Christopher L. Pin

The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity

The pancreas is a complex organ that consists of separate endocrine and exocrine cell compartments. Although great strides have been made in identifying regulatory factors responsible for endocrine pancreas formation, the molecular regulatory circuits that control exocrine pancreas properties are just beginning to be elucidated. In an effort to identify genes involved in exocrine pancreas function, we have examined Mist1, a basic helix-loop-helix transcription factor expressed in pancreatic acinar cells. Mist1-null (Mist1KO) mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation. The exocrine disorganization is accompanied by increases in p8, RegI/PSP, and PAP1/RegIII gene expression, mimicking the molecular changes observed in pancreatic injury. By 12 m, Mist1KO mice develop lesions that contain cells coexpressing acinar and duct cell markers. Analysis of the factors involved in cholecystokinin (CCK) signaling reveal inappropriate levels of the CCK receptor A and the inositol-1,4,5-trisphosphate receptor 3, suggesting that a functional defect exists in the regulated exocytosis pathway of Mist1KO mice. Based on these observations, we propose that Mist1KO mice represent a new genetic model for chronic pancreas injury and that the Mist1 protein serves as a key regulator of acinar cell function, stability, and identity.

Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma

Despite the prevalence of oncogenic Kras mutations in the earliest stages of pancreatic ductal adenocarcinoma, the cellular compartment in which oncogenic Kras initiates tumorigenesis remains unknown. To address this, we have gene targeted KrasG12D into the open reading frame of Mist1, a basic helix-loop-helix transcription factor that is expressed during pancreatic development and required for proper pancreatic acinar organization. Although the pancreata of Mist1(KrasG12D/+) mutant mice predictably exhibited acinar metaplasia and dysplasia, the frequent death of these mice from invasive and metastatic pancreatic cancer with mixed histologic characteristics, including acinar, cystic, and ductal features, was unexpected and in contrast to previously described mutant mice that ectopically expressed the Kras oncogene in either acinar or ductal compartments. Interestingly, many of the mutant mice developed hepatocellular carcinoma, implicating Mist1(KrasG12D/+) cells in both pancreatic and hepatic neoplasia. Concomitant Trp53+/- mutation cooperated with Mist1(KrasG12D/+) to accelerate lethality and was associated with advanced histopathologic findings, including parenchymal liver metastasis. These findings suggest that Mist1-expressing cells represent a permissive compartment for transformation by oncogenic Kras in pancreatic tumorigenesis.

Fibroblast Growth Factor 21 Reduces the Severity of Cerulein-Induced Pancreatitis in Mice

BACKGROUND & AIMS Fibroblast growth factor 21 (FGF21) acts as a hormonal regulator during fasting and is involved in lipid metabolism. Fgf21 gene expression is regulated by peroxisome proliferator-activated receptor (PPAR)-dependent pathways, which are enhanced during pancreatitis. Therefore, the aim of this study was to investigate FGF21s role in pancreatic injury. METHODS Fgf21 expression was quantified during cerulein-induced pancreatitis (CIP) or following mechanical or thapsigargin-induced stress through Northern blot analysis, in situ hybridization, and quantitative reverse transcription polymerase chain reaction. FGF21 protein was quantified by Western blot analysis. Isolated acinar cells or AR42J acinar cells were treated with recombinant FGF21 protein, and extracellular regulated kinase 1/2 activation was examined. The severity of CIP was compared between wild-type mice and mice overexpressing FGF21 (FGF21Tg) or harboring a targeted deletion of Fgf21 (Fgf21(-/-)). RESULTS Acinar cell Fgf21 expression markedly increased during CIP and following injury in vitro. Purified FGF21 activated the extracellular regulated kinase 1/2 pathway in pancreatic acinar cells. The severity of CIP is inversely correlated to FGF21 expression because FGF21Tg mice exhibited decreased serum amylase and decreased pancreatic stellate cell activation, whereas Fgf21(-/-) mice had increased serum amylase and tissue damage. The expression of Fgf21 was also inversely correlated to expression of Early growth response 1, a proinflammatory and profibrotic transcription factor. CONCLUSIONS These studies suggest a novel function for Fgf21 as an immediate response gene protecting pancreatic acini from overt damage.

Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis

Mist1 is a basic helix‐loop‐helix transcription factor that represses E‐box‐mediated transcription. Previous studies have suggested that the Mist1 gene is expressed in a wide range of tissues, although a complete characterization of Mist1 protein accumulation in the adult organism has not been described. In an effort to identify specific cell types that contain the Mist1 protein, antibodies specific for Mist1 were generated and used in Western blot and immunohistochemical assays. Our studies show that the Mist1 protein is present in many different tissues but that it is restricted to cell types that are exclusively secretory in nature. Pancreatic acinar cells, serous or seromucous cells of the salivary glands, chief cells of the stomach, and secretory cells of the prostate and seminal vesicle show high levels of Mist1 protein, whereas nonserous exocrine cells, including the mucus‐producing cells of the salivary glands, remain Mist1 negative. These results identify Mist1 as the first transcription factor that exhibits this unique serous‐specific expression pattern and suggest that Mist1 may have a key role in establishing and maintaining a pathway responsible for the exocytosis of serous secretions. Anat Rec 259:157–167, 2000.

