Am J Physiol Endocrinol Metab 306: E916–E928, 2014. First published February 18, 2014; doi:10.1152/ajpendo.00559.2013.

Silencing of the Fibroblast growth factor 21 gene is an underlying cause of acinar cell injury in mice lacking MIST1 Charis L. Johnson,1,2 Rashid Mehmood,1,2 Scott W. Laing,1,3 Camilla V. Stepniak,1,2 Alexei Kharitonenkov,5 and Christopher L. Pin1– 4 1

Children’s Health Research Institute, London, Ontario, Canada; Departments of 2Paediatrics, 3Physiology and Pharmacology, and 4Oncology, University of Western Ontario, London, Ontario, Canada; and 5Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana Submitted 9 October 2013; accepted in final form 11 February 2014

Johnson CL, Mehmood R, Laing SW, Stepniak CV, Kharitonenkov A, Pin CL. Silencing of the Fibroblast growth factor 21 gene is an underlying cause of acinar cell injury in mice lacking MIST1. Am J Physiol Endocrinol Metab 306: E916 –E928, 2014. First published February 18, 2014; doi:10.1152/ajpendo.00559.2013.—Fibroblast growth factor 21 (FGF21) is a key regulator of metabolism under conditions of stress such as starvation, obesity, and hypothermia. Rapid induction of FGF21 is also observed in experimental models of pancreatitis, and FGF21 reduces tissue damage observed in these models, suggesting a nonmetabolic function. Pancreatitis is a debilitating disease with significant morbidity that greatly increases the risk of pancreatic ductal adenocarcinoma. The goals of this study were to examine the regulation and function of FGF21 in acinar cell injury, specifically in a mouse model of pancreatic injury (Mist1⫺/⫺). Mist1⫺/⫺ mice exhibit acinar cell disorganization, decreased acinar cell communication and exocytosis, and increased sensitivity to cerulein-induced pancreatitis (CIP). Examination of Fgf21 expression in Mist1⫺/⫺ mice by qRT-PCR, Northern blot, and Western blot analyses showed a marked decrease in pancreatic Fgf21 expression before and after induction of CIP compared with C57Bl/6 mice. To determine whether the loss of FGF21 accounted for the Mist1⫺/⫺ phenotypes, we generated Mist1⫺/⫺ mice overexpressing human FGF21 from the ApoE promoter (Mist1⫺/⫺ApoE-FGF21). Reexpression of FGF21 partially mitigated pancreatic damage in Mist1⫺/⫺ tissue based on reduced intrapancreatic enzyme activation, reduced expression of genes involved in fibrosis, and restored cell-cell junctions. Interestingly, alteration of Fgf21 expression in Mist1⫺/⫺ tissue was not simply due to a loss of direct transcriptional regulation by MIST1. Chromatin immunopreciptation indicated that the loss of Fgf21 in the Mist1⫺/⫺ pancreas is due, in part, to epigenetic silencing. Thus, our studies identify a new role for FGF21 in reducing acinar cell injury and uncover a novel mechanism for regulating Fgf21 gene expression. epigenetics; gene silencing; MIST1; pancreatitis; histone methylation FIBROBLAST GROWTH FACTOR 21 (FGF21) is an important regulator of metabolism, belonging to the endocrine family of FGFs, which includes FGF15/19 and FGF23 (1, 24). FGF21 increases insulin production and glucose uptake (52), promotes ketogenesis (2, 18), and enhances conversion of white to brown adipose tissue (14). FGF21 expression and secretion are triggered by a number of adverse environmental events including fasting (13), alterations in diet leading to increased fatty acid oxidation (4), obesity (9), or hypothermia (14). We have shown that FGF21 is increased in the pancreas during ceruleininduced pancreatitis (CIP) and L-arginine induced pancreatitis

Address for reprint requests and other correspondence: C. Pin, Dept. of Paediatrics, Univ. of Western Ontario, Children’s Health Research Institute, 5th Floor, Victoria Research Laboratories, London, ON, Canada N6C 2V5, (e-mail: [email protected]). E916

and can target pancreatic acinar cells in culture (23). Overexpressing FGF21 reduces the severity of CIP, and Fgf21⫺/⫺ mice showed enhanced inflammation and fibrosis during CIP, highlighting a protective role for FGF21 in restricting injury during pancreatitis (23). However, there are still limited data regarding the regulation and function of FGF21 in the exocrine pancreas in vivo. Pancreatitis is a debilitating disease affecting more than 100,000 individuals each year in North America. While excessive alcohol consumption and gallstone obstruction of the pancreatic duct are known causes of pancreatitis, a significant genetic component influences both idiopathic and environmentally-related cases of pancreatitis (28, 51). Several rodent models have been established to identify early initiating events for pancreatitis, including supramaximal secretagogue stimulation, ductal ligation, and L-arginine injection (5, 26, 38, 44). By combining these experimental models with genetically modified mouse lines, investigators have identified several genes that either provide protection against pancreatitis or promote increased severity and susceptibility to pancreatitis. In some cases, these lines exhibit dramatically different molecular and cellular responses to induced pancreatic injury, providing insight into pathways that promote injury that may be targeted therapeutically. Our laboratory has established a knockout mouse line that mimics many of the early events observed in pancreatitis. These animals harbor a gene deletion of Mist1 (Mist1⫺/⫺). MIST1 is expressed in developing and mature acinar cells of the pancreas as well as other serous exocrine glands (41). Mist1⫺/⫺ pancreatic tissue shows premature activation of digestive enzymes, altered Ca2⫹ signaling, cellular disorganization, and increased activation and expression of genes linked to pancreatic injury (33, 42, 43). Mist1⫺/⫺ acinar cells also exhibit increased proliferation rates (20, 42) and are significantly more likely to undergo acinar-to-duct cell metaplasia (22), providing a potential link to pancreatic ductal adenocarcinoma (PDAC). We have recently shown loss of MIST1 accumulation in human samples from chronic pancreatitis and PDAC (22). These observations indicate that loss of MIST1 is a key event in progressing from healthy to diseased pancreatic tissue. Whether FGF21 is involved in these processes was examined in this study. MIST1 is a basic helix-loop-helix (bHLH) transcription factor (TF), targeting E boxes (CANNTG) within the genome (30). Unlike most bHLH proteins that heterodimerize with ubiquitously expressed bHLH factors, MIST1 preferentially binds target genes as a homodimer (54). Cyclic amplification and selection of targets (CASTing) revealed a preference for

