CGMP-DEPENDENT PROTEIN KINASE (PKG) EXPRESSION

CGMP 依赖性蛋白激酶 (PKG) 表达

• 批准号: 6228966
• 负责人: Thomas M. Lincoln
• 金额: $ 35.88万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6228966
• 关键词: binding proteins; blood vessels; cGMP dependent protein kinase; cofactor; enzyme activity; enzyme induction /repression; gel mobility shift assay; genetic promoter element; muscle cells; nitric oxide; northern blottings; nucleic acid sequence; phenotype; protein protein interaction; transcription factor; yeast two hybrid system

DESCRIPTION (Verbatim from the application): The underlying pathologic basis in vascular disorders such as atherosclerosis and restenosis following injury is the migration of medial vascular smooth muscle cells (VSMC) to the intima where they proliferate and secrete abundant amounts of extracellular matrix proteins. The mechanism underlying the modulation of the phenotype of VSMC from specialized contractile cells to fibroproliferative cells is key to our understanding of the genesis of vascular diseases. A number of studies have underscored the importance of endothelial-derived nitric oxide (NO) in maintaining a relatively quiescent and differentiated VSMC phenotype both in vivo and in vitro. The enzyme that mediates these beneficial effects of endothelial-derived NO is the Type I cyclic GMP-dependent protein kinase (PKG Ia). However, the expression of PKG Ia is reduced in proliferating neointimal VSMC in vivo and in cells cultured in vitro where they modulate to the secretory phenotype. Since restoration of PKG expression in vitro restores the contractile phenotype, then understanding the mechanism regulating PKG Ia expression is key to understanding and attenuating phenotypic modulation. Recent studies from our laboratory have shown that inflammatory cytokines, NO donor drugs and cyclic nucleotide analogs suppress PKG Ia expression in cultured VSMC. Furthermore, a 500 bp promoter for PKG Ia responds to NO and cyclic nucleotide analogs with a decrease in activity as assessed by in vitro transfection assays. However, the NO/cyclic nucleotide-responsive elements in the PKG Ia promoter have not been defined nor has the molecular basis for tissue-specific suppression of PKG expression been analyzed. Therefore, the specific aims of this proposal are to characterize the elements in the PKG Ia promoter that mediate NO/cyclic nucleotide-mediated suppression of PKG expression, and to identify tissue-specific co-factors that mediate the effects of NO/cyclic nucleotide suppression of PKG expression in VSMC. These studies will be done using in vitro transfection assays to analyze the cis-acting elements in the cloned PKG Ia promoter and electromobility shift assays (EMSA) to define the transcription factors regulated by NO/cyclic nucleotides. Yeast two-hybrid methods will be used to identify and characterize specific binding proteins that interact with NO/cyclic nucleotide-regulated transcription factors in a tissue specific manner. These studies are the first to characterize the mechanisms regulating Type I PKG expression in mammalian cells, and will be critical for our understanding of the mechanisms underlying phenotypic modulation in VSMC. Ultimately, new approaches for understanding and treating vascular diseases can be undertaken based on this new knowledge.

描述（申请中的逐字记录）：潜在的病理基础 血管疾病，如动脉粥样硬化和损伤后的再狭窄 内侧血管平滑肌细胞 (VSMC) 迁移至内膜 它们增殖并分泌大量的细胞外基质蛋白。 VSMC表型调节的机制 专门的收缩细胞到纤维增殖细胞是我们的关键 了解血管疾病的起源。多项研究已 强调了内皮源性一氧化氮（NO）在 维持相对静止和分化的 VSMC 表型 体内和体外。介导这些有益作用的酶 内皮源性 NO 是 I 型环 GMP 依赖性蛋白激酶 (PKG 亚）。然而，PKG Ia 的表达在增殖的新内膜中降低。 VSMC 在体内和体外培养的细胞中调节 分泌表型。由于体外 PKG 表达的恢复恢复了 收缩表型，然后了解调节 PKG Ia 的机制 表达是理解和减弱表型调节的关键。 我们实验室最近的研究表明，炎症细胞因子、NO 供体药物和环核苷酸类似物抑制 PKG Ia 表达 培养的VSMC。此外，PKG Ia 的 500 bp 启动子响应 NO 和 体外评估活性降低的环核苷酸类似物 转染测定。然而，NO/环核苷酸反应元件 PKG Ia 启动子尚未定义，也没有分子基础 分析了 PKG 表达的组织特异性抑制。因此， 该提案的具体目标是描述 PKG Ia 中的要素特征 介导 NO/环核苷酸介导的 PKG 抑制的启动子 表达，并鉴定介导影响的组织特异性辅助因子 NO/环核苷酸对 VSMC 中 PKG 表达的抑制。这些研究 将使用体外转染测定来分析顺式作用 克隆的 PKG Ia 启动子和电动迁移率分析 (EMSA) 中的元件 定义受NO/环核苷酸调节的转录因子。酵母 双杂交方法将用于识别和表征特异性结合 与 NO/环核苷酸调节转录相互作用的蛋白质 以组织特定方式的因素。这些研究首次 描述哺乳动物中调节 I 型 PKG 表达的机制 细胞，对于我们理解其背后的机制至关重要 VSMC 中的表型调节。最终，理解和理解的新方法 可以基于这一新知识来治疗血管疾病。