Dexamethasone effects on somatostatin receptors in pancreatic acinar AR4-2J cells

The effects of glucocorticoids on somatostatin binding and cAMP response in the rat pancreatic acinar carcinoma AR4-2J cell line were examined. Dexamethasone treatment reduced the number of somatostatin receptors 2.5 fold without any change in receptor affinity. In addition, dexamethasone increased the sensitivity of the cells to somatostatin-inhibited cAMP formation and restored the biphasic pattern of cAMP response to somatostatin previously observed in normal pancreatic acinar cells. Such effect may be associated with the glucocorticoid-promoted cellular pancreatic differentiation of AR4-2J cells.     

Effects of cholecystokinin on cytosolic calcium in human pancreatic cancer cells.

Cholecystokinin (CCK) has been shown to increase cytosolic calcium and stimulate enzyme release from pancreatic acinar cells and a rat acinar cell line, AR42J. CCK is also trophic to normal pancreas and pancreatic cancer; however, the cellular mechanisms which regulate CCK-stimulated growth are unknown. The effect of CCK on intracellular calcium was evaluated in four human pancreatic cancer cell lines known to grow in response to CCK but not secrete enzymes (SW-1990, MIA PaCa-2, BXPC-3 and PANC-1) and a rat acinar cell line (AR42J) shown to secrete enzymes but not grow with CCK. By using single cell fluorescence microscopy in fura-2 loaded cells, intracellular calcium [Ca2+]i was measured. After obtaining baseline fluorescent cell images, synthetic CCK-octapeptide (CCK8) was added to the cells and images of cell fluorescence captured. [Ca2+]i of the rat acinar cells increased (603%) over the baseline within the first minute after the addition of CCK (4.10(-13) M to 4.10(-10) M) in 77% of cells tested. In contrast [Ca2+]i failed to significantly change in the human cancer cells treated with CCK. To further localize the defect in hormone signal transduction in cancer cells, cells were suspended in low calcium media and the plasma membranes were selectively permeabilized with digitonin. Media free calcium concentration was continuously monitored by fura-2 fluorescence. Addition of inositol 1,4,5-trisphosphate (IP3) resulted in a marked increase in medium calcium concentration indicating IP3 was capable of releasing calcium from intracellular stores in both the AR42J rat acinar cell line and in the human pancreas cancer cell lines. In conclusion, CCK does not increase cytosolic calcium in human pancreatic cancer cells in contrast to rat acinar cells although all contain IP3-sensitive intracellular Ca2+ pools. Our results suggest that growth promoting and secretory effects of CCK on pancreatic cells may occur via two independent signalling pathways.     

Functional and molecular characterization of CCK receptors in the rat pancreatic acinar cell line AR 4-2J.

Competitive inhibition binding studies on membranes from the rat pancreatic AR 4-2J cell line revealed the predominance (80%) of low selectivity CCK receptors (KD of 1 nM and 4 nM for, respectively, CCK-8 and gastrin-17I (G-17I] over selective receptors (20% with a KD of 1 nM and 1 microM for, respectively, CCK-8 and G-17I). Amylase secretion was stimulated by low concentrations of CCK-8, G-17I and CCK-4. G-17I-induced amylase secretion was unaffected by 100 nM of the selective peripheral CCK-A receptor antagonist L-364,718, suggesting that amylase hypersecretion followed non-selective CCK receptor activation, a function normally assumed by selective CCK-A receptors in rat pancreatic acini. Direct ultraviolet irradiation of AR 4-2J cell membranes preloaded with 125I-BH-CCK-33 or 125I(Leu)G(2-17)I resulted in covalent cross-linking with, respectively, a 90 kDa protein and a 106 kDa protein, both distinct from the 81 kDa CCK binding species revealed in normal rat pancreatic membranes. Gpp[NH]p increased the dissociation rate of CCK-8 and G-17I from AR 4-2J cell membranes, indicating a coupling of receptors with guanyl nucleotide regulatory protein(s) G. [32P]ADP-ribosylation of AR 4-2J cell membranes allowed to detect the presence of two Gs alpha (the 50 kDa form predominating over the 45 kDa form) and one Gi alpha (41 kDa). However, Gi and Gs may not be involved in gastrin stimulation of amylase secretion, as Bordetella pertussis toxin and cholera toxin pretreatment of cells did not suppress G-17I-dependent amylase secretion.     

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells.

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (Kd approximately 1 nM) VIP receptors with a mass of 67 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of approximately 44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues on its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.     