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PARTIAL RESCUE OF MIST1⫺/⫺ PANCREATIC PHENOTYPES BY FGF21

MIST1 to bind E boxes containing a central TA sequence, whereas other bHLH dimers prefer GC-rich sequences (50). Recently, it has been suggested that MIST1 acts as a scaling factor, a type of TF that enhances rather than activates gene expression (36). Scaling factors are believed to target genes that contribute to a common cellular pathway. MIST targets several genes involved in the exocytosis pathway (including Rab3d and Rab26), and Mist1⫺/⫺ acinar cells have a reduced exocytotic ability (21, 33, 48). However, several phenotypic consequences tied to the loss of MIST1 argue against a role simply as a scaling factor, including the complete absence of Cx32 accumulation in Mist1⫺/⫺ acinar cells (43) and the increased proliferation of acinar cells (20). In addition, the events that promote increased susceptibility for pancreatitis in Mist1⫺/⫺ mice are unclear. Therefore, it is important to delineate events that are directly tied to loss of MIST1 function vs. those that arise as a consequence of the cellular phenotypes observed in Mist1⫺/⫺ mice. We have shown that the unfolded protein response UPR, including activating transcription factor 4, is suppressed in Mist1⫺/⫺ pancreatic tissue during CIP (11), suggesting that an absence of FGF21 signaling may underlie the acinar cell damage exhibited by Mist1⫺/⫺ mice. Recently, ATF4 has been identified as a regulator of Fgf21 expression (6). Given the link between the UPR and both FGF21 and MIST1, we suggest that altered FGF21 signaling may account for some of the phenotypes observed in Mist1⫺/⫺ mice. In this study, we examined pancreatic FGF21 accumulation in the absence of MIST1 and identified a dramatic loss of FGF21 expression in Mist1⫺/⫺ mice, likely due to epigenetic silencing of the Fgf21 gene as opposed to the loss of direct MIST1 transcriptional activity. By generating Mist1⫺/⫺ mice that expressed high levels of FGF21, we showed that FGF21 reduced pancreatic injury in Mist1⫺/⫺ mice. These results demonstrate a novel physiological role for FGF21 in acinar cell biology. MATERIALS AND METHODS

Unless indicated, all reagents and consumables were obtained from Fisher Scientific (Burlington, ON). Animals. Procedures were approved by the University of Western Ontario Animal Care Committee (Protocol No. 2008-116). Mice containing a deletion of Mist1 (Mist1⫺/⫺) or Fgf21 (Fgf21⫺/⫺) or overexpressing the human FGF21 from the ApoE promoter (ApoEFGF21) have been described (14, 25, 42) and were all on a C57/Bl6 background. Mist1⫺/⫺ mice were mated to ApoE-FGF21 mice to produce the Mist1⫹/⫺ApoE-FGF21 genotype. Mist1⫹/⫺ApoE-FGF21 mice were crossed back to Mist1⫺/⫺ mice to generate Mist1⫺/⫺ApoEFGF21 mice. CIP was initiated as described (27). Each mouse received intraperitoneal injections of cerulein (50 ␮g/kg) each hour until being euthanized. Mice were euthanized 1 or 4 h after initial cerulein injection. Tissue isolation and histology. Mouse pancreata were fresh-frozen in cryomatrix and sectioned at 6 ␮m. Cryostat sections were processed for immunofluorescence (IF) as described (21). The following primary antibodies were used: mouse anti-connexin 32 (Cx32, 1:500; Millipore, Temecula, CA), rabbit anti-carboxypeptidase (CPA; 1:500; AbD Serotec, Oxford, UK), rabbit anti-Ki67 (1:500; Abcam, Cambridge, MA), mouse anti-phospho-histone H3 (pH3, 1:250; Millipore), mouse anti-␤-catenin (1:250; BD Transduction Laboratories, Mississauga, ON, Canada), and mouse anti-smooth muscle actin (SMA, 1:300; Sigma, St. Louis, MO). Secondary antibodies included anti-mouse FITC and anti-rabbit FITC (1:250; Jackson ImmunoResearch, West

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Grove, PA). Immunofluorescence was visualized with a Leica DMIOS upright microscope or an Olympus Fluoview FV1000 confocal microscope. Images were captured using the Open Lab 3.1.5 imaging program (Quorom Technologies, Guelph, ON, Canada) or FV 10-ASW 1.6 (FluoView 1000 software, Olympus) and Image-Pro Analyzer (v. 6.2, Media Cybernetics) software, respectively. Alternatively, sections were stained with haematoxylin and eosin to assess morphology. Chromatin immunopreciptation. Chromatin immunoprecipitation (ChIP) was performed on chromatin isolated from pancreatic tissue of male mice as described (12). The purified rabbit anti-MIST1 antibody used for ChIP was designed from ProSci (Poway, CA), while antibodies for H3K4Me3 and H3K27Me3 were obtained from Millipore (Temecula, CA). qPCR was performed using the GoTaq PCR Mastermix system (Promega, Madison WI). Samples were evaluated using the ABI Prism 7900HT Sequence Detection System and Vii A7 RUO software (Applied Biosystems, Foster City, CA). Average CT values for individual ChIP and IgG controls were expressed as a percentage of starting chromatin samples (input). The primers used for amplification spanned the Fgf21 promoter (see Table 1 for primers). Cell culture and luciferase assay. HEK-293 cells were cultured for 24 h and then transfected with an Fgf21 promoter-luciferase reporter (⫺1497Fgf21-pGL2; courtesy Steve Kliewer, UT Southwestern), pGL2 (no promoter) or pGK-luciferase plasmids all in combination with pcDNA3.1, pcDNA3.1-Mist1, or pcDNA3.1-Mist1-mut. Cells were transfected with 450 ng using Effectene (Qiagen, Toronto, ON, Canada). Cells were cotransfected with PGK-Renilla luciferase to normalize for transfection efficiency. For the luciferase assay, cells were lysed 30 h posttransfection, and the ratio between firefly and Renilla luciferase signals was measured using a dual luciferase assay kit (Promega) according to the manufacturer’s instructions. RNA was isolated from acinar (rat AR42J and mouse 266.6), pancreatic derived (rat ARIP), pancreatic ductal adenocarcinoma (human PANC1), and nonpancreatic (mouse NIH3T3 and human HEK293) cells. AR42J cells were grown in F12K nutrient mixture ⫹ 20% fetal bovine serum (FBS); all other cells were grown in DMEM ⫹10% FBS. Acinar cell culture. Pancreata were dissected and acini isolated as described previously (22). In some instances, isolated acini were processed for RNA or protein within an hour of isolation. The acini were plated within a collagen matrix (VWR; Mississauga, ON, Canada) of 1:1 solution of rat tail collagen with DMEM containing 1% stock penicillin-streptomycin. Cultures were fed DMEM containing 1% penicillin-streptomycin, 0.25 ␮g/ml amphotericin B, 0.1 mM IBMX, and 1 ␮M dexamethasone. The medium was changed every 2 days, and cultures were followed for 9 days. To assess cyst formation, the number of clusters containing cysts was determined from 50 randomly identified clusters. Protein isolation and Western blot analysis. Tissue protein extraction, protein electrophoresis, and immunoblotting were performed as described (23). Antibodies used were rabbit anti-CPA (1:2,000; AbD Serotec, Oxford, UK) or goat anti-FGF21 (1:500; R&D Systems, Minneapolis, MN). Secondary antibodies used were anti-rabbit HRP (1:10,000) or anti-goat HRP (1:2,000, Jackson Laboratories). RNA isolation, real-time qRT-PCR, and Northern blot analysis. RNA was isolated from whole tissue or cell lines using TRIzol (Invitrogen, Burlington, ON, Canada), following the manufacturer’s instructions. Northern blot analysis was performed as described (27). Blots were hybridized overnight at 42°C with a [␣-32P]dCTP-labeled probe (gene:I.M.A.G.E ID no. Fgf21: 6774352) or 18S rRNA. Realtime qRT-PCR was performed on cDNA samples prepared as described (22). Using mitochondrial ribosomal protein L1 (Mrpl1) as a normalization control, ViiA 7 RUO software (Applied Biosystems) was used to calculate the amount of RNA relative to untreated or saline treatment for the equivalent time points. Primer sequences are available in Table 1. Statistics. For all statistical analysis, a one-way ANOVA was performed using GraphPad Prism 5 followed by Tukey’s multiple

