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Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis.

Karki A, Humphrey SE, Steele RE, Hess DA, Taparowsky EJ, Konieczny SF - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, the Purdue Center for Cancer Research, and the Bindley Bioscience Center, Purdue University, West Lafayette, IN, 47907-2057, United States of America.

ABSTRACT
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 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.

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Establishing the Mist1CreERT/lox model system.(A) Schematic of Tam treatment and time course analysis for Mist1CreERT/lox mice. (B) Immunoblot demonstrating the absence of MIST1 protein in pancreata from Tam-treated Mist1CreER/lox mice. HSP90 was used as a loading control. (C) IF staining with anti-MIST1 confirming that the vast majority of acinar cells are MIST1 negative 7d post-Tam treatment. (D) RT-qPCR analysis of MIST1 gene targets revealing loss of MIST1 regulation post-Tam. (E) CX32 gap junctions are readily detected in pancreata from -Tam treated Mist1CreERT/lox mice but are completely absent in +Tam samples. *p ≤ 0.05; ***p ≤ 0.001.
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pone.0145724.g003: Establishing the Mist1CreERT/lox model system.(A) Schematic of Tam treatment and time course analysis for Mist1CreERT/lox mice. (B) Immunoblot demonstrating the absence of MIST1 protein in pancreata from Tam-treated Mist1CreER/lox mice. HSP90 was used as a loading control. (C) IF staining with anti-MIST1 confirming that the vast majority of acinar cells are MIST1 negative 7d post-Tam treatment. (D) RT-qPCR analysis of MIST1 gene targets revealing loss of MIST1 regulation post-Tam. (E) CX32 gap junctions are readily detected in pancreata from -Tam treated Mist1CreERT/lox mice but are completely absent in +Tam samples. *p ≤ 0.05; ***p ≤ 0.001.

Mentions: Previous studies reported that Mist1 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 s, 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 Mist1lox/lox mouse line (S3 Fig). Mist1CreERT/+ mice were crossed to Mist1lox/+ animals to generate Mist1CreERT/lox offspring where one Mist1 allele expressed CreERT2 while the other Mist1 allele, engineered with an N-terminal BT-tag and a C-terminal MYC-tag, was flanked by LoxP sites (S3 Fig). Treatment of 8 wk Mist1CreERT/lox mice with tamoxifen (Tam) led to efficient recombination and rapid loss of MIST1 protein in 99.6% acinar cells as early as 24h post-Tam (Fig 3A–3C). 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 Mist1CreERT/lox mice (Fig 3E).


Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis.

Karki A, Humphrey SE, Steele RE, Hess DA, Taparowsky EJ, Konieczny SF - PLoS ONE (2015)

Establishing the Mist1CreERT/lox model system.(A) Schematic of Tam treatment and time course analysis for Mist1CreERT/lox mice. (B) Immunoblot demonstrating the absence of MIST1 protein in pancreata from Tam-treated Mist1CreER/lox mice. HSP90 was used as a loading control. (C) IF staining with anti-MIST1 confirming that the vast majority of acinar cells are MIST1 negative 7d post-Tam treatment. (D) RT-qPCR analysis of MIST1 gene targets revealing loss of MIST1 regulation post-Tam. (E) CX32 gap junctions are readily detected in pancreata from -Tam treated Mist1CreERT/lox mice but are completely absent in +Tam samples. *p ≤ 0.05; ***p ≤ 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696804&req=5

pone.0145724.g003: Establishing the Mist1CreERT/lox model system.(A) Schematic of Tam treatment and time course analysis for Mist1CreERT/lox mice. (B) Immunoblot demonstrating the absence of MIST1 protein in pancreata from Tam-treated Mist1CreER/lox mice. HSP90 was used as a loading control. (C) IF staining with anti-MIST1 confirming that the vast majority of acinar cells are MIST1 negative 7d post-Tam treatment. (D) RT-qPCR analysis of MIST1 gene targets revealing loss of MIST1 regulation post-Tam. (E) CX32 gap junctions are readily detected in pancreata from -Tam treated Mist1CreERT/lox mice but are completely absent in +Tam samples. *p ≤ 0.05; ***p ≤ 0.001.
Mentions: Previous studies reported that Mist1 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 s, 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 Mist1lox/lox mouse line (S3 Fig). Mist1CreERT/+ mice were crossed to Mist1lox/+ animals to generate Mist1CreERT/lox offspring where one Mist1 allele expressed CreERT2 while the other Mist1 allele, engineered with an N-terminal BT-tag and a C-terminal MYC-tag, was flanked by LoxP sites (S3 Fig). Treatment of 8 wk Mist1CreERT/lox mice with tamoxifen (Tam) led to efficient recombination and rapid loss of MIST1 protein in 99.6% acinar cells as early as 24h post-Tam (Fig 3A–3C). 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 Mist1CreERT/lox mice (Fig 3E).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, the Purdue Center for Cancer Research, and the Bindley Bioscience Center, Purdue University, West Lafayette, IN, 47907-2057, United States of America.

ABSTRACT
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 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.

Show MeSH
Related in: MedlinePlus