Differentiation Of Pancreatic Acinar Cells Research Articles

Recurrent gastric amphicrine tumor with neuroendocrine and pancreatic acinar cell differentiation and somatic MEN1 inactivation arisen during immunotherapy.

Amphicrine neoplasms (ANs) are poorly understood epithelial malignancies composed of cells with co-existing exocrine-neuroendocrine features. Here, we report a recurrent mucin-producing gastric amphicrine tumor co-expressing neuroendocrine (chromogranin-A, synaptophysin, and CD56) and pancreatic acinar cell (BCL10 and trypsin) markers, arisen in a 64-year-old woman during adjuvant immunotherapy for melanoma. Ki-67 was < 2%. The gastric background context was atrophic gastritis. Next-generation sequencing showed MEN1 mutation (p.P71fs*42) coupled with loss of heterozygosity. The key lessons were as follows: (1) gastric ANs can show the co-existence of exocrine mucin-producing elements with neuroendocrine and pancreatic acinar differentiation; (2) they may represent a new entity arising in the context of atrophic gastritis and during immunotherapy; (3) they should be considered in the diagnostic workup of gastric neuroendocrine tumors; and (4) their molecular profile can show striking similarities with well-differentiated neuroendocrine tumors. These findings may be of help to improve the knowledge and the biological taxonomy of ANs.

Heparanase 2 (Hpa2)- a new player essential for pancreatic acinar cell differentiation

Heparanase 2 (Hpa2, HPSE2) is a close homolog of heparanase. Hpa2, however, lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase enzymatic activity. Mutations of HPSE2 were identified in patients diagnosed with urofacial syndrome (UFS), a rare genetic disorder that exhibits abnormal facial expression and bladder voiding dysfunction, leading to renal damage and eventually renal failure. In order to reveal the role of HPSE2 in tissue homeostasis, we established a conditional Hpa2-KO mouse. Interestingly, the lack of Hpa2 was associated with a marked decrease in the expression of key pancreatic transcription factors such as PTF1, GATA6, and Mist1. This was associated with a two-fold decrease in pancreas weight, increased pancreatic inflammation, and profound morphological alterations of the pancreas. These include massive accumulation of fat cells, possibly a result of acinar-to-adipocyte transdifferentiation (AAT), as well as acinar-to-ductal metaplasia (ADM), both considered to be pro-tumorigenic. Furthermore, exposing Hpa2-KO but not wild-type mice to a carcinogen (AOM) and pancreatic inflammation (cerulein) resulted in the formation of pancreatic intraepithelial neoplasia (PanIN), lesions that are considered to be precursors of invasive ductal adenocarcinoma of the pancreas (PDAC). These results strongly support the notion that Hpa2 functions as a tumor suppressor. Moreover, Hpa2 is shown here for the first time to play a critical role in the exocrine aspect of the pancreas.

Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development

The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease.

Let-7b and miR-495 Stimulate Differentiation and Prevent Metaplasia of Pancreatic Acinar Cells by Repressing HNF6

Diseases of the exocrine pancreas are often associated with perturbed differentiation of acinar cells. MicroRNAs (miRNAs) regulate pancreas development, yet little is known about their contribution to acinar cell differentiation. We aimed to identify miRNAs that promote and control the maintenance of acinar differentiation. We studied mice with pancreas- or acinar-specific inactivation of Dicer (Foxa3-Cre/Dicer(loxP/-) mice), combined (or not) with inactivation of hepatocyte nuclear factor (HNF) 6 (Foxa3-Cre/Dicer(loxP/-)/Hnf6-/- mice). The role of specific miRNAs in acinar differentiation was investigated by transfecting cultured cells with miRNA mimics or inhibitors. Pancreatitis-induced metaplasia was investigated in mice after administration of cerulein. Inhibition of miRNA synthesis in acini by inactivation of Dicer and pancreatitis-induced metaplasia were associated with repression of acinar differentiation and with induction of HNF6 and hepatic genes. The phenotype of Dicer-deficient acini depends on the induction of HNF6; overexpression of this factor in developing acinar cells is sufficient to repress acinar differentiation and to induce hepatic genes. Let-7b and miR-495 repress HNF6 and are expressed in developing acini. Their expression is inhibited in Dicer-deficient acini, as well as in pancreatitis-induced metaplasia. In addition, inhibiting let-7b and miR-495 in acinar cells results in similar effects to those found in Dicer-deficient acini and metaplastic cells, namely induction of HNF6 and hepatic genes and repression of acinar differentiation. Let-7b, miR-495, and their targets constitute a gene network that is required to establish and maintain pancreatic acinar cell differentiation. Additional studies of this network will increase our understanding of pancreatic diseases.