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Table 1. Primers used for qRT-PCR and ChIP-qPCR Gene

Mist1 Fgf21 (⫺3291) (⫺3399 ⫺3183) Fgf21 (⫺2006) (⫺2113 ⫺1898) Fgf21 (⫺670) (⫺777 ⫺562) Fgf21 (⫺76) (⫺172 ⫹20) Fgf21 (⫹1285) (⫹1177 ⫹1393) Ppp1r15a mFgf21 hFGF21 Mrpl1 Asb11 Gjb1 Pde1a Hdac1 Hdac3 Hdac6 Kmt2a Kmt2b Kmt2c Kmt2e Atf4 Beta actin

Sequence

Size (bp)

Forward: 5=-GTGGTGGCTAAAGCTACGTG-3= Reverse: 5=-GACTGGGGTCTGTCAGGTGT-3= Forward: 5=-CCTCCTGGGGTGATTGTCC-3= Reverse: 5=-TGTGCTCTCTGCCGGCTC-3= Forward: 5=-AGGCTCAGAGACCGGTGGG-3= Reverse: 5=-GCTTCTGGGTACCAGTTGG-3= Forward: 5=-CCAGGTTCCTTCTTAGTACCC-3= Reverse: 5=-CTGAGGTAGGAGACTGAAGTC-3= Forward: 5=-TTCATTCAGACCCCTGTTGGA-3= Reverse: 5=-AGAAGACACTAAGGCTGTCTGG-3= Forward: 5=-ACAGCCTCACTTTGATCCTG-3= Reverse: 5=-CTCTGGGGGCAGGAATCC-3= Forward: 5=-TCGCGTACTTGCTCGGAAAT-3= Reverse:-3=-ACTCAATCTGCGCCAACATC-3= Forward: 5=-ACAGATGACGACCAAGACACTG-3= Reverse: 5=-GTCCTCCAGCAGCAGTTCTC-3= Forward: 5=-ACAGATGATGCCCAGCAGACAG-3= Reverse: 5=-AGTGGAGCGATCCATACAGG-3= Forward: 5=-TTGGATATGCCAAGTGACCA-3= Reverse: 5=-GCTTCTGCCGTTTGAGTTTC-3= Forward: 5=-TGCATGGAGATTCTGCTGAC-3= Reverse: 5=-GGTATCCAGCCACTGACCAT-3= Forward: 5=-TGGGGAGGGATGTGGGCAAG-3= Reverse: 5=-GGCCTTGGGAACTGGGGACT-3= Forward: 5=-GAAACACCTCCAAAGACCCA-3= Reverse: 5=-TTGCGTGTGAAAGTTGAAGC-3= Forward: 5=-CGGGTCATGACTGTGTCCTT-3= Reverse: 5=-GGCACAGTGAGAAATCGTGA-3= Forward: 5=-CTCGCAGTGGGTAGTTCACA-3= Reverse: 5=-CTCGCAGTGGGTAGTTCACA-3= Forward: 5=-TCCTCAGCTGTGTTGACCTG-3= Reverse: 5=-TCAGAAGGGAAACAGGCAGT-3= Forward: 5=-CAGCATCAGGCTACAAAGCA-3= Reverse: 5=-GGCACAGTGAGAAATCGTGA-3= Forward: 5=-AGACGATGACACCATGCAAA-3= Reverse: 5=-TCACCTTGGTGATCTTGCTG-3= Forward: 5=-TGTTGTCTCAGGGAGCACAG-3= Reverse: 5=-AGGAGCTTGGTCGATGTGTT-3= Forward: 5=-TGATGGACCTGCTTCCTGGTT-3= Reverse: 5=-TCTGGCAAAAGCCTGCAAAA-3= Forward: 5=-CCACCATGGCGTATTAGAGG-3= Reverse: 5=-CAACACTGCTGCTGGATTTC-3= Forward: 5=-TGTGTGGATCGGTGGCTCCATCCTGGCC-3= Reverse: 5=-CTGCGCAAGTTAGGTTTTGTCAAAG-3=

comparison post hoc test. Data are presented as means ⫾ SE; n values are indicated in the context of each experiment. RESULTS

Mist1⫺/⫺ acinar cells show reduced Fgf21 expression before and after induction of CIP. Mist1⫺/⫺ mice show increased sensitivity to pancreatic injury (27), but it is unclear what molecular changes in these mice account for this sensitivity. We have shown that FGF21 reduces the severity of CIP in mice, acinar cells express ␤-klotho, the specific receptor for FGF21, and treatment of isolated acinar cells with FGF21 results in ERK1/2 activation within minutes (23). Recent studies indicate that the Fgf21 gene is a target of ATF4 transcriptional regulation (6) and our previous analysis showed that ATF4 expression is reduced in Mist1⫺/⫺ pancreatic tissue (11). Therefore, Fgf21 expression was examined before and after initiating CIP in wild-type (WT) and Mist1⫺/⫺ mice (Fig. 1). Under nontreated conditions, the expression of Fgf21 was significantly reduced (24 ⫾ 5.2% of WT levels) in Mist1⫺/⫺

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pancreatic tissue (Fig. 1A). In the liver, another tissue expressing Fgf21 but not Mist1, Fgf21 levels were higher in Mist1⫺/⫺ mice (164 ⫾ 13.8% of WT levels), indicating the reduction in Fgf21 expression was tissue specific in Mist1⫺/⫺ mice. No difference in circulating FGF21 was observed between genotypes (data not shown). We next compared pancreatic Fgf21 expression 1 and 4 h into CIP between WT and Mist1⫺/⫺ mice using qRT-PCR (Fig. 1B). Since there are reports that ␤-actin expression increases during CIP (19) and we have observed elevations in ␤-actin in Mist1⫺/⫺ mice (42), we normalized Fgf21 levels to Mrpl1. Within 1 h, a marked increase in Fgf21 accumulation was observed in WT mice (81 ⫾ 23.8-fold increase relative to saline-treated mice), whereas Mist1⫺/⫺ pancreatic tissue showed no such increase. Four hours into CIP, the expression of Fgf21 was 28.3 ⫾ 9.6-fold higher relative to saline-treated mice. Although statistically higher than in saline-treated Mist1⫺/⫺ mice, Fgf21 levels in Mist1⫺/⫺ pancreatic tissue were still lower than levels in saline-treated WT mice (Fig.

