KCα抑制生肌素转位及nAChR基因表达

烟碱样乙酰胆碱受体(nAChR)的表达调控受神经电活动影响,电刺激引起肌细胞膜去极化可抑制nAChR的表达.以往的研究表明,Ca2+和PKC以及生肌素在其中发挥着重要的作用.然而,目前尚不清楚究竟是哪种PKC亚型参与此过程,PKC激活对特异转录因子生肌素浆核转位有何影响?为探讨PKC在去极化-nAChR转录偶联中的作用,构建了含nAChRγ亚基启动子的绿色荧光蛋白(GFP)表达载体pEGFP-γ,将其分别与4种cPKC(PKCα、PKCβⅠ、PKCβⅡ、PKCγ)真核表达载体共转染C2C12肌细胞.结果发现PKCβⅠ、PKCβⅡ对nAChRγ启动子驱动的GFP报告基因表达没有影响(P＞0.05),PKCγ对报告基因表达有抑制作用(P＜0.05),PKCα则有明显抑制作用(P＜0.01).采用4种cPKC真核表达载体与GFP-生肌素融合蛋白表达载体(pGFP-myog)共转染C2C12肌细胞,观察了不同亚型PKC表达对生肌素浆至核转位的影响,发现只有强制性表达外源性PKCα可明显抑制生肌素向核中转位,而PKCβⅠ、PKCβⅡ及PKCγ对生肌素浆核转位没有明显抑制作用.结果提示,PKCα通过抑制生肌素转位是阻遏nAChR基因表达机制之一.     

去极化时Ca~(2+) 内流引起蛋白激酶C_α浆膜转位

 为观察胞外Ca2 + 内流和肌浆网Ca2 + 释放两种来源的Ca2 + 对cPKCα转位激活的影响 ,揭示PKC在去极化 nAChR转录偶联中的作用 ,构建了pPKCα EGFP N1融合蛋白真核基因表达载体 .转染C2C1 2肌细胞后 ,采用激光共聚焦显微镜记录了KC1或咖啡因处理所引起的细胞Ca2 + 波变化及PKCα GFP融合蛋白在细胞内的分布 .结果提示 ,只有用KC1处理引起细胞膜去极化时 ,伴随Ca2 +内流 ,才能观察到PKCα GFP绿色荧光在细胞内发生的细胞浆至细胞膜分布变化 .然而 ,采用肌浆网Ca2 + 通道激动剂咖啡因刺激肌细胞 ,使肌浆网中Ca2 + 释放 ,未见PKCα GFP绿色荧光在浆、膜分布发生任何变化 .结果提示 ,去极化时外Ca2 + 内流可引起PKCα转位激活 ,肌浆网Ca2 + 释放对PKCα的转位激活没有影响 .     

生肌素引起组蛋白H3和H4高度乙酰化及染色质溶解性增加

为研究生肌素在染色质重建过程中的作用 ,采用生肌素真核表达载体转染C2C12肌细胞 ,细胞核经微球菌核酸酶消化后 ,提取DNA进行SDS PAGE分析 .生肌素转染的细胞 ,其核小体 (染色质 )在Mg2 + 溶液中的溶解性明显增加 ,提示染色质组蛋白乙酰化程度提高 .组蛋白经TAU SDS(2 D)双相凝胶电泳分析 ,发现在转染生肌素真核表达载体 2 4h后 ,组蛋白H4的乙酰化修饰程度最高 .采用抗乙酰化组蛋白H3和H4抗体进行的Western印迹分析进一步证明了乙酰化的发生 .上述变化与染色质的活跃程度相关 .RT PCR结果显示 ,生肌素的靶基因烟碱样乙酰胆碱受体 (nAChR)α亚基和肌酸激酶 (MCK)基因在转染后表达水平提高 .结果提示 ,强制性表达外源性生肌素可引起肌细胞核染色质重建 ,进而激活靶基因     

电刺激引发骨骼肌细胞钙振荡对nAChRγ启动子活性的影响

利用不同电刺激条件模拟神经电活动 ,研究C2C12细胞 (小鼠骨骼肌成肌细胞系 )内Ca2 + 激活的不同形式及其对nAChR基因表达活性的影响 .电刺激分化 1d的C2C12细胞 ,用共聚焦显微镜记录不同电刺激参数引起的细胞内Ca2 + 信号变化 ,共观察到 3种不同形式的Ca2 + 信号 ,称之为整体Ca2 + 振荡、局部Ca2 + 振荡和局部Ca2 + 振荡诱发的整体Ca2 + 振荡 .在此基础上 ,又构建nAChRγ亚基启动子萤光素酶报告基因质粒并转染C2C12细胞 ,进一步检测不同形式Ca2 + 引起细胞萤光素酶活性的变化 ,测定细胞内PKC活性的改变 .不同Ca2 + 振荡引起细胞内PKC活性升高幅度不同 ;电刺激C2C12细胞可导致nAChRγ基因表达活性降低 ,3种Ca2 + 振荡对nAChRγ基因启动子活性的影响无显著差异 .结果表明 ,电刺激可引起肌细胞产生不同形式Ca2 + 信号 ,但不同形式Ca2 + 信号对nAChRγ启动子活性的抑制无明显差异 ,提示Ca2 + 依赖的PKC途径可能不是nAChR基因表达下调的唯一途径     

