CGMP-DEPENDENT PROTEIN KINASE (PKG) EXPRESSION

CGMP 依赖性蛋白激酶 (PKG) 表达

• 批准号: 6490755
• 负责人: Thomas M. Lincoln
• 金额: $ 21.53万
• 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 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490755
• 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（逐字从应用程序中）： 受伤后动脉粥样硬化和再狭窄等血管疾病是 内侧血管平滑肌细胞（VSMC）迁移到内膜 它们扩散并分泌大量的细胞外基质蛋白。 VSMC表型调制的基础机制 专门的收缩细胞到纤维增生细胞是我们的关键 了解血管疾病的起源。许多研究有 强调了内皮衍生的一氧化氮（NO）的重要性 保持相对静止和分化的VSMC表型都在 体内和体外。介导这些有益作用的酶 内皮衍生的NO是I型环状GMP依赖性蛋白激酶（PKG） ia）。然而，pkg ia的表达降低了新的新膜状 VSMC在体内和在体外培养的细胞中调节为 分泌表型。由于体外恢复PKG表达的恢复 收缩表型，然后了解调节PKG IA的机制 表达是理解和衰减表型调制的关键。 我们实验室的最新研究表明，炎性细胞因子，否 供体药物和环状核苷酸类似物抑制PKG IA表达 培养的VSMC。此外，PKG IA的500 bp启动子响应否和 循环核苷酸类似物，其活性降低如在体外评估 转染测定法。但是，NO/循环核苷酸响应元件 PKG IA启动子尚未定义，也没有分子基础 分析了对PKG表达的组织特异性抑制。因此， 该建议的具体目的是表征PKG IA中的要素 介导无/环状核苷酸介导的PKG抑制的启动子 表达，并鉴定组织特异性的辅助因素，以介导该作用 VSMC中PKG表达的NO/环状核苷酸抑制。这些研究 将使用体外转染测定法进行分析顺式作用 克隆的PKG IA启动子和电动性转移测定法（EMSA）中的元素（EMSA） 定义由NO/环状核苷酸调节的转录因子。酵母 两种杂交方法将用于识别和表征特定的结合 与NO/环状核苷酸调节转录相互作用的蛋白质 特定于组织的因素。这些研究是第一个 表征调节哺乳动物I型PKG表达的机制 细胞，对于我们对基本机制的理解至关重要 VSMC中的表型调制。最终，新的理解方法和 可以根据这种新知识进行治疗血管疾病。