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Fig. 1. Fibroblast growth factor 21 (Fgf21) expression is suppressed in Mist1⫺/⫺ pancreatic tissue. A: real-time qRT-PCR for Fgf21 expression in wild-type (WT) and Mist1⫺/⫺ pancreatic (n ⫽ 5) and liver (n ⫽ 6 for WT and n ⫽ 4 for Mist1⫺/⫺) tissue from 4-mo-old mice. Values are normalized to Mrpl1 expression. *P ⬍ 0.05, **P ⬍ 0.01. B: similar analysis 1 and 4 h after inducing pancreatitis. Values are shown normalized to Mrpl1 expression; n ⫽ 3 in all cases except 4-h-treated Mist1⫺/⫺ mice (n ⫽ 5). C: representative Northern blot for Fgf21 or 18S 4 h after initiating treatment with saline (SAL) and cerulein (CIP) in WT and Mist1⫺/⫺ mice. Each lane represents an individual mouse. D: representative Western blot analysis for FGF21 1 or 4 h into CIP treatment for WT and Mist1⫺/⫺ mice. Control (⫺) samples were isolated from saline-treated mice 4 h into treatment. E: qRT-PCR for Fgf21 expression in WT and Mist1⫺/⫺ pancreatic acinar cells immediately after dissociation; n ⫽ 3. *P ⬍ 0.01.

1B). Surprisingly, statistical analysis suggested that this increase in WT mice was not significant, whereas the increase in Fgf21 expression between untreated and 4-h-treated Mist1⫺/⫺ mice was significant. To provide a more definitive result regarding Fgf21 mRNA levels during CIP, we performed Northern blot analysis and compared Fgf21 levels to 18S (Fig. 1C). This analysis confirmed a dramatic increase in Fgf21 expression 4 h into CIP but not saline treatment, but only in WT animals. Western blot analysis confirmed a marked increase in FGF21 expression only in WT tissue within 4 h of initiating CIP (Fig. 1D). Interestingly, we did not observe an increase in circulating FGF21 levels in WT mice during this time (data not shown), suggesting that the production of FGF21 is localized to the pancreas. The inability of Mist1⫺/⫺ acinar cells to activate Fgf21 expression was also observed under other conditions that promoted increased Fgf21 accumulation. Analysis of Fgf21 expression immediately following isolation (1 h) of acinar cells showed dramatic increases in Fgf21 in WT tissue relative to tissue expression and an almost complete absence of Fgf21 accumulation in Mist1⫺/⫺ acini (Fig. 1E). Combined, these results suggest that the FGF21 signaling axis was compromised in the absence of MIST1. Restoring FGF21 expression in the Mist1⫺/⫺ background reduces pancreatic damage. To date, there has been little evidence for a role for FGF21 signaling in pancreatic acinar cells. However, the absence of FGF21 may account for the pancreatic abnormalities observed in Mist1⫺/⫺ pancreatic tissue. To examine this possibility, Mist1⫺/⫺ mice were crossed

with mice that overexpress human FGF21 from the ApoE promoter (Mist1⫺/⫺ApoE-FGF21) (25). To date, all of the reported FGF21 effects have been through endocrine mechanisms. Therefore, it is possible that FGF21 targets acinar cells through an endocrine action. Although we did observe increased Fgf21 mRNA in the liver, this did not lead to increased circulating FGF21. This would suggest that there is not an increase in endocrine FGF21 activity in Mist1⫺/⫺ mice. FGF21 is expressed at high levels in the liver of Mist1⫺/⫺ApoEFGF21 mice leading to 150- to 300-fold higher levels of circulating FGF21 (25). Interestingly, RT-PCR readily detected human FGF21 in the pancreas (Fig. 2A) as well as the parotid salivary gland, brain, and lung (Fig. 2B) of Mist1⫺/⫺ApoE-FGF21 mice, suggesting that the ApoE promoter is not specific to the liver in the context of this transgenic line. Therefore, even if FGF21 actions on acinar cells are through a paracrine effect, we should still observe an effect on the Mist1⫺/⫺ phenotype. We initially examined two of the more striking phenotypes of the Mist1⫺/⫺ mouse, altered acinar morphology and premature enzyme activation, both of which are indicative of pancreatic injury and predispose mice to pancreatitis. WT acinar cells are highly organized structures with the endoplasmic reticulum and nuclei located at the basal aspect of the cells (Fig. 2Ci), and the loss of MIST1 leads to disruptions in zymogen granule localization, nuclear dysplasia (nonuniform nuclear size), and abnormal cellular architecture (Fig. 2Cii) (42). H&E analysis was inconclusive regarding any difference in acinar cell organization between Mist1⫺/⫺ApoE-FGF21 and

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Fig. 2. Restored FGF21 expression reduces acinar cell injury in Mist1⫺/⫺ mice. A and B: RT-PCR with primers specific for human FGF21, mouse Fgf21, or ␤-actin mRNA in adult mouse pancreatic tissue (A) or other tissues from ApoE-FGF21 mice (B). SG, parotid salivary gland; SV, seminal vesicle. Negative control (⫺) was water. C: representative H&E histology on 4-moold pancreatic tissue from mice. An acinus has been outlined in each section. Scale bar, 25 ␮M. D: Western blot analysis for procarboxypeptidase (proCPA) and the cleaved active carboxypetidase (CPA). Densitometry was performed comparing the ratio of active (CPA) over inactive (proCPA) enzyme. *P ⬍ 0.05; n ⫽ 3.

