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BBA - Molecular Cell Research (v.1744, #1)
Uptake of LPS/ E. coli/latex beads via distinct signalling pathways in medfly hemocytes: the role of MAP kinases activation and protein secretion by Irene Lamprou; Sotiris Tsakas; Georgios L. Theodorou; Marina Karakantza; Maria Lampropoulou; Vassilis J. Marmaras (pp. 1-10).
In response to LPS/ E. coli treatment, extracellular signal-regulated kinase (ERK) is activated in medfly hemocytes. To explore the molecular mechanisms underlying LPS/ E. coli/latex beads endo- and phagocytosis, we studied the signalling pathways leading to p38 and c-jun N-terminal kinase (JNK) activation. JNK and p38-like proteins were initially identified within medfly hemocytes. Flow cytometry analysis revealed that mitogen-activated protein kinases (MAPK) are required for phagocytosis. Inhibition of specific MAPK signalling pathways, with manumycin A, toxin A, cytochalasin D and latrunculin A, revealed activation of p38 via Ras/Rho/actin remodelling pathway and activation of JNK that was independent of actin cytoskeleton reorganization. ERK and p38 pathways, but not JNK, appeared to be involved in LPS-dependent hemocyte secretion, whereas all MAPK subfamilies seemed to participate in E. coli-dependent secretion. In addition, flow cytometry experiments in hemocytes showed that the LPS/ E. coli-induced release was a prerequisite for LPS/ E. coli uptake, whereas latex bead phagocytosis did not depend on hemocyte secretion. This is a novel aspect, as in mammalian monocytes/macrophages LPS/ E. coli-triggered release has not been yet correlated with phagocytosis. It is of interest that these data suggest distinct mechanisms for the phagocytosis of E. coli and latex beads in medfly hemocytes.
Keywords: Abbreviations; MAPK; mitogen-activated protein kinase; ERK; extracellular-signal regulated kinase; MEK; MAPK/ERK Kinases; GRB2; growth factor receptor-bound protein 2; JNK; c-jun N-terminal kinaseMAP kinase; Protein secretion; Phagocytosis; LPS; E. coli; Insect immunity
Identification of a novel phosphorylation site in ataxin-1 by Cynthia A. Vierra-Green; Harry T. Orr; Huda Y. Zoghbi; Deborah A. Ferrington (pp. 11-18).
Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease resulting from an expanded CAG repeat in the SCA1 gene that leads to an expanded polyglutamine tract in the gene product. Previous studies have demonstrated that serine at site 776 is phosphorylated [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Studies of ataxin-1 S776 and serine mutated to an alanine, A776, have also shown differential protein–protein interactions and reduced neurodegeneration [H.K. Chen, P. Fernandez-Funez, S.F. Acevedo, Y.C. Lam, M.D. Kaytor, M.H. Fernandez, A. Aitken, E.M. Skoulakis, H.T. Orr, J. Botas, H.Y. Zoghbi, Interaction of Akt_phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.]. However, mutation of the site serine 776 to an alanine did not abolish all phosphorylation of the protein ataxin-1, suggesting the presence of additional phosphorylation sites [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mutational analysis demonstrated a novel phosphorylation site at serine 239 of ataxin-1.
Keywords: SCA1; Ataxin-1; Phosphorylation; MALDI-TOF MS
Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells by Takeshi Kubota; Yutaka Shindo; Kentaro Tokuno; Hirokazu Komatsu; Hiroto Ogawa; Susumu Kudo; Yoshiichiro Kitamura; Koji Suzuki; Kotaro Oka (pp. 19-28).
To determine the nature of intracellular Mg2+ stores and Mg2+ release mechanisms in differentiated PC12 cells, Mg2+ and Ca2+ mobilizations were measured simultaneously in living cells with KMG-104, a fluorescent Mg2+ indicator, and fura-2, respectively. Treatment with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), increased both the intracellular Mg2+ concentration ([Mg2+]i) and the [Ca2+]i in these cells. Possible candidates as intracellular Mg2+ stores under these conditions include intracellular divalent cation binding sites, endoplasmic reticulum (ER), Mg-ATP and mitochondria. Given that no change in [Mg2+]i was induced by caffeine application, intracellular IP3 or Ca2+ liberated by photolysis, it appears that no Mg2+ release mechanism thus exists that is mediated via the action of Ca2+ on membrane-bound receptors in the ER or via the offloading of Mg2+ from binding sites as a result of the increased [Ca2+]i. FCCP treatment for 2 min did not alter the intracellular ATP content, indicating that Mg2+ was not released from Mg-ATP, at least in the first 2 min following exposure to FCCP. FCCP-induced [Mg2+]i increase was observed at mitochondria localized area, and vice versa. These results suggest that the mitochondria serve as the intracellular Mg2+ store in PC12 cell. Simultaneous measurements of [Ca2+]i and mitochondrial membrane potential, and also of [Ca2+]i and [Mg2+]i, revealed that the initial rise in [Mg2+]i followed that of mitochondrial depolarization for several seconds. These findings show that the source of Mg2+ in the FCCP-induced [Mg2+]i increase in PC12 cells is mitochondria, and that mitochondrial depolarization triggers the Mg2+ release.
