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Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Krasmutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras(Krasmut) in mature pancreatic cells develop PDAC at a low frequency. Independent of cell of origin, a second genetic hit (loss of tumor suppressor TP53or PTEN) is important for development of PDAC in mice. We hypothesized ectopic expression and elevated levels of oncogenic mutant Kraswould promote PanIN arising in pancreatic ducts. To test our hypothesis, the significance of elevating levels of K-Ras and Ras activity has been explored by expression of a CAG driven LGSL-KrasG12Vallele (cKras) in pancreatic ducts, which promotes ectopic Krasexpression. We predicted expression of cKrasin pancreatic ducts would generate neoplasia and PDAC. To test our hypothesis, we employed tamoxifen dependent CreERT2mediated recombination. Hnf1b:CreERT2;KrasG12V(cKrasHnf1b/+) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKrasin low (cKrasLow), moderate (cKrasMod), and high (cKrasHigh) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKrasHighmice. cKrasModmice had grades of Pancreatic Intraepithelial Neoplasia (PanIN), recapitulating early and advanced PanIN observed in human PDAC. Proteomics analysis revealed significant differences in PTEN/AKT and MAPK pathways between wild type, cKrasLow, cKrasMod, and cKrasHighmice. In conclusion, in this study, we provide evidence that ectopic expression of oncogenic mutant K-Ras in pancreatic ducts generates early and late PanIN as well as PDAC. This Ras rheostat model provides evidence that AKT signaling is an important early driver of invasive ductal derived PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Krasmutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras(Krasmut) in mature pancreatic cells develop PDAC at a low frequency. Independent of cell of origin, a second genetic hit (loss of tumor suppressor TP53or PTEN) is important for development of PDAC in mice. We hypothesized ectopic expression and elevated levels of oncogenic mutant Kraswould promote PanIN arising in pancreatic ducts. To test our hypothesis, the significance of elevating levels of K-Ras and Ras activity has been explored by expression of a CAG driven LGSL-KrasG12Vallele (cKras) in pancreatic ducts, which promotes ectopic Krasexpression. We predicted expression of cKrasin pancreatic ducts would generate neoplasia and PDAC. To test our hypothesis, we employed tamoxifen dependent CreERT2mediated recombination. Hnf1b:CreERT2;KrasG12V(cKrasHnf1b/+) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKrasin low (cKrasLow), moderate (cKrasMod), and high (cKrasHigh) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKrasHighmice. cKrasModmice had grades of Pancreatic Intraepithelial Neoplasia (PanIN), recapitulating early and advanced PanIN observed in human PDAC. Proteomics analysis revealed significant differences in PTEN/AKT and MAPK pathways between wild type, cKrasLow, cKrasMod, and cKrasHighmice. In conclusion, in this study, we provide evidence that ectopic expression of oncogenic mutant K-Ras in pancreatic ducts generates early and late PanIN as well as PDAC. This Ras rheostat model provides evidence that AKT signaling is an important early driver of invasive ductal derived PDAC.

The exocrine pancreas is the organ with the highest level of protein synthesis in the adult—each day the pancreas produces litres of fluid filled with enzymes that are capable of breaking down nearly all organic substances. For optimal health, the pancreas must produce sufficient enzymes of the right character to match the dietary intake. Disruption of normal pancreatic function occurs primarily as a result of dysfunction of the acinar cells that produce these digestive enzymes, and can lead to acute or chronic diseases. For many years, the prevailing dogma has been that inappropriate intracellular activation of the digestive enzymes produced by acinar cells was the key to pancreatic inflammatory diseases, as digestive enzymes themselves are potentially harmful to the cells that secrete them. However, we now know that many stressors can affect pancreatic acinar cells, and that these stressors can independently trigger pancreatic pathology through various mechanisms. This Review focuses on protein synthesis and active digestive enzymes—two key stressors faced by the acinar cell that are likely to be the major drivers of pathology encountered in the pancreas.

Pancreatic stellate cells are source of dense fibrotic stroma, a constant pathological feature of chronic pancreatitis and pancreatic adenocarcinoma. We observed correlation between levels of cyclooxygenase 2 (COX-2) and its product prostaglandin E2(PGE2) and the extent of pancreatic fibrosis. The aims of this study were to delineate the effects of PGE2on immortalized human pancreatic stellate cells (HPSCs) and to identify the receptor involved.