Mist1⫺/⫺ pancreatic tissue (Fig. 2Ciii). However, reexpression of FGF21 partially mitigated the premature activation of procarboxypeptidase (ProCPA) to active CPA (Fig. 2D). Densitometric analysis of Western blots for CPA revealed 5.3-fold more active CPA in Mist1⫺/⫺ relative to WT tissue (n ⫽ 3). Reexpression of FGF21 reduced the level of active CPA in Mist1⫺/⫺ApoE-FGF21 pancreatic extracts to 2.5-fold over WT levels (Fig. 2D). This suggests that the extent of pancreatic injury observed in Mist1⫺/⫺ mice is reduced following restoration of FGF21. To confirm that reexpression of FGF21 reduced acinar cell injury in Mist1⫺/⫺ mice, we assessed accumulation of cell-cell junctions between acinar cells. We had previously shown that Mist1⫺/⫺ mice have dramatically reduced accumulation of Cx32 specifically in acinar cells (43), and loss of Cx32 increases pancreatic injury during CIP (15). Restoration of intercellular junctions would indicate reduced acinar cell damage. IF confirmed that Cx32 accumulation was completely abolished within acinar tissue of Mist1⫺/⫺ mice (Fig. 3A). However, punctate accumulation of Cx32 was restored in Mist1⫺/⫺-ApoE-FGF21 tissue (Fig. 3A), suggesting that cellular architecture was improved by FGF21. Cx32 accumulation was not disrupted in Fgf21⫺/⫺ pancreatic tissue, indicating that

the loss of Cx32 in Mist1⫺/⫺ mice was not solely due to loss of FGF21 signaling. Analysis of Gjb1 levels (gene encoding Cx32) by qRT-PCR revealed dramatically reduced accumulation in both Mist1⫺/⫺ and Mist1⫺/⫺-ApoE-FGF21 tissue relative to WT mice (Fig. 3B), indicating that restored Cx32 accumulation was not due to restoration of Gjb1 gene expression. These results indicate that FGF21 can improve the cellular damage resulting from loss of MIST1. We next assessed ␤-catenin accumulation within acinar cells, which is normally localized to the apical-lateral border (Fig. 3C) and marks adherens junctions. Previous studies indicate that Mist1⫺/⫺ acini have reduced ␤-catenin accumulation (42), which we confirmed here. Similar to the Cx32 localization, Mist1⫺/⫺ApoE-FGF21 acini showed some maintenance of ␤-catenin at the apical border of acini (Fig. 3C). Another indicator of pancreatic damage is the presence of interstitial stellate cells. Stellate cells are activated by injury and express ␣SMA (smooth muscle actin). The number of active stellate cells reflects the extent of tissue damage (47). We have shown that the absence of MIST1 leads to increased SMA production and stellate cell numbers, whereas FGF21 (23) overexpression during CIP reduces stellate cell activation. Examination of SMA⫹ cells by IF revealed a consistent

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Fig. 3. Acinar cell junctions are maintained by forced expression of FGF21 in Mist1⫺/⫺ mice. A: representative immunifluorescence (IF) for connexin 32 (Cx32) accumulation (green; arrows) on pancreatic sections from 4-mo-old pancreatic tissue. Nuclei were counterstained with DAPI (blue). B: real-time qRT-PCR comparing Gjb1 expression between genotypes. *P ⬍ 0.01 between WT and Mist1⫺/⫺ or Mist1⫺/⫺ApoE-FGF21; n ⫽ 7. C: confocal IF microscopy for ␤-catenin in pancreatic tissue. Individual acini are delineated by a line; apical localization of ␤-catenin is indicated by closed white arrows. Open arrow, localization of ␤-catenin to duct cells in Mist1⫺/⫺ acini. In all cases, Mist1⫺/⫺ApoE-FGF21 (Mist1⫺/⫺FGF21⫹).

reduction in the number of stellate cells in Mist1⫺/⫺ApoEFGF21 pancreatic tissue relative to Mist1⫺/⫺ tissue and similar to WT tissue (Fig. 4, A–C). Data mining of previous arrays comparing WT and Mist1⫺/⫺ tissue identified several genes that affect stellate cell biology, including Pde1a (Phosphodiesterase 1a). Pde1a promotes fibroblast activation and extracellular matrix remodeling, leading to increased ␣SMA (53). Pde1a is increased 5 ⫾ 0.6-fold in Mist1⫺/⫺ mice and is significantly reduced in Mist1⫺/⫺ApoE-FGF21 mice, although not back to WT levels (Fig. 4D). The increase in Pde1a expression in Mist1⫺/⫺ mice is not simply due to an absence of FGF21 signaling as Fgf21⫺/⫺ mice show no difference in Pde1a levels relative to WT mice. Combined, the results confirm that FGF21 mitigates, to a certain extent, the acinar cell damage caused by the absence of MIST1. The reduction of Pde1a gene expression following FGF21 reexpression in Mist1⫺/⫺ mice confirms that some molecular, and potentially phenotypic, events that occur in these mice are not directly due to altered transcriptional regulation by MIST1. Therefore, we examined additional phenotypes that do not appear to be related to MIST1’s regulation of exocytosisspecific genes. Mist1⫺/⫺ pancreata show increased acinar cell proliferation (20), which we confirmed through IF for Ki-67, a marker for proliferating cells (Fig. 5A). Mist1⫺/⫺ApoE-FGF21 acini showed similar proliferation rates relative to WT tissue and reduced levels compared with the Mist1⫺/⫺ mice. Similar results were observed following phospho-H3 analysis (Fig. 5A). The reduced proliferation rate for Mist1⫺/⫺ApoE-FGF21 acinar cells could be a byproduct of the reduced injury follow-

ing FGF21 reexpression but may also suggest a more stable cellular phenotype. We have shown that Mist1⫺/⫺ acini rapidly revert to a progenitor-like state following induction of pancreatic injury or culturing in vitro (22). From our previous work, we identified decreased expression of Asb11 (Ankyrin-SOCS binding 11) an essential component of canonical delta-Notch signaling (8). Overexpression of Asb11 in neuronal tissue maintains progenitor populations in the brain (7, 45). If Asb11 also maintains pancreatic progenitor populations, then increased Asb11 in Mist1⫺/⫺ tissue would be consistent with the observation that Mist1⫺/⫺ acini are less differentiated and have greater potential to undergo acinar-to-duct cell metaplasia (ADM). Analysis of Asb11 expression revealed a marked increase in Mist1⫺/⫺ mice (7.1 ⫾ 0.6-fold relative to WT levels), which was reduced back to WT levels (2.6 ⫾ 0.6-fold) in Mist1⫺/⫺ApoE-FGF21 tissue (Fig. 5B; P ⬎ 0.05 between WT and Mist1⫺/⫺ApoE-FGF21 mice), supporting a potential effect of FGF21 on the differentiation potential of Mist1⫺/⫺ acinar cells. We then compared the effects of culturing on Mist1⫺/⫺ and Mist1⫺/⫺ApoE-FGF21 acini, focusing on their ability to form cyst-like structures, which represent ADM. Contrary to our expectations, Mist1⫺/⫺ApoE-FGF21 acini rapidly developed cysts upon culturing. While 10.6 ⫾ 0.1% of Mist1⫺/⫺ acini and 3.3 ⫾ 0.7% of WT acini developed cysts by 3 days, 28.0 ⫾ 1.6% of the Mist1⫺/⫺ApoE-FGF21 acinar cell clusters contained cyst structures at the same time (Fig. 5C). By day 5, 92.7 ⫾ 1.2% of Mist1⫺/⫺ApoE-FGF21 acini contained a cyst structure, compared with only 61.3 ⫾ 4.5 and 16.7 ⫾ 4.7% for Mist1⫺/⫺ and WT cultures, respectively. Similar experiments with Fgf21⫺/⫺ acini indicate that these

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Fig. 5. Restored FGF21 levels reduce proliferation but not acinar-to-duct cell metaplasia in Mist1⫺/⫺ acini. A: quantification of Ki-67- or phospho-histone 3 (pH3)-positive acinar cells as %total cell number from different genotypes (n ⫽ 3). B: qRT-PCR for Asbl1 accumulation in pancreatic tissue isolated from 4-mo-old mice; n ⫽ 7. *P ⬍ 0.05, ***P ⬍ 0.001. C: quantification of cyst-forming structures in acinar cell cultures over 9 days from the different genotypes (n ⫽ 3). In both cases, *P ⬍ 0.05 between Mist1⫺/⫺ and Mist1⫺/⫺ApoE-FGF21 cultures, **P ⬍ 0.05 between WT and Mist1⫺/⫺ or Mist1⫺/⫺ApoE-FGF21 cultures. For all assays, acronyms are WT and Mist1⫺/⫺ApoEFGF21 (Mist1⫺/⫺FGF21⫹).

