Abstract:

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.

Key words: TNF-α, Hepatorenal syndrome, Human mesangial cells, Protein kinase C, Phosphatidylcholine-specific phospholipase C