Treatment Of Acinar Cells Research Articles

Comprehensive analysis of microRNA signature of mouse pancreatic acini: overexpression of miR-21-3p in acute pancreatitis

In the current study, we have characterized the global miRNA expression profile in mouse pancreatic acinar cells and during acute pancreatitis using next-generation RNA sequencing. We identified 324 known and six novel miRNAs that are expressed in mouse pancreatic acinar cells. In the basal state, miR-148a-3p, miR-375-3p, miR-217-5p, and miR-200a-3p were among the most abundantly expressed, whereas miR-24-5p and miR-421-3p were the least abundant. Treatment of acinar cells with caerulein (100 nM) and taurolithocholic acid 3-sulfate [TLC-S (250 μM)] induced numerous changes in miRNA expression profile. In particular, we found significant overexpression of miR-21-3p in acini treated with caerulein and TLC-S. We further looked at the expression of miR-21-3p in caerulein, l-arginine, and caerulein + LPS-induced acute pancreatitis mouse models and found 12-, 21-, and 50-fold increased expression in the pancreas, respectively. In summary, this is the first comprehensive analysis of global miRNA expression profile of mouse pancreatic acinar cells in normal and disease conditions. Our analysis shows that miR-21-3p expression level correlates with the severity of the disease.

Abstract A09: Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia

Abstract Almost all reported cases of pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, harbor an oncogenic Kras mutation. Recently, targeting oncogenic Kras to specific cell types in the adult pancreas has shown the acinar compartment to be more susceptible to neoplastic transformation than ductal cells. Acinar to ductal metaplasia (ADM) is a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasias, a precursor lesion to pancreatic cancer. Acinar cells harvested from mice expressing mutant Kras undergo spontaneous ADM when embedded in 3D collagen cultures, however, we and others have found the epidermal growth factor receptor (EGFR) is required for ADM induced by mutant Kras expression in vitro. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has also been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EGFRf/f; Ptf1a+/Cre (EKO) mice, which harbor a pancreas specific deletion of EGFR. Surprisingly, we found that expression of N2ICD failed to induce ADM in acinar cells from EKO mice. We also observed that co-expression of a constitutively active p110α mutant or mutant Kras with N2ICD was able to rescue ADM in acinar cells from EKO mice, though neither was sufficient to drive ADM in the absence of N2ICD. Additionally, treatment of acinar cells from WT mice with either the p110α inhibitor Pik75 or the expression of a dominant negative mutant of the PI3K downstream target Rac1 reduced Erk1/2 phosphorylation, whereas expression of constitutively active p110α increased the level of Erk1/2 phosphorylation. These results demonstrate the importance of EGFR signaling in ADM, and identify the need to determine the mechanism of crosstalk between the PI3K and MAPK pathways in acinar cells. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A09. doi: 10.1158/1557-3125.RASONC14-A09

KRAS above and beyond - EGFR in pancreatic cancer

KRAS above and beyond - EGFR in pancreatic cancer

Role of parathyroid hormone-related protein in the pro-inflammatory and pro-fibrogenic response associated with acute pancreatitis

Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1–36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1–36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7–34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.

Do cytokines have a role in the regulation of lacrimal gland acinar cell ion transport and protein secretion?

The present study explored the potential involvement of cytokines (IL-1 alpha, IL-1 beta, IL-6, and IFN-gamma) in the regulation of basic physiological processes carried out by lacrimal acinar cells. Overall, evidence gathered supports the hypothesis that cytokines may be involved in the regulation of lacrimal secretory processes. When combined with earlier studies, these data suggest that acute treatment (i.e., 20 min) of acinar cells with cytokines does not significantly impact either basal or carbachol-stimulated ion transport or protein secretion. However, chronic treatment of cultured acinar cells with cytokines does appear, in some instances, to influence these cell functions. For example, 24 h treatment of acinar cells with IL-6 or IFN-gamma did not alter basal or carbachol-stimulated protein (beta-hexosaminidase) secretion, whereas a combination of IL-1 alpha and IL-1 beta decreased carbachol-stimulated beta-hexosaminidase secretion by 80%. The mechanism behind this effect is unknown, but it appears to stem from an increase in the basal secretion rather than a decrease in the stimulated secretion. Future studies will attempt to identify the mechanism behind the actions of IL-1 alpha and IL-1 beta, in addition to testing other pertinent cytokines (e.g., TNF-alpha, TGF-beta). The study of cytokine involvement in the regulation of lacrimal acinar cell physiology has received relatively little attention. Thus, substantial gaps remain concerning the identity of cytokines, their source(s) and interplay, receptor distribution, second messenger involvement, and ultimate influence over the secretion of aqueous tears.