Fig. 4. Reexpression of FGF21 reduces stellate cell activation found in Mist1⫺/⫺ pancreatic tissue. A–C: representative IF for smooth muscle actin cells (green; arrow) in 4-mo-old Mist1⫺/⫺ (A), Mist1⫺/⫺ApoE-FGF21 (B; Mist1⫺/⫺FGF21⫹), WT pancreatic tissue (C). Nuclei were counterstained with DAPI (blue). Arrows indicate stellate cells. Scale bar, 25 ␮m; n ⫽ 3. D: qRT-PCR for Pde1a accumulation in WT, Mist1⫺/⫺, Mist1⫺/⫺ApoEFGF21 (Mist1⫺/⫺FGF21⫹), and Fgf21⫺/⫺ pancreatic tissue; n ⫽ 7. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

acini behave like WT cultures, with only 12.9 ⫾ 7.9% of acini developing cysts after 5 days in culture. Therefore, although reexpressing FGF21 partially mitigates acinar injury, supporting a novel role for FGF21 in pancreas biology, it does not completely revert all phenotypes associated with the Mist1⫺/⫺ phenotype. The Fgf21 gene is epigenetically reprogrammed in Mist1⫺/⫺ pancreatic tissue. Since MIST1 is a transcription factor, we investigated its ability to directly regulate Fgf21 gene expression. Examination of the Fgf21 promoter from the translational start site (ATG) to the beginning of Fucosyltransferase 1

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(closest gene upstream of Fgf21 on mouse chromosome 7) identified 16 putative E box sequences (CANNTG; Fig. 6A), the DNA consensus site through which bHLH transcription factors bind (37). While several E boxes contain an internal GC sequence that is often associated with the binding of bHLH proteins, none contain the internal TA sequence that MIST1 preferentially binds. Comparative genomics using the UCSC Genome Browser (www.genome.ucsc.edu) identified two sig-

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nificant areas of conservation among mouse, rat, human, chimp, and dog sequences upstream of the transcriptional start site (Fig. 6B). Region I (⫺38 to ⫺150) showed 74% identity across species, with one E box (position ⫺83 to ⫺88) found in all five species. A second, 59-bp sequence (Region II; position ⫺1013 to ⫺1071), contained 79% identity between species, and an E box (⫺1062 to ⫺1067) conserved across all five species (Fig. 6C). A third E box within the 5=-UTR (⫹21 to

Fig. 6. Areas of conservation in the Fgf21 promoter. A: sequence of the upstream region from the Fgf21 translation start site. The first exon of the Fgf21 gene is highlighted in gray. Regions I and II are highlighted in green, and E boxes are underlined and in bold. E boxes conserved among mouse, rat, human, and dog are highlighted in yellow. B: Using the UCSC Genome browser, the Fgf21 promoter was compared among mouse, rat, human, chimp, and dog. Homology maps are shown in green for each species. Highly conserved regions of sequence identity are indicated in blue, with two regions showing significant identity (⬎70%) across all species. Sequences for Regions I (⫺38 to ⫺150 relative to transcriptional start site) and II (⫺1013 to ⫺1071) within the Fgf21 promoter are shown with putative E boxes highlighted.

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⫹26) is also conserved across species (Fig. 6A). None of the remaining E boxes were conserved. To determine whether MIST1 is recruited to the Fgf21 promoter, we assessed MIST1 occupancy along the Fgf21 gene using ChIP on pancreatic tissue. MIST1 occupancy was readily detected near the Fgf21 transcriptional start site (TSS; ⫺76 bp relative to TSS; 42.9 ⫾ 29.6-fold relative to IgG), which corresponds to Region I, as well as 3.2 kb upstream of the Fgf21 TSS (35.5 ⫾ 13.0-fold relative to IgG; Fig. 7A). No enrichment for MIST1 was observed either within the Fgf21 gene (⫹1.3 kb relative to TSS) or around Region II. As a control, similar ChIP experiments with Mist1⫺/⫺ tissue showed no MIST1 enrichment along the length of the gene. Therefore, MIST1 physically binds upstream of the Fgf21 TSS. To ascertain whether MIST1 can affect Fgf21 expression, we transiently transfected an Fgf21 promoter-luciferase reporter containing ⫺1497 to ⫹4, relative to the Fgf21 TSS with or without MIST1 into HEK-293 cells (Fig. 7B). As a control for DNA binding, we performed a similar transfection with a mutant form of MIST1 (mutMIST1), which contains a mutation in the basic region and prevents binding of MIST1 to DNA (29), The ⫺1497Fgf21 promoter contains both conserved regions and is responsive to factors promoting Fgf21 expression including PPAR␣ (18). We chose HEK-293 cells as they expressed Fgf21 but not Mist1, which would allow us to determine MIST1’s ability to enhance expression (Fig. 7C). Surprisingly, luciferase activity was decreased when MIST1 was cotransfected into the cells (Fig. 7B). Conversely, the mutMIST1 did not reduce luciferase activity under similar conditions. Next, we compared Fgf21 and Mist1 expression in cell lines and tissues to determine whether Fgf21 expression correlated with MIST1 activity. Fgf21-expressing cell lines such as NIH3T3 and HEK-293 do not express MIST1 (Fig. 7C), while a number of MIST1-expressing cells (AR42J; Fig. 7C) and tissues, including salivary glands and seminal vesicles

