Abstract
We investigated the mechanism of interleukin-6 (IL-6) synthesis induced by tumor necrosis factor-alpha (TNF) in osteoblast-like MC3T3-E1 cells. TNF stimulated the synthesis of IL-6 dose dependently in the range between 1 and 30 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the TNF-induced synthesis of IL-6. 1-Oleoyl-2-acetylglycerol, a specific activator of PKC, inhibited the TNF-induced IL-6 synthesis. The stimulative effect of TNF was markedly increased in the PKC down-regulated cells. TNF produced diacylglycerol. TNF had little effect on the formation of inositol phosphates and choline. On the contrary, TNF significantly stimulated the formation of phosphocholine dose dependently. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed the TNF-induced diacylglycerol production. The TNF-induced IL-6 synthesis was significantly enhanced by D-609. TNF induced sphingomyelin hydrolysis. Neither C2-ceramide nor sphingosine but sphingosine 1-phosphate significantly stimulated the synthesis of IL-6. PKC down-regulation amplified the IL-6 synthesis by sphingosine 1-phosphate. These results strongly suggest that sphingosine 1-phosphate may act as a second messenger for TNF-induced IL-6 synthesis and that TNF autoregulates IL-6 synthesis due to PKC activation via phosphatidylcholine-specific phospholipase C in osteoblast-like cells.
Highlights
We investigated the mechanism of interleukin-6 (IL-6) synthesis induced by tumor necrosis factor-␣ (TNF) in osteoblast-like MC3T3-E1 cells
Effect of Calphostin C or Staurosporine on Synthesis of IL-6 Induced by TNF in MC3T3-E1 Cells—protein kinase C (PKC) is well known to play a pivotal role in the regulation of various cellular functions [7]
Staurosporine amplified the IL-6 synthesis by TNF. It seems that the TNF-induced synthesis of IL-6 is inhibited by PKC, which is activated by TNF itself
Summary
Materials—myo-[3H]Inositol (90 Ci/mmol), [methyl-3H]choline chloride (85 Ci/mmol), and mouse IL-6 enzyme immunoassay kit were purchased from Amersham Japan (Tokyo, Japan). Assay for IL-6 —The cultured cells were stimulated by TNF, C2ceramide, sphingosine, or sphingosine 1-phosphate in 1 ml of ␣-MEM containing 0.3% FCS for the indicated periods. Phosphocholine was eluted by 10 ml of water, and choline was eluted with 10 ml of 1 M HCl. Measurement of DAG Production—The cultured cells were incubated in the assay buffer containing 0.01% bovine serum albumin at 37 °C for 20 min, and stimulated by TNF for the indicated periods. The labeled cells were stimulated by TNF in the assay buffer containing 0.01% bovine serum albumin for the indicated periods. All data are presented as the mean Ϯ S.D. of triplicate determinations
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