Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis

Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis—events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional null mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.


Introduction
The majority of the exocrine pancreas consists of acinar cells which are tasked with synthesizing, modifying, packaging and secreting vast quantities of pro-digestive enzymes (zymogens) into the duodenum to maintain metabolic homeostasis for the organism [1][2][3][4]. The ability of is a transient event. As cells recover from AP damage, the Mist1 locus is transcriptionally reactivated and MIST1 protein levels are restored. Despite this re-expression, analysis of conditional Mist1 knock-out (Mist1 cKO) mice revealed that Mist1-deficient pancreata responded similarly to AP treatment as control animals, with an initial damage phase that was rapidly followed by recovery. We next examined if sustained Mist1 expression (iMist1) in genetically engineered mice could alleviate AP-induced damage. Surprisingly, in iMist1 animals, AP produced a dramatic phenotype of significant tissue damage followed by cell death in cells that expressed iMist1. Despite the extreme damaged response in iMist1 pancreata, the pancreas partially recovered by regenerating healthy acini from the small minority of acinar cells that failed to activate the iMist1 transgene. We conclude that silencing Mist1 expression is a critical event for acinar cells to survive an AP episode where down regulating MIST1 activity may allow cells to suppress their secretory function and permit a window of cell proliferation. However, to fully re-establish a functional acinar cell capable of efficient exocytosis, the Mist1 gene must be reactivated to scale up the appropriate intracellular machinery that generates secretory vesicles, expands the ER and establishes cell communication via gap junction signaling. The importance of MIST1 to these events suggests that devising strategies to modulate transcriptional networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.

Mouse Strains and Genotyping
Mist1 CreERT/+ and LSL-Mist1 myc (iMist1 myc ) mice have been described previously [26,33,58]. Mist1 lox/+ mice were produced by generating a Mist1 targeting vector containing loxP sites flanking the entire Mist1 coding region within exon 2 [62]. In addition, a small biotin-tag [63] and MYC-tag were added to the N-terminus and C-terminus of the MIST1 open reading frame, respectively. ES cell electroporation and blastocyst injections were performed by the Purdue University Transgenic Mouse Core Facility. Mist1 conditional knock-out (Mist1 cKO) mice (Mist1 CreERT/lox ) were produced by crossing Mist1 CreERT/+ mice to Mist1 lox/+ animals. Induction of CreER T2 activity was accomplished by administrating tamoxifen (200 μl of 20 mg/ml) via oral gavage to adult mice (6-8 wk). Genotyping primer sets are listed in S1 Table. All experiments were performed with mice on a C57BL/6 background and all animal studies were conducted in strict compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Purdue University IACUC guidelines. The protocol was approved by the IACUC Committee of Purdue University (Approval Number 1110000037).

Histology and Immunohistochemistry
Mouse pancreata were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and stained using conventional histological techniques. Tissue sections (5 μm) were deparaffinized and retrieved using the 2100-Retriever (Electron Microscopy Sciences, Hatfield, PA) with antigen unmasking solution (Vector Laboratories, Burlingame, CA). For IHC, sections were incubated in 3% H 2 O 2 for 5 min to block endogenous peroxidase activity followed by 1 hr in M.O.M. blocking reagent (Vector Laboratories, Burlingame, CA). Tissue sections were incubated in primary antibodies for 1 hr at room temperature. Biotinylated secondary antibodies were used at 1:200 dilution for 20 min at room temperature. IHC development was performed using Vector reagents and DAB (diamonibenzidine) peroxidase substrate (Vector Labs, Burlingame, CA). Secondary antibodies for immunofluorescence utilized avidin-conjugated Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 555, Oregon Green 488, and Alexa Avidin Cy5.5 (Invitrogen, Camarillo, CA). Detailed information on the primary antibodies used in this study is provided in S2 Table   Immunoblots Pancreata samples were lysed using a Tissue Tearor Homogenizer (Biospec Products, Inc) in ice-cold RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors and sodium orthovanadate. Protein extracts (30 μg) were resolved on 12% SDS-PAGE and transferred onto PVDF membranes (Bio Rad, Hercules, CA). Membranes were blocked overnight at 4°C in 5% non-fat dry milk prepared in Tris-buffered saline plus 0.1% Tween 20. Membranes were incubated in primary antibodies at room temperature for 1 hr followed by three 10 min washes and then incubated in horseradish peroxidase (HRP) conjugated secondary antibodies at 1:5000 dilution at room temperature for 30 min. Immunoblots were visualized on X-ray films using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, Waltham, MA) and quantified using ImageJ (normalized to the HSP90 or S6 signal) or visualized and quantified on a ChemiDoc Touch Imaging System (Bio Rad, Hercules, CA) using the HSP90 or S6 signal for normalization.