(Fig. 7D), do not express Fgf21. These findings suggest that although MIST1 binds the Fgf21 promoter, on its own it does not activate Fgf21 expression. Therefore, other factors must explain the loss of Fgf21 expression in Mist1⫺/⫺ pancreatic tissue. One possibility is that the absence of MIST1 leads to epigenetic reprogramming of the Fgf21 gene. Epigenetic reprogramming involves modifications to the histone complex or methylation of cytosine residues in CpG islands within gene promoters. Bisulfite sequencing, which distinguishes methylated from nonmethylated nucleic acids, showed no change in methylation at two putative CpG islands in the Fgf21 promoter between WT and Mist1⫺/⫺ pancreatic tissue (data not shown). Conversely, changes in methylation of histone 4 (H4) at lysine (K) residues 4 and 27, were readily observed by ChIP analysis (Fig. 8). Trimethylation (Me3) of H3K4 is associated with active promoters, whereas H3K27Me3 is associated with suppression of gene expression (17, 40). In WT mice, the Fgf21 promoter is enriched for both histone modifications (Fig. 8A). The presence of both active and repressive epigenetic marks is common for promoters that are rapidly activated, since they are maintained in a poised position (39). In Mist1⫺/⫺ pancreatic tissue, there is a clear shift away from enrichment for H3K4Me3 and increased enrichment of H3K27Me3, suggesting the Fgf21 gene is being suppressed (Fig. 8B). Similar analysis of the Fgf21 gene in liver shows no such change in the epigenetic program of the Fgf21 gene between WT and Mist1⫺/⫺ mice (Fig. 8C). Analysis of a closely related gene (Ppp1r15a) showed no change in epigenetic programming based on H3K4Me3 or H3K27Me3 enrichment (Fig. 8D). Whereas previous studies indicate that Fgf21 may be a target for histone deacetylases (32), ChIP-PCR showed no differences between genotypes for acetylated H3 (Fig. 8E). Importantly, this change in epigenetic programming of the Fgf21 gene was not due to differential expression of epigenetic

Fig. 7. MIST1 does not activate Fgf21 expression. A: ChIP-qPCR analysis for MIST1 enrichment on the Fgf21 promoter in mouse pancreatic tissue. Locations of expected amplicons are indicated as the midpoint of the amplicon, and values for MIST1 and control IgG antibodies are shown relative to input (n ⫽ 3). Primer sequences can be found in Table 1. B: luciferase activity of the ⫺1491Fgf21 promoter normalized to PGK Renilla luciferase activity following transfection into HEK-293 cells with an empty pcDNA3.1 vector or one expressing MIST1 or a mutated MIST1 (mutMIST1) that does not bind DNA. *P ⬍ 0.05; n ⫽ 4. C and D: representative RT-PCR amplification of Mist1 and Fgf21 in cell lines (C) and various mouse tissues (D). Note that primers for Fgf21 and Mist1 will amplify mouse, human, and rat cDNA sequences. Mrpl1 was used as a control. Pan, pancreas; SG, parotid salivary gland; ST, stomach; SV, seminal vesicle; Kid, kidney. Cell lines include AR42J (rat acinar cells), 266.6 (mouse acinar cells), ARIP (rat nonacinar cells derived from pancreas), NIH3T3 (mouse fibroblasts), HEK-293 (human embryonic kidney cells), and Panc1 (human PDAC cells). WT and Mist1⫺/⫺ (M1⫺) pancreatic RNA were included as controls. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00559.2013 • www.ajpendo.org

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Fig. 8. The Fgf21 gene is silenced in Mist1⫺/⫺ pancreatic tissue. A: schematic representation of the Fgf21 gene on mouse chromosome 7. The Fgf21 gene consists of 3 exons with the protein coding region in black. Ppp1r15a gene is found some 90 kb downstream of the Fgf21 gene. Amplicons for ChIP-qPCR of the Fgf21 (I and II) or Ppp1r15a (III) genes are indicated, and the direction of transcription is indicated by arrows. B: ChIP-qPCR for H3K4Me3 (H3K4) or H3K27Me3 (H3K27) enrichment at regions downstream (I) or upstream (II) of the Fgf21 transcriptional start site in WT and Mist1⫺/⫺ pancreatic tissue (n ⫽ 3; P ⬍ 0.05). Similar analysis in (C) WT and Mist1⫺/⫺ liver tissue (n ⫽ 2), or (D) for Ppp1r15a within the pancreas (n ⫽ 3). In all cases, error bars represent SE ⫾ mean. E: representative ChIP for acetylated H3 occupancy of the Fgf21 promoter in WT (⫹/⫹) and Mist1⫺/⫺ (⫺/⫺) pancreatic tissue.

modifying genes in the absence of MIST1. qRT-PCR showed no alterations in lysine-specific methyltransferase (Kmt2) or histone deacetylase (Hdac) genes between WT and Mist1⫺/⫺ pancreatic tissue (Fig. 9, A and B). Combined, these data suggest that the loss of Fgf21 expression is due to alterations in the epigenetic program, thereby changing the expression state of the gene from poised to suppressed. DISCUSSION

To date, the majority of studies regarding FGF21 have focused on its endocrine effects specifically related to hepatocytes or adipocytes. However, our previous work suggested a direct role for FGF21 in reducing acinar cell injury in response to experimentally induced pancreatitis. The goal of this study was to determine whether altered FGF21 signaling played a role in the phenotypes associated with loss of MIST1. MIST1 is a key factor in stabilizing the acinar cell phenotype and reducing the susceptibility to pancreatitis and pancreatic ductal adenocarcinoma (21, 22, 46). We focused on FGF21, since it may be regulated by the unfolded protein response, which is altered before and after induction of CIP in Mist1⫺/⫺ tissue (22). We found a dramatic reduction in Fgf21 expression in Mist1⫺/⫺ mice and that reexpression of FGF21 reduced the

acinar cell injury found in these animals. Interestingly, we also demonstrated that the loss of Fgf21 was not due strictly to loss of MIST1 transcriptional activity but rather to epigenetic reprogramming that silences the Fgf21 gene specifically in Mist1⫺/⫺ acinar cells. Combined, our study results shed light on the functional changes that occur in the absence of MIST1 that may contribute to disease and provide novel insight into the regulation and function of FGF21 in the exocrine pancreas. Effectors of MIST1 function may either be direct targets of its transcriptional activity or be altered due to the phenotypes that arise from the loss of MIST1. Studies to date have focused on the direct transcriptional targets of MIST1 and have identified several genes involved in acinar cell biology, including Gjb1 (encodes Cx32), Atp2c2 (encodes SPCA2), Rab3d, and Rab26 (16, 21, 43, 48). The fact that all of these genes can be linked to acinar cell exocytosis has led to a model in which MIST1 acts as a scaling factor, a type of transcription factor that enhances rather than initiates gene expression of a number of genes associated with a particular cell function (36). Our results lend support to this model in two ways. First, Mist1⫺/⫺ mice show no Cx32 accumulation specifically in MIST1expressing tissues, suggesting that MIST1 is absolutely re-

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Fig. 9. Expression of epigenetic modifying genes does not change between WT and Mist1⫺/⫺ pancreatic tissue. qRT-PCR analysis for (A) lysine (K)-specific methyltransferases Kmt2a, Kmt2b, Kmt2c, and Kmt2e, or (B) histone deacetylases Hdac1, Hdac3, and Hdac6 between WT and Mist1⫺/⫺ pancreatic tissue. C: similar analysis comparing activating transcription factor 4 (Atf4) between WT and Mist1⫺/⫺ pancreatic tissue following 4 h of saline or cerulein treatment or immediately after isolation of acinar cells (ACC). Expression is relative to Mrpl1; n ⫽ 4.

