Transcriptional regulation of CRP1 in smooth muscle

描述(申请人提供)：血管平滑肌细胞是血管壁的重要组成部分，具有非凡的适应能力。这些细胞表现出一系列的表型，这些表型依赖于转录程序的选择性利用。虽然平滑肌调节对于正常的血管功能是必不可少的，但它们的适应能力与血管闭塞疾病的病理呈负相关。因此，控制基因转录导致结果表型的分子机制在生物学和临床上都是相关的。作为阐明控制平滑肌转录的关键特征的途径，我们的研究集中在前体细胞向成熟平滑肌的发育和分化上。分化伴随着一组精确的收缩所需基因的精心激活。虽然明确的调控元件和相应的转录因子被认为在分化中起作用，但它们是如何与传递选择性基因表达的信号耦合的还不是很清楚。我们假设，细胞外信号，如转化生长因子-β(转化生长因子-β)，使用细胞内途径和转录因子的不同组合来传递平滑肌限制性表达。转化生长因子-β已成为调控平滑肌细胞表型的主要候选者。该方案的目的是利用富含半胱氨酸蛋白1(CRP1)基因的平滑肌特异性表达来研究顺式作用元件和反式作用因子，这些作用因子传递选择性转录活性以响应特定的信号事件。我们已经确定了CRP1基因的一个独特的调控区，该区域仅在动脉平滑肌细胞中驱动表达。我们打算利用这一调控元件来表征CRP1基因在分化的平滑肌细胞中表达的重要转录途径。其具体目的是：1)确定转化生长因子-β1(TGF-β1)激活CRP1基因的转录机制。2)确定血清反应因子(SRF)的功能活性与调控平滑肌基因表达的信号事件之间的关系。3)通过靶向诱变明确Carg元件对CRP1表达的必要性。这些研究意义重大，因为它们将超越对转录调节因子的分析，并确定介导反式激活的确切途径，以解决有关平滑肌细胞表型表现的基本问题。

DESCRIPTION (provided by applicant): Vascular smooth muscle cells are a vital component of the blood vessel wall, possessing extraordinary adaptive abilities. These cells display a range of phenotypes that are dependent upon the selective utilization of transcriptional programs. While smooth muscle modulation is essential for normal blood vessel function, their adaptive abilities are adversely associated with the pathologies of vascular occlusion diseases. Consequently, the molecular mechanisms governing gene transcription leading to resultant phenotypes are both biologically and clinically relevant. As an inroad to elucidating critical features that control smooth muscle transcription, our studies are focused on the development and differentiation of precursor cells into mature smooth muscle. Differentiation is accompanied by the orchestrated activation of a precise set of genes required for contraction. Though definitive regulatory elements and corresponding transcription factors are recognized to have a role in differentiation, how they are coupled to signals that convey selective gene expression is not well understood. We hypothesize that extracellular signals, like transforming growth factor-beta (TGF-¿), use a distinct combination of intracellular pathways and transcription factors to impart smooth muscle-restricted expression. TGF-¿ has emerged as primary candidate for governing smooth muscle cell phenotypes. The aims of this proposal are designed to employ the smooth muscle-specific expression of the cysteine-rich protein 1 (CRP1) gene to investigate the cis-acting elements, and trans- acting factors that convey selective transcriptional activity in response to defined signaling events. We have identified a unique regulatory region of the CRP1 gene that drives expression exclusively in arterial smooth muscle cells. We intend to use this regulatory element to characterize essential transcriptional pathways important for the expression of the CRP1 gene in differentiated smooth muscle cells. The specific aims are: 1) To define the transcriptional mechanisms underlying the activation of the CRP1 gene by transforming growth factor-beta-1 (TGF-¿1). 2) To determine the relationship between the functional activity of serum response factor (SRF), and signaling events that govern smooth muscle gene expression. 3) To unequivocally determine the necessity of the CArG element for expression of CRP1 by targeted mutagenesis. These studies are significant, as they will extend beyond the analysis of the transcriptional regulators, and determine the exact pathways that mediate transactivation, to address fundamental questions regarding the manifestation of smooth muscle cell phenotypes.