The Study Of TNFα-induced IP₃R1 Expression And The Relevant Signal Transduction In Human Mesangial Cells

AimThe reduction in glomerular filtration rate (GFR) is the key pathogenesis of hepatorenal syndrome (HRS). Glomerular mesangial cells (GMCs) are closely associated with glomerular filtration area. Cell contraction is closely related to changes in the intracellular calcium concentration ([Ca2+]i). The inositol 1,4,5-trisphosphate receptors (IP3Rs) are the main intracellular Ca2+ release channels. Obviously high plasma level of tumor necrosis factor-α(TNFα) is also found in HRS. There is little known about the precise mechanisms of TNFαin decreased GFR. Is there some interaction between the increased TNFαlevels and the contraction of GMCs? Theoretically, the change in IP3R1 expression level has been linked to regulation of [Ca2+] i.In order to explore the possible pathogenesis, we evalued the IP3R1 mRNA and proetin levels of HMCs treated by TNFαwith Real time quantitative PCR and Western blot methods. Furthermore, the corresponding singaling mechanisms by which TNFαinfluenced on the IP3R1 expression was also tested with various singnal transduction inhibitors and domain negative constructs transient transfection methods. Identification of TNFα-dependent IP3R1 genes singnal transduction may also help to better elucidate the biological functions of these key enzymes and this pathway may become an important target for future therapies in TNFα-mediated sever hepatitis.Materials and methods1,Materials Primary culturing HMCs and Mesangial cell medium (MCM 4201) were obtained from the Science Cell Research Laboratories (San Diego, CA). HMCs were grown in MCM according to the supplier's instructions. These cells are characterized by the manufacturer using morphological appearances and immunofluorescent method with various antibodies, including anti-Thy-1 and fibronectin.2,Methods(1) Groups depending on the requirements of experiments1) Primarily cultured GMCs were divided into TNFα-treated 0h,2h,4h,8h,24h2) Primarily cultured GMCs were divided into TNFα-treated 4h,8h,12h,24h3) the HMCs model of Pretreatment with PMA①TNFα-treated Oh group (control group, TO group)②TNFα-treated 24h group (T24h group)③PMA+TNFαgroup (PMA+T group)4) the HMCs model 1 of pretreatment with various inhibitors①TNFα-treated 0 h group (T0 group)②TNFα-treated 8 h group (T8h group)③safingol-treated group (Sa group)④PP1-treated group (PP1 group)⑤rottlerin-treated group (rot group)⑥U73122-treated group (U group)⑦D609-treated group (D group)⑧TNFα-safingol cotreated group (S+T group)⑨TNFα-PP1 cotreated group (PP1+T group)⑩TNFα-rottlerin cotreated group (rot+T group)(?)TNFα-U73122 cotreated group (U7+T group)(?)TNFα-D609 cotreated group (D+T group)5) the HMCs model 2 of pretreatment with various inhibitors①TNFα-treated 0 h group (T0 group)②TNFα-treated 24 h group (T24h group)③TNFα-safingol cotreated group (S+T group)④TNFα-PP1 cotreated group(PP1+T group)⑤TNFα-rottlerin cotreated group (rot+T group)⑥TNFα-U73122 cotreated group (U7+T group) ⑦TNFα-D609 cotreated group (D+T group)6) the HMCs model of blocking PLC and PKCasignaling pathway①TNFα-treated 0 h group (T0 group)②TNFα-treated 24 h group (T24h group)③TNFα-safingol cotreated group (S+T group)④TNFα-U73122 cotreated group (U7+T group)⑤TNFα-D609 cotreated group (D+T group)7) the HMCs model of transient transfection①the nomal group (control group)②TNFα-treated 8 h or 24 hgroup (T8h or T24h group)③pCDNA3.0+TNFα④pHACE-WT-PKCα+TNFα⑤pHACE-DN-PKCα+TNFα8) the HMCs model of Pretreatment with anti-TNFR1 antibody or anti-TNFR1 antibody①the nomal group (control group)②TNFα-treated 8 h or 24 hgroup (T8h or T24h group)③TNFR1-TNFαcotreated group (TR1+T)④TNFR2-TNFαcotreated group (TR2+T)(2) The effects of TNFαon the leval of IP3R1 mRNA determined by Quantitative real-time polymerase chain reaction and IP3R1 protein determined by western blot(3) Effects of pretreatment with PMA on TNFα-induced IP3R1 protein expression(4) Effects of cell signaling inhibitors on TNFα-induced IP3R1 mRNA and protein expression(5) Effects of TNFαon intracellular PKCαsignaling pathways determined by western blot and non-radioactive protein kinase c assay.