RNA Expression Analysis
Total cellular RNA from pancreata was isolated using the E.Z.N.A midi kit (Qiagen, Valencia, CA). For quantitative RT-PCR analysis, reverse transcription using 1 μg RNA was performed with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA), followed by gene amplification using FastStart Universal SYBR Green (Roche Applied Science, Indianapolis, IN) and a Roche LightCycler 96 thermocycler (Roche Diagnostics Corporation, Indianapolis, IN). All individual reactions were performed in duplicate and all genes were normalized to 18S ribosomal RNA or to the ribosomal transcript Rplp0. Quantitative RT-PCR primers are listed in S3 Table. Graph-Pad Prism 6 (La Jolla, CA) was used to generate graphs included in this study. Statistical analyses are presented as standard error of the mean. P values were determined using two-tailed unpaired tests.

Microscopy and Image Analysis
All H&E, IHC and IF images were taken using an Olympus BX51 upright microscope and a DP80 high resolution camera (Olympus Life Science). Images representing INSULIN+, AMY-LASE+, MYC+, MYC-, BrdU+, BrdU-, etc. areas were quantified using ImageJ (NIH) from 12-15 random 10x fields from ! 3 sections at different pancreas depths per mouse. For each image, individual pixels were converted to μm to establish the appropriate area in μm 2 . All calculations were performed in GraphPad Prism 6. Statistical analyses are presented using standard error of the mean. P values were determined using two-tailed unpaired tests.
To evaluate the importance of MIST1 during acinar metaplasia, we characterized Mist1 expression during the damage and subsequent recovery phases of AP, a known driver of PDAC tumor development [15,18,19,21]. For these studies, Mist1 CreERT/+ mice were used as controls as all subsequent mouse lines contained the Mist1 CreERT knock-in allele [26,58]. Standard caerulein treatment ( Fig 1A) of 8 week Mist1 CreERT/+ mice led to significant and rapid damage to the exocrine acinar cells. As early as 6h post-AP, acinar lumens were distended and zymogen granules were rapidly lost ( Fig 1B and S1A Fig). By 1d post-AP, significant increases in edema and inflammatory cell infiltrates were observed, accompanied by extensive formation of KERATIN19 (K19)+/AMYLASE (AMY)+ ADM lesions. Expression of CLUSTERIN, a known marker of acinar cell damage [67,68], also was greatly elevated at 6h post-AP (Fig 1B,1C and S1A,S1B Identical ADM responses were obtained with caerulein-treated wild-type mice (data not shown). Despite significant development of ADM lesions upon AP induction, AP metaplasia was transient as lesions resolved 4d-10d post-AP. In all cases, Clusterin, K19 and Sox9 transcript and protein levels returned to their low control states while acinar markers (Amylase, Trypsinogen, Carboxypeptidase) re-established high expression thresholds ( Fig  1B-1D and S1A,S1B Fig).
The major phenotype associated with caerulein-induced AP is loss of acinar cell integrity [22,[69][70][71]. Because the transcription factor MIST1 is critical for maintaining acinar cell polarity and function, we examined if MIST1 protein accumulation was altered in AP mice. As shown in Fig 2A and 2B, high levels of MIST1 protein were detected in all control acinar cells, whereas duct and islet cells remained MIST1 negative (S2 Fig). However, in AP mice, Mist1 transcripts and protein were rapidly lost in damaged acinar cells (Fig 2A-2C). The absence of MIST1 was observed 6h-2d post-AP during the period corresponding to the major time frame for ADM lesion induction. Nonetheless, as mouse acinar cells recovered (4d-10d post-AP), Mist1 transcript and protein levels greatly increased, achieving levels that were comparable to those observed in control acinar cells. The transient change in Mist1 transcripts and protein during the AP response was also reflected in the expression profiles of known MIST1 target genes [33,44,56]. Transcripts from MIST1-induced genes Atp2c2, Copz2 and Rab3d were reduced during the 6h-2d post-AP period while transcripts from MIST1-repressed genes (e.g.,

Rnd2
) were up-regulated ( Fig 2D). Thus, transient silencing of Mist1 influences a number of key events associated with acinar cell integrity. These results suggest that the process of silencing and then re-expressing Mist1 may be critical in allowing the exocrine pancreas to properly recover from an acute pancreatitis episode.