The Absence of MIST1 Leads to Increased Ethanol Sensitivity and Decreased Activity of the Unfolded Protein Response in Mouse Pancreatic Acinar Cells

BackgroundAlcohol abuse is a leading cause of pancreatitis in humans. However, rodent models suggest that alcohol only sensitizes the pancreas to subsequent insult, indicating that additional factors play a role in alcohol-induced pancreatic injury. The goal of this study was to determine if an absence of MIST1, a transcription factor required for complete differentiation of pancreatic acinar cells in mice, increased the sensitivity to alcohol.MethodsTwo to four month-old mice lacking MIST1 (Mist1−/−) or congenic C57 Bl6 mice were placed on a Lieber-DeCarli diet (36% of total kcal from ethanol and fat), a control liquid diet (36% kcal from fat) or a regular breeding chow diet (22% kcal from fat). After six weeks, pancreatic morphology was assessed. Biochemical and immunofluorescent analysis was used to assess mediators of the unfolded protein response (UPR).ResultsEthanol-fed Mist1−/− mice developed periductal accumulations of inflammatory cells that did not appear in wild type or control-fed Mist1−/− mice. Wild type mice fed diets high in ethanol or fat showed enhancement of the UPR based on increased accumulation of peIF2α and spliced XBP1. These increases were not observed in Mist1−/− pancreatic tissue, which had elevated levels of UPR activity prior to diet exposure. Indeed, exposure to ethanol resulted in a reduction of UPR activity in Mist1−/− mice.ConclusionsOur findings suggest that an absence of MIST1 increases the sensitivity to ethanol that correlated with decreased activity of the UPR. Therefore, events that affect the expression and/or function of MIST1 may be confounding factors in pancreatitis.

Differentiation of Pancreatic Acinar Cells to Hepatocytes Requires an Intermediate Cell Type

The appearance of hepatic foci in pancreas has been well-documented in animal experiments and in patients with pancreatic cancer. We previously demonstrated that transdifferentiation of pancreatic exocrine cells to hepatocytes required members of the CCAAT enhancer binding protein family. Although the molecular basis of hepatic transdifferentiation is understood, the early cellular events remain to be defined. Dexamethasone and oncostatin M were used to induce transdifferentiation of primary cultures of mouse acinar cells and exocrine cell lines into hepatocytes. Fluorescent-activated cell sorting was used to identify intermediate cell types and side-population characteristics. Cre-loxP-based lineage tracing was used to investigate whether acinar cells contribute directly to hepatocytes via intermediates that express adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2). Lineage tracing studies showed that hepatocytes were derived directly from pancreatic cells via ABCG2-expressing intermediates. Exposure of cells to insulin increased Akt phosphorylation, ABCG2 expression, and hepatic transdifferentiation. Inhibition of the phosphoinositide 3-kinase pathway, through addition of LY294002 or overexpression of a dominant-negative form of Akt, was sufficient to prevent transdifferentiation. When ABCG2-expressing cells were incubated with glucagon-like-peptide 1 or epidermal growth factor, the intermediate cells could differentiate into insulin-producing beta-like cells. The phosphoinositide 3-kinase pathway is important in the transdifferentiation of acinar cells to hepatocytes and those hepatocytes arise from acinar cells via ABCG2-expressing intermediates. Furthermore, ABCG2-expressing cells are multipotent and able to differentiate into hepatocytes and insulin-producing beta cells.