Keywords: Abbreviations; caged Ca; 2+; nitrophenyl EGTA; JC-1; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; FCCP; carbonyl cyanide; p; -(trifluoromethoxy) phenylhydrazone; KMG-104; 1-(2,7-difluoro-6-hydroxy-3-oxo-3; H; -xanthen-9-yl)-4-oxo-4; H; -quinolizine-3-carboxylic acid; EPMA; electron probe microanalysis; IP; 3; inositol 1,4,5-trisphosphate; Ψ; Mt; mitochondrial membrane potential; PD; Parkinson's diseaseMagnesium; Mitochondria; KMG; Photolysis; FCCP; Fluorescent imaging
Calcyclin (S100A6) expression is stimulated by agents evoking oxidative stress via the antioxidant response element by Wiesława Leśniak; Anna Szczepańska; Jacek Kuźnicki (pp. 29-37).
Calcyclin (S100A6) is a cell-specific, calcium binding protein of the S100 family whose expression is augmented in many types of cancer. By means of luciferase activity assays, RT-PCR and Northern blot hybridization, we established that transcription of S100A6 gene is increased by agents known to evoke oxidative stress. Mutation of the antioxidant response element (ARE) located at position −290/−281 of the calcyclin gene promoter, and overlapping the E-box sequence recognized by the upstream stimulatory factor (USF), led to inhibition of calcyclin gene promoter activity stimulated by cadmium ions. Electrophoretic mobility shift assays (EMSA) with the −302/−260 calcyclin gene promoter fragment revealed, apart from USF binding, the presence of another protein complex (N) shown by competitive EMSA to be bound to ARE. DNA affinity chromatography followed by Western blot showed the binding of Nrf2 transcription factor to the immobilized calcyclin gene promoter fragment and concomitant appearance of complex N in EMSA of the eluted fractions. The results indicate that agents evoking oxidative stress activate calcyclin gene via the ARE sequence in its promoter.
Keywords: Abbreviations; ARE; antioxidant response element; HO-1; heme oxygenase-1; CacyBP/SIP; calcyclin binding protein/Siah interacting protein; EpRE; electrophile response element; USF; upstream stimulatory factor; EMSA; electrophoretic mobility shift assayCalcyclin (S100A6); Cadmium; Oxidative stress
Uneven cellular expression of recombinant α2A-adrenoceptors in transfected CHO cells results in loss of response in adenylyl cyclase inhibition by Susann Björk; Minna Vainio; Mika Scheinin (pp. 38-46).
Two populations of Chinese hamster ovary (CHO) cells expressing similar numbers of recombinant human alpha2A-adrenergic receptors (α2A-AR) showed different capacity to inhibit adenylyl cyclase (AC) activity. Cells transfected with an integrating vector exhibited agonist-dependent inhibition of forskolin-stimulated AC, whereas cells transfected with a non-integrating episomal vector showed no inhibition. Fluorescent microscopy and flow cytometry revealed a very uneven receptor distribution in the episomally transfected cell population. Monoclonal cell populations were expanded from this parent population. Most clones lacked significant amounts of receptors, while a few expressed receptors at high density; these exhibited efficient agonist-dependent inhibition of forskolin-stimulated AC activity. Thus, dense receptor expression in only a few cells is not sufficient to evoke a significant inhibitory response in a functional assay where AC is stimulated in all cells. Consequently, a false negative result was produced. Furthermore, the cell population transfected with an integrating vector showed loss of homogeneity with increasing passage number.
Keywords: Abbreviations; α; 2; -AR; alpha2-adrenergic receptor; AC; adenylyl cyclase; cAMP; cyclic adenosine 3′,5′ monophosphate; CHO; Chinese hamster ovary; FCS; fetal calf serum; FITC; fluorescein isothiocyanate; IBMX; isobutylmethylxanthine; G protein; guanine nucleotide binding protein; GPCR; G protein-coupled receptor; HA; haemagglutinin; NA; noradrenaline; PTX; pertussis toxinAlpha2-adrenoceptor; cAMP; Expression vector; Functional assay; Recombinant receptor; Transfection
Bovine type I collagen inhibits Raw264.7 cell proliferation through phosphoinositide 3-kinase- and mitogen-activated protein kinase-dependent down-regulation of cyclins D1, A and B1 by Min Kyung Cho; Seung Hoon Suh; Chang Ho Lee; Sang Geon Kim (pp. 47-57).