quired for initiating Gjb1 expression in these cells, which would argue against the scaling factor model. However, reexpression of FGF21 restores Cx32 plaque formation in the absence of MIST1 without increasing Gjb1 expression levels. This indicates that absence of FGF21 signaling in Mist1⫺/⫺ tissue is not the cause of decreased Gjb1 gene expression, supported by the finding that Cx32 expression and localization in Fgf21⫺/⫺ acini is similar to that observed in WT mice. In addition, the complete absence of Cx32 gap junction plaques in Mist1⫺/⫺ acini is only partly due to the loss of MIST1 enhancing Gjb1 expression, since no change in mRNA expression is observed at a time when Cx32⫹ plaques are apparent in Mist1⫺/⫺FGF21⫹ acini. We suggest that the pancreatic injury found in Mist1⫺/⫺ mice affects Cx32 stability and targeting. The maintenance of adherens junction, based on ␤-catenin localization, also supports reduced acinar cell damage. The reduction in pancreatic injury following FGF21 reexpression would allow for both Cx32 and ␤-catenin to be properly targeted to the cell membrane. Second, a number of phenotypic changes observed in Mist1⫺/⫺ mice, including increased cell proliferation and stellate cell activation, are not directly tied to acinar cell exocytosis. Previous work by our group and others has identified a number of genes involved in these processes that are differentially expressed in Mist1⫺/⫺ mice. Reexpression of FGF21 in Mist1⫺/⫺ mice restores the acinar cell proliferation rate to WT levels and reduces the number of activated stellate cells, indicative of reduced tissue damage. We also showed partial (Pde1a and Asbl1) restoration in expression levels following FGF21 reexpression, indicating that many differentially expressed genes within the Mist1⫺/⫺ tissue are not necessarily direct targets of MIST1. Therefore, it is clear that the Mist1⫺/⫺

phenotype is progressive and that the extensive injury observed in these animals is not solely due to cellular processes regulated by MIST1. This study also confirms a role for FGF21 signaling in pancreatic acinar cell biology. Although our results do not distinguish an endocrine vs. a paracrine function in acinar cell signaling, they indicate that FGF21 mitigates acinar cell injury to some extent. More importantly, our results provide novel insight into the regulation of FGF21 in pancreatic acinar cells. While MIST1 can directly bind the Fgf21 gene, it appears that it is not an activator of Fgf21 expression. We have identified decreased levels of MIST1 at times during CIP when Fgf21 levels are increasing (data not shown), which actually suggests a repressive role for MIST1 in Fgf21 regulation. Fgf21 has been identified as a target for ATF4, which is expressed in pancreatic acinar cells and further increased during CIP. We have shown that Atf4 is not activated by CIP in Mist1⫺/⫺ mice (27), supporting a potential mechanistic link between ATF4 and Fgf21. However, Atf4 levels are not affected in untreated Mist1⫺/⫺ mice, where Fgf21 levels are lower than in WT tissue. In addition, Atf4 levels do not increase following acinar cell isolation from WT pancreatic tissue, whereas Fgf21 levels do (compare Fig. 9C with Fig. 1E). Therefore, we predicted that an alternative mechanism of repression must exist. We show that the Fgf21 gene is epigenetically silenced specifically in pancreatic tissue of Mist1⫺/⫺ mice on the basis of increased enrichment of H3K27Me3 and loss of H3K4Me3. Other studies suggest that the Fgf21 gene is maintained in a repressed state by histone deacetylase (HDAC) 3 (32) or that E4BP4 can promote Fgf21 silencing through recruitment of H3K9 methylases (49). However, this is the first report of an epigenetic reprogramming of Fgf21 in vivo. To date, no such

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regulation has been reported in the liver or white adipose tissue during chronic exposure to adverse conditions. Trimethylation of H3K27 is mediated by the polycomb repressor complex (PRC) 2 (3), and Enhancer of Zeste (EZH) 2, a component of the PRC2 complex (10), is believed to be an important regulator of acinar cell regeneration following pancreatitis (34). EZH2 is also increased in PDAC (31). Importantly, examination of a number of genes encoding epigenetic modifying proteins showed no difference in expression between WT and Mist1⫺/⫺. These findings suggest that MIST1 is likely not directly regulating the expression of epigenetic modifying genes but rather that the acinar cell environment in Mist1⫺/⫺ acinar cells promotes reprogramming of genes, possibly through the differential recruitment of epigenetic modifying proteins. We have recently shown that epigenetic reprogramming, based on H3K4Me3, occurs within Mist1⫺/⫺ pancreata, affecting the activation of a subset of genes in response to CIP (35). If the chronic cell stress observed in Mist1⫺/⫺ acinar cells is the underlying stimulus to Fgf21 silencing, this suggests the potential of FGF21 being silenced by other environmental influences that promote cell stress, such as obesity. We are currently examining whether silencing of the Fgf21 gene occurs in nongenetic models of chronic injury as well as the mechanism behind the silencing. In conclusion, we have identified repression of an important signaling molecule, FGF21, resulting from the loss of MIST1 function. The loss of FGF21 contributes to increased pancreatic injury observed in these mice and highlights a potential target for mitigating damage in the tissue. Future studies will focus on translating this work to other mouse and human models of pancreatitis and define the mechanisms by which Fgf21 silencing occurs. ACKNOWLEDGMENTS We thank Gabor Varga, Sonali Mohanty, and Yufeng Li for helpful discussions regarding the content of this manuscript, and Anja Koëster for originally generating the FGF21-ApoE mouse line. We also thank Agnes Kowalik, Sami Chadi, Lucimar Ferreira, and Kira Misuraca, who provided experimental help or animal husbandry for the work, and Steve Kliewer for providing valuable reagents. We also thank Gabriel DiMattia and Thomas Drysdale for their continual intellectual support on this and other projects ongoing in C. L. Pin’s laboratory. Finally, this work would not have been possible with the support of the Children’s Health Foundation. GRANTS This work was supported by operating grant MOE 58083 from the Canadian Institutes of Health Research, as well as local support from the Children’s Health Research Institute and the Lawson Health Research Institute. R. Mehmoud was supported by a postdoctoral fellowship from the Children’s Health Foundation. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: C.L.J., R.M., S.W.L., and C.V.S. performed experiments; C.L.J., R.M., S.W.L., C.V.S., A.K., and C.L.P. analyzed data; C.L.J., R.M., S.W.L., A.K., and C.L.P. interpreted results of experiments; C.L.J., R.M., S.W.L., and C.L.P. prepared figures; C.L.J., R.M., and C.L.P. drafted manuscript; C.L.J., R.M., A.K., and C.L.P. edited and revised manuscript; C.L.J., R.M., A.K., and C.L.P. approved final version of manuscript; A.K. and C.L.P. conception and design of research.

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