【作者】 张玲；

【导师】 熊志刚；

【作者基本信息】 安徽医科大学 ， 药理学， 2020， 博士

【摘要】 蛋白质磷酸化(Protein phosphorylation)是最常见的蛋白质翻译后修饰方式之一,是一种普遍的生命活动调节方式,在生理和病理过程中都起着重要作用。像许多其他蛋白质一样,膜受体和离子通道已成为蛋白质磷酸化的重要目标。组织酸中毒是许多病理状态的普遍特征。酸敏感离子通道(acid-sensing ion channels,ASICs)在中枢以及外周神经系统中高度表达,在对细胞外酸性环境的感知中起着至关重要的作用。ASIC1a是中枢神经元的主要功能性亚基。ASIC1a的激活在缺血酸中毒介导的神经毒性中起关键作用,是脑缺血神经元损伤的潜在治疗靶点。蛋白激酶C(protein kinase C,PKC)的活性和功能与许多生理过程和病理状态有关,然而,PKC磷酸化修饰是否调节ASICs,尤其是ASIC1a的蛋白表达及通道功能尚不明确。本研究利用原代培养小鼠皮质神经元和小鼠神经母细胞瘤细胞系NS20Y细胞,通过Western blot分析PKC磷酸化对ASIC1a蛋白表达的影响,采用全细胞膜片钳技术检测细胞ASIC电流的变化,并使用恶性胶质瘤细胞A172,分别对PKC激动剂和抑制剂参与调控细胞迁移能力进行分析。根据佛波酯(phorbol 12-myristate 13-acetate,PMA,一种PKC激动剂)调控ASIC1a蛋白表达的时效量效关系测定结果,本实验中采用PMA 200 n M孵育细胞。结果显示,相比于对照组,PMA 200 n M孵育6 h显著增加了NS20Y细胞和原代培养小鼠皮质神经元细胞ASIC1a蛋白的表达以及ASIC电流密度。与PMA相反,PKC抑制剂钙磷蛋白C(Calphostin C,200n M)孵育6 h和24 h均显著降低了NS20Y细胞和原代培养小鼠皮质神经元细胞中ASIC1a蛋白表达和ASIC电流密度。与Calphostin C相似,同工酶特异性PKC和PKC I抑制剂Go6976(1 M)孵育24 h能显著降低NS20Y细胞中ASIC1a的蛋白表达,提示PKC参与了PKC调控ASIC1a蛋白表达的过程。ASIC1a的激活在酸中毒介导的细胞损伤中起重要作用,为了探究PKC磷酸化在酸诱导的细胞损伤中的作用,本课题通过检测乳酸脱氢酶的释放和细胞活力MTT实验分析,结果显示PKC抑制剂Go6976可减轻酸中毒介导的NS20Y细胞损伤,而PKC激动剂PMA可增强酸中毒介导的NS20Y细胞损伤。此外,本研究通过单层细胞划痕实验,发现弱酸(p H7.0 ECF)环境可促进恶性胶质瘤A172细胞的迁移,且ASIC1a特异性抑制剂Psalmotoxin 1(Pc Tx1,20 n M)可抑制弱酸引起的细胞迁移能力的增强,提示ASIC1a通道的激活参与了酸刺激下细胞的迁移。进一步探究发现,PKC激动剂PMA(200 n M)能进一步增强弱酸刺激下细胞的迁移能力,而PKC抑制剂Go6976(1 M)显著抑制了弱酸刺激下A172细胞迁移能力的增强。同样Pc Tx1可抑制这种细胞迁移能力的改变,提示PKC磷酸化引起的细胞迁移能力的改变与ASIC1a的通道功能密切相关。机制上,本课题通过使用蛋白质合成抑制剂环已酰亚胺(Cycloheximide,CHX,20μM)阻止新的蛋白质合成,发现PKC抑制剂Calphostin C仍然能够促进ASIC1a蛋白的降解,并且蛋白酶体抑制剂MG-132(10 M)可阻止Calphostin C诱导的ASIC1a蛋白的降解,表明泛素-蛋白酶体系统(ubiquitinproteasome system,UPS)降解途径介导了Calphostin C诱导的ASIC1a的蛋白降解过程。最后,为了确定PKC磷酸化改变ASIC1a蛋白表达是否需要核因子-κB(nuclear factor kappa B,NF-κB)的激活信号转导通路,本课题使用NF-κB上游I-κB激酶(inhibitor ofκB kinase,IKK)-特异性抑制剂IMD-0354(1 M)共同处理细胞,通过Western blot分析,发现PKC介导的ASIC1a蛋白表达的变化在与IKK-抑制剂共存时被抑制,表明NF-κB信号通路参与了PKC磷酸化调控ASIC1a蛋白表达的过程。综上结果表明,ASIC1a的蛋白表达和通道功能受PKC及其下游信号通路的密切调控。更多还原