Generation and Characterization of Mist1 lox/lox Mice
Previous studies reported that Mist1 null animals exhibited a pronounced AP phenotype, suggesting that the absence of MIST1 sensitizes acinar cells to an AP episode [70,72,73]. However, because these studies could only use germ line Mist1 nulls, it was not possible to establish if the enhanced AP phenotype was due to embryonic loss of MIST1 protein or was the result of inducing AP in already damaged adult pancreata. Thus, to directly test if MIST1 protein is required for acute pancreatitis recovery, we generated and characterized a conditional . Deletion of Mist1 also led to significant changes in the expression patterns of MIST1 target genes. As predicted, expression of Atp2c2 and Cx32 decreased while Rnd2 gene transcripts (which are normally repressed by MIST1 protein) increased following Tam treatment (Fig 3D). Similarly, MIST1-regulated CX32 gap junctions [33,49] were rapidly lost upon Tam treatment of Mist1 CreERT/lox mice (Fig 3E). To determine if AP-induction in Mist1 CreERT/lox animals produced a recovery delay when compared to Mist1 CreERT/+ mice, Mist1 CreERT/lox animals were treated with Tam (to delete the Mist1 coding region) (Mist1 cKO) and then induced with caerulein to generate an AP response ( Fig 4A). As expected, control and AP-treated Mist1 cKO mice failed to express MIST1 protein ( Fig 4C). Caerulein injections in Mist1 cKO animals elicited strong edema, inflammatory cell infiltrates and extensive ADM lesions as early as 6h post-AP (Fig 4B and S4A Fig). ADM was accompanied by significant increases in Clusterin, K19 and Sox9 transcript and protein levels with a concomitant decrease in Amylase and Carboxypeptidase levels (Fig 4B-4D and S4A,S4B Fig). As with Mist1 CreERT/+ mice, the ADM phenotype was transient and the Mist1 cKO pancreas returned to a relatively normal status by 10d post-AP, although Mist1 cKO acini remained defective in acinar cell polarity and organization due to the absence of MIST1 protein. Surprisingly, with the exception of sustained elevated SOX9 protein levels at 10d post-AP, there was little difference between the AP responses for Mist1 CreERT/+ (Fig 1) and Mist1 cKO animals (Fig 4).
The ability of Mist1 cKO pancreata to recover from an acute pancreatitis episode with the same kinetics as Mist1 CreERT/+ mice was surprising given previous reports showing that Mist1 null pancreata exhibited an enhanced AP response [70,72,73]. The main difference between the two models is that with germline Mist1 -/mice, the pancreas is significantly disorganized and defective by 8 wk of age [48]. In contrast, Mist1 CreERT/lox mice allow us to delete the Mist1 allele in adult animals and induce AP prior to the development of overt pancreas damage caused by the absence of MIST1. Thus, to establish if short versus long-term loss of MIST1 activity differentially influences AP responses, Mist1 CreERT/lox animals were given Tam and then treated with caerulein at 1 week post-Tam or 8 week post-Tam. As shown in Fig 4E, even in the absence of AP, Mist1 cKO pancreata at 8 week post-Tam exhibited early signs of ADM, with large increases in SOX9 and K19 protein levels (compare -AP 1 week versus 8 week). The increase in ductal gene expression reflected the ADM damage response that was observed in adult Mis-t1 CreERT/CreERT (Mist1 null) animals where the Mist1 locus was absent in the germline. Interestingly, AP episodes in 1 week versus 8 week post-Mist1 deletion did not reveal a significant difference in how the pancreas responded to this acute damage (Fig 4E). In all cases, 1 week and 8 week post-tam treated mice still managed to recover from the bulk of AP-induced damage by 10d post-AP (data not shown). Taken together, we conclude that the absence of MIST1 protein in adult acinar cells has little impact in allowing cells to recover from acute pancreatitis.