Trefoil factor 1 (TFF1) is a stable secretory protein expressed widely in the gastrointestinal mucosa that is also expressed in pancreatic ductal adenocarcinoma (PDAC). In the current study, we documented the extent and timing of TFF1 expression and investigated the effects of TFF1 on PDAC cells and stellate cells, the primary cells of the PDAC stroma.

This study defines the specific spatial and temporal characteristics of intracellular Ca2+signaling events, together with their downstream consequences in pancreatic stellate cells during transformation from the quiescent to the activated state, both in culture and in situ, in a disease model of chronic pancreatitis and pancreatic cancer.

The specific characteristics of intracellular Ca2+signaling and the downstream consequences of these events were investigated in mouse pancreatic stellate cells (PSC) in culture and in situ using multiphoton microscopy in pancreatic lobules. PSC undergo a phenotypic transformation from a quiescent state to a myofibroblast-like phenotype in culture. This is believed to parallel the induction of an activated state observed in pancreatic disease such as chronic pancreatitis and pancreatic cancer. By day 7 in culture, the complement of cell surface receptors coupled to intracellular Ca2+signaling was shown to be markedly altered. Specifically, protease-activated receptors (PAR) 1 and 2, responsive to thrombin and trypsin, respectively, and platelet-derived growth factor (PDGF) receptors were expressed only in activated PSC (aPSC). PAR-1, ATP, and PDGF receptor activation resulted in prominent nuclear Ca2+signals. Nuclear Ca2+signals and aPSC proliferation were abolished by expression of parvalbumin targeted to the nucleus. In pancreatic lobules, PSC responded to agonists consistent with the presence of only quiescent PSC. aPSC were observed following induction of experimental pancreatitis. In contrast, in a mouse model of pancreatic disease harboring elevated K-Ras activity in acinar cells, aPSC were present under control conditions and their number greatly increased following induction of pancreatitis. These data are consistent with nuclear Ca2+signaling generated by agents such as trypsin and thrombin, likely present in the pancreas in disease states, resulting in proliferation of "primed" aPSC to contribute to the severity of pancreatic disease.

Background: Prostaglandin E2 (PGE2) levels are frequently elevated in colorectal carcinomas. PGE2 is perceived via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is most relevant. PGE2/EP4-receptor interaction activates CREB via the ERK/MEK pathway. However, the downstream target genes activated by this pathway remained to be investigated.Methodology/Prinicipal Findings: Here, we have identified S100P (an EF-hand calcium binding protein) as a novel downstream target. We show by realtime RT-PCR that S100P mRNA levels are elevated in 14/17 (82%) colon tumor tissues as compared to paired adjacent normal colonic tissues. S100P expression is stimulated in the presence of PGE2 in a time dependent manner at mRNA and protein levels in colon, breast and pancreatic cancer cells. Pharmacological and RNAi-mediated inhibition of the EP4 receptor attenuates PGE2-dependent S100P mRNA induction. RNAi-mediated knockdown of CREB inhibits endogenous S100P expression. Furthermore, using luciferase reporter analysis and EMSA we show that mutation and/or deletion of the CRE sequence within the S100P promoter abolished PGE2-mediated transcriptional induction. Finally, we demonstrate that RNAi-mediated knockdown of S100P compromised invadopodia formation, colony growth and motility of colon cancer cells. Interestingly, endogenous knock down of S100P decreases ERK expression levels, suggesting a role for ERK in regulating S100P mediated cell growth and motility.Conclusions/Significance: Together, our findings show for the first time that S100P expression is regulated by PGE2/EP4-receptor signaling and may participate in a feedback signaling that perpetuates tumor cell growth and migration. Therefore, our data suggest that dysregulated S100P expression resulting from aberrant PGE2/EP4 receptor signaling may have important consequences relevant to colon cancer pathogenesis.

The pancreas is the primary organ responsible for the digestion of food. Pancreatic acinar cells are specialized for the production of digestive enzymes, and these cells have a higher rate of protein synthesis than all other adult human tissues. Digestive enzymes are produced in the endoplasmic reticulum (ER), a multifunctional organelle responsible for the synthesis and correct folding of proteins in the secretory pathway. Disturbances of ER function lead to stress-response mechanisms that can restore homeostasis but can also, if uncontrolled, cause disease. Pancreatic acinar cells are particularly susceptible to ER perturbations, and mechanisms that relieve ER stress are necessary for normal pancreatic development. Furthermore, ER stress occurs during acute pancreatitis, and may also be present in pancreatic cancer. However, the specific roles of ER stress-response mechanisms in these diseases are unknown.

