Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation.

OBJECTIVE: Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. METHODS AND RESULTS: Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. CONCLUSIONS: PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

Evidence that phospholipase-C-dependent, calcium-independent mechanisms are required for directional migration of T-lymphocytes in response to the CCR4 ligands CCL17 and CCL22.

Macrophage-derived chemokine [CC chemokine ligand 22 (CCL22)] and thymus- and activation-regulated chemokine (CCL17) mediate cellular effects, principally by binding to their receptor CC chemokine receptor 4 (CCR4) and together, constitute a multifunctional chemokine/receptor system with homeostatic and inflammatory roles within the body. This study demonstrates that CCL22 and CCL17 stimulate pertussis toxin-sensitive elevation of intracellular calcium in the CEM leukemic T cell line and human peripheral blood-derived T helper type 2 (Th2) cells. Inhibition of phospholipase C (PLC) resulted in the abrogation of chemokine-mediated calcium mobilization. Chemokine-stimulated calcium responses were also abrogated completely by the inhibition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor-mediated calcium release. Chemotactic responses of CEM and human Th2 cells to CCL17 and CCL22 were similarly abrogated by inhibition of PLC and inhibition of novel, Ca2+-independent/diacylglycerol-dependent protein kinase C (PKC) isoforms. Inhibition of Ins(1,4,5)P3 receptor-mediated calcium release from intracellular stores had no effect on chemotactic responses to CCR4 ligands. Taken together, this study provides compelling evidence of an important role for PLC and diacylglycerol-dependent effector mechanisms (most likely involving novel PKC isoforms) in CCL17- and CCL22-stimulated, directional cell migration. In this regard, CCL22 stimulates phosphatidylinositol-3 kinase-independent phosphorylation of the novel delta isoform of PKC at threonine 505, situated within its activation loop--an event closely associated with increased catalytic activity.

Involvement of Listeria monocytogenes phosphatidylinositol-specific phospholipase C and host protein kinase C in permeabilization of the macrophage phagosome.

We have previously shown that phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Listeria monocytogenes activates a host protein kinase C (PKC) cascade which promotes escape of the bacterium from a macrophage-like cell phagosome. Here, we provide evidence linking bacterial PI-PLC and host PKC beta to phagosome permeabilization, which precedes escape.

Regulation of sialyl-Lewis x epitope expression by TNF-alpha and EGF in an airway carcinoma cell line.

Sialyl-Lewis x epitopes and MUC5AC protein are known to be overexpressed in mucins secreted by patients suffering from various respiratory diseases. To investigate the mechanisms by which airway inflammatory agents mediate the expression of sialyl-Lewis x epitopes and MUC5AC mucin, we examined the effects of tumor necrosis factor (TNF)-alpha and epidermal growth factor (EGF) in the human lung carcinoma cell line, NCI-H292. Basal expression levels of hST3GalIV, FUT3 and C2/4GnT mRNA, involved in the biosynthesis of sialyl-Lewis x, were higher than those of other glycosyltransferases in NCI-H292 cells. TNF-alpha induced expression of hST3GalIV, FUT3, C2/4GnT and MUC5AC mRNAs in NCI-H292 cells. When cells were pretreated with U73122, a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor, the expression of these glycosyltransferase mRNAs was suppressed. Treating cells with EGF induced the down-regulation of these glycosyltransferase mRNAs and sialyl-Lewis x epitopes, while inducing an increase in expression of MUC5AC mRNA. These EGF-mediated effects on the glycosyltransferase and MUC5AC mRNAs were blocked when cells were first exposed to AG1478, an EGF receptor tyrosine kinase inhibitor. These findings suggest that the expression of sialyl-Lewis x epitopes, which is regulated separately from the expression of MUC5AC protein, may be controlled through pathways such as the EGF receptor tyrosine kinase and PI-PLC signaling cascades in NCI-H292 cells.

Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Deltaplc1, Deltaarg82, and Deltakcs1 deletion strains, but not the Deltaipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation.

Phosphatidylinositol-specific phospholipase C of Bacillus anthracis down-modulates the immune response.

Phosphatidylinositol-specific phospholipases (PI-PLCs) are virulence factors produced by many pathogenic bacteria, including Bacillus anthracis and Listeria monocytogenes. Bacillus PI-PLC differs from Listeria PI-PLC in that it has strong activity for cleaving GPI-anchored proteins. Treatment of murine DCs with Bacillus, but not Listeria, PI-PLC inhibited dendritic cell (DC) activation by TLR ligands. Infection of mice with Listeria expressing B. anthracis PI-PLC resulted in a reduced Ag-specific CD4 T cell response. These data indicate that B. anthracis PI-PLC down-modulates DC function and T cell responses, possibly by cleaving GPI-anchored proteins important for TLR-mediated DC activation.

Fusion activity of lipid-anchored envelope glycoproteins of herpes simplex virus type 1.

Expression of the herpes simplex virus type 1 (HSV-1) glycoproteins gB, gD, gH, and gL is necessary and sufficient to cause cell fusion. To identify the requirements for a membrane-spanning domain in HSV-1 glycoprotein-induced cell fusion, we created gB, gD, and gH mutants with transmembrane and cytoplasmic domains replaced by a glycosylphosphatidylinositol (gpi)-addition sequence. The corresponding gBgpi, gDgpi, and gHgpi proteins were expressed with wild-type efficiency at the cell surface and were linked to the plasma membrane via a gpi anchor. The gDgpi mutant promoted cell fusion near wild-type gD levels when co-expressed with gB, gH, and gL in a cell-mixing fusion assay, indicating that the gD transmembrane and cytoplasmic domains were not required for fusion activity. A plasma membrane link was required for fusion because a gD mutant lacking a transmembrane and cytoplasmic domain was nonfunctional for fusion. The gDgpi mutant was also able to cooperate with wild-type gB, gH, and gL to form syncytia, albeit at a size smaller than those formed in the wild-type situation. The gBgpi and gHgpi mutants were unable to promote fusion when expressed with the other wild-type viral glycoproteins, highlighting the requirement of the specific transmembrane and cytoplasmic domains for gB and gH function.