Preventing Mist1 Gene Silencing Significantly Alters the Acinar AP Response
Our studies have shown that Mist1 expression is transiently silenced during the peak of AP damage and that Mist1 re-expression is not required for the pancreas to recover from an AP episode. Nonetheless, given the importance of MIST1 to normal acinar cell polarity and secretory function [33, 46-49, 72, 74], we investigated if sustained MIST1 protein expression could be used to limit the initial AP damage response. Previous studies have shown that formation of ADM and PanIN lesions is significantly attenuated when Mist1 expression is maintained in the presence of oncogenic KRAS G12D [26,39]. Therefore, we hypothesized that a similar lessening of AP damage might be achieved by maintaining MIST1 transcriptional activity. For these studies, we utilized a Cre-inducible LSL-Mist1 myc (iMist1 myc ) transgenic mouse model (S5 Fig) [33] and generated Mist1 CreERT/+ /iMist1 myc offspring. Administering Tam to Mist1 CreERT/+ / iMist1 myc mice induced iMist1 myc transgene expression in 94.7% pancreatic acinar cells ( Fig  5A-5C). Despite elevated levels of MIST1, Mist1 CreERT/+ /iMist1 myc mice exhibited a completely normal pancreas phenotype with no significant changes in the expression of acinar and ductal genes (Fig 5D, data not shown) [33].
We next induced iMist1 myc expression by treating Mist1 CreERT/+ /iMist1 myc mice with Tam, followed by PBS (control) or caerulein to initiate an AP phenotype (Fig 6A). Surprisingly, instead of attenuating the AP response, Mist1 CreERT/+ /iMist1 myc mice exhibited enhanced  (Fig 6B and S6A Fig). By 2d-4d post-AP, the majority of acini structures were grossly altered with disorganized and distended lumens, a severe absence of eosinophilic zymogens, sustained elevated CLUSTERIN levels, and a large accumulation of infiltrating cells that included CD45-+ immune cell populations (Fig 6B,6C and S6A-S6C Fig). During this period, the epithelial tissue mass was largely replaced by VIMENTIN+ and alpha-SMOOTH MUSCLE ACTIN (SMA) + stromal cells (Fig 6C,6D and S6C Fig). The tissue also exhibited an increased islet density as the normal tissue mass that occupied space between available islets decreased, leaving the majority of the pancreas consisting of ductal, stromal and islet cells (Figs 6C and 7A). Protein immunoblots and RT-qPCR analyses revealed a typical AP damage profile with accumulation of CLUSTERIN protein, decreased expression of acinar gene products and increased Sustained Mist1 Expression Blocks AP Recovery expression of duct gene products over the 6h-2d post-AP period (Fig 7B-7D). However, by 7d-10d post-AP, despite reduced CLUSTERIN levels, ADM markers did not recover. Acinar genes (Amy, Cpa) remained suppressed while duct genes (K19, Sox9) continued to be expressed ( Fig  7B-7D). Further analysis of these animals revealed a greatly decreased AMY+ acinar cell mass.  At 2d and 4d post-AP, the vast majority of AMY+ acini co-expressed K19 in ADM structures (Fig 7D). Similarly, MIST1+ acinar cells were greatly decreased while stromal cells became more prominent within the exocrine tissue (Figs 6C, 7A, 7D and S6C Fig).
The inability of iMist1 myc mice to recover from AP damage by 7d prompted us to examine animals at extended times. During 7d-10d post-AP, iMist1 myc pancreata were grossly reduced in size (S7A Fig) with no evidence of normal acini structures. Instead, the tissue was composed of loose connective tissue containing VIMENTIN+ fibroblasts, CD45+ immune cells and areas of edema (S7A Fig). Within the remaining pancreas tissue, we observed small pockets of epithelial ADM structures that exhibited elevated levels of CLUSTERIN and retained co-expression of AMY and K19 (Fig 8A and S7A and S8 Figs). However, the number of AMY+ acinar cells greatly decreased over this time period with only small groupings of acinar cells remaining at 7d post-AP (Fig 8B and 8C). During this time frame there was a significant increase in cleaved CASPASE 3+/AMY+ epithelial cells, suggesting that cell death was primarily responsible for the vivid loss of acini structures (Fig 9A and S9 Fig). Over the ensuing 3-8 weeks post-AP iMist1 myc pancreata underwent a significant recovery as healthy acinar tissue began to appear in the disrupted organs (S7B Fig). Areas of ADM were replaced with relatively normal acini that were AMY+ and CLUSTERIN negative (Fig 8A-8C). Interestingly, lineage-tracing revealed that the majority of the recovered acini were MIST1 myc negative. This was particularly evident in the later (3-8 wk post-AP) times. Quantification of these tissues showed that approximately 75% of AMY+ acinar cells did not express the iMIST1 myc protein (Fig 9B). The increase in AMY+/MYC-acinar cells was exclusively due to an increase in cell proliferation of the MYC-population. At 3w post-AP there was an 18.7-fold increase in BrdU-labelled cells when compared to control pancreas samples. Importantly, of the regenerating cell population >90% BrdU+ cells were MYC- (Fig 9C). At 8w post-AP pancreata also accumulated small amounts of adipose tissue that typically associated with the periphery of the organ (Fig 8A). However, the fat cells were always MYC-, demonstrating that they did not arise via an acinar cell transdifferentiation event. Taken together, these results show that sustained MIST1 protein is detrimental to AP recovery and that the iMist1 myc pancreas recovers from an AP episode by relying on the small percentage of acinar cells that failed to initially activate iMist1 myc expression, allowing this population to re-enter a proliferative state and repopulate the organ. We conclude that sustained Mist1 expression does not alleviate the initial AP damage and instead is detrimental to maintaining a healthy acinar cell state under AP conditions.

