BBA - Molecular Cell Research (v.1693, #3)

The effects of cyclic ADP-ribose (cADPR) and the immunosuppressant drug FK506 on microsomal Ca2+ release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. After a steady state of 45Ca2+ uptake into the microsomal vesicles, ryanodine or caffeine was added. Preincubation of the vesicles with cADPR (0.5 μM) shifted the dose–response curve of ryanodine- or caffeine-induced 45Ca2+ release from the vesicles to the left. Preincubation with cADPR shifted the dose–response curve of the FK506-induced 45Ca2+ release upward. Preincubation with FK506 (3 μM) shifted the dose–response curve of the ryanodine- or caffeine-induced 45Ca2+ release to the left by the same extent as that in the case of cADPR. FK506 shifted the dose–response curve of the cADPR-induced 45Ca2+ release upward. The presence of both cADPR and FK506 enhanced the ryanodine (30 μM)- or caffeine (10 mM)-induced 45Ca2+ release by the same extent as that in the case of cADPR alone or FK506 alone.These results indicate that cADPR and FK506 modulate the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the mechanism. In addition, there is a possibility that the mechanisms of modulation by cADPR and FK506 are the same.
Keywords: Caffeine; Cyclic ADP-ribose; FK506; Ryanodine; Ryanodine receptor; Pancreatic acinar cell;

The histone deacetylase inhibitor trichostatin A (TSA) has been previously shown to block cellular growth in G2 and induce apoptosis in human pancreatic cancer cell lines. In order to better understand this phenomenon, we have analyzed the gene expression profiles in PaCa44 cells after treatment with TSA using microarrays containing 22,283 probesets. TSA was found to cause both the induction and repression of a large number of genes, although the number whose expression was up-regulated was greater than the number of genes that were down-regulated. When a threshold value of 3 was used as a cutoff level, a total of 306 (3.4%) of the detectable genes had altered expression. When categorized according to cellular function, the differentially expressed genes were found to be involved in a wide variety of cellular processes, including cell proliferation, signaling, regulation of transcription, and apoptosis. Moreover, Sp1/Sp3 transcription factor binding sites were significantly more abundant among TSA-induced genes. One prominent feature was the increased ratio between the levels of expression of pro-apoptotic (BIM) and anti-apoptotic (Bcl-XL and Bcl-W) genes. This result was confirmed in eight additional pancreatic cancer cell lines after treatment with TSA, suggesting that this event may be a strong determinant for the induction of apoptosis by TSA.
Keywords: Trichostatin A; Pancreatic cancer; Expression profiling;

Calmodulin binds to and inhibits the activity of phosphoglycerate kinase by Michael A. Myre; Danton H. O'Day (177-183).
Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis. Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK. Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind. The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency. Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition. Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells.
Keywords: PGK; Calmodulin-binding protein; Enzyme activity; W-7; CaMBOT; Dictyostelium;

Combined effect of testosterone and apocynin on nitric oxide and superoxide production in PMA-differentiated THP-1 cells by Packiasamy A.R. Juliet; Toshio Hayashi; Sumi Daigo; Hisako Matsui-Hirai; Asaka Miyazaki; Akiko Fukatsu; Jun Funami; Akihisa Iguchi; Louis J. Ignarro (185-191).
Human inducible nitric oxide synthase (iNOS) is most readily observed in macrophages from patients with inflammatory diseases like atherosclerosis. The aim of the present study was to find out the combined effect of male sex hormone; testosterone and apocynin (NADPH oxidase inhibitor) on cytokine-induced iNOS production. THP-1 cells were differentiated into macrophages by phorbol myristate acetate (PMA). Expression of iNOS was induced by the addition of cytokine mixture? Testosterone was added at different concentrations (10−6–10−12 M) with apocynin (1 mM). Testosterone (10−8, 10−10 M) inhibited NO x production in cytokine-added THP-1 cells which was further confirmed by quantikine assay of iNOS protein and RT-PCR analysis. Testosterone treatment decreased 40% of superoxide anion production. Testosterone showed inhibition of NADPH oxidase, especially expression of p67phox and p47phox (cytosol subunits). Addition of testosterone with apocynin further decreased the expression of p67phox and p47phox subunits of NADPH oxidase. The findings of the present study suggest that, testosterone; the male androgen plays an important role in the prevention of atherogenesis. Even though apocynin does not have any role on NO production, addition of apocynin together with testosterone is effective in suppressing iNOS activity.
Keywords: Testosterone; Apocynin; iNOS; Superoxide; Aromatase; Monocyte;

