Phospholipase C-δ 1 regulates interleukin-1Β and tumor necrosis factor-α mRNA expression
Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794, USA.Experimental Cell Research (Impact Factor: 3.25). 06/2012; 318(16):1987-93. DOI: 10.1016/j.yexcr.2012.06.010
Phospholipase C-δ(1) (PLCδ(1)) is a widely expressed highly active PLC isoform, modulated by Ca(2+) that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLCδ(1) modulated expression of the pro-inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLCδ(1) was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLCδ(1) knockdown enhanced expression IL-1β and tumor necrosis factor-α (TNF-α) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLCδ(1) knock down caused persistently high Nfκb levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLCδ(1) knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1β and TNF-α mRNA's induced by PLCδ(1) knockdown. Our results show that loss of PLCδ(1) enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nfκb pathway. Our findings are consistent with the idea that PLCδ(1) is a suppressor of PKC activity.
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ABSTRACT: Toll-like receptors (TLRs) are key components of the innate immune system, functioning as pattern recognition receptors that recognise a wide range of microbial pathogens. TLRs represent a primary line of defence against invading pathogens in mammals, plants and insects. Recognition of microbial components by TLRs triggers a cascade of cellular signals that culminates in the activation of NFkappaB which leads to inflammatory gene expression and clearance of the infectious agent. The history of NFkappaB began with the TLR4 ligand lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, since this was the stimulus first used to activate NFkappaB in pre-B-cells. However, since those early days it has been a circuitous route, made possible by drawing on information provided by many different fields, that has led us not only to the discovery of TLRs but also to an understanding of the complex pathways that lead from TLR ligation to NFkappaB activation. In this review we will summarize the current knowledge of TLR-mediated NFkappaB activation, and also the recent discoveries that subtle differences in kappaB binding sequences and NFkappaB dimer formation result in specific gene expression profiles.Biochemical Pharmacology 11/2006; 72(9):1102-13. DOI:10.1016/j.bcp.2006.07.010 · 5.01 Impact Factor
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ABSTRACT: Phospholipase Cβ (PLCβ) isoforms, which are under the control of Gαq and Gβγ subunits, generate Ca2+ signals induced by a broad array of extracellular agonists, whereas PLCδ isoforms depend on a rise in cytosolic Ca2+ for their activation. Here we find that PLCβ2 binds strongly to PLCδ1 and inhibits its catalytic activity in vitro and in living cells. In vitro, this PLC complex can be disrupted by increasing concentrations of free Gβγ subunits. Such competition has consequences for signaling, because in HEK293 cells PLCβ2 suppresses elevated basal [Ca2+] and inositol phosphates levels and the sustained agonist-induced elevation of Ca2+ levels caused by PLCδ1. Also, expression of both PLCs results in a synergistic release of [Ca2+] upon stimulation in A10 cells. These results support a model in which PLCβ2 suppresses the basal catalytic activity of PLCδ1, which is relieved by binding of Gβγ subunits to PLCβ2 allowing for amplified calcium signals.Journal of Biological Chemistry 02/2005; 280(2):1438-47. DOI:10.1074/jbc.M407593200 · 4.57 Impact Factor
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ABSTRACT: In the present study, we studied the potential regulation by rat myometrial alpha1-adrenergic receptors (alpha1-AR) of the newly identified Gh alpha protein/phospholipase C delta 1 (PLC delta 1) signaling pathway and compared myometrial inositol phosphates (InsP) production and activity of the uterine circular muscle in response to alpha1-AR activation between mid-pregnancy and term. For this, we quantified the level of rat myometrial alpha1-AR coupling to Gh alpha protein by photoaffinity-labeling, the cytosolic amount of PLC delta 1 enzyme by immunoblotting, and the expression level of alpha1-AR subtypes by RT-PCR. The results showed an increased level of alpha1-AR/Gh alpha protein coupling and the amount of PLC delta 1 at term (+147 and +65% respectively, versus mid-pregnancy). This was correlated with an up-regulation of alpha 1d-AR subtype (+70% versus mid-pregnancy). Incubation of myometrial strips with phenylephrine (Phe), a global alpha1-agonist, increased InsP production in a dose-dependent manner at both mid-pregnancy and term, but with an enhanced potency (tenfold decrease in EC(50) value) at term. Phe also dose-dependently induced contraction of the circular muscle at both mid-pregnancy and term. However, unlike InsP response, no amelioration of potency was observed at term. Similar results were obtained with the endogenous agonist norepinephrine. Our results show, for the first time, that rat myometrial alpha 1d-AR/Gh alpha/PLC delta 1 signaling pathway is up-regulated at term. This is associated with an increased potency of alpha1-AR to elicit InsP production but not uterine contraction at this period. It is thus hypothesized that alpha1-AR, through activation of Gh alpha/PLC delta 1 system, are not primarily involved in the initiation of labor but may rather regulate responses such as myometrial cell proliferation or hypertrophy.Reproduction (Cambridge, England) 02/2008; 135(1):55-62. DOI:10.1530/REP-07-0332 · 3.17 Impact Factor
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