK also increased expression and NFATc1 binding to the promoter of Leukemia Inhibitory Protein (Lif). Lif has recently been shown to regulate microvessel density and VEGF expression in retinopathy32. Direct or indirect role of Rcan1 in regulation of angiogenesis is certainly a promising area for future research.

Finally, our work here shows that transgenic overexpression of Rcan1 is sufficient to completely block pancreatic growth. The pre-emptive expression of Rcan1 clamps the CN-NFAT-Rcan1 signal in a permanent “off” position, independent of CCK. This tissue-specific genetic approach circumvents side effects of pharmacological agents like immune suppression, nephro and neurotoxicity, whereas inducible overexpression preempts possible developmental defects or compensatory changes. Rcan1 also inhibited many, but not all of the genes blocked by CN inhibitor FK506. Interestingly, Rcan1 appeared to significantly inhibit only CN-dependent genes which we also computationally predicted to be NFAT-regulated. NFAT-specific skewing of the inhibitory effect of Rcan1 on CN has been suggested elsewhere, but primarily based on biochemical evidence33.

Recent advances in manipulation of cell fate and plasticity of pancreatic tissue brought renewed promise of treatments for pancreatitis, pancreatic cancer and diabetes. Our work and the expression profiles of pancreatic development34, pancreatitis35 and pancreatic cancer36 are grounds for further integrative research. Relevantly, NFATc1 has been shown to be inappropriately activated in pancreatic cancer37. The role of Rcan1 has yet to be examined, but may well prove to be important based on significant downregulation of its message in at least two microarrays of human pancreatic cancer samples3839.

In summary, we examined the expression profile of an early point of CCK-mediated pancreatic growth. We computationally and experimentally examined several growth related genes, focusing on endogenous CN regulator Rcan1. Rcan1 showed a strong, sustained expression throughout the growth response and significantly inhibited CCK-simulated CN-NFAT signaling. Most notably, inducible, exocrine-specific expression of Rcan1 in vivo completely blocked acinar cell hyperplasia and the accompanying CCK-driven pancreatic growth. Our work identifies an important new feedback mechanism that delimits adaptive pancreatic growth and provides firm evidence that CN-NFAT axis is necessary for this hormonally-mediated, adaptive response.

Supplementary Material






Grant Support: The research was supported by NIH grants DK 59578 (JAW) and P30 DK-34933 (Michigan Gastrointestinal Peptide Center). GTG was supported by Systems and Integrative Biology Training Grant (T32 GM008322) and the Medical Scientist Training Program. Additional grant support included DK52067 (CDL), R21 DK068414 (BJ), DK-0077423 (SJC) and HL072016 (BAR). We also used the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center funded by NIH5P60 DK20572.

We thank Linda Samuelson (University of Michigan) for CCK-deficient mice, Bradley Nelson for help with immunohistochemistry and Scott Tomlins and the Arul Chinnayian laboratory (University of Michigan) for assistance with bioinformatics.


Financial Disclosure: None

No conflicts of interest exist

Role in manuscript: GTG = study design, data acquisition/analysis/interpretation, drafted and revised manuscript, intellectual content, statistical analysis; SJC = data acquisition, intellectual content, revised manuscript; BJ = intellectual content, revised manuscript; SAE = data analysis/interpretation, technical support, intellectual content; CDL = intellectual content, revised manuscript, obtained funding; BAR = study design, intellectual content, technical support, revised manuscript; JAW = study design, revised manuscript, intellectual content, obtained funding

