摘要
目的
肝肾综合征(HRS)的主要发病机制是肾小球滤过率(GFR)明显下降。已知肾小球系膜细胞(GMCs)收缩可引起肾小球滤过面积减少。HRS患者血中肿瘤坏死因α(TNFα)水平异常升高,TNFα是否参与了GMCs收缩引起的GFR下降尚不清楚。胞浆游离Ca2+浓度([Ca2+]i)升高与GMCs收缩密切相关。1,4,5-三磷酸肌醇受体(IP3Rs)是细胞内重要的Ca2+释放通道。因此GMCs中IP3Rs表达的多少与其对缩血管物质的敏感性有着密切联系。本研究用TNFα处理原代培养的人肾小球系膜细胞(HMCs),实时定量PCR和免疫印迹检测IP3R1mRNA和蛋白的表达；并应用信号转导抑制剂及瞬时转染活性失活的负显性突变体质粒检测对TNFα对IP3R1mRNA和(或)蛋白的影响,明确IP3R1的关键上游信号蛋白并探究肿瘤坏死因受体(TNFR)在此信号通路中的作用。初步明确TNFα促进IP3R1表达的信号通路,以期为肝肾综合征GFR下降的发生机制和防治思路提供理论依据。材料与方法
1、材料
人肾小球系膜细胞(HMC)和人肾小球系膜细胞培养液(MCM)购至美国sccience cell公司；TNFα,兔抗人TNFR1抗体、兔抗人TNFR2抗体,R&D公司；兔抗人I型IP3R抗体,Chemicon International公司；兔抗人PKCα抗体,p-PKCα(thr637)抗体,PKC总抗体购至Celll singnaling technology公司；兔抗人β-actin多克隆抗体,Santa Cruz公司；anti-HA抗体购至Sigma公司；辣根过氧化物酶标记山羊抗兔二抗,Santa Cruz; PP1、rottleri、U73122、D609和Safingol购至Calbiochen公司；PepTagRAssay for Non-Radioactive Detection of Protein Kinase C Kit, promega公司；Enhanced chemiluminescence (ECL)试剂盒,pierce公司；Trizol试剂,Invitrogen公司；引物合成,Takara公司；逆转录试剂盒ExScriptTM RT Reagent Kit、PCR试剂盒SYBR(?) premix EX TaqTM(TaKaRa)、Takara公司；人野生型PKCα(pHACE-WT-PKCα),活性失活的PKCα(pHACE-DN-PKCα)质粒及pCDNA3.0质粒由Dr. Jae-Won Soh, Inha University in Korea.惠赠(已经大连宝生物工程有限公司测序分析)。去内毒素质粒小提及中提试剂盒,天能公司；转染试剂盒：Liprofetamin-2000, invitrogen公司。
2、方法
(1)分组：
1) TNFα不同时间点处理细胞分组1:TNFα处理0h、2h、4h、8h、24h组。
2) TNFα不同时间点处理细胞分组2:TNFα处理0h、4h、8h、24h组。
3)PMA预处理HMC细胞模型：①TNFα处理Oh组(T0h组)、
②TNFα处理24h组(T24h组)、
③PMA+TNFα处理组(PMA+T组)
4)多种抑制剂预处理HMC细胞模型1
①TNFα处理0h组(T0h组)、②TNFα处理8h组(T8h组)
②safingol处理组(Sa组)、④PP1组(PP1组)、
⑤rottlerin组(rot组)、⑥U73122组(U组)、
⑦D609组(D组)⑧Sa+T组、⑨PP1+T组、
⑩rot+T组、(?)U7+T组、(?)D+T组。
5)多种抑制剂预处理HMC细胞模型2
①TNFα处理0h组(T0h组)、②TNFα处理24h组(T24h组)、
③Sa+T组、④PP1+T组、⑤rot+T组、⑥U7+T组、⑦D+T组
6)阻断PLC, PKCα信号通路细胞模型：
①TNFα处理0h组(T0h组)、②TNFα处理8h组(T8h组)、
③safingol+TNFα(Sa+T组)、④U73122+TNFα组(U+T组)、
⑤D609+TNFα组(D+T组)。
7)瞬时转染细胞模型：
①正常对照组(control组)②TNFα刺激8h组或24h(T8h或T24h)
③pCDNA3. 0+TNFα④pHACE-WT-PKCα+TNFα⑤HACE-DN-PKCα+TNFα
8) TNFR1抗体或TNFR2抗体预处理HMC细胞模型：
①正常对照组(T0h组)②TNFα刺激8h组或24h组(T8h或T24h组)
③TNFR1抗体+TNFα组(TR1+T组)④TNFR2抗体+TNFα组(TR2+T组)
(2)应用Western blot、Real-time PCR方法检测TNFα对HMCs中IP3R1mRNA和蛋白表达的影响。
(3)应用PMA预处理HMCs, Western blot检测对TNFα诱导IP3R1蛋白表达的影响。
(4)应用多种信号转导抑制剂预处理HMCs, Western blot和Real-time PCR方法检测TNFα对HMCs中IP3R1mRNA和蛋白表达的影响。
(5)应用Western blot,非放射性PKC测活方法检测TNFα对HMCs中PKCα的活化和(或)PKCα蛋白表达的影响。
(6)瞬时转染野生型和活性失活PKCα质粒,Western blot和Real-time PCR方法检测TNFα对HMCs中IP3R1mRNA和蛋白表达的影响。
(7)应用TNFR1抗体或TNFR2抗体预处理HMCs, Western blot检测对TNFα诱导IP3R1蛋白和p-PKCα蛋白表达的影响。
结果
1、实时定量PCR检测各组细胞IP3R1mRNA表达
与TOh比较,TNFα处理组IP3R1mRNA的表达明显增加,2h到8h IP3R1mRNA稳定逐渐增加,于8h达高峰(P<0.001)。
2、Western Blot检测各组细胞IP3R1蛋白表达
与TOh比较,TNFα处理组IP3R1的表达明显增加,4h开始升高,至24h IP3R1蛋白升高达高峰,统计学有性显著差异(P<0.001)。
3、PMA下调内源性蛋白激酶C (PKC)对TNFα诱导IP3R1蛋白表达的影响
与T24h相比,PMA+T组,IP3R1蛋白表达明显下降(P<0.001)。PMA刺激HMCs24小时后,总PKC的表达与对照组相比明显下降。
4、多种信号抑制剂对TNFα诱导IP3R1mRNA的影响