Expression of the extracellular matrix (ECM) protein tenascin-C is induced in fibroblasts by growth factors as well as by tensile strain. Mechanical stress can act on gene regulation directly, or indirectly via the paracrine release of soluble factors by the stimulated cells. To distinguish between these possibilities for tenascin-C, we asked whether cyclic tensile strain and soluble factors, respectively, induced its mRNA via related or separate mechanisms. When cyclic strain was applied to chick embryo fibroblasts cultured on silicone membranes, tenascin-C mRNA and protein levels were increased twofold within 6 h compared to the resting control. Medium conditioned by strained cells did not stimulate tenascin-C mRNA in resting cells. Tenascin-C mRNA in resting cells was increased by serum; however, cyclic strain still caused an additional induction. Likewise, the effect of TGF-β1 or PDGF-BB was additive to that of cyclic strain, whereas IL-4 or H2O2 (a reactive oxygen species, ROS) did not change tenascin-C mRNA levels. Antagonists for distinct mitogen-activated protein kinases (MAPK) inhibited tenascin-C induction by TGF-β1 and PDGF-BB, but not by cyclic strain. Conversely, a specific inhibitor of Rho-dependent kinase strongly attenuated the response of tenascin-C mRNA to cyclic strain, but had limited effect on induction by growth factors. The data suggest that regulation of tenascin-C in fibroblasts by cyclic strain occurs independently from soluble mediators and MAPK pathways; however, it requires Rho/ROCK signaling.
Keywords: Extracellular matrix; Tenascin-C; Mechanical stress; TGF-β; PDGF; Gene regulation; Signaling;

Early insulin signaling cascade in a model of oxidative skeletal muscle: mouse Sol8 cell line by Rodney A. Hill; A. Lulu Strat; Nikki J. Hughes; Theresa J. Kokta; Michael V. Dodson; Arieh Gertler (205-211).
Cell models provide important tools to investigate the mechanisms modulating the insulin-signaling cascade. Insulin interaction and subsequent signaling of cells is complex and regulated at multiple levels: receptor abundance, binding dynamics, phosphorylation/dephosphorylation of tyrosine and serine/threonine residues, and subsequent interactions of key intracellular messengers. We report early insulin signaling events in the mouse Sol8 myogenic cell line. Sol8 cells responded to insulin by increasing total IRS-1, p85 PI3-kinase and tyrosine phosphorylated IRS-1 (pY-IRS-1) at 10 min (P<0.05), but not at 1 min of insulin stimulation. The dose–response relationships at 10-min insulin (10 to 300 nM) stimulation showed that IRS-1 and pY-IRS-1 responded to 100 and 300 nM insulin, and the p85 PI3-kinase response peaked at 30 nM insulin. PI3-kinase appeared to be present in high abundance and, in response to insulin, recruitment to the insulin receptor tyrosine kinase (IR) of IRS-1 and PI3-kinase was observed. The increase in IRS-1 detected in IR immunoprecipitates was twofold, while the corresponding increase in PI3-kinase was threefold, suggesting direct recruitment of PI3-kinase to the IR. PI3-kinase detected in IRS-1 immunoprecipitates in response to insulin increased 1.7-fold. An ultimate target of this pathway, GLUT4 recruitment to the PM, was delayed (30 min), the increase in GLUT4 being of similar magnitude (1.6-fold) to the early signaling events. Saturation binding analysis indicated that IR in the plasma membrane was not down-regulated in response to insulin. The present study suggests that early signaling events in the insulin cascade are invoked in Sol8 myogenic cells and that this cell line provides a useful model to study insulin signaling.
Keywords: Signal transduction; Insulin; Insulin receptor; IR; IRS-1; PI3-kinase; Sol8; Muscle;

Author Index (215-216).

Cumulative Contents (217-218).