Transcriptional Profiling of Antigen-Dependent Murine B Cell Differentiation and Memory Formation

Humoral immunity is characterized by the generation of Ab-secreting plasma cells and memory B cells that can more rapidly generate specific Abs upon Ag exposure than their naive counterparts. To determine the intrinsic differences that distinguish naive and memory B cells and to identify pathways that allow germinal center B cells to differentiate into memory B cells, we compared the transcriptional profiles of highly purified populations of these three cell types along with plasma cells isolated from mice immunized with a T-dependent Ag. The transcriptional profile of memory B cells is similar to that of naive B cells, yet displays several important differences, including increased expression of activation-induced deaminase and several antiapoptotic genes, chemotactic receptors, and costimulatory molecules. Retroviral expression of either Klf2 or Ski, two transcriptional regulators specifically enriched in memory B cells relative to their germinal center precursors, imparted a competitive advantage to Ag receptor and CD40-engaged B cells in vitro. These data suggest that humoral recall responses are more rapid than primary responses due to the expression of a unique transcriptional program by memory B cells that allows them to both be maintained at high frequencies and to detect and rapidly respond to antigenic re-exposure.

Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1

Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26 (Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, we examined Cx26 and Cx32 gene activities in mice lacking the basic helix-loop-helix transcription factor Mist1 (Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice revealed a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription was greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remained unaffected. However, in the absence of Cx32 protein, Cx26 did not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirmed that Mist1 transcriptionally regulates expression of the Cx32 gene. We conclude that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted.

Mist1 is necessary for the establishment of granule organization in serous exocrine cells of the gastrointestinal tract

Establishing a pool of granules at the luminal border is a key step during exocrine cell development in the pancreas and is necessary for efficient release of digestive enzymes through regulated exocytosis. Several proteins have been linked to maintaining granule organization, but it is unclear which regulatory mechanisms are necessary to establish organization. Based on temporal and spatial expression, the transcription factor Mist1 is an excellent candidate, and analysis of mice that do not express Mist1 (Mist1KO) reveal disrupted cell morphology in adult pancreatic acini. To address Mist1s role in establishing granule location, we have characterized the organization of pancreatic acini throughout development in Mist1KO mice. Using various histological approaches, we have determined that correct granule organization is never established in pancreatic acini of Mist1KO mice. Further examination indicates that this disruption in granule targeting may be the primary defect in Mist1KO mice as granule organization is affected in other serous exocrine cells that normally express Mist1. To identify a mechanistic link between granule targeting and the loss of Mist1 function, intercellular junctions and the expression of Rab3D were assessed. While both of these factors are affected in Mist1KO mice, these changes alone do not account for the disorganization observed in Mist1KO tissues. Therefore, we conclude that Mist1 is necessary for complete differentiation and maturation of serous exocrine cells through the combined regulation of several exocrine specific genes.

Distal regulatory elements control MRF4 gene expression in early and late myogenic cell populations

MRF4 is a muscle‐specific transcription factor that belongs to a family of basic helix‐loop‐helix proteins known as the myogenic regulatory factors (MRFs). In vitro studies have shown that expression of the MRF4 gene is controlled by a proximal promoter element (−336 to +71) that binds the muscle‐specific transcription factors MEF2 and myogenin to activate transcription. To examine further the regulatory elements necessary for endogenous MRF4 gene expression during development, transgenic mice were generated that contained either a proximal MRF4 promoter‐LacZ reporter gene (−336 MRF4‐nLacZ) or a MRF4‐LacZ reporter gene containing 8.5 kb of 5′ flanking sequence (−8500 MRF4‐nLacZ). Characterization of individual transgenic mouse lines throughout development revealed that expression of both transgenes is restricted to skeletal muscle tissue. However, unlike previous in vitro data, the proximal promoter transgene exhibits only limited transcriptional activity at all developmental time points, whereas the −8500 MRF4‐nLacZ lines fully recapitulate the later developmental expression patterns and exhibit transcription in myotomal cells during somitic differentiation. Tissue culture analysis of myogenic cells isolated from E12.5, E16.5, and adults confirmed that the −8500 MRF4‐nLacZ transgene is expressed in greater than 90% of the myotubes for all myogenic populations. These results indicate that 8.5 kb of MRF4 5′ flanking sequence contains all the regulatory elements necessary for late MRF4 expression and that at least some of these elements lie upstream of the −336 proximal promoter. It is also likely that distant upstream regulatory sequences control early somitic MRF4 expression. These findings, coupled with previous in vitro studies, suggest that the early and late developmental expression patterns of the MRF4 gene are controlled by distinct sets of regulatory elements. Dev. Dyn. 208:299–312, 1997.

Deletion of Panx3 Prevents the Development of Surgically Induced Osteoarthritis

Osteoarthritis (OA) is a highly prevalent, disabling joint disease with no existing therapies to slow or halt its progression. Cartilage degeneration hallmarks OA pathogenesis, and pannexin 3 (Panx3), a member of a novel family of channel proteins, is upregulated during this process. The function of Panx3 remains poorly understood, but we consistently observed a strong increase in Panx3 immunostaining in OA lesions in both mice and humans. Here, we developed and characterized the first global and conditional Panx3 knockout mice to investigate the role of Panx3 in OA. Interestingly, global Panx3 deletion produced no overt phenotype and had no obvious effect on early skeletal development. Mice lacking Panx3 specifically in the cartilage and global Panx3 knockout mice were markedly resistant to the development of OA following destabilization of medial meniscus surgery. These data indicate a specific catabolic role of Panx3 in articular cartilage and identify Panx3 as a potential therapeutic target for OA. Lastly, while Panx1 has been linked to over a dozen human pathologies, this is the first in vivo evidence for a role of Panx3 in disease.Key messagePanx3 is localized to cartilage lesions in mice and humans.Global Panx3 deletion does not result in any developmental abnormalities.Mice lacking Panx3 are resistant to the development of osteoarthritis.Panx3 is a novel therapeutic target for the treatment of osteoarthritis.

Silencing of the Fibroblast growth factor 21 gene is an underlying cause of acinar cell injury in mice lacking MIST1

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.