Pancreatic tau related maps: Biochemical and immunofluorescence analysis in a tumoral cell line

In the present study, we report the existence of four tau-related microtubule-associated proteins (MAPs) of 48, 50, 55 and 58 kDa in a pancreatic exocrine cell line (AR4-2J). Using immunofluorescence, we demonstrate that these tau-related MAPs are associated with microtubules in AR4-2J cells. That colocalization is particularly striking on microtubules bundles in cellular extensions and is the first evidence for tau-related MAPs colocalization with microtubules in non-neuronal cells. As it has been often discussed for neuronal tau, the localization of tau-related proteins in AR4-2J cells suggests that these proteins may be involved in microtubule bundling.     

Human brain endothelial cells are responsive to adenosine receptor activation

The blood–brain barrier (BBB) of the central nervous system (CNS) consists of a unique subset of endothelial cells that possess tight junctions which form a relatively impervious physical barrier to a large variety of blood components. Until recently, there have been no good in vitro models for studying the human BBB without the co-culture of feeder cells. The hCMEC/D3 cell line is the first stable, well-differentiated human brain endothelial cell line that grows independently in culture with characteristics that closely resemble those of resident human brain endothelial cells. As our previously published findings demonstrated the importance of adenosine receptor (AR) signaling for lymphocyte entry into the CNS, we wanted to determine if human brain endothelial cells possess the capacity to generate and respond to extracellular adenosine. Utilizing the hCMEC/D3 cell line, we determined that these cells express CD73, the cell surface enzyme that converts extracellular AMP to adenosine. When grown under normal conditions, these cells also express the A₁, A_2A, and A_2B AR subtypes. Additionally, hCMEC/D3 cells are responsive to extracellular AR signaling, as cAMP levels increase following the addition of the broad spectrum AR agonist 5′-N-ethylcarboxamidoadenosine (NECA). Overall, these results indicate that human brain endothelial cells, and most likely the human BBB, have the capacity to synthesize and respond to extracellular adenosine.     

An amphicrine pancreatic cell line: AR42J cells combine exocrine and neuroendocrine properties.

The permanent cell culture line AR42J, derived from a rat pancreatic acinar carcinoma, is widely used for functional studies of exocrine pancreatic acinar cells. We now present evidence that these cells are amphicrine in that they contain zymogen granules as well as small (40-80 nm) neuroendocrine (NE) vesicles and typical neurotransmitters. Using the small NE vesicle-specific markers synaptophysin and "protein S.V.2", including synaptophysin cDNA probes, we have found that AR42J cells synthesize these proteins and contain vesicles harboring these proteins with biophysical properties similar to those of small NE vesicles. NE properties of these cells are further indicated by the presence of considerable amounts of stored amino acids (gamma-aminobutyric acid (GABA), glycine, glutamate) and by the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase. Finally, intermediate filament (IF) protein typing showed only cytokeratins 8 and 18, indicating that AR42J cells possess an IF protein complement indistinguishable from that of acinar and islet cells. Our results document the unusual case of a permanent cell line with combined exocrine and neuroendocrine properties that may be indicative of a derivation from a cell with multipotential character.     

An immunocytochemical and electron-microscopical study of endocrine cells in the gut and pancreas of a stomachless teleost fish,Barbus conchonius (Cyprinidae)

Summary Four immunoreactive endocrine cell types can be distinguished in the pancreatic islets of B. conchonius: insulin-producing B cells, somatostatin-producing A₁ (= D) cells, glucagon-producing A₂ cells and pancreatic poly-peptide-producing PP cells. The principal islet of this species contains only a few PP cells, while many PP cells are present in the smaller islets. Except for the B cell all pancreatic endocrine cell types are also present in the pancreatic duct.At least six enteroendocrine cell types are present in the gut of B. conchonius: 1. a cell type (I) with small secretory granules, present throughout the intestine, and possibly involved in the regulation of gut motility; 2. a C-terminal gastrin immunoreactive cell, probably producing a caerulein-like peptide; these cells are located at the upper parts of the folds, especially in the proximal part of the intestinal bulb; 3. a met-enkephalin-immunoreactive cell, present throughout the first segment; 4. a glucagon-immunoreactive cell, which is rare in the first segment; 5. a PP-immunoreactive cell, mainly present in the first half of the first segment; 6. an immunoreactive cell, which cannot at present be specified, located in the intestinal bulb. The latter four cell types are mostly located in the basal parts of the folds, although some PP-immunoreactive cells can also be found in the upper parts.Most if not all enteroendocrine cells are of the open type. The possible functions of all enteroendocrine cell types are discussed.Abbreviations BPP bovine pancreatic polypeptide - CCK cholecystokinin - GEP gastro-entero-pancreatic - GIP gastric inhibitory peptide or glucose-dependent insulin releasing peptide - PPP pig pancreatic polypeptide - VIP vasoactive intestinal polypeptide     