The receptor for advanced glycation end-products (RAGE) is a multifunctional receptor with multiple ligands that is known to play a key role in several diseases, including diabetes, arthritis, and Alzheimer's disease. Recent evidence indicates that this receptor also has an important role in cancer. RAGE ligands, which include the S100/calgranulins and high-mobility group box 1 (HMGB1) ligands, are expressed and secreted by cancer cells and are associated with increased metastasis and poorer outcomes in a wide variety of tumors. These ligands can interact in an autocrine manner to directly activate cancer cells and stimulate proliferation, invasion, chemoresistance, and metastasis. RAGE ligands derived from cancer cells can also influence a variety of important cell types within the tumor microenvironment, including fibroblasts, leukocytes, and vascular cells, leading to increased fibrosis, inflammation, and angiogenesis. Several of the cells in the tumor microenvironment also produce RAGE ligands. Most of the cancer-promoting effects of RAGE ligands are the result of their interaction with RAGE. However, these ligands also often have separate intracellular roles, and some may interact with other extracellular targets, so it is not currently possible to assign all of their effects to RAGE activation. Despite these complications, the bulk of the evidence supports the premise that the ligand ? RAGE axis is an important target for therapeutic intervention in cancer.

To describe advances in the development of biomarkers for pancreatic cancer over the past year.

Serum biomarkers for early diagnosis of pancreatic adenocarcinoma are not currently available. We recently observed elevated expression of CEACAM1 in pancreatic adenocarcinomas and sought to determine whether serum CEACAM1 levels were elevated in pancreatic cancer patients.

Long-term ethanol consumption does not cause acute pancreatitis but rather sensitizes the pancreas to subsequent insults. The mechanisms responsible for this sensitization are unknown. To determine whether alterations in pancreatic gene expression might participate in ethanol-mediated sensitization, we performed gene-profiling analysis.

To establish a primary culture model for murine pancreatic acinar cells and to investigate the effects of different culture conditions on the phenotype and plasticity of these cells in extended culture.

Tissue desmoplasia occurs in a number of disease states, but its molecular basis is poorly understood. To determine which genes are overexpressed in cells contained within the desmoplastic stroma of pancreatic adenocarcinoma and chronic pancreatitis, we undertook genetic profiling of microdissected tissue samples of pancreatic adenocarcinoma, chronic pancreatitis, normal pancreas, and pancreatic cancer cell lines. We observed that samples of both pancreatic adenocarcinoma and chronic pancreatitis showed elevated expression of many shared genes compared with the normal pancreas. We hypothesized that these common genes likely important in stromal production and/or function could be identified using a strategy that involved comparisons between pancreatic adenocarcinoma, chronic pancreatitis, normal pancreas, and pancreatic cancer cell lines.

Raf-1 kinase inhibitory protein (RKIP) was recently identified as a physiologic endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway. The expression and role of RKIP within the pancreas are unknown.

We hypothesized that genes expressed in pancreatic acinar cells during the initiation of acute pancreatitis determine the severity of the disease. Therefore, we utilized microarrays to identify those genes commonly induced in rat pancreatic acinar cells within 1–4 h in two in vivo models, caerulein and taurocholate administration. This strategy yielded 51 known genes representing a complex array of molecules, including those that are likely to either reduce or increase the severity of the disease. Novel genes identified in the current study included ATF3, BRF1, C/EBPβ, CGRP, EGR-1, ephrinA1, villin2, ferredoxin, latexin, lipocalin, MKP-1, NGFI-B, RhoA, tissue factor (TF), and syndecan. To validate these microarray results, the role of EGR-1 was further investigated using quantitative RT-PCR, Western blotting, and immunocytochemistry. EGR-1 expression occurred within acinar cells and correlated with the development of caerulein-induced acute pancreatitis in rats. Furthermore, the levels of the inflammation-related genes MCP-1, PAI, TF, IL-6, and ICAM-1 and the extent of lung inflammation were reduced during the initiation of caerulein-induced acute pancreatitis in EGR-1-deficient mice. Thus this study identified EGR-1 and several other novel genes likely to be important in the development and severity of acute pancreatitis.