Abstract A71: Distinct functions of epithelial growth factor receptor (EGFR) pathways in pancreatic acinar cell differentiation and initiation of pancreatic ductal adenocarcinoma

Abstract Significance: Pancreatic ductal adenocarcinoma (PDA) is one of the most fatal malignancies, partially due to the lack of early detection methods. Background: Efforts to understand the early stages of PDA progression have led to identification of Kras mutations as the initiating event and pancreatic intraepithelial neoplasias (PanINs) as one of the cellular precursors of PDA. Recently, several labs have demonstrated that PanINs can be generated from pancreatic acinar cells upon expression of Kras mutants in mouse models. This dramatic shift from differentiated acinar cells to duct-like PanIN cells, generally termed acinar-ductal metaplasia (ADM), is accelerated by loss of Mist1, an acinar-restricted basic helix-loop-helix transcription factor. Despite this recent progress, the transcription and signaling networks that regulate ADM remain largely unknown. Results: To study these networks, we employed a collagen-based 3-dimensional (3D) culture model of ADM, where primary acinar cells can be transformed into duct-like cysts when treated with transforming growth factor-alpha (TGF-α). A similar acinar-to-ductal conversion was observed with primary acinar cells that express the KrasG12D mutant. Initial characterization of the ADM process revealed that the MAPK pathway is required in both settings. However, EGFR activity is required for TGF-α-induced conversion but not for KrasG12D-induced conversion. Consistent with previous data generated from mouse models, loss of Mist1 accelerates KrasG12D-induced conversion to ductal cysts in vitro. This accelerated ADM formation can be reversed by inhibition of EGFR, without affecting the basal level conversion induced by KrasG12D, suggesting that loss of Mist1 promotes transformation through an EGFR-dependent pathway. Further investigation has revealed that Mist1 directly activates transcription of the epidermal growth factor (EGF) gene, which encodes a classic ligand of EGFR. Conclusion: These data suggest that EGFR has a complex role in this ADM model. EGFR activity either promotes maintenance of acinar differentiation, or cooperates with KrasG12D-induced transformation, possibly depending on the functional specificity of different ligands. Citation Information: Cancer Res 2009;69(23 Suppl):A71.

Colonic carcinoma with a pancreatic acinar cell differentiation. A case report

A case of a colonic carcinoma showing a pancreatic acinar cell differentiation is described for the first time. A 65-year-old woman underwent surgical resection for an ulcerated protruding tumour of 4 x 2.5 cm in size on the anterior wall of the sigmoid colon. Histologically, tumour cells were organized in acinar structures resembling pancreatic acini and in solid nests and ribbons or diffusely infiltrated as poorly cohesive cells. Lymph nodes and femur metastases displayed the same histological features. The ultrastructural analysis of the primary tumour indicated the presence of zymogen-like granules in the cytoplasm of tumour cells. Immunohistochemically, both acinar and diffuse patterns of growth showed an intense staining for trypsin, chymotrypsin and BCL10 and a weaker immunoreactivity for lipase and carboxyl ester hydrolase. Most tumour cells were cytokeratin 20, CDX2 and p53 positive; whereas, mucin (MUC)2 immunoreactivity was observed only in the signet ring cells present in the diffuse pattern and chromogranin A in rare isolated tumour cells. No immunoreactivity was observed for cytokeratin 7, MUC1, MUC5AC, pancreatic amylase or PDX1. There was no evidence of a pancreatic acinar cell carcinoma or of heterotopic pancreatic tissue. A colonic origin ought to be suspected when a metastatic carcinoma of unknown primary shows an acinar differentiation.

Pancreatic-type acinar cell carcinoma of the stomach beneath a focus of pancreatic metaplasia of the gastric mucosa

Acinar cell carcinoma is an uncommon type of carcinoma of the pancreas that can exceptionally arise in ectopic pancreatic tissue. Herein, we report a case of a 52-year-old man with pancreatic-type acinar cell carcinoma of the stomach and concomitant pancreatic metaplasia of the adjacent nonneoplastic gastric mucosa. There was neither clinical nor radiographic evidence of a tumor in the pancreas itself. A subtotal gastrectomy was performed. Macroscopically, an ulcerated tumor, measuring 4 x 1.7 cm, was found in the distal antrum. Microscopically, the biopsy and the surgical specimen revealed a neoplasm with a predominantly trabecular architecture composed of moderately atypical cells with finely dispersed chromatin and indistinct nucleoli. The neoplastic cells and those of the adjacent metaplastic mucosa were both strongly immunoreactive for alpha-1-antitrypsin, consistent with pancreatic acinar cell differentiation. Ectopic pancreatic-type acinar cell carcinoma is an extremely rare condition, having been previously reported only in 5 occasions, none of them in association with pancreatic acinar cell metaplasia of the gastric mucosa.

