Smad2 and Smooth Muscle Differentiation from Neural Crest Stem Cells

描述（由申请人提供）：本提案的主要目的是阐明控制血管平滑肌细胞（VSMC）从神经嵴干细胞（NCSCs）分化的分子机制。异常的VSMC分化有助于许多主要的心血管疾病，包括最常见的先天性心脏病（CHD）。体内和体外研究均表明，TGF-信号在VSMC向NCSCs分化过程中起着至关重要的作用。然而，TGF-在这一过程中复杂活性的分子机制在很大程度上仍然未知。通过体外模型系统，我们发现TGF-下游中间体Smad2，而不是Smad3，是TGF诱导的VSMC从NCSCs分化的关键；而Smad3，而不是Smad2，对于VSMC从间充质来源（非ncsc）的C3H10T1/2细胞分化是重要的。这些发现有力地支持了Smad2在从NCSCs向VSMC分化过程中作为TGF-重要特异性介质的假设。更重要的是，我们发现Smad2而非Smad3是NCSCs中VSMC标记基因激活所必需的。我们还发现心肌素相关转录因子B （MRTFB）参与了NCSCs中VSMC特异性基因的激活。为了探讨TGF-调控VSMC向NCSCs分化的机制，我们将重点关注两个具体目标。在Aim 1中，我们将研究Smad2功能在VSMC从NCSCs分化中的分子机制。我们将确定Smad2如何调节VSMC标记基因的转录激活，并确定Smad2是否与MRTFB相互作用以控制标记基因启动子。在Aim 2中，我们将使用神经嵴组织特异性敲除（Wnt1-Cre）小鼠来验证Smad2是体内VSMC从NCSCs分化所必需的假设。Smad2在NCSCs中的特异性将通过SM22-Cre小鼠在非ncsc起源的VSMC中突变Smad2基因进一步测试。NCSC分化是一个受TGF-及其家族成员等多种因素调控的复杂过程。通过生物化学、分子、细胞和发育生物学技术的结合，深入研究Smad2在VSMC分化中的功能。本研究的完成将显著推进我们对TGF-调控NCSC向VSMC分化的分子机制的理解，并有助于开发治疗包括冠心病在内的人类心血管疾病的新疗法。此外，由于Smad2特异于与NCSC分化的VSMC，而Smad3特异于来自中胚层的VSMC，我们的研究可能会为专门针对不同来源的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Smad2功能的研究将显著促进我们对神经嵴细胞平滑肌分化分子机制的理解。这一结果将为先天性心脏病和一些遗传疾病（如遗传性出血性毛细血管扩张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。

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.