Pancreatic secretion can be influenced by cholecystokinin (CCK) either directly via actions on acinar cells or indirectly via actions on nerves. The presence and functional roles of CCK receptors on human pancreatic acinar cells remains unclear. In the current study human pancreatic acini were isolated and then treated with CCK-8, gastrin and/or carbachol. Functional parameters were measured including intracellular [Ca2+] and amylase secretion. It was observed that human acini did not respond to CCK agonists but did respond to carbachol with robust increases in functional parameters. Adenoviral-mediated gene transfer of CCK1 or CCK2 receptors to the human cells resulted in cell responses to CCK agonists. In order to determine the reason for the lack of responsiveness of the human acini, expression of receptor mRNAs was determined using quantitative RT-PCR and localized by in situ hybridization. mRNA levels for CCK1 receptors were ∼30 times lower than those of CCK2 receptors, which were ∼10 times lower than those of m3 Ach receptors as measured by quantitative PCR. Neither CCK1 nor CCK2 receptors were localized in adult human pancreas by in situ hybridization. These results indicate that human pancreatic acinar cells do not respond directly to CCK receptor activation and this is likely due to an insufficient level of receptor expression.

Rab3 proteins are believed to play an important role in regulated exocytosis and previous work has demonstrated the presence of Rab3D on pancreatic zymogen granules. To further understand the function of Rab3D in acinar cell exocytosis, adenoviral constructs were prepared encoding hemagglutinin-tagged wild type Rab3D and three mutant forms, N135I and T36N (both deficient in guanine nucleotide binding) and Q81L (deficient in GTP hydrolysis), which also expressed enhanced green fluorescent protein driven by a separate promoter. When isolated mouse pancreatic acini were cultured with 5 × 106pfu/ml adenovirus, nearly 100% of acini were infected as visualized by expression of green fluorescent protein. Cultured acini showed a biphasic dose-response to cholecystokinin (CCK); basal amylase secretion was 1.8 ± 0.3%/30 min, peak release was 7.3 ± 0.2%/30 min at 30 pmCCK and reduced secretion was observed at higher CCK concentrations. Control β-galactosidase virus infection had no effect on either basal or CCK-induced secretion in the titer range from 0.5 to 10 × 106pfu/ml. While the expression of Rab3D and Rab3D Q81L had no effect on amylase secretion, Rab3D N135I and T36N functioned as dominant negative mutants and inhibited CCK-induced amylase release by 40–50% at all points on the CCK dose-response curve from 3 to 300 pm. Inhibition was stronger during the first 5 min (71 ± 5%) than over 30 min (36%±5%). Similar inhibition was found using other agonists including bombesin, carbachol, A23187, and cAMP. Localization of adenoviral expressed Rab protein showed wild type Rab3D localized to zymogen granules. The two dominant negative mutants did not localize to granules and were primarily in the basolateral region of the cell. Since both dominant negative Rab3D mutants had no effect on intracellular calcium increase induced by CCK, it is unlikely that they acted at receptors or transmembrane signaling. These results suggest that Rab3D plays an important role in regulating the terminal steps of acinar exocytosis and that this effect is greatest on the early phase of amylase release.

The cholecystokinin (CCK) analog JMV-180 acts as a partial agonist in rats and a full agonist in mice. Whether this functional variability is due to species differences in CCK receptor structure or to alterations in the cellular environment is unknown. To address this question, an adenoviral construct encoding the rat CCKAreceptor (AdCCKAR) was used to express the rat receptor in acini from CCKAreceptor-deficient mice (CCKAR −/−). Infection of CCKAR −/− acini in vitrowith pAdCCKAR led to a time-dependent increase in125I-CCK8binding. The affinity for JMV-180 of the adenovirally transferred rat and the endogenous mouse CCKAreceptors was not different. In native mouse acini, JMV-180 acted as a full agonist (both stimulation and inhibition of amylase release). In contrast, in mouse acini expressing pAdCCKAR JMV-180 acted as a partial agonist (only stimulation of amylase release). In addition, the pattern of protein synthesis induced by JMV-180 in CCKAR −/− mouse acini infected with AdCCKAR resembled the pattern observed in wild-type rats (lack of inhibition) rather than the respective pattern in wild-type mice (inhibition). These data suggest that species differences in the CCKAreceptor of rats and mice account for the observed divergence in the acinar cell response to JMV-180.

