Regulator of Calcineurin 1 Controls Growth Plasticity of Adult Pancreas

doi:10.1053/j.gastro.2010.04.050

Gastroenterology

Volume 139, Issue 2, August 2010, Pages 609-619.e6

https://doi.org/10.1053/j.gastro.2010.04.050 Get rights and content

Background & Aims

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

Methods

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 Regulator of Calcineurin 1 (Rcan1) was achieved by breeding elastase-Cre(estrogen receptor [ER]) transgenics with “flox-on” Rcan1 mice.

Results

CN inhibitor FK506 blocked expression of 38 genes, as confirmed by quantitative polymerase chain reaction. The CN-dependent genes were linked to growth-related processes, whereas their promoters were enriched in NFAT and NFAT/AP1 sites. Multiple NFAT targets, including Rcan1, Rgs2, HB-EGF, Lif, and Gem, were validated by chromatin immunoprecipitation. One of these, a CN feedback inhibitor Rcan1, was induced >50 fold during 1–8 hours 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 quantitative polymerase chain reaction. Most importantly, the increase in exocrine pancreas size, protein/DNA content, and acinar proliferation were all blocked in Rcan1 overexpressing mice.

Conclusions

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.

Section snippets

Materials

The PI camostat was provided by Ono Pharmaceuticals (Osaka, Japan). TaqMan reverse transcription and Expand polymerase chain reaction (PCR) kits were purchased from Roche (Basel, Switzerland). Antibodies and all other reagents are listed in Supplementary Materials and Methods.

Animals and Treatment

Animal experiments, including PI feeding and FK506 injections, are outlined in Supplementary Materials and Methods or performed as previously described.⁸ To overexpress Rcan1 in pancreatic acini, we bred FLAG-Rcan1 (Rcan1)

RNA Microarray Analysis of PI-Induced and CN-Dependent Genes

We performed genome-wide expression profiling of the 2-hour 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 refed PI-containing chow for 2 hours and injected with either 3 mg/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

Discussion

Physiologic growth of the pancreas takes place in response to high protein diet, hyperphagia, pregnancy, and lactation.²⁰ 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 1 of these genes, Rcan1, functions as a CN/NFAT-dependent feedback inhibitor that

Acknowledgments

The authors 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.

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Cited by (29)

