PKCα signaling pathway involves in TNF-α-induced IP3R1 expression in human mesangial cells / 世界急诊医学杂志（英文）

BACKGROUND: This study aimed to explore the effects of TNF-α on the expression of IP3R1 mRNA and protein in human mesangial cells (HMCs), and to elucidate the mechanism of TNF-α relating to IP3R1 expression in the occurrence of hepatorenal syndrome (HRS). METHODS: HMCs were stimulated by tumor (TNF-α) with 100 ng/mL for different hours (2, 4, 8, and 24 hours). The expression changes of IP3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting. Several inhibitors including D609, U73122, PP1, safingol, rottlerin and non-radioactive protein kinase C (PKC) were used to examine the mechanism of signal transduction of TNF-α-regulated IP3R1 in HMCs. RESULTS: The levels of IP3R1 mRNA at 2 hours after TNF-α exposure were significantly enhanced and peaked at 8 hours in HMCs (P<0.01), then descended at 24 hours (P<0.01). The levels of IP3R1 protein at 4 hours after TNF-α exposure were obviously increased and peaked at 24 hours after TNF-α exposure (P<0.01). Compared to the control group, safingol (PKCα inhibitor) and D609 (phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-α-induced expression of IP3R1 mRNA (3.30±0.81 vs. 1.95±0.13,P<0.05; 2.10±0.49,P<0.01) and IP3R1 protein (3.09±0.13 vs. 1.86+0.39,P<0.01; 1.98±0.02,P<0.01). TNF-α promoted PKCα activation with maximal PKCα phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay, and the effect was markedly attenuated by pretreatment with D609 or safingol. CONCLUSION: TNF-α increased the expression of IP3R1 and this was mediated, at least in part, through the PC-PLC/PKCα signaling pathways in HMCs.

Effect of Chinese propolis on PC-PLC activity and TLR4 expression in LPS-treated vascular endothelial cells / 中国病理生理杂志

AIM: To investigate the effect of Chinese propolis on the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and the expression of Toll-like receptor 4 (TLR4) in LPS-treated vascular endothelial cells (VECs). METHODS: Confluent VECs were stimulated with LPS at the concentration of 100 μg/L in the presence of 0.5% fetal bovine serum. The cells were treated with Chinese propolis at the concentration of 12.5 mg/L for 12 h and 24 h. The viability of VECs and the level of nitric oxide (NO) were detected by sulforhodamine B (SRB) assay and chemical method, respectively. The activity of PC-PLC was measured using L-α-phosphatidylcholine as substrate. The protein levels of TLR4, nuclear factor-Κb p65 (NF-Κb p65) and p53 were determined by Western blotting. The level of intracellular reactive oxygen species (ROS) was examined using a fluorescent probe, 2',7'-dichlorodihydrofluorescin (DCHF). For the measurement of mitochondrial membrane potential, the fluorescent dye JC-1 was used. RESULTS: Treatment with Chinese propolis for 24 h had no effect on the viability of VECs. However, the levels of NO and ROS were significantly decreased by Chinese propolis. PC-PLC activity and NF-Κb p65 expression were significantly depressed by Chinese propolis treated for 12 h, and the expression of TLR4 and p53 were dramatically decreased by Chinese propolis treated for 12 and 24 h. No effect of Chinese propolis on mitochondrial membrane potential was observed. CONCLUSION: Chinese propolis depresses the activity of PC-PLC and the expression of TLR4, and then inhibits the downstream signal molecules such as NF-Κb p65, p53, ROS and NO in VECs.

D60-sensitive tyrosine phosphorylation is involved in Fas-mediated phospholipase D activation

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.

D60-sensitive tyrosine phosphorylation is involved in Fas-mediated phospholipase D activation

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.