Activation of Gqprotein-coupled receptors can either stimulate or inhibit cell growth. Previously, these opposite effects were explained by differences in the cell models. Here we show that activation of m3 muscarinic acetylcholine receptors ectopically expressed in NIH3T3 cells can cause stimulation and inhibition of growth in the same cell. A clonal cell line was selected from cells that formed foci agonist dependently (3T3/m3 cells). In quiescent 3T3/m3 cells, carbachol stimulated DNA synthesis. In contrast, when 3T3/m3 cells were growing, either due to the presence of serum or after transformation with oncogenic v-src, carbachol inhibited growth. This inhibition was not due to reduction of extracellular signal-regulated kinase activity because carbachol induced extracellular signal-regulated kinase phosphorylation in both quiescent and growing 3T3/m3 cells. Investigating the cell cycle mechanisms involved in growth inhibition, we found that carbachol treatment decreased cyclin D1 levels, increased p21cip1expression, and led to hypophosphorylation of the retinoblastoma gene product (Rb). Proteasome inhibitors blocked the carbachol-induced degradation of cyclin D1. Effects on p21cip1were blocked by a protein kinase C inhibitor. Thus, m3 muscarinic acetylcholine receptors couple to both growth-stimulatory and -inhibitory signaling pathways in NIH3T3 cells, and the observed effects of receptor activation depend on the context of cellular growth.

The uptake of 125I-labeled epidermal growth factor ( 125I-EGF) by mouse pancreatic acini was inhibited (40–50%) by the secretagogue cholecystokinin octapeptide (CCK 8). Analysis of competitive binding data showed that the apparent Kdof EEG binding increased 135% while the binding capacity was only slightly altered (30% increase). That the effect of CCK 8on acini was mediated by intracellular Ca 2+was indicated by the following: (i) Inhibition of 125I-EGF binding to acini was dose-dependent and paralleled the known abilities of CCK 8, its analogs, and the cholinergic secretagogue carbachol to induce Ca 2+efflux from acini; and (ii) addition of the Ca 2+ionophore A23187 also inhibited 125I-EGF binding. In addition, EGF association with A431 cells was also inhibited by A23187 in the presence but not the absence of Ca 2+.

The uptake of 125I‐labeled epidermal growth factor (125I‐EGF) by mouse pancreatic acini was inhibited (40–50%) by the secretagogue cholecystokinin octapeptide (CCK8). Analysis of competitive binding data showed that the apparent Kdof EEG binding increased 135% while the binding capacity was only slightly altered (30% increase). That the effect of CCK8on acini was mediated by intracellular Ca2+was indicated by the following: (i) Inhibition of 125I‐EGF binding to acini was dose‐dependent and paralleled the known abilities of CCK8, its analogs, and the cholinergic secretagogue carbachol to induce Ca2+efflux from acini; and (ii) addition of the Ca2+ionophore A23187 also inhibited 125I‐EGF binding. In addition, EGF association with A431 cells was also inhibited by A23187 in the presence but not the absence of Ca2+.

To investigate the role of the carboxyl terminus in the regulation of the bombesin (BN) receptor, we constructed two chimeric receptors with carboxyl termini transferred from either m3 muscarinic cholinergic (m3 ACh) (BMC) or cholecystokinin A (CCKA) (BCC) receptors and expressed them in Chinese hamster ovary cells. Previous studies showed that agonist treatment caused rapid internalization of CCKAbut not m3 ACh receptors in these cells. In the current study we conducted separate analyses of ligand and receptor internalization and analyzed receptor recycling. Ligand internalization was assessed using acid washing. BN and CCKAreceptors internalized ligand with 80 ± 3 and 85 ± 7% in an acid-resistant compartment at equilibrium. Ligand internalization of chimeric receptors generally assumed the properties of the donor receptors. Thus, BCC receptors internalized ligand to a similar extent as wild-type CCKAreceptors (75 ± 3%), whereas, BMC receptors showed reduced ligand internalization (38 ± 1%). Receptor internalization was more directly assessed by determining agonist-induced loss of surface binding. BN and CCKAreceptors were largely internalized (56 ± 8 and 50 ± 7%, respectively). BCC receptors were also extensively internalized (82 ± 3%). In contrast, BMC receptors were minimally internalized (22 ± 8%). Receptor recycling was assessed as recovery from agonist induced loss of binding. BN, CCKA, and BMC receptors showed rapid recycling. In contrast, BCC receptors did not recycle. These data indicate that carboxyl-terminal structures determine both internalization of ligand-receptor complexes and subsequent receptor recycling.