低氧预适应增高小鼠脑组织内cPKCγ的膜转位水平

本实验拟通过观察重复性低氧对经典型蛋白激酶C(cPKC)膜转位水平(激活程度)的影响,初步探讨cPKC特定亚型在脑低氧预适应发生过程中的作用。按我室已建立的小鼠低氧预适应模型方法,制备重复性低氧1-4次的小鼠(H1-H4)。应用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白印迹(Western bolt)等生化技术,并结合Gel Doc凝胶成像系统,半定量检测小鼠海马和大脑皮层组织内cPKCα和γ的膜转位水平。实验结果表明,随低氧次数(H1-H4)的增加,小鼠海马组织内cPKCγ的膜转位水平明显增高,且在H2、H3和H4组的变化具有统计学显著意义(P<0.05,n=6);同样,大脑皮层内cPKCγ膜转位水平也随低氧次数的增加(H1-H4)而明显增高,且在H2、H3和H4组的变化具有统计学显著意义(P<0.05,n=6):而cPKCα亚型无论在大脑皮层还是在海马组织内的膜转位变化均无统计学意义。上述观察结果提示,cPKCγ膜转位可能在脑低氧预适应的发生发展过程中发挥着重要作用;但cPKCβ Ⅰ、β Ⅱ以及其它新奇型和非典型PKC特定亚型的变化还有待于进一步的研究和探讨。     

蛋白激酶C亚型在HL-60细胞诱导分化中的变化

用全反式维甲酸(ATRA)或佛波酯(PMA)处理人早幼粒白血病细胞(HL-60)3天,用形态学,NBT还原实验,特异性和非特异性酯酶测定,证明细胞分别向粒细胞或单核/巨噬细胞分化。通过免疫组化法观察了蛋白激酶C(PKC)α,βⅠ和βⅡ亚型在分化后的变化。结果显示,ATRA可引起HL-60细胞PKCα,βⅠ和βⅡ的含量升高,分别为对照的5.0,2.8和4.2倍,并存在从胞膜向胞质转位。PMA则使PKCα和βⅡ的表达水平下降,而PKCβⅠ增高,且三种亚型均在核内有不同程度的表达。结果提示HL-60细胞向粒细胞的分化可能需要PKCα和PKCβ评的持续激活,而PKC的核内转位可能是HL-60细胞分化为单核/巨噬细胞的重要环节。     

肌肉LIM蛋白增强生肌素对AChRγ启动子的反式激活作用

采用RT PCR技术克隆了大鼠肌肉LIM蛋白 (MLP) 6 40bp的全长cDNA序列 .以此cDNA为探针进行的Northern印迹表明 ,MLP于C2C12细胞在分化的第 3d至第 5d表达 .将MLPcDNA亚克隆至pcDNA3,构建真核表达质粒pcDNA3 MLP ,同时构建AChRγ启动子序列 (96 0bp)调控的荧光素酶报告基因真核表达质粒pGL3 γ .C2C12细胞转染及荧光素酶活性分析表明 ,复合转染pcD NA3 MLP和pGL3 γ的分化肌细胞表达的荧光素酶活性约为对照的 4倍 ;而在 3T3或未分化肌细胞复合转染pcDNA3 MLP和pGL3 γ均未检出报告基因表达 ,说明MLP可促进生肌素对AChRγ亚基基因启动子的反式激活作用 .     