【Abstract】 Protein phosphorylation is one of the most common post-translational modification of proteins.It is a universal mode of regulating life activities and plays an important role in both physiological and pathological conditions.Like many other proteins,membrane receptors and ion channels have become major targets for protein phosphorylation.Tissue acidosis is a common feature in many pathological conditions.Acid-sensing ion channels(ASICs)are highly expressed in peripheral sensory and central neurons and play a critical role in the perception of a wide range of p H changes in various conditions related to tissue acidosis.ASIC1 a is a major functional ASIC subunit in central neurons and a potential therapeutic target for neuronal injury,which activation plays a key role in acidosis-mediated neurotoxicity.Protein kinase C(PKC)activity or function has been proved to be associated with many physiological processes and pathological conditions,however,whether PKC activation regulates ASIC1 a protein expression and channel function remains ill defined.In this study,the effects of PKC phosphorylation on ASIC 1a protein expression and ASIC channel function were detected through Western blot,patch-clamp and cell migration analysis in primary cultured mouse cortical neurons,and in NS20 Y cells,a neuronal cell line,as well as in A172 cells,a human malignant glioma cell line.According to the pre-experimental results,cells were first incubated with phorbol-12-myristate-13-acetate(PMA,a PKC activator)at 200 n M.Compared with the control group,treatment with PMA for 6 h significantly increased ASIC1 a protein expression and ASIC current density in NS20 Y cells and in primary cultured mouse cortical neurons,while incubation with PMA for 24 h did not change ASIC1 a protein expression and ASIC current density.In contrast,treatment with Calphostin C(200 n M),a non-selective PKC inhibitor,for 6 h or longer decreased ASIC1 a protein expression and ASIC currents.Similar to Calphostin C,incubation with Go6976(1 M),a specific PKC and I inhibitor,for 24 h also reduced ASIC1 a protein expression,suggesting that PKC is involved in regulating ASIC1 a protein expression.Since activation of ASIC1 a plays an important role on acidosis-mediated cell injury,we explored the potential effect of PKC activator and inhibitor on acid-induced cell injury by measuring lactate dehydrogenase(LDH)release and MTT cell viability assay.We found that treatment of cells with PMA(200 n M)increased LDH release and aggravated acidosisinduced decrease in cell viability,while treatment of cells with Go6976(1 M)reduced LDH release and attenuated acidosis-induced decrease in cell viability in NS20 Y cells,which is consistent with their effects on ASIC1 a protein expression and channel function.In addition,we studied the effect of PKC activation on acid-mediated change of cell migration.We first confirmed that,comparing with p H7.4 extracellular fluid(ECF)incubation,weak acid(p H7.0 ECF)incubation could promote A172 cells migration in a monolayer scratch assay,and ASIC1 a specific inhibitor Pc Tx1(20 n M)could prevent this enhanced migration,confirming that activation of ASIC1 a channel is related to acid-induced cell migration.Next,A172 cells were incubated with PMA(200n M)or Go6976(1 M)respectively,followed by incubation with p H7.4 or p H7.0 ECF for 3 h.The data showed that incubation with PMA dramatically promoted the migration of A172 cells,while incubation with Go6976 significantly inhibited the migration of A172 cell at 24 h.Similarly,Pc Tx1 prevents the changes in cell migration,suggesting that PKC-induced change in cell migration is mediated,at least partially,by ASIC1 a.To examine the mechanism of PKC-mediated changes in ASIC1 a protein expression,a protein synthesis inhibitor Cycloheximide(CHX,20 M)was used to prevent new protein synthesis in NS20 Y cells.We found that the reduction in ASIC1 a protein expression by PKC inhibition involves a change in ASIC1 a protein degradation,which is mediated by ubiquitin proteasome system(UPS)-dependent degradation pathway,as MG-132(10 M),a proteasome inhibitor,can prevent Calphostin C-induced down-regulation of ASIC1 a protein expression.Last,we showed the evidence that PKC regulation of ASIC1 a protein expression involves NF-κB signaling pathway.A specific inhibitor IMD-0354(1 M),a nuclear factor kappa B(NF-κB)upstream inhibitor of kappa B kinase-(IKK-),was used to co-treated cells.Western blot analysis showed that PKC-mediated changes in ASIC1 a protein expression were totally inhibited in the presence of IMD-0354.Together,these results indicate that ASIC1 a protein expression and channel function are closely regulated by the activity of protein kinase C and its downstream signaling pathway(s).更多还原