(6) Effects of transient transfection of HMCs with a dominant-negative PKCαconstruct on TNFα-induced IP3R1 mRNA and protein expression.(7) Effects of anti-TNFR1/2 antibody on TNFα-induced IP3R1 expression and PKCαactivity in HMCsResults1. Real time PCR analysis of IP3R1 mRNA We found an increase of IP3RI mRNA expression in TNFα-treated 2 h-24 h groups compared with control group. The maximal effect was seen at 8 h (P<0.01).2. Western blot analysis of IP3R1 proteinOur results showed low level expression of IP3R1 protein in control group. The expression of the IP3R1 protein obviously increased in TNFα-treated 4 h-24 h groups. The maximal effect was seen at 24 h group (P<0.01)3. Effects of PKC downregulation by PMA on TNFα-induced IP3R1 protein expression.Expression of the IP3R1 protein was significantly reduced in HMCs that were pretreated by PMA prior to TNFαexposure compared with TNFαstimulation alone (P <0.01).4. Effects of cell signaling inhibitors on TNFα-induced IP3R1 mRNA and protein expression.IP3R1 protein expression was significantly attenuated in the group that was pretreated with D-609 or safingol prior to TNFαexposure compared with TNFαstimulation alone (P<0.01). wherears the effects of TNFαwas not affected by U73122, rottlerin or PP1 pretreament.5. Effects of cell signaling inhibitors on TNFα-induced IP3R1 proteinIP3R1 protein expression was significantly attenuated in the group that was pretreated with D-609 or safingol prior to TNFαexposure compared with TNFαstimulation alone (P<0.01). wherears the effects of TNFαwas not affected by U73122, rottlerin or PP1 pretreament.6. Effects of TNFαon intracellular PKCαsignaling pathwaysTNFαpromoted of PKCαwith maximal phosphorylation that occurred 8 h post-stimulation while the total PKCαprotein expression did not differ between the different groups. 7. Effects of cell signaling inhibitors on TNFα-induced PKCαactivationExpression of the p-PKCαprotein was significantly reduced in HMCs that were pretreated by D609 and safingol prior to TNFαexposure compared with TNFαstimulation alone (P<0.01). wherears the effects of TNFαwas not affected by U73122.8. PKCαactivity assaysThere was mild activity of PKCαin control group. TNFαcould strikingly increase the activity of PKCαand the maximal effect was seen at 8 h. PKCαactivation was attenuated by the pretreatment of HMCs with D609 and safingol.9. Effects of transient transfection of HMCs with a dominant-negative PKCαconstruct on TNFα-induced IP3R1 mRNA and protein expression.Compared with TNFαstimulation alone, IP3R1 mRNA by the use of qRT-PCR and protein expression by western blot were significantly decreased in HMCs transfected with the pHACE-DN-PKCαconstruct (P<0.01), but increased in cells transfected with the pHACE-WT-PKCαconstruct in response to TNFα.10. Effects of anti-TNFR1/2 antibody on TNFα-induced IP3R1 expression and PKCαactivity in HMCsIP3R1 expression were marked reduced in the groups pretreated with anti-TNFR1 or anti-TNFR2 antibodies to various degrees (P=0.002; P=0.02). while phosphorylated PKCαexpression was significantly blocked only by anti-TNFR1 pretreatment (P<0.01).Conclusion1,TNFαhas a potent ability to increase the protein expression of IP3R1 in HMCs. The increased protein expression of IP3R1 may be due to increased synthesis of IP3R1 protein as we found an increase in mRNA levels prior to observing an increase in protein levels. 2,PKCαactivity induced by TNFαwas closely associated with TNFα-induced IP3R1 upregulation in HMCs.3. TNFα/TNFR1/PC-PLC dependent but not TNFR2 or PI-PLC might mediate TNFα-enhanced PKCαactivity in HMCs.4% TNFαincreased the expression of IP3R1 from HMCs, and this was mediated, at least in part, through the TNFR1/PC-PLC/PKCαand TNFR2 signaling pathways.