与T8h组比较,Sa+T和D+T组IP3R1 mRNA的表达明显下降,有统计学差异(P<0.05),而U+T, PP1+T, rot+T组IP3R1 mRNA的表达无明显变化(P>0.05)。
5、多种信号抑制剂对TNFα诱导IP3R1蛋白表达的影响
与T24h组比较,Sa+T和D+T组,IP3R1蛋白的表达明显下降(P<0.001),有统计学意义,而U+T, PP1+T, rot+T组对TNFα上调IP3R1蛋白的表达无明显影响(P>0.05)。
6、Western blot分析PKCα和p-PKCα蛋白表达
PKCα蛋白表达：HMCs中PKCα蛋白呈高水平表达,但PKCα蛋白的表达在TNFα处理各组与对照组间无显著差异(P>0.05)。
p-PKCα蛋白表达：与T0h比较,TNFα刺激8h组p-PKCα蛋白表达明显增加(P<0.01),有统计学差异。
7、Western blot分析抑制剂对TNFα活化PKCα的影响
与T8h比较,Sa+T和D+T组p-PKCα蛋白表达显著减少(P<0.01),且差异具有统计学意义。
8、PKCα活性检测
TNFα处理8h PKCα活性最强(P<0.01),统计学有显著性差异。而与T8h比较,Sa+T和D+T组PKCα活性明显减弱。
9、实时定量PCR和Western blot检测瞬时转染野生型和活性失活PKCα质粒后IP3R1mRNA和蛋白的表达
与T8h组比较,pHACE-WT-PKCα+TNFα组IP3R1mRNA的表达明显增加,统计学上有显著差异(P<0.01)；而pHACE-DN-PKCα+TNFα组IP3R1mRNA的表达明显降低(P<0.01)。与T24h组比较,pHACE-WT-PKCα+TNFα组IP3R1蛋白表达明显增加,统计学上有显著差异(P<0.001)；而pHACE-DN-PKCα+TNFα组IP3R1mRNA的表达明显降低,统计学上有显著差异(P<0.001)。
10、应用TNFR1抗体或TNFR2抗体预处理对p-PKCα和IP3R1蛋白表达的影响
与T8h组比较,TRl抗体+TNFα组p-PKCα蛋白表达明显下降,统计学分析有显著性差异(P<0.001)；而TR2抗体+TNFα组,p-PKCα蛋白表达无明显变化。与T24h组比较,TR1抗体+TNFα组和TR2抗体+TNFα组IP3R1蛋白表达均有不同程度的下降,并且统计学分析均有显著差异(P=0.002；P=0.02)。
结论
1、TNFα处理的HMCs中,IP3R1mRNA和蛋白的表达明显增加。
2、TNFα处理的HMCs中,PKCα存在明显激活现象并与TNFα上调IP3R1蛋白表达密切相关。
3、TNFα通过TFNR1/PC-PLC途径活化PKCα.
4、TNFα通过TFNR1/PC-PLC/PKCα和TNFR2独立的两条信号通路上调IP3R1的表达的。
Aim
The 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 methods
1、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 experiments
1) Primarily cultured GMCs were divided into TNFα-treated 0h,2h,4h,8h,24h
2) Primarily cultured GMCs were divided into TNFα-treated 4h,8h,12h,24h
3) the HMCs model of Pretreatment with PMA
①TNFα-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 inhibitors
①TNFα-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 inhibitors
①TNFα-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 pathway
①TNFα-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 transfection
①the nomal group (control group)
②TNFα-treated 8 h or 24 hgroup (T8h or T24h group)③pCDNA3.0+TNFα
④pHACE-WT-PKCα+TNFα⑤pHACE-DN-PKCα+TNFα
8) the HMCs model of Pretreatment with anti-TNFR1 antibody or anti-TNFR1 antibody
①the 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 HMCs
Results
1. 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 protein
Our 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 protein
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.
6. Effects of TNFαon intracellular PKCαsignaling pathways
TNFαpromoted 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 PKCαactivation
Expression 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 assays
There 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 HMCs
IP3R1 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).
Conclusion
1、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.
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