Notch Signaling Is Required for Exocrine Regeneration After Acute Pancreatitis

The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

Activated Notch1 prevents differentiation of pancreatic acinar cells and attenuate endocrine development

Mice carrying loss-of-function mutations in certain Notch pathway genes display increased and accelerated pancreatic endocrine development, leading to depletion of precursor cells followed by pancreatic hypoplasia. Here, we have investigated the effect of expressing a constitutively active form of the Notch1 receptor (Notch1 ICD) in the developing pancreas using the pdx1 promoter. At e10.5 to e12.5, we observe a disorganized pancreatic epithelium with reduced numbers of endocrine cells, confirming a repressive activity of Notch1 upon the early differentiation program. Subsequent branching morphogenesis is impaired and the pancreatic epithelium forms cyst-like structures with ductal phenotype containing a few endocrine cells but completely devoid of acinar cells. The endocrine cells that do form show abnormal expression of cell type-specific markers. Our observations show that sustained Notch1 signaling not only significantly represses endocrine development, but also fully prevents pancreatic exocrine development, suggesting that a possible role of Notch1 is to maintain the undifferentiated state of common pancreatic precursor cells.

Upregulation of dynamin II expression during the acquisition of a mature pancreatic acinar cell phenotype.

Members of the dynamin superfamily are GTPases which have been shown to support receptor-mediated endocytosis in vivo and bind to growth factor receptor-associated proteins in vitro. In acinar cells of the pancreas, receptor-mediated endocytosis is very important for the recycling of membranes after secretory granule release. Therefore, characterization of the molecular machinery responsible for this process is critical for a better understanding of this phenomenon. In this study we sought to determine the expression pattern of the endocytic GTPase dynamin II during pancreatic acinar cell differentiation in developing rat embryos and in dexamethasone-treated AR42J cells using Western blot, Northern blot, and immunocytochemical analyses. During pancreatic development, dynamin immunoreactivity is almost undetectable until day E17 but undergoes significant upregulation in acinar cells starting at E18. In addition, the levels of dynamin mRNA and protein in AR42J cells increase approximately threefold during dexamethasone-induced acinar differentiation. The increase in dynamin levels that occurs in both embryonic pancreatic cells and dexamethasone-treated AR42J cells correlates with the establishment of a more differentiated acinar phenotype. Therefore, these results suggest a potential role for dynamin in supporting receptor-mediated endocytosis in mature pancreatic acinar cells.

Growth and Differentiation of Pancreatic Acinar Cells

Dexamethasone (DEX) inhibits growth and induces differentiation in rat pancreatic acinar AR42J cells. We wished to determine whether growth and differentiation are mutually exclusive in AR42J cells and whether DEX effects on growth and differentiation are mutually dependent or independent. Inhibition of DNA synthesis, assessed by [3H]thymidine incorporation, was detectable after 6 h, half-maximal after 12 h, and complete after 18-h DEX treatment, at which time incorporation was reduced to 9.0% of control. The half-maximal effective dose for inhibition of DNA synthesis was 0.5 nM, and maximal inhibition was achieved with 10 nM DEX. This dose-response was similar to that previously reported for DEX-induced parameters of differentiation. The rank order of potency for inhibition of DNA synthesis by various steroid hormones was DEX greater than corticosterone greater than aldosterone greater than progesterone. Hydroxyurea or serum starvation inhibited growth to the same extent as DEX but did not induce differentiation. Moreover, hydroxyurea or serum starvation did not block the ability of DEX to induce differentiation. Addition of either EGF or insulin significantly reversed the growth inhibitory effects of submaximal (1 nM) DEX. In cultures released from growth inhibition, 1 nM DEX increased cellular amylase content 5.9- to 6.5-fold, similar to the amylase increase in growth-inhibited cultures. Therefore, growth inhibition and differentiation are independent delayed events regulated by DEX in AR42J cells.