LRP16对乳腺癌MCF-7细胞增殖的影响

用Northern印迹方法检测雌二醇 (17β E2 )对LRP16mRNA表达的时间及剂量依赖性调控作用 .构建LRP16基因启动子序列调控的萤光素酶报告子 (pS0 ) ,并与雌激素受体α和 β(ERα和ERβ)表达载体共转染COS 7和MCF 7细胞后测定萤光素酶活性 .将LRP16基因的表达载体转染MCF 7细胞 ,测定过表达LRP16对细胞的生长特性的影响 .17β E2 使MCF 7细胞中LRP16mRNA表达水平增加 ,增加幅度未显示出 17β E2 培养时间和剂量的依赖性 .pS0 与ERα表达载体共转染细胞的相对萤光素酶活性较非共转染组 (对照组 )及pS0 ERβ表载体共转染组升高 5～ 10倍 .LRP16基因过表达促进MCF 7细胞的增殖 .研究表明 ,雌激素可能通过ERα上调乳腺癌MCF 7细胞LRP16基因的表达并促进细胞增殖     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

CNTF对NMDA引起大鼠海马神经元蛋白激酶C核转位的影响

目的：探讨睫状神经营养因子(CNTF)对N-甲基-D-天冬氨酸(NMDA)引起大鼠海马神经元蛋白激酶C(PKC)发生核转位的影响。方法：以NMDA及CNTF处理原代培养的大鼠海马神经元,PKCγ、免疫细胞化学并结合图象分析方法测定PKC阳性神经元胞核的灰度。结果：①给予不同浓度NMDA处理不同时间后,神经元核内有不同程度的PKCγ及PKCε表达,其中以100μmol／L NMDA 30min组表现尤为显著;②CNTF 500μmol／L NMDA组神经元胞核PKC灰度与对照组相似。结论：NMDA可引起海马神经元PKCγ、PKCε的核转位,而CNTF则抑制其转位的发生,提示CNTF对海马神经元的保护作用与抑制PKC的核转位右关。     

鸡AChRγ亚基基因远上游增强子样作用依赖生肌因子

先前曾报告鸡ＡＣｈＲγ亚基基因５＇连接区－５３８／－２８８片段（含一对Ｅ盒子）与生肌调节因子（ｍｙｏｇｅｎｉｎ）的相互作用,为进一步观察ＤＮＡ－ｍｙｏｇｅｎｉｎ相互作用对γ基因转录激活功能的影响,利用瞬时表达体系研究了鸡ｎＡＣｈＲγ亚基基因５＇端－５３８／－２８８片段调控机能。ＣＡＴ分析表明：γ基因－５３８／－２８８片段可显著激活ｔｋ启动子在分化的Ｃ２Ｃ１２肌细胞中的转录活性,但不能激活在未分化Ｃ２Ｃ１２肌细胞中的转录活性。这种组织特异性转录激活作用与距离无关,因而是一个典型的增强子;－５３８／－２８８片段的增强子样作用既依赖于两毗邻Ｅ盒子的完整性,又依赖于ｍｙｏｇｅｎｉｎ等生肌调节因子的存在。强化表达ｍｙｏｇｅｎｉｎ可激活ｐＢＬＣＡＴ２－γ（－５３８／－４３３）和ｐＢＬＣＡＴ２－γ（－４３２／－２８８）（各含一个Ｅ盒子）在分化肌细胞中的表达,究竟是克隆片段中的独立Ｅ盒子对这种表达起贡献还是出现在克隆位点附近的反向Ｅ盒子（ＧＴＣＧＡＣ）或ｔｋ启动子中的另一个Ｅ盆子起作用？尚不十分清楚。     

异源基因α1,3半乳糖转移酶与增强型绿色荧光蛋白融合对荧光蛋白表达的影响

目的 研究异源(猪)基因α1,3半乳糖转移酶(3GT)与增强型绿色荧光蛋白(EGFP)基因形成的融合蛋白对其荧光表达量的影响.方法 BamHI,EcoRI酶切pcDNA3.1-α1,3GT重组载体后,回收含α1,3GT的片段,与BamHI、EcoRI酶切回收的pEGFP-N1载体连接,并酶切、测序鉴定重组真核表达载体p...     

Metabotropic signaling cascade involved in α4β2 nicotinic acetylcholine receptor-mediated PKCβII activation

Nicotinic acetylcholine receptors (nAChRs) belong to the ionophore receptor family, which regulates plasma membrane conductance to Na⁺, K⁺, and Ca²⁺ ions. Some studies, however, have shown that nAChRs also employ second messengers for intracellular signaling. We previously showed that α4β2 nAChR mediates the translocation of protein kinase CβII (PKCβII) from the cytoplasm to the plasma membrane, which is a typical activation marker for PKCβII. In this study, we investigated the molecular mechanisms underlying PKCβII activation through α4β2 nAChR. α4β2 nAChR is the most abundant nAChR subtype and is implicated in various brain functions and diseases. Putative α4β2 nAChR signaling components were identified by knockdown or chemical inhibition of candidate proteins, and the signaling cascade was deduced by protein interactions in predicted cellular components. α4β2 nAChR-mediated PKCβII translocation was found to occur in an ionophore activity-independent manner. Nicotinic stimulation of α4β2 nAChR activated Src in a β-arrestin1 and 14–3-3η-dependent manner. Activated Src phosphorylated the tyrosine residue(s) on Syk molecules, which in turn interacted with phospholipase C γ1 to trigger the translocation of PKCβII to the cell membrane by elevating cellular diacylglycerol levels. The activated PKCβII in turn exerted a positive feedback effect on Src activation, suggesting that α4β2 nAChR signaling is amplified by a positive feedback loop. These findings provide novel information for unveiling the previously unclear metabotropic second messenger-based signal transduction pathway of nAChRs.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

