Phospholipase C-δ(1) regulates interleukin-1β and tumor necrosis factor-α mRNA expression.
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.
Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794, USA.
Eric
E
Chung
Paul
P
Jakinovich
Aekyung
A
Bae
Mario
M
Rebecchi
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D007211:Indoles / Q000494:pharmacology; D053583:Interleukin-1beta / Q000235:genetics* / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D054803:Phospholipase C delta / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
D007211:Indoles; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D008301:Maleimides; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; C116174:bisindolylmaleimide II; D048051:p38 Mitogen-Activated Protein Kinases; D054803:Phospholipase C delta; D002118:Calcium