Regulating Insulin-Receptor-Gene Expression by Differentiation and Hormones

Insulin regulates cell function by first binding to the insulin receptor (IR) localized on the cell surface. With the cloning of IR cDNA and the IR-gene promoter, the regulation of the IR gene during differentiation and by various hormones can be studied. Muscle is a major target tissue for insulin action. BC3H1 cells, a mouse muscle cell line in culture, are a model cell type for studying insulin action. Differentiation in these cells results in a 5- to 10-fold increase in IR binding and a 5- to 10-fold increase in IR content. Studies of IR mRNA by Northern and slot-blot analyses reveal a 10-fold increase in IR mRNA after differentiation. These studies indicate that there is a selective increase in IR-gene expression during muscle differentiation. A similar increase in IR-gene expression is observed for the IR during pancreatic acinar cell differentiation. Glucocorticoids increase IR content in several target tissues. Studies in cultured IM-9 lymphocytes indicate that glucocorticoids induce a 5-fold increase in IR mRNA levels. Studies of IR mRNA half-life indicate that glucocorticoids do not alter IR mRNA stability. When the transcription of the IR is measured by elongation assays, glucocorticoids directly stimulate IR transcription 5- to 10-fold. The effect is detectable within 30 min of glucocorticoid treatment and is maximal within 2 h. Therefore, these studies demonstrate that the IR gene is under the direct regulation of glucocorticoids. Insulin downregulates the IR in various target tissues. Prior studies indicate that this downregulation was partly because of accelerated IR degradation. Studying AR42J pancreatic acinar cells, we also found that insulin accelerates IR degradation. Moreover, in these cells, insulin decreases IR biosynthesis by approximately 50%. Studies of IR mRNA indicate there is a concomitant decrease in IR mRNA levels after insulin treatment. Thus, insulin decreases IR-gene expression. The genomic structure of the IR promoter has been elucidated. Primer extension and nuclease S1 analysis indicate that IR mRNA has multiple start sites. The promoter fragment was ligated to a promoterless "reporter" plasmid containing the bacterial gene chloramphenicol acetyltransferase (CAT). When this plasmid is transfected into cultured cells, CAT activity is detected, indicating promoter activity. Various portions of a genomic fragment were ligated to a promoter to study glucocorticoid regulation of the IR promoter. These studies indicate that IR-gene expression is regulated by differentiation and hormonal agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Papillary cystic tumor of the pancreas. An analysis of cellular differentiation by electron microscopy and immunohistochemistry.

Three cases of clinically benign pancreatic papillary cystic tumors in young female patients were studied by immunohistochemistry and electron microscopy in order to define the cellular nature of this type of neoplasm. Two of the tumors showed focal cytokeratin- and desmoplakin-positivity as evidence of focal epithelial differentiation, while the tumor cells were in all cases positive for vimentin--the intermediate filament protein typical of (but not specific for) mesenchymal cells. Electron microscopy showed some cell-cell junctions, but there was no evidence of acinar or islet cell differentiation. The tumors were at least focally positive for neuron-specific enolase, and small clusters of polypeptide hormone immunoreactive cells were present in all cases (glucagon 3/3, somatostatin 2/3, insulin 2/3). However, the tumors were negative for synaptophysin and neurofilament proteins, unlike most islet cell tumors. Trypsin and chymotrypsin immunoreactivity was found in all tumors, but because many nonpancreatic carcinomas were also positive, we doubt whether these two enzyme proteins can act as specific markers for pancreatic acinar cell differentiation. Two of the tumors that were studied immunohistochemically for the presence of nuclear estrogen receptors, were negative. Therefore no proof of the suggested hormone dependence of this tumor could be obtained. We conclude that papillary cystic tumor is a neoplasm of primitive pancreatic epithelial cells, that may exhibit focal endocrine cell differentiation.

Hepatocyte-like Cells within the Pancreas of Rats Fed Methyl-Deficient Diets

Administration of a methyl-group-deficient diet with or without an initial single injection of diethylnitrosamine to male Fischer 344 rats resulted in the development of cells structurally similar to hepatocytes that lay in small clusters around one or more islets of Langerhans in the pancreas. These cells were detected in 15/256 rats fed various methyl-deficient diets in study 1 and in 7/28 rats fed a severely methyl-group-deficient diet in study 2. The results of special staining procedures for the detection of glycogen, albumin and particulate iron uptake indicated that at least some of these cells shared features common to liver cells. These results, as well as previously reported observations of similar cells in hamsters treated with ethionine, provide additional evidence that the lack of available physiological methyl donors contributes to altered differentiation of pancreatic acinar cells.