Bovine type I collagen (BIC), which is widely used as a fibrous extracellular matrix component in cell culture models, inhibits the progression of melanoma cell cycle via p27 up-regulation. BIC also induces nitric oxide synthase in macrophages through JunB/AP-1 and NF-κB activation. Given the previous observations, this study investigates the effect of BIC on the cell cycle progression and regulatory function of Raw264.7 macrophage cells and the responsible signaling pathways. Cell cycle analysis revealed that BIC completely suppressed proliferation of Raw264.7 cells with inhibition of the percentage of cells in the S phase and the reciprocal decrease in the G0/G1 phase. DNA synthesis was also inhibited by BIC, as evidenced by a decrease in the cellular incorporation of [3H]thymidine. The G1/S arrest induced by BIC was reversed by chemical inhibition of phosphatidylinositol 3-kinase (PI3-kinase) or overexpression of the p85 subunit of PI3-kinase. Either PD98059 or stable transfection with mitogen-activated protein kinase kinase-1 [MKK1(−)] or c-Jun N-terminal kinase 1 [JNK1(−)] also released the cell cycle arrest. Immunoblot analyses revealed that the levels of cyclins D1, A and B1 were partly or completely down-regulated by BIC, but cyclin E, p21 and p27 were minimally changed. Chemical inhibition and dominant negative mutant overexpression experiments revealed that either PI3-kinase inhibition or JNK1(−) transfection prevented the decreases in cyclin D1, A and B1 by BIC, indicating that the PI3-kinase and JNK1 pathways were associated with disruption of the cyclins. The pathway involving MKK1-extracellular signal-regulated kinase-1/2 (ERK1/2) was responsible for the suppression of cyclin A and B1, but not that of cyclin D1. The present study showed that BIC inhibited proliferation of Raw264.7 cells and that the pathways involving PI3-kinase and mitogen-activated protein kinases regulate the cell cycle arrest.
Keywords: Abbreviations; BIC; bovine type I collagen; Cdk; cyclin-dependent kinases; DAPI; 4′,6-diamidino-2-phenylindole; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; JNK; c-Jun N-terminal kinase; MAP kinase; mitogen-activated protein kinase; PI3-kinase; phosphatidylinositol 3-kinaseBovine type I collagen; PI3-kinase; ERK; JNK; Cyclin; Cell cycle arrest
Increase of intracellular glutathione by low-level NO mediated by transcription factor NF-κB in RAW 264.7 cells by Risa Kurozumi; Shuji Kojima (pp. 58-67).
The mechanism underlying the elevation of intracellular glutathione (GSH) in RAW 264.7 cells exposed to low concentrations of sodium nitroprusside (SNP), a well-known nitric oxide (NO) donor, was investigated. The peak of intracellular GSH was reached at 6 h after exposure of the cells to SNP (0.1–0.5 mM), and this was preceded by the induction of mRNA for γ-glutamylcysteine synthetase (γ-GCS; the rate-limiting enzyme of de novo GSH synthesis), which peaked at 3 h. N-α-Tosyl-l-phenylalanine chloromethyl ketone (TPCK) and caffeic acid phenethyl ester (CAPE), specific inhibitors of NF-κB, significantly suppressed the SNP-induced elevation of GSH protein and γ-GCS mRNA, while curcumin, an inhibitor of AP-1, was less effective. Electrophoretic mobility shift assay (EMSA) showed that SNP exposure markedly increased the DNA binding of NF-κB, but not that of AP-1. Deletion or mutagenesis of the NF-κB site in the γ-GCS gene promoter abolished the SNP-induced up-regulation of GSH protein and γ-GCS mRNA.These results suggest that the elevation of intracellular GSH in RAW 264.7 cells exposed to low concentrations of SNP occurs through the operation of the de novo GSH pathway, and is mediated by transcriptional up-regulation of the γ-GCS gene, predominantly at the NF-κB binding site in its promoter.
Keywords: Sodium nitroprusside; Nitric oxide; γ-GCS mRNA; NF-κB
Outer membrane protein 25-a mitochondrial anchor and inhibitor of stress-activated protein kinase-3 by Naomi W. Court; Evan Ingley; S. Peter Klinken; Marie A. Bogoyevitch (pp. 68-75).
Stress-activated protein kinase-3 (SAPK3) is unique amongst the mitogen-activated protein kinase (MAPK) family with its C-terminal 5 amino acids directing interaction with the PDZ domain-containing substrates α1-Syntrophin and SAP90/PSD95. Here, we identify three additional PDZ domain-containing binding partners, Lin-7C, Scribble, and outer membrane protein 25 (OMP25). This latter protein is localised together with SAPK3 at the mitochondria but it is not a SAPK3 substrate. Instead, OMP25 inhibits SAPK3 activity towards PDZ domain-containing substrates such as α1-Syntrophin and substrates without PDZ domains such as the mitochondrial protein Sab. This is a new mechanism for the regulation of SAPK3 and suggests that its intracellular activity should not be solely assessed by its phosphorylation status.
Keywords: Abbreviations; MAPK; mitogen-activated protein kinase; SAPK; stress-activated protein kinase; OMP25; outer membrane protein 25; PDZ; PSD-95/Discs-large/ZO-I; PSD-95; post-synaptic density-95; SAP90; synapsin-associated protein 90Stress-activated protein kinase-3; OMP25; Mitochondria; PDZ domain
The plasticity of p19ARF null hepatic stellate cells and the dynamics of activation by Verena Proell; Mario Mikula; Eva Fuchs; Wolfgang Mikulits (pp. 76-87).
In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, α-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-β1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120ctn and β-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19ARF deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.
Keywords: Hepatic stellate cell; TGF-β; Fibrogenesis; Myofibroblast