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 > corticosterone > aldosterone > 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.

The effects of dexamethasone on ornithine decarboxylase gene expression were examined in rat pancreatic AR42J cells. Dexamethasone increased ornithine decarboxylase activity and messenger RNA (mRNA) concentrations in a time-dependent manner, with a maximal effect at 12 hours (207% ± 63% and 327% ± 34% of control, respectively; n = 5). Ornithine decarboxylase mRNA levels returned to control values at 48 hours, whereas ornithine decarboxylase activity was decreased to 41% ± 8% of control (n = 3). Dexamethasone induction of ornithine decarboxylase mRNA was dose dependent, with half-maximal effects at 10−8mol/L (210% ± 20% of control; n = 4) and maximal effects at 10−7mol/L (327% ± 26% of control; n = 4). The glucocorticoid antagonist RU 38486 blocked the dexamethasone effects in a dose-dependent manner, with maximal effects occurring at 10−7mol/L (120% ± 18% of control; n = 3). When protein synthesis was blocked by addition of cycloheximide, ornithine decarboxylase mRNA levels remained unchanged in response to glucocorticoids, indicating a primary effect of dexamethasone. Furthermore, cycloheximide by itself had no significant effect on ornithine decarboxylase mRNA levels. Inhibition of transcription with actinomycin D showed a half-life for ornithine decarboxylase mRNA of approximately 240 minutes. Ornithine decarboxylase mRNA stability was not affected by dexamethasone pretreatment for 12 hours. Therefore, these data suggest that dexamethasone regulates ODC gene expression via glucocorticoid receptor-mediated gene transcription. Furthermore, translational mechanisms seem to be involved in glucocorticoid-regulated ornithine decarboxylase induction.

We examined the role of glucocorticoids in the regulation of pancreatic glandular kallikrein gene expression in vivo and in vitro. Adult male rats were adrenalectomized (Adx). Corticosterone pellets were administered to maintain either physiologic (Adx 1+) or high physiologic (Adx 3+) plasma corticosterone levels. Pancreatic kallikrein mRNA levels were examined by Northern hybridization and quantitated by slot-blot hybridization. Adrenalectomy resulted in a 75% ± 14% (n = 4) increase in kallikrein mRNA as compared with sham-operated controls. This increase was completely reversed by exogenous corticosterone replacement to normal physiologic concentrations. Replacement with high corticosterone levels (Adx 3+) resulted in a decrease of kallikrein mRNA levels to 53% ± 4% (n = 4) of controls. A significant negative correlation was observed between individual kallikrein mRNA levels and plasma corticosterone (r = − 0.81, n = 12). In vitro, using the rat pancreatic acinar cell line AR42J, dexamethasone decreased kallikrein mRNA steady-state levels in a time- and dose-dependent manner. These data, therefore, indicate that physiologic concentrations of plasma corticosterone decrease pancreatic kallikrein mRNA levels in vivo, and that this is a direct effect on pancreatic acinar cells.

An in vivo translation system, the Xenopus laevisoocyte, was employed to study the synthesis and secretion of pancreatic proteins. RNA was purified from normal and diabetic rat pancreas and normal rat liver by use of guanidine isothiocyanate lysis and cesium chloride gradient centrifugation. The presence of functional mRNA was documented by translation in a reticulocyte lysate that yielded precursors of all major secretory proteins, i.e., slightly higher M, than proteins synthesized in situ by pancreatic acini. Mature X. laevisoocytes were then microinjected with either total RNA or purified mRNA. When oocytes were subsequently incubated with “S-methionine,” pancreatic secretory proteins or hepatic albumin could be immunoprecipitated from oocyte lysate with specific polyclonal antibodies against amylase, trypsin, ribonuclease, and albumin. Amylase was shown to be enzymatically active. Moreover, oocytes released pancreatic secretory proteins into the medium when injected with pancreatic RNA in a time-dependent manner. Only the mature form of amylase was secreted and secretion was not regulated by secretagogues. When a comparison was made after injection of RNA from diabetic pancreas known to contain altered amounts of individual mRNAs, there was a decrease in amylase and an increase in trypsinogen synthesis in oocytes that was comparable to the results of cell free translation. The oocyte expression system, therefore, should be useful not only for studies of protein synthesis but also for processing and secretion.