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


1. Brannon PM. Adaptation of the exocrine pancreas to diet. Annu Rev Nutr. 1990;10:85–105. [PubMed]
2. Sato N, et al. Different effects of oral administration of synthetic trypsin inhibitor on the pancreas between cholecystokinin-A receptor gene knockout mice and wild type mice. Jpn J Pharmacol. 2002;89:290–295. [PubMed]
3. Tashiro M, Samuelson LC, Liddle RA, Williams JA. Calcineurin mediates pancreatic growth in protease inhibitor-treated mice. Am J Physiol Gastrointest Liver Physiol. 2004;286:G784–G790. [PubMed]
4. Wu H, Peisley A, Graef IA, Crabtree GR. NFAT signaling and the invention of vertebrates. Trends Cell Biol. 2007;17:251–260. [PubMed]
5. Molkentin JD, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998;93:215–228. [PMC free article] [PubMed]
6. Wilkins BJ, et al. Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy. Circ Res. 2004;94:110–118. [PubMed]
7. Tashiro M, Samuelson LC, Liddle RA, Williams JA. Calcineurin mediates pancreatic growth in protease inhibitor-treated mice. Am J Physiol Gastrointest Liver Physiol. 2004;286:G784–790. [PubMed]
8. Gurda GT, Guo L, Lee SH, Molkentin JD, Williams JA. Cholecystokinin Activates Pancreatic Calcineurin-NFAT Signaling In Vitro and In Vivo. Mol Biol Cell. 2008;19:198–206. [PMC free article] [PubMed]
9. Rothermel BA, Vega RB, Williams RS. The role of modulatory calcineurin-interacting proteins in calcineurin signaling. Trends Cardiovasc Med. 2003;13:15–21. [PubMed]
10. Oh M, et al. Calcineurin is necessary for the maintenance but not embryonic development of slow muscle fibers. Mol Cell Biol. 2005;25:6629–6638. [PMC free article] [PubMed]
11. Ji B, et al. Robust acinar cell transgene expression of CreErT via BAC recombineering. Genesis. 2008;46:390–395. [PMC free article] [PubMed]
12. Madison BB, et al. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J Biol Chem. 2002;277:33275–33283. [PubMed]
13. Le Page SL, Bi Y, Williams JA. CCK-A receptor activates RhoA through G alpha 12/13 in NIH3T3 cells. Am J Physiol Cell Physiol. 2003;285:C1197–C1206. [PubMed]
14. Guo L, et al. Induction of early response genes in trypsin inhibitor-induced pancreatic growth. Am J Physiol Gastrointest Liver Physiol. 2007;292:G667–677. [PubMed]
15. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–386. [PubMed]
16. Crozier SJ, et al. CCK-induced pancreatic growth is not limited by mitogenic capacity in mice. Am J Physiol Gastrointest Liver Physiol. 2008 [PubMed]
17. Carpenter AE, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7:R100. [PMC free article] [PubMed]
18. Storey JD, Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad Sci U S A. 2003;100:9440–9445. [PubMed]
19. Yang J, et al. Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles. Circ Res. 2000;87:E61–68. [PubMed]
20. Green GM, et al. Role of cholecystokinin in induction and maintenance of dietary protein-stimulated pancreatic growth. Am J Physiol. 1992;262:G740–746. [PubMed]
21. Bi Y, Page SL, Williams JA. Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion. Am J Physiol Gastrointest Liver Physiol. 2005;289:G561–570. [PubMed]
22. Han B, Ji B, Logsdon CD. CCK independently activates intracellular trypsinogen and NF-kappaB in rat pancreatic acinar cells. Am J Physiol Cell Physiol. 2001;280:C465–472. [PubMed]
23. Wente W, et al. Fibroblast growth factor-21 improves pancreatic beta-cell function and survival by activation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways. Diabetes. 2006;55:2470–2478. [PubMed]
24. Means AL, et al. Overexpression of heparin-binding EGF-like growth factor in mouse pancreas results in fibrosis and epithelial metaplasia. Gastroenterology. 2003;124:1020–1036. [PubMed]
25. Ogata H, et al. Deletion of the SOCS3 gene in liver parenchymal cells promotes hepatitis-induced hepatocarcinogenesis. Gastroenterology. 2006;131:179–193. [PubMed]
26. Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol. 2004;5:836–847. [PubMed]
27. Jensen JN, et al. Recapitulation of elements of embryonic development in adult mouse pancreatic regeneration. Gastroenterology. 2005;128:728–741. [PubMed]
28. Hogan PG, Chen L, Nardone J, Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 2003;17:2205–2232. [PubMed]
29. Chinenov Y, Kerppola TK. Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene. 2001;20:2438–2452. [PubMed]
30. Johnson EN, et al. NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells. J Biol Chem. 2003;278:1686–1692. [PMC free article] [PubMed]
31. Minami T, et al. Vascular endothelial growth factor- and thrombin-induced termination factor, Down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis. J Biol Chem. 2004;279:50537–50554. [PubMed]
32. Kubota Y, Hirashima M, Kishi K, Stewart CL, Suda T. Leukemia inhibitory factor regulates microvessel density by modulating oxygen-dependent VEGF expression in mice. J Clin Invest. 2008 [PMC free article] [PubMed]
33. Chan B, Greenan G, McKeon F, Ellenberger T. Identification of a peptide fragment of DSCR1 that competitively inhibits calcineurin activity in vitro and in vivo. Proc Natl Acad Sci U S A. 2005;102:13075–13080. [PubMed]
34. Gu G, et al. Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development. 2004;131:165–179. [PubMed]
35. Ji B, et al. Pancreatic gene expression during the initiation of acute pancreatitis: identification of EGR-1 as a key regulator. Physiol Genomics. 2003;14:59–72. [PubMed]
36. Grutzmann R, et al. Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes. Oncogene. 2005;24:5079–5088. [PubMed]
37. Buchholz M, et al. Overexpression of c-myc in pancreatic cancer caused by ectopic activation of NFATc1 and the Ca2+/calcineurin signaling pathway. Embo J. 2006;25:3714–3724. [PubMed]
38. Buchholz M, et al. Transcriptome analysis of microdissected pancreatic intraepithelial neoplastic lesions. Oncogene. 2005;24:6626–6636. [PubMed]
39. Logsdon CD, et al. Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer. Cancer Res. 2003;63:2649–2657. [PubMed]