ABSTRACT
Objective To investigate the effects of specific protein 1 ( Sp1 ) on the TNF-αin-duced expression of inositol 1, 4, 5 trisphosphate receptor type 1 ( IP3R1 ) in human mesangial cells ( HMCs) and to further elucidate the molecular mechanism regarding the decreased glomerular filtration rate ( GFR ) during hepatorenal syndrome .Methods Quantitative real-time polymerase chain reaction and Western blot assay were used to analyze the effects of TNF-αon the expression of IP3R1 at mRNA level and the expression of IP3R1 and Sp1 at protein level in HMCs , respectively.HMCs were transfected with a re-combinant plasmid PGL3-IP3R1 promoter to determine the effects of TNF-αon the activity of IP3R1 promot-er.HMCs were treated with Mithramycin A , an inhibitor of Sp1 binding, and transfected with Sp1-siRNA plasmid respectively to evaluate the expression of IP 3R1 regulated by TNF-α.The role of TNFR1 and TNFR2 in the TNF-αinduced expression of Sp 1 and IP3R1 proteins were detected by Western blot .Results TNF-αincreased the expression of IP3R1 at mRNA level and the expression of IP3R1 and Sp1 at protein lev-el in HMCs.Moreover, the activity of IP3R1 promoter in HMCs was remarkably increased by TNF-αas well.TNF-αinduced expression of IP3R1 was inhibited by Mithramycin A in a concentration dependent manner.HMCs transfected with Sp1-siRNA plasmid showed a significantly decreased expression of IP 3R1 protein.Both anti-TNFR1 and anti-TNFR2 antibodies blocked the TNF-αinduced IP3R1 expression, while only anti-TNFR1 antibodies inhibited the TNF-αinduced Sp1 expression.Conclusion TNF-αmight in-crease the expression of IP3R1 through the TNFR1/Sp1 signaling pathways in HMCs .
ABSTRACT
Objective To explore the effects of TNF-α on the expression of IP33R1 mRNA and protein in human mesangial cells (HMCs) and elucidate the mechnism of TNF-α indnces the IP3R1 expression in the occurrence of hepatorenal syndrome (HRS).Methods HMCs was stimulated by tumor (TNF-α) with 100 ng/mL for different hours (2,4,8,24 hours).The expression change of IP3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblot assay.Several inhibitors including D609,U73122,PP1,Safingol,Rottlerin and non-radioactive PKC assay to examine the mechanism of signal transduction of TNF-α-regulated IP3R1 in HMCs.Results The levels of IP3R1 mRNA at 2 h post-TNF-α exposure were significantly enhanced and reached peak at 8 h in HMCs (P ＜ 0.01),then descened at 24 h (P ＜ 0.01).The levels of IP3R1 protein at 4 h post-TNF-α exposure were obviously increased and reached peak at 24 h post-TNF-α exposure (P ＜ 0.01).Compared with the control group,safingol (PKC-α inhibitor) and D609 (PC-PLC inhibitor) each significantly suppressed TNF-α-induced expression of IP3R1 mRNA (3.30 ± 0.81) vs.(1.95 ± 0.130,P ＜ 0.05 ; (2.10 ± 0.49),P ＜ 0.01 andIP3R1 protein (3.09±0.13) vs.(1.86+0.39),P＜0.01; (1.98±0.02),P＜0.01.TNF-αpromoted autophosphorylation,and hence the activation,of PKC-α with maximal phosphorylation that occurred 8 h post-stimulation measured by non-radioactive PKC assay,and the effect was marked attenuated by pretreated with D609 or safingol.Conclusions TNF-α increased the expression of IP3R1 and this was mediated,at least in part,through the PC-PLC/PKC-α signaling pathways in HMCs.
ABSTRACT
Objective To explore the effects of TNF-α on the expression of IP3 R1 mRNA and protein in human mesangial cells (HMCs) and elucidate the role of protein kinase C (PKC) in this signal pathway.Methods Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effects of TNF-α on IP3R1 mRNA and protein expression.Depletion PKC,the selective inhibitor of PKCα Safingol and inhibitor of PKCδ Rottlerin,overexpression of dominant negative mutant of PKC to examine the mechanism of signal transduction of TNF-α-regulated IP3 R1 in HMCs.PKCα activation was assayed by Western blot.Results TNF-α increased IP3R1 mRNA and protein expression in HMCs,effects that were blocked by prolonged incubted chronic PMA,Safingol and also by domain negative PKCα construct.TNF-α promoted PKCα activation with maximal PKCα phosphorylation that occurred 8 h post-stimulation.Conclusion TNF-α increased the expression of IP3 R1 and this was mediated through the PKCα activation signaling pathways in HMCs.