RCAN1 is a marker of oxidative stress, induced in acute pancreatitis
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Citation Excerpt :
It has been shown to be regulated in neuronal cells by both CN-NFAT signaling, NFκB [28] and oxidative stress [29]. The specific role of Rcan1 in the pancreas is not as well studied, however overexpression of Rcan1 in pancreatic β-cells has been shown to cause hypoinsulinemia, β-cell dysfunction and diabetes [30] and CN-NFAT signaling induced Rcan1 expression in acinar cells, following damage-induced regeneration [15]. One of the most commonly used in vivo models of AP is the caerulein induction model [31], where hyper-stimulation with caerulein induces zymogen activation [32], ROS production and AP [33,34].
To date, there still is a lack of specific acute pancreatitis markers and specifically an early marker that can reliably predict disease severity. The inflammatory response in acute pancreatitis is mediated in part through oxidative stress and calcineurin-NFAT (Nuclear Factor of Activated T-cells) signaling, which is inducing its own negative regulator, regulator of calcineurin 1 (RCAN1). Caerulein induction is a commonly used in vivo model of experimental acute pancreatitis. Caerulein induces CN-NFAT signaling, reactive oxygen species and inflammation.
To screen for potential markers of acute pancreatitis, we used the caerulein model of experimental acute pancreatitis (AP) in C57Bl/6 J mice. Pancreata from treated and control mice were used for expression profiling. Promising gene candidates were validated in cell culture experiments using primary murine acinar cells and rat AR42J cells. These candidates were then further tested for their usefulness as biomarkers in mouse and human plasma.
We identified a number of novel genes, including Regulator of calcineurin 1 (Rcan1) and Sestrin 2 (Sesn2) and demonstrated that they are induced by oxidative stress, by stimulation with H₂O₂ and by inhibiting caerulein stimulated expression with the antioxidant N-acetylcysteine. We found Rcan1 protein to be significantly elevated in AP-induced mouse plasma as well as in plasma from AP patients.
We demonstrated that Rcan1 is regulated by oxidative stress and identified RCAN1 as a potential diagnostic marker of AP.
NFATc1 links EGFR signaling to induction of sox9 transcription and acinar-ductal transdifferentiation in the pancreas
2015, Gastroenterology
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The identification of Cox2 as an EGFR/NFAT target already points toward critical functions of the EGFR/NFAT cascade for the integration of inflammatory signals during carcinogenesis. In particular, during pancreatic cancer formation, NFAT proteins have been shown to play a central role in the integration of environmental signals into oncogenic transcriptional processes that mediate proliferation, cellular differentiation, and adaptation to inflammation.21,22,28,30 In this study, we showed that sustained pancreatic inflammation induced a strong up-regulation of NFATc1 in metaplastic areas of both murine and human chronic pancreatitis models.
Oncogenic mutations in KRAS contribute to the development of pancreatic ductal adenocarcinoma, but are not sufficient to initiate carcinogenesis. Secondary events, such as inflammation-induced signaling via the epidermal growth factor receptor (EGFR) and expression of the SOX9 gene, are required for tumor formation. Herein we sought to identify the mechanisms that link EGFR signaling with activation of SOX9 during acinar–ductal metaplasia, a transdifferentiation process that precedes pancreatic carcinogenesis.
We analyzed pancreatic tissues from Kras^G12D;pdx1-Cre and Kras^G12D;NFATc1^Δ/Δ;pdx1-Cre mice after intraperitoneal administration of caerulein, vs cyclosporin A or dimethyl sulfoxide (controls). Induction of EGFR signaling and its effects on the expression of Nuclear factor of activated T cells c1 (NFATc1) or SOX9 were investigated by quantitative reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemical analyses of mouse and human tissues and acinar cell explants. Interactions between NFATc1 and partner proteins and effects on DNA binding or chromatin modifications were studied using co-immunoprecipitation and chromatin immunoprecipitation assays in acinar cell explants and mouse tissue.
EGFR activation induced expression of NFATc1 in metaplastic areas from patients with chronic pancreatitis and in pancreatic tissue from Kras^G12D mice. EGFR signaling also promoted formation of a complex between NFATc1 and C-JUN in dedifferentiating mouse acinar cells, leading to activation of Sox9 transcription and induction of acinar–ductal metaplasia. Pharmacologic inhibition of NFATc1 or disruption of the Nfatc1 gene inhibited EGFR-mediated induction of Sox9 transcription and blocked acinar–ductal transdifferentiation and pancreatic cancer initiation in mice.
EGFR signaling induces expression of NFATc1 and Sox9, leading to acinar cell transdifferentiation and initiation of pancreatic cancer. Strategies designed to disrupt this pathway might be developed to prevent pancreatic cancer initiation in high-risk patients with chronic pancreatitis.
Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin
2013, Journal of Biological Chemistry
Citation Excerpt :
Each of the inhibitors reduced the activation of our reporter system driven by the NFAT promoter, suggesting that TLCS injury is mediated by calcineurin. Further, infecting cells with an adenovirus that overexpresses an endogenous inhibitor of calcineurin, Mcip1, also known as regulator of calcineurin 1 (Rcan1) (33), showed similar results (Fig. 1F). We demonstrate that TLCS promotes transcriptional activation via a calcineurin-specific pathway.
Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca²⁺. Thus, it would be clinically relevant to know the targets of this aberrant Ca²⁺ signal. We hypothesized that the Ca²⁺-activated phosphatase calcineurin is such a Ca²⁺ target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca²⁺. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca²⁺ generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.
NFATc3 regulates trypsinogen activation, neutrophil recruitment, and tissue damage in acute pancreatitis in mice
2012, Gastroenterology
The signaling mechanisms that regulate trypsinogen activation and inflammation in acute pancreatitis (AP) are unclear. We explored the involvement of the calcium- and calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in development of AP in mice.
We measured levels of myeloperoxidase and macrophage inflammatory protein 2 (CXCL2), trypsinogen activation, and tissue damage in the pancreas 24 hours after induction of AP by retrograde infusion of taurocholate into the pancreatic ducts of wild-type, NFAT luciferase reporter (NFAT-luc), and NFATc3-deficient mice. We isolated acinar cells and measured NFAT nuclear accumulation, trypsin activity, and expression of NFAT-regulated genes.
Infusion of taurocholate increased the transcriptional activity of NFAT in the pancreas, aorta, lung, and spleen of NFAT-luc mice. Inhibition of NFAT with A-285222 blocked taurocholate-induced activation of NFAT in all organs. A-285222 also reduced taurocholate-induced increases in levels of amylase, myeloperoxidase, and CXCL2; activation of trypsinogen; necrosis of acinar cells; edema; leukocyte infiltration; and hemorrhage in the pancreas. NFATc3-deficient mice were protected from these effects of taurocholate. Similar results were obtained using an l-arginine–induced model of AP. Reverse-transcription polymerase chain reaction and confocal immunofluorescence analyses showed that NFATc3 is expressed by acinar cells. NFATc3 expression was activated by stimuli that increase intracellular calcium levels, and activation was prevented by the calcineurin blocker cyclosporin A or A-285222. Activation of trypsinogen by secretagogues in acinar cells was prevented by pharmacologic inhibition of NFAT signaling or lack of NFATc3. A-285222 also reduced expression of inflammatory cytokines such as CXCL2 in acinar cells.
NFATc3 regulates trypsinogen activation, inflammation, and pancreatic tissue damage during development of AP in mice and might be a therapeutic target.
Evaluating the mode of action of perfluorooctanoic acid-induced liver tumors in male Sprague-Dawley rats using a toxicogenomic approach
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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by NIH grants DK 59578 (to J.A.W.) and P30 DK-34933 (Michigan Gastrointestinal Peptide Center); by Systems and Integrative Biology Training Grant (T32 GM008322; to G.T.G.) and the Medical Scientist Training Program (G.T.G.); by DK52067 (to C.D.L.), R21 DK068414 (to B.J.), DK-0077423 (to S.J.C.), and HL072016 (B.A.R.); and for use of the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center funded by NIH5P60 DK20572.

