NO & AP-1 MODULATE TNF-ACTIVATED LUNG ENDOTHELIAL PROTEI

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• 批准号: 6389855
• 负责人: ARNOLD JOHNSON
• 金额: $ 23.97万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6389855
• 关键词: AP1 protein; antisense nucleic acid; enzyme activity; gel mobility shift assay; genetic transcription; molecular pathology; nitric oxide; northern blottings; nuclear runoff assay; protein kinase C; respiratory epithelium; tissue /cell culture; transfection; tumor necrosis factor alpha; vascular endothelium; western blottings

The central hypothesis of this proposal is that, in the Tumor Necrosis Factor-alpha (TNF)-activated pulmonary endothelium, reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite (ONOO-) modulate the prolonged (greater than or equal to 4 hrs.) activation of Protein Kinase C-alpha (PKC-alpha), at least in part, by stimulating activator protein-1 (AP-1)-mediated transcription of PKCalpha. The pathogenetic sequence begins with RNS (either ONOO-, NO or both) activating guanylate cyclase, resulting in the generation of cGMP. The stimulated cGMP-PKG pathway phosphorylates the AP-1 subcomponents cFos and/or cJun, leading to AP-1 activation. The activated AP- 1 induces transcription of the PKCalpha gene. The increased transcription results in increased translation of PKCalpha mRNA into C Kinase protein, promoting the prolonged activation of PKC, with subsequent well-studied injurious effects on the pulmonary endothelium. The specific aims of the current proposal are: (1) to study whether TNF induces transcription of the pulmonary endothelial PKCalpha gene, (2) to determine the roles of RNS generation and AP-1 activation in the TNF induction of message for pulmonary endothelial PKCalpha, (3) to investigate whether the cGMP-PKG-mediated phosphorylation induced by TNF and RNS activates AP-1, and (4) to probe the roles of RNS, AP-1 and PKCalpha message in the prolonged activation of pulmonary endothelial PKCalpha induced by TNF. The hypothesis will be tested using human and bovine pulmonary microvessel endothelial cells. TNF-induced PKC-alpha transcription is studied using Nuclear Run-On and Northern Blot of cytosolic RNA. AP-1 activation is investigated using the Electrophoretic Mobility Shift Assay. Transfection with AP-1 reporter plasmids is used to verify role of RNS in TNF-induced AP-1 activation in gene regulation. Antisense oligonucleotides (anti-cJun, anti-PKC-alpha) is used to verify the AP-1-mediated maintenance of TNF-induced PKC-alpha transcription and activity. The role of RNS and PKG is investigated using (1) direct assay and (2) agonists and antagonists of RNS and PKG. Synthesis of PKC-alpha is studied using immunoprecipitation and Western Blot of 35S-labeled PKC, and activity of PKC is assessed by PKC translocation and phosphorylation of PKC-alpha specific substrate.

该提议的中心假设是，在肿瘤坏死因子因子-Alpha（TNF）活化的肺内皮中，反应性氮种（RN），例如一氧化物（NO）（NO）和过氧硝酸盐（ON-ON-）调节延长的延长（大于或相等）（大于或等于4 Hrs prots蛋白酶）。 （PKC-Alpha），至少部分通过刺激激活剂蛋白-1（AP-1）介导的PKCALPHA转录。 致病序列始于RN（ONOO-，NO或两者）激活鸟苷酸环化酶，从而产生CGMP。 刺激的CGMP-PKG途径磷酸化AP-1亚组成部分和/或CJUN，导致AP-1激活。 活化的AP-1诱导PKCALPHA基因的转录。 增加的转录导致PKCALPHA mRNA转化为C激酶蛋白，从而促进PKC的延长激活，随后对肺部内皮的有害作用进行了充分研究。 The specific aims of the current proposal are: (1) to study whether TNF induces transcription of the pulmonary endothelial PKCalpha gene, (2) to determine the roles of RNS generation and AP-1 activation in the TNF induction of message for pulmonary endothelial PKCalpha, (3) to investigate whether the cGMP-PKG-mediated phosphorylation induced by TNF and RNS激活AP-1，（4）探测RN，AP-1和PKCALPHA消息在TNF诱导的肺内皮PKCALPHA的长时间激活中的作用。 该假设将使用人和牛肺微血管内皮细胞进行检验。 使用核RNA的核跑和北印迹研究了TNF诱导的PKC-Alpha转录。 使用电泳迁移率转移测定法研究了AP-1激活。 用AP-1报告基因质粒转染用于验证RN在TNF诱导的AP-1激活中的作用。 反义寡核苷酸（抗CJUN，抗PKC-Alpha）用于验证TNF诱导的PKC-Alpha转录和活性的AP-1介导的维持。 使用（1）直接测定和（2）RNS和PKG的激动剂和拮抗剂研究了RN和PKG的作用。 使用免疫沉淀和35S标记的PKC的蛋白质印迹研究了PKC-α的合成，PKC的活性通过PKC易位和PKC-Alpha特异性底物的磷酸化评估。