A microplate binding assay for the somatostatin type-2 receptor (SSTR2)

The clinical importance of somatostatin type-2 receptors (SSTR2) and the study of novel analogues of somatostatin such as OctreoScan or [Tyr3]-octreotide containing DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal chelator led us to develop a methodology to monitor the expression of SSTR2 on tumours of pancreatic origin (e.g. rat AR4-2J cancer cells). Usual binding assay protocols using the commercial [125I][Tyr1]-somatostatin radioligand failed, even in the presence of a cocktail of protease inhibitors with a broad spectrum of activity, possibly due to the high susceptibility of this tracer to proteases expressed in pancreatic cells. We prepared our own radioligand [125I][Tyr2]-octreotide which was shown to be much more resistant to degradation after incubation with AR4-2J plasma membranes. As expected, the increased stability of [125I][Tyr3]-octreotide was associated with good binding to SSTR2. Addition of appropriate protease inhibitors further increased the specific binding of [125I][Tyr3]-octreotide to AR4-2J plasma membranes without affecting the stability of the tracer, suggesting that the protease inhibitors also protect the integrity of SSTR2. Optimal conditions (time, temperature, medium) were developed for a binding assay in 96-well plates using AR4-2J plasma membranes in order to make the assay suitable for high-throughput analysis. This protocol was the basis for studying the in vivo regulation of SSTR2 expression in AR4-2J cells implanted into scid mice after exposure to different compounds.     

Membrane lipid composition of pancreatic AR42J cells: modification by exposure to different fatty acids

Dietary fat type influences fatty acids in rat pancreatic membranes, in association with modulation of secretory activity and cell signalling in viable acini. We aimed to confirm whether AR42J cells are a valid model to study the interactions between lipids and pancreatic acinar cell function. For this purpose we have (i) compared the baseline fatty acid composition of AR42J cells with that of pancreatic membranes from rats fed a standard chow; (ii) investigated if fatty acids in AR42J membranes can be modified in culture; and (iii) studied if similar compositional variations that can be evoked in rats when dietary fat type is altered occur in AR42J cells. Weaning Wistar rats were fed for 8 weeks either a commercial chow (C) or semi-purified diets containing virgin olive oil (VOO) or sunflower oil (SO) as fat source. AR42J cells were incubated for 72 hrs in medium containing unmodified fetal calf serum (FCS, AR42J-C cells), FCS enriched with 18:1 n-9 (AR42J-O cells), or FCS enriched with 18:2 n-6 (AR42J-L cells). Fatty acids in crude membranes from rat pancreas and AR42J cells were determined by gas-liquid chromatography. Differences in membrane fatty acids between C rats and AR42J-C cells can be explained in part by variations in the amount of fatty acids in the extracellular environment. Supplementation of FCS with 18:1 n-9 or 18:2 n-6 changed the fatty acid spectrum of AR42J cells in a manner that resembles the pattern found, respectively, in VOO and SO rats, although AR42J-L cells were unable to accumulate 20:4 n-6. The AR42J cell line can be a useful tool to assess the effect of membrane compositional changes on acinar cell function. However, differences in baseline characteristics, and perhaps fatty acid metabolism, indicate that results obtained in AR42J cells should be confirmed with experiments in the whole animal.     

Bombesin binding and biological effects on pancreatic acinar AR42J cells

The effects of bombesin on amylase release and the receptor binding of 125I-[Tyr4]bombesin in the rat pancreatic acinar carcinoma cell line AR42J were examined. Bombesin-like peptides stimulated amylase release from AR42J cells in a dose-dependent manner; a maximal 2-fold stimulation occurred at a bombesin concentration of 300 pM. Binding of 125I-[Tyr4]-bombesin to AR42J cells was specific, saturable and temperature dependent. The relative potencies with which various structurally related peptides stimulated amylase release correlated well with their relative abilities to compete for the bombesin receptor.     

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH₂ terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells. erythropoietin receptor; cell signaling; mitogen-activated protein kinase induction     

LIN28B regulates androgen receptor in human trophoblast cells through Let‐7c

LIN28B is an RNA‐binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let‐7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let‐7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first‐trimester trophoblast cell line, ACH‐3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral‐based vectors. LIN28B knockdown in ACH‐3P cells resulted in significantly decreased levels of AR and increased levels of Let‐7 miRNAs. Moreover, treatment of ACH‐3P cells with Let‐7c mimic, but not Let‐7e or Let‐7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin‐induced syncytialization and Let‐7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW‐1 and a significant decrease in AR in ACH‐3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let‐7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.     