PKC对人mdr1基因转录调控的研究

研究了ＰＫＣα、β１和β２亚型对多药耐药基因ｍｄｒ１转录的调控作用．以ｍｄｒ１基因上游调控序列驱动的虫荧光素酶报告基因表达载体和ＰＫＣ基因表达载体共同转染ＣＯＳ７细胞后,测定虫荧光素酶报告基因表达水平．实验结果表明,ＰＫＣα、β１及β２对ｍｄｒ１基因的转录有下调作用,ＰＭＡ可进一步加强这一作用;在此转染体系中,ＰＫＣ抑制剂ｓｔａｕｒｏｓｐｏｒｉｎｅ也可抑制ｍｄｒ１基因的转录,但在只转染ｍｄｒｌｕｃ的细胞中,ｓｔａｕｒｏｓｐｏｒｉｎｅ对ｍｄｒ１的转录则没有影响,提示ｓｔａｕｒｏｓｐｏｒｉｎｅ仅在ＰＫＣ过量表达的细胞中抑制ｍｄｒ１基因的转录．     

Regulatory domain of human protein kinase Cα dominantly inhibits protein kinase Cβ-I regulated growth and morphology in Saccharomyces cerevisiae

This study demonstrates that the isolated regulatory (R) domain (amino acids 1–270) of human protein kinase Cα (PKCα) is a potent inhibitor of PKCβ-I activity in a yeast expression system. The PKCα R domain fused to glutathione-S-transferase competitively inhibited the activity of yeast-expressed rat PKCβ-I in vitro (Ki = 0.2 μM) and was 400-fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19–36 from PKCα. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMP-dependent protein kinase. The PKCα R domain (without glutathione-S-transferase [GST]) also was tested for its ability to inhibit PKCβ-I activity in vivo, in a yeast strain expressing rat PKCβ-I. Upon treatment with a PKC-activating phorbol ester, yeast cells expressing rat PKCβ-I were growth-inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKCβ-I blocked the phorbol ester-induced inhibition of yeast cell growth and the phorbol ester-dependent alterations in yeast cell morphology. These results indicate that the R domain of PKCα acts as a dominant inhibitor of PKC activity in vivo and thus provides a useful genetic tool to assess the roles of PKC in various signal transduction processes. © 1996 Wiley-Liss, Inc.     

Epithelial-mesenchymal transdifferentiation of renal tubular epithelial cells induced by urinary proteins requires the activation of PKC-α and βI isozymes

Proteinuria is a common feature for almost all glomerular diseases and reflects the severity of the glomerular lesion. The presence of a large amount of proteins in tubular fluid, however, may also contribute to the development of RIF (renal interstitial fibrosis). Endocytosis of albumin in proximal tubular cells triggers PKC (protein kinase C)-dependent generation of reactive oxygen species and secretion of chemokines. As a family including 12 isozymes, which PKC isozymes participate in RIF is still unclear. EMT (epithelial-mesenchymal transdifferentiation) of RTECs (renal tubular epithelial cells) plays a crucial role in the progress of RIF induced by proteinuria. In the present study, we investigated the role of classical PKC isozymes in the proteinuria-induced EMT of RTECs. Employing immunochemical staining, we found that PKC-α, -βI and -βII were expressed in glomerulus and in RTECs in both normal and diseased renal tissues, while PKC-γ was only expressed in podocytes in the glomerulus. Treatment of HK-2 cells with extracted urinary proteins resulted in EMT, as evidenced by morphological changes, decreased E-cadherin expression, increased α-SMA (α-smooth muscle actin) expression, as well as production of type I collagen and fibronectin. Western blot analysis of PKC isozymes in the cytosolic compared with membrane fraction revealed translocation of PKC-α and -βI, but not PKC-βII, in HK-2 cells undergoing EMT. Pretreatment with selective PKC-α inhibitor G-6976 or PKC-β inhibitor significantly attenuated EMT induced by urinary proteins. In summary, the present study suggested that PKC-α and -βI play critical roles in the EMT of RTECs in response to urinary proteins.