Discussion
MIST1 is a bHLH transcription factor expressed exclusively in exocrine secretory cells, including pancreatic acinar, salivary acinar and stomach zymogenic cells [48,51,53,74,75]. A number of studies have shown that MIST1 is critical to establishing intracellular apical-basal polarity, appropriate secretory vesicle formation, expansion of the ER and the ability of cells to exhibit proper regulated exocytosis of pro-digestive enzymes [33, 46-49, 56, 74]. Additionally, MIST1 is necessary for maintaining appropriate protein synthesis and processing rates when cells are under ER stress [45,52]. In all cases, defects in MIST1 activity greatly impact the secretory function of these organs.
The importance of the MIST1 transcriptional network also has been defined in pancreatic and stomach cancer. In both systems, silencing of Mist1 gene expression is an early event associated with metaplasia of stomach zymogenic and pancreatic acinar cells [26,27,[58][59][60]. Indeed, Mist1 silencing is one of the first events associated with Kras-induced pancreatic ductal adenocarcinoma (PDAC) with MIST1 negative acinar cells exhibiting early activation of EGFR signaling and downstream MAPK pathways [26,27]. Similarly, Mist1-deficient acinar cells are highly sensitized to Kras transformation, suggesting that MIST1 plays a tumor suppressive role in the adult pancreas [26,39]. In support of this hypothesis, sustained Mist1 expression in the presence of oncogenic KRAS G12D dramatically prevents PanIN/PDAC development [39]. A similar phenotype has been shown for the bHLH transcription factor PTF1A where deletion of Ptf1a also sensitizes cells to PDAC formation [40]. Thus, bHLH factors are essential for maintaining quiescent, healthy acinar cells. Indeed, altering the bHLH transcriptional network can force human PDAC tumor cells to redifferentiate into functional acinar cells [76]. Lineage tracing strategies have confirmed that mouse and human PDAC can develop from adult acinar cells upon Kras G12D and other oncogenic or tumor suppressor gene mutations [58,77,78]. However, despite the presence of a KRAS G12D driver, most acinar cells remain refractile to transformation unless secondary stressors are placed upon the cells [28,79]. Although loss of Mist1 can be a secondary driver to PDAC development, there is little evidence that homozygous deletion of Mist1 alleles occurs in PDAC patients. Instead, other pathways that result in decreased Mist1 expression could be responsible for enhancing PDAC development. For this reason, we investigated how pancreatitis, a known risk factor for PDAC [12][13][14], influences Mist1 gene expression and activity and ultimately the development of ADM lesions, the precursors to PanIN/PDAC progression. Our studies revealed that the Mist1 locus is transiently silenced during the initial damage stage of AP. The Mist1 gene continues to be repressed as acinar cells enter an early recovery phase during which a significant increase in cell proliferation aids the organ in regenerating. However, as this recovery continues, Mist1 transcripts and protein return to normal levels, allowing the restored acinar tissue to resume normal secretory activity. This is in contrast to instances where AP damage is combined with KRAS G12D . In this setting, ADM and PanIN lesions never recover Mist1 expression, suggesting that KRAS signaling events permanently inhibit the Mist1 gene in a cancer setting.
