Smad2 and Smooth Muscle Differentiation from Neural Crest Stem Cells

Smad2 和神经嵴干细胞的平滑肌分化

• 批准号: 7842097
• 负责人: Shiyou Chen
• 金额: $ 24.35万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842097
• 关键词: Accounting; Actins; Affect; Arteries; Biochemical; Biological; Biological Models; Blood Vessels; Carbachol; Cardiac; Cardiovascular Abnormalities; Cardiovascular Diseases; Cardiovascular system; Cell Differentiation process; Cells; Complement Factor B; Complex; Data; Development; Disease; Embryonic Development; Family member; Gene Activation; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Hereditary Disease; Hereditary hemorrhagic telangiectasia; Human; In Vitro; Intervention; Knock-out; Knockout Mice; Knowledge; Lead; Light; Live Birth; Mediating; Mediator of activation protein; Mesenchymal; Mesoderm; Molecular; Mus; Muscarinic Agonists; Mutate; Neural Crest; Neural Crest Cell; Pathogenesis; Pharmaceutical Preparations; Phenotype; Play; Process; Proteins; Publishing; Role; Serum Response Factor; Signal Transduction; Signaling Molecule; Smad Proteins; Smad protein; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Specificity; Stem cells; System; Techniques; Testing; Therapeutic Agents; Therapeutic Intervention; Tissues; To specify; Transcriptional Activation; Transduction Gene; Vascular Smooth Muscle; congenital heart disorder; gastrulation; in vitro Model; in vivo; myocardin; novel; novel therapeutics; prenatal; progenitor; promoter; public health relevance; receptor; stem cell differentiation; transcription factor

DESCRIPTION (provided by applicant): The primary objective of this proposal is to elucidate the molecular mechanism governing vascular smooth muscle cell (VSMC) differentiation from neural crest stem cells (NCSCs). Abnormal VSMC differentiation contributes to a number of major cardiovascular disorders including the most prevalent forms of congenital heart disease (CHD). Both in vivo and in vitro studies have demonstrated that TGF- signaling is critical for VSMC differentiation from NCSCs. However, the molecular mechanisms underlying the complex activities of TGF- on this process remain largely unknown. By utilizing in vitro model systems we found that the TGF- downstream intermediate Smad2, but not Smad3, is critical for TGF--induced VSMC differentiation from NCSCs; while Smad3, but not Smad2, is important for VSMC differentiation from mesenchymal- originated (non-NCSC) C3H10T1/2 cells. These findings strongly support the hypothesis that Smad2 acts as an important and specific mediator of TGF- in VSMC differentiation from NCSCs. More importantly, we made the novel observation that Smad2, but not Smad3, is required for VSMC marker gene activation in NCSCs. We also found that myocardin-related transcription factor B (MRTFB) contributes to VSMC specific gene activation in NCSCs. To investigate the mechanism by which TGF- regulates VSMC differentiation from NCSCs, we will focus on two Specific Aims. In Aim 1, we will study the molecular mechanisms underlying the Smad2 function in VSMC differentiation from NCSCs. We will determine how Smad2 regulates transcriptional activation of VSMC marker genes, and determine if Smad2 interacts with MRTFB in controlling marker gene promoters. In Aim 2, we will test the hypothesis that Smad2 is essential for VSMC differentiation from NCSCs in vivo, using a neural crest tissue-specific knockout (Wnt1-Cre) mouse. The specificity of Smad2 in NCSCs will be further tested by mutating Smad2 gene in VSMC of non-NCSC origins by using a SM22-Cre mouse. NCSC differentiation is a complex process modulated by multiple factors, including TGF- and its family members. Through a combination of biochemical, molecular, cellular and developmental biological techniques, the function of Smad2 in VSMC differentiation will be thoroughly studied. Accomplishment of the proposed studies will significantly advance our understanding of the molecular mechanism by which TGF- regulates NCSC differentiation to VSMC and contribute to the development of novel therapeutics for the treatment of human cardiovascular diseases including CHD. Moreover, since Smad2 is specific to the VSMC differentiated from NCSC while Smad3 is specific to the VSMC from mesoderm, our studies will likely provide new revenues for drug intervention specifically targeting at diseases that are caused by abnormal function of VSMCs from different origins. PUBLIC HEALTH RELEVANCE Vascular smooth muscle differentiation is closely related to several major cardiovascular diseases including congenital heart disease. The proposed studies focusing on the function of Smad2 will significantly advance our understanding of the molecular mechanisms governing the smooth muscle differentiation from neural crest cells. The results will shed new light on the pathogenesis of congenital heart disease and a number of genetic diseases such as hereditary hemorrhagic telangiectasia type 1 and type 2, which may ultimately lead to therapeutic intervention.

描述（由申请人提供）：本提案的主要目的是阐明从神经嵴干细胞（NCSC）分化为血管平滑肌细胞（VSMC）的分子机制。异常VSMC分化导致许多主要心血管疾病，包括最常见的先天性心脏病（CHD）。体内和体外研究均表明，TGF-β信号传导对于NCSC向VSMC分化至关重要。然而，TGF-β在这一过程中复杂活性的分子机制仍不清楚。通过利用体外模型系统，我们发现TGF-下游中间体Smad 2，而不是Smad 3，对于TGF-诱导的VSMC从NCSC分化是关键的;而Smad 3，而不是Smad 2，对于VSMC从间充质来源的（非NCSC）C3 H10 T1/2细胞分化是重要的。这些发现有力地支持了Smad 2在NCSC的VSMC分化中作为TGF-β的重要和特异性介导剂的假设。更重要的是，我们进行了新的观察，Smad 2，而不是Smad 3，是所需的VSMC标记基因在NCSC激活。我们还发现，心肌素相关转录因子B（MRTFB）有助于NCSC中VSMC特异性基因激活。为了研究TGF-β 1调节NCSCs分化为VSMC的机制，我们将集中于两个具体的目的。在目的1中，我们将研究Smad 2在NCSC向VSMC分化中的分子机制。我们将确定Smad 2如何调节VSMC标记基因的转录激活，并确定Smad 2是否与MRTFB在控制标记基因启动子中相互作用。在目标2中，我们将使用神经嵴组织特异性敲除（Wnt 1-Cre）小鼠来检验Smad 2对于VSMC从NCSC体内分化所必需的假设。将通过使用SM 22-Cre小鼠突变非NCSC来源的VSMC中的Smad 2基因来进一步测试Smad 2在NCSC中的特异性。NCSC分化是一个复杂的过程，受多种因素调节，包括TGF-β及其家族成员。本研究将结合生物化学、分子生物学、细胞生物学和发育生物学技术，对Smad 2在VSMC分化中的作用进行深入研究。这些研究的完成将大大促进我们对TGF-β调节NCSC向VSMC分化的分子机制的理解，并有助于开发治疗包括CHD在内的人类心血管疾病的新疗法。此外，由于Smad 2特异于从NCSC分化的VSMC，而Smad 3特异于来自中胚层的VSMC，因此我们的研究可能会为专门针对由不同来源的VSMC功能异常引起的疾病的药物干预提供新的收入。血管平滑肌分化与包括先天性心脏病在内的几种主要心血管疾病密切相关。对Smad 2功能的研究将极大地促进我们对神经嵴细胞向平滑肌细胞分化的分子机制的理解。这些结果将为先天性心脏病和一些遗传性疾病（如遗传性出血性毛细血管扩张症1型和2型）的发病机制提供新的线索，最终可能导致治疗干预。