We compared the cellular events induced by hyperstimulation of rats with caerulein which induces acute pancreatitis, to bombesin, which does not induce pancreatitis. Both secretogogues induced the intracellular activation of trypsinogen and the colocalization of lysosomal hydrolases and zymogen granules within 10–15 minutes. These data indicate that these parameters, previously thought to be crucial initiating events of pancreatitis, are not definitive cellular markers of the disease. We then compared the abilities of the two secretagogues to activate stress-activated protein kinase (SAPK). Significant effects of caerulein hyperstimulation on SAPK activity were observed within 5 minutes, the maximum (57-fold) activation was evident after 15 minutes, and levels remained above control for at least 3 hours. In comparison, hyperstimulation with bombesin induced a maximal 5-fold increase of SAPK activity which returned to basal within one hour. These data indicate that SAPK activity is the earliest and best correlated cellular marker associated with secretagogue-induced pancreatitis.

Opercular membranes isolated from the freshwater-adapted euryhaline teleost, Sarotherodon mossambicus, and mounted in Ussing-style chambers, have low conductance and current and do not actively transport chloride. In contrast, membranes isolated from seawater-adapted S. mossambicus have high conductance and generate large currents representing net chloride extrusion. Full development of this chloride secretion process requires 1–2 weeks, the time-course of which provides the first unambiguous measurement of changes in net extrarenal salt secretion associated with a teleost’s adaptation to seawater. Tissues from seawater-adapted fish contain typical chloride cells, when observed with the electron microscope, which appear as large cells with fluorescence microscopy after staining with dimethylamino-styrylaethylpyridiniumiodine. These cells are absent from the freshwater tissue, although rudimentary chloride cells are present, appearing as small cells with fluorescence microscopy. Following seawater transfer, the number of chloride cells increases only during the first 3 days. Subsequent chloride cell hypertrophy is highly correlated with the quantity of chloride extrusion. These data strongly implicate the chloride cell as the salt-secretory cell-type. When cortisol was injected into freshwater fish, chloride cell density increased but chloride secretion was not activated. It appears that development of salt extrusion involves increased numbers (controlled, at least in part, by cortisol) and differentiation of chloride cells, including activation of membrane active-transport sites. The opercular membrane from S. mossambicus provides a valuable model for studying these physiological and morphological events.

Stimulation of pancreatic acini from male Sprague-Dawley rats by both cholecystokinin (CCK)-8 and anisomycin caused an increase in p46jnkand p55mapkactivities. Both forms of c-Jun amino-terminal kinase (JNK) were slightly activated at 5 min, reached a maximum at 30 min, and remained significantly increased at 60 min of CCK stimulation. By contrast, p42mapkwas activated fully by 5 min. In pancreatic acini stimulated with different concentrations of CCK for 30 min, the minimal and maximal JNK responses were observed at 30 pM and 100 nM CCK, respectively; p42mapkactivation was, as previously reported, much more sensitive, with maximal activation by 1 nM CCK. Carbachol and bombesin also stimulated JNK activity, while vasoactive intestinal peptide did not. Neither activating protein kinase C nor increasing intracellular Ca2+significantly activated JNK. In in vivoexperiments, rats were infused intravenously for 5 and 15 min with a secretory (0.1 μg/kg/h) or supramaximal (10 μg/kg/h) dose of the CCK analog caerulein (CER). Secretory doses of CER induced a 4-fold increase of both forms of JNK in pancreatic tissue at 5 and 15 min, while at the same time points, supramaximal stimulation with CER caused 4- and 27-fold increases, respectively, of these kinase activities. The secretory dose of CER slightly increased the activities of both forms of mitogen-activated protein kinase, while the supramaximal dose induced a 10-fold increase of p42mapkat 5 min. In conclusion, JNKs and mitogen-activated protein kinases are rapidly activated in rat pancreatic acini stimulated with CCK as well as in pancreatic tissue during in vivostimulation with CER. The large response to supramaximal CER stimulation may be of importance in the early pathogenesis of acute pancreatitis.

DKK3 is produced by the stroma in pancreatic cancer, promotes tumor progression and resistance to therapy, and is a therapeutic target.