Despite re-expression of Mist1 following an AP episode, MIST1 is not necessary for acinar cells to recover from AP damage. Mist1 cKO acini recovered with similar kinetics as observed for Mist1 +/+ and Mist1 +/acinar cells, although Mist1 cKO cells continued to exhibit the secretory defects ascribed to Mist1 deficient cells. The similar response of Mist1 +/and Mist1 cKO pancreata to AP was surprising given that previous studies have shown that Mist1 -/-(Mist1 KO ) Sustained Mist1 Expression Blocks AP Recovery mice display an increased sensitivity to AP with amplified damage responses and a delay in regeneration [70]. Related studies have shown that Mist1 KO pancreata are highly prone to ethanol-induced pancreas damage [80], suggesting that the absence of MIST1 sensitizes acinar cells to general stress/insult events. The apparent disparity between these reports and our current results is likely due to differences in the Mist1 model systems. In the case of Mist1 KO mice, the developing and adult pancreas always lacks MIST1 protein, leading to a significantly damaged acinar cell state in post-weaned animals [33,48]. Indeed, the enhanced stress and cell damage associated with Mist1 KO pancreata highly sensitizes the organ to KRAS G12D -induced transformation events [26,39]. In contrast, Mist1 cKO animals allow for the conditional deletion of the Mist1 loci in adult animals so that episodes of AP occur in Mist1 null, but otherwise healthy cells. This new model allows for the direct examination of the role of MIST1 in AP recovery in the absence of the long-term stress and injury conditions associated with germ-line Mist1 KO mice. Thus, we show that deleting Mist1 just prior to induced AP has little effect on pancreas recovery, suggesting that the increased sensitivity of Mist1 KO pancreata to AP was likely due to the prior damaged status of the Mist1 KO organ. In support of this hypothesis, Mist1 cKO mice expressed increased ADM markers over time that approached levels observed in Mist1 KO animals. Interestingly, Mehmood et al. [73] recently showed that germ-line Mist1 KO pancreata are enriched for H3K4Me3 active epigenetic marks on select genes that function within pancreatitis and PDAC pathways. Several of these genes are differentially expressed in Mist1 KO animals in response to AP damage [73], demonstrating that the chronic damage and stress associated with germ-line MIST1 deficiency results in key epigenetic changes that prime cells to increased sensitivity to AP and PDAC tumor formation. Thus, we now show that the absence of MIST1 per se is not sufficient to produce the increased sensitivity to disease states. Rather, it is the general damage and stress conditions associated with germ-line Mist1 KO acinar cells that lead to increased AP responses and PDAC development.
Given that MIST1 is critical for maintaining a healthy acinar cell state and Mist1 gene expression is transiently silenced during AP episodes, we investigated if sustained MIST1 activity could attenuate the initial damage response. Surprisingly, acinar cells that were prevented from down-regulating Mist1 gene expression in the early stages of AP underwent CASPASE-3 dependent apoptosis, leaving the organ grossly reduced in size with large numbers of infiltrating immune and stromal cells occupying vast areas of the pancreas. Over the initial weeks post-AP, the number of AMYLASE expressing acinar cells declined dramatically and most of the remaining cells were assembled into small acini that lacked large accumulations of zymogen granules. Sustained iMist1 expression also kept the majority of rare surviving cells in a quiescent state, most likely due to MIST1 controlling high p21 Cip1/Waf1 levels [46]. This is in sharp contrast to what has been shown for PanIN/PDAC formation in Mist1 KO /Kras G12D pancreata [26,39]. Here, sustained iMIST1 activity prevents PanIN development but with no signs of cell death [39]. Thus, downstream KRAS signaling pathways likely provide a survival benefit to acinar cells that retain MIST1 protein during initial ADM transitions.
Despite these widespread deficiencies, iMist1 organs did slowly recover functional acini over time with lineage-tracing confirming that the majority of acinar cells at 8 weeks post-AP were descendants of the small percentage of cells that failed to activate expression of the LSL-Mist1 myc transgene during the initial tamoxifen induction. These normal (MYC-) acinar cells that silenced Mist1 expression during AP were able to reactive the endogenous Mist1 gene and recover from damage. Indeed, these cells regenerated and repopulated much of the damaged pancreas in this model system. We propose that silencing Mist1 expression is a critical event that permits acinar cells to survive an AP episode (Fig 10). Down-regulating MIST1 activity may allow cells to suppress secretory functions and p21 Cip1/Waf1 levels and permit a window of cell proliferation. Once established, the Mist1 gene is then reactivated so that cells have the appropriate intracellular machinery to assemble their secretory vesicles, expand the ER, communicate via CX32-containing gap junctions, and resume efficient exocytosis functions. Thus, AP damage and recovery phases involve key transcriptional networks that control the terminal differentiation and maturation status of these specialized secretory cells. Future studies will be geared towards understanding the regulatory mechanisms that control Mist1 expression in both AP and PDAC disease states with a long-term goal of devising strategies to modulate transcriptional networks that could alleviate clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.