Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

Effect of flutamide on survival in patients with pancreatic cancer: results of a prospective, randomised, double blind, placebo controlled trial

Objectives: To assess whether flutamide (Drogenil), a pure androgen receptor blocking agent, improves survival in patients with pancreatic carcinoma and thus whether testosterone is a major growth factor for this tumour.

Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at ≈6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 × 3.2 × 1.2 μm3) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455–476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, ≈66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.

Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages

Tumor necrosis factor receptor (TNFR) p55-knockout (KO) mice are susceptible profoundly to Salmonella infection. One day after peritoneal inoculation, TNFR-KO mice harbor 1,000-fold more bacteria in liver and spleen than wild-type mice despite the formation of well organized granulomas. Macrophages from TNFR-KO mice produce abundant quantities of reactive oxygen and nitrogen species in response to Salmonella but nevertheless exhibit poor bactericidal activity. Treatment with IFN-γ enhances killing by wild-type macrophages but does not restore the killing defect of TNFR-KO cells. Bactericidal activity of macrophages can be abrogated by a deletion in the gene encoding TNFα but not by saturating concentrations of TNF-soluble receptor, suggesting that intracellular TNFα can regulate killing of Salmonella by macrophages. Peritoneal macrophages from TNFR-KO mice fail to localize NADPH oxidase-containing vesicles to Salmonella-containing vacuoles. A TNFR-KO mutation substantially restores virulence to an attenuated mutant bacterial strain lacking the type III secretory system encoded by Salmonella pathogenicity island 2 (SPI2), suggesting that TNFα and SPI2 have opposing actions on a common pathway of vesicular trafficking. TNFα–TNFRp55 signaling plays a critical role in the immediate innate immune response to an intracellular pathogen by optimizing the delivery of toxic reactive oxygen species to the phagosome.

ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma

DPC4 is known to mediate signals initiated by type β transforming growth factor (TGFβ) as well as by other TGFβ superfamily ligands such as activin and BMP (bone morphogenic proteins), but mutational surveys of such non-TGFβ receptors have been negative to date. Here we describe the gene structure and novel somatic mutations of the activin type I receptor, ACVR1B, in pancreatic cancer. ACVR1B has not been described previously as a mutated tumor-suppressor gene.

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor-α

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-γ (INF-γ) or tumor necrosis factor-α (TNF-α) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-γ plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-α antibody, suggesting the release and autocrine action of TNF-α. Treatment with INF-γ plus TNF-α also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell–cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18α-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-γ plus LPS or INF-γ plus TNF-α. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppessor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: Sensitivity of human thymocytes

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

Correlation between Binding Affinity and Necrosis-Inducing Activity of Mutant AVR9 Peptide Elicitors1

The race-specific peptide elicitor AVR9 of the fungus Cladosporium fulvum induces a hypersensitive response only in tomato (Lycopersicon esculentum) plants carrying the complementary resistance gene Cf-9 (MoneyMaker-Cf9). A binding site for AVR9 is present on the plasma membranes of both resistant and susceptible tomato genotypes. We used mutant AVR9 peptides to determine the relationship between elicitor activity of these peptides and their affinity to the binding site in the membranes of tomato. Mutant AVR9 peptides were purified from tobacco (Nicotiana clevelandii) inoculated with recombinant potato virus X expressing the corresponding avirulence gene Avr9. In addition, several AVR9 peptides were synthesized chemically. Physicochemical techniques revealed that the peptides were correctly folded. Most mutant AVR9 peptides purified from potato virus X::Avr9-infected tobacco contain a single N-acetylglucosamine. These glycosylated AVR9 peptides showed a lower affinity to the binding site than the nonglycosylated AVR9 peptides, whereas their necrosis-inducing activity was hardly changed. For both the nonglycosylated and the glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membrane-localized binding site and their necrosis-inducing activity in MoneyMaker-Cf9 tomato was found. The perception of AVR9 in resistant and susceptible plants is discussed.

An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling.

Signals emanating from CD40 play crucial roles in B-cell function. To identify molecules that transduce CD40 signalings, we have used the yeast two-hybrid system to done cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer protein, designated TRAF5, has been molecularly cloned. TRAF5 has a tumor necrosis factor receptor-associated factor (TRAF) domain in its carboxyl terminus and is most homologous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, a previously identified CD40-associated factor. The amino terminus has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other members of the TRAF family protein except for TRAF1. In vitro binding assays revealed that TRAF5 associates with the cytoplasmic tail of CD40, but not with the cytoplasmic tail of tumor receptor factor receptor type 2, which associates with TRAF2. Based on analysis of the association between TRAF5 and various CD40 mutants, residues 230-269 of CD40 are required for the association with TRAF5. In contrast to TRAF3, overexpression of TRAF5 activates transcription factor nuclear factor kappa B. Furthermore, amino-terminally truncated forms of TRAF5 suppress the CD40-mediated induction of CD23 expression, as is the case with TRAF3. These results suggest that TRAF5 and TRAF3 could be involved in both common and distinct signaling pathways emanating from CD40.