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.     

Interplay between steroid receptors and neoplastic progression in sarcoma tumors

Steroid hormones are expressed at low levels in mesenchymal cells and are highly expressed in soft tissue sarcoma. In human soft tissue fibrosarcoma cell line (HT-1080), the epidermal growth factor (EGF) stimulates the express of matrix metal (MMPs) expression through a Src-dependent mechanism. In human fibrosarcomas, increased expression of MMPs correlates with the metastatic progression. Our recent data in human breast cancer cell line MCF-7, demonstrates that EGF stimulates estradiol receptor (ER) phosphorylation on tyrosine at position 537 thereby promoting the association of a complex among EGF receptor (EGFR), androgen receptor (AR), ER, and Src that activates EGF-dependent signaling pathway. In the present study, we demonstrate that, in HT-1080 cells, the Src kinase activity is involved in EGFR phosphorylation and this activity is regulated by an interplay between Src, steroid receptors, and EGFR. In these cells, estradiol (E(2) )/ER and synthetic androgen (R1881)/AR trans-activate EGFR leading to the downstream signaling and to ERK activation. Indeed, the association between ER/AR and EGFR enhances metastatic progression of fibrosarcoma tumors. A population pilot study performed on 16 patients with soft tissue neoplasias highlights that MMPs expression correlates with progression of anaplastic sarcoma as well as overexpression of EGFR. These findings suggest that there is a crosstalk among AR, ER, and EGFR that lead to src activation also in fibrosarcoma cells.     

Immunological Detection of Isoforms of the Somatostatin Receptor Subtype, SSTR2

Abstract: Somatostatin (SRIF) induces its diverse physiological actions through interactions with different receptor subtypes. Multiple SRIF receptor subtypes have recently been cloned. To analyze the physical properties of receptor subtype SSTR2, two different peptide-directed antibodies were generated against SSTR2. Antibody “2e3,” directed against the peptide SSCTINWPGESGAWYT (residues 191–206), corresponding to a region in the predicted third extracellular domain of mouse SSTR2, and antibody “2i4,” directed against the peptide SGTEDGERSDS (residues 333–343) from the predicted cytoplasmic tail of mouse SSTR2, were developed. In Chinese hamster ovary (CHO) cells stably expressing the mouse SSTR2 gene (CHOB), the antibody 2e3 recognized specifically a protein of 93-kDa protein by immunoblotting. No specific immunoreactivity was detected by 2e3 in nontransfected CHO cells or CHO cells stably expressing vector alone or human SSTR1 or mouse SSTR3 genes. The antibody 2i4 specifically immunoprecipitated SSTR2 solubilized from CHOB cells that could be labeled with the SSTR2-specific ligand ¹²⁵I-MK-678. Furthermore, both 2e3 and 2i4 specifically immunoprecipitated 93-kDa [³⁵S]methionine-labeled proteins from CHOB cells, indicating that they recognize the same proteins. In contrast to studies in CHOB cells, immunoblotting studies showed that 2e3 detected specifically a single 148-kDa protein from different regions of the rat brain that have previously been shown to express high levels of SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. In contrast, no immunoreactivity was detected in rat kidney, liver, or lung, which do not express SSTR2. No 93-kDa protein was detected specifically in the rat brain. The 148-kDa protein detected by 2e3 is an SRIF receptor because 2e3 and 2i4 specifically immunoprecipitated solubilized rat brain SRIF receptors that could be reversibly labeled with ¹²⁵I-MK-678. As in rat brain, 2e3 interacted specifically with a single 148-kDa protein in rat pituitary, in the rat pancreatic cell line AR42J, and in the HEK 293 cell line derived from human kidney, all of which express SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. These findings indicate that rat brain and pituitary, as well as a pancreatic and a kidney cell line, express primarily a form of SSTR2 different from CHOB cells. The multiple forms of SSTR2 may result from differential post-translational processing of SSTR2 because 2e3 immunoprecipitated 41-kDa in vitro translation products generated from mRNA extracted from CHOB and AR42J cells. This 41-kDa protein has the predicted size of unprocessed SSTR2. These results demonstrate that 2e3 and 2i4 antibodies interact specifically with SSTR2. Detection of two different size proteins by the SSTR2 peptide-directed antibodies suggests the existence of multiple forms of SSTR2.