View full text

Basic—Liver, Pancreas, and Biliary TractRegulator of Calcineurin 1 Controls Growth Plasticity of Adult Pancreas

Background & Aims

Methods

Results

Conclusions

Section snippets

Materials

Animals and Treatment

RNA Microarray Analysis of PI-Induced and CN-Dependent Genes

Discussion

Acknowledgments

Jpn J Pharmacol

Trends Cell Biol

Cell

Trends Cardiovasc Med

J Biol Chem

Gastroenterology

Gastroenterology

Gastroenterology

J Biol Chem

J Biol Chem

Adaptation of the exocrine pancreas to diet

Annu Rev Nutr

Calcineurin mediates pancreatic growth in protease inhibitor-treated mice

Am J Physiol Gastrointest Liver Physiol

Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy

Circ Res

Calcineurin mediates pancreatic growth in protease inhibitor-treated mice

Am J Physiol Gastrointest Liver Physiol

Cholecystokinin activates pancreatic calcineurin-NFAT signaling in vitro and in vivo

Mol Biol Cell

Calcineurin is necessary for the maintenance but not embryonic development of slow muscle fibers

Mol Cell Biol

Robust acinar cell transgene expression of CreErT via BAC recombineering

Genesis

CCK-A receptor activates RhoA through G α 12/13 in NIH3T3 cells

Am J Physiol Cell Physiol

Induction of early response genes in trypsin inhibitor-induced pancreatic growth

Am J Physiol Gastrointest Liver Physiol

Basic—Liver, Pancreas, and Biliary Tract
Regulator of Calcineurin 1 Controls Growth Plasticity of Adult Pancreas