Control of cholecystokinin receptor dephosphorylation in pancreatic acinar cells

Phosphorylation of cell surface receptors regulates the physiological response to many hormones and neurotransmitters. We have demonstrated that the receptor for cholecystokinin (CCK), the major secretagogue for the exocrine pancreas, is phosphorylated on serines in response to CCK (Klueppelberg, U.G., Gates, L.K., Gorelick, F.S., and Miller, L.J. (1991) J. Biol. Chem. 266, 2403-2408). Receptor phosphorylation was transient, even in the continued presence of agonist, and suggested a role for pancreatic protein phosphatases (PP) in regulating receptor phosphorylation in the intact cell. Treatment of acinar cells with okadaic acid increased the extent and duration of receptor phosphorylation induced by CCK. Receptor phosphorylated in the intact cell in response to CCK was used as substrate to analyze protein phosphatases in pancreatic acinar cell extracts. The predominant CCK receptor phosphatase activity was found in the cytosol and was potently inhibited by okadaic acid (IC50 = 0.2 nM). This phosphatase activity was unaffected by inhibitor-2, Ca2+, or Mg2+, suggesting that the major receptor phosphatase was PP-2A. Stimulation of cells with CCK resulted in a 3-fold increase in protein phosphatase activity toward the CCK receptor, at the same time that phosphorylase a phosphatase activity was unchanged. This increase in receptor phosphatase activity was reflected only in the cytosol, with the particulate activity unchanged. Consistent with this subcellular localization, the hormone-regulated receptor phosphatase activity was PP-2A, with chromatographic separation demonstrating activity in both PP-2A1 and PP-2A2 forms. These data suggest that CCK coordinates the activity of both protein kinases and phosphatases acting on the CCK receptor to control the extent and duration of receptor phosphorylation in the pancreatic acinar cell.

Excess divalent cations activate Ca(2+)-mobilizing receptors in pancreatic acinar cells.

In single, enzymatically dissociated, rat pancreatic acinar cells, external application of excess divalent cations (Ca2+, Sr2+, Ba2+, Ni2+ and Mg2+ over 50 mM) induced Ca(2+)-dependent current responses monitored with the whole-cell recording technique. Inclusion of either EGTA, heparin or GDP[beta S] in the internal solution or treatment of acinar cells with a phorbol ester abolished the divalent-cation-induced responses. In contrast, internal inositol trisphosphate (InsP3) or GTP[gamma S] potentiated the responses. The results indicate that excess divalent cations activate membrane surface receptors or receptor/effector complexes, thereby inducing InsP3-mediated Ca2+ mobilization. The mechanism may be due to modulation of the receptors by changes in electrical profile through indirect action of divalent cations on membrane surface charges, i.e. neutralization of anionic charges. This proposal was supported by the evidence that the trivalent cation, La3+, and the polyvalent cation, protamine, both at much lower concentrations, could induce Ca(2+)-dependent responses, which were abolished by internal application of heparin, GDP[ beta S] or a high concentration of EGTA or by protein kinase C activation with a phorbol ester.

Relationship between cytosolic Ca 2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca 2+] i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca 2+ antagonist, 8-( N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca 2+ chelator, 1,2-bis( O-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca 2+] i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02–0.5 μM) of ionomycin, a Ca 2+ ionophore, caused increases in [Ca 2+] i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12- O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca 2+] i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca 2+] i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.