Achaete-scute-like-3 (ASCL3) Regulates Vascular Muscle Cell Proliferation

描述（由申请方提供）：在发育过程中参与细胞命运决定并控制细胞增殖、分化、凋亡和迁移的许多级联反应受碱性-螺旋-环-螺旋类（bHLH）转录因子（TF）调节，包括bHLH的无毛鳞片家族中的转录因子，其在无脊椎动物和人类中高度保守。虽然哺乳动物的ASCL 1已在神经生物学的广泛研究的对象，但非常少的信息是可用于家庭的其他成员，ASCL 2至5，特别是普遍表达的ASCL 3的作用。我们的初步数据表明，ASCL 3，ASCL 2和ASCL 1在血管平滑肌细胞（VSMC）中表达。聚焦于ASCL 3，我们发现其表达在用血管紧张素II刺激增殖时增加，并且在较小程度上PDGF-BB，而在Notch 1活化或BMP 2处理的生长抑制的背景下减少。此外，ASCL 3的稳定过表达导致体外VSMC增殖增加，并且在体内血管损伤后，ASCL 3在新生内膜中明显上调。这些数据表明，ASCL 3积极促进VSMC的增殖，在病理性血管重塑的发展中具有潜在的作用，将其定义为“血管重塑”因子。这些观察结果导致了我们的中心假设，即ASCL 3是一种调节VSMC增殖的促生长bHLH转录因子。 为了解决这一假设的影响和预测，我们建议采取： 具体目标1：通过（1.1）分析响应于体外不同刺激的ASCL 3表达，表征与VSMC增殖相关的ASCL 3表达谱，关注血管病理学中的相关因素，以将其与（1.2）分析体内血管重塑中的ASCLS表达相关联，和 具体目标2：通过表征（2.1）ASCLS功能获得和丧失对VSMC增殖的影响以及（2.2）天然来源的调控VSMC增殖的ASCL 3靶基因的鉴定，分析ASCL 3对VSMC体外增殖的影响。 这些具体的目标将解决使用免疫化学和定量分析的表达在体内和体外结合生长因子的相关血管重塑以及在获得和损失的功能，通过遗传和生化方法的背景下。该提议的发展将ASCL 3定义为在血管增殖和重塑中具有积极作用的新型bHLH TF。这项工作必将超越血管生物学领域，阐明这种普遍存在的TF在细胞增殖中的一般作用，并有可能影响其他领域，其中失调的细胞生长是所涉及的生物过程的根源，因为它是癌症或发育生物学的情况。

DESCRIPTION (provided by applicant): Numerous cascades involved in cell fate determination during development and governing cell proliferation, differentiation, apoptosis and migration are regulated by transcription factors (TF) in the basic-helix-loop-helix class (bHLH), including those in the achaete-scute family of bHLH, which has been highly conserved from invertebrates to humans. Although the mammalian ASCL1 has been the object of extensive studies in neurobiology, yet very little information is available on the role of the other members of the family, ASCL2 to 5, particularly the ubiquitously expressed ASCL3. Our preliminary data show that ASCL3, ASCL2 and ASCL1 are expressed in vascular smooth muscle cells (VSMC). Focusing on ASCL3, we found that its expression is increased upon stimulation of proliferation with angiotensin II, and to a lesser extent PDGF-BB, while being reduced in the context of growth inhibition by Notch1 activation or BMP2 treatment. Furthermore, stable over-expression of ASCL3 leads to increased proliferation of VSMC in vitro and ASCL3 is clearly up-regulated in neointima upon vascular lesion in vivo. These data suggest that ASCL3 contributes actively to proliferation in VSMC with a potential role in development of pathological vascular remodeling, defining it as a "vasculopatic" factor. These observations led to our central hypothesis that ASCL3 is a growth-promoting bHLH transcription factor regulating VSMC proliferation. To address the implications and predictions of this hypothesis we propose to undertake: Specific aim 1: Characterization of ASCL3 expression profiles in relation to VSMC proliferation through (1.1) analysis of ASCL3 expression in response to different stimuli in vitro, focusing on factors of relevance in vascular pathologies to correlate it to the (1.2) analysis of ASCLS expression in vascular remodeling in vivo, and Specific aim 2: Analysis of the effects of ASCL3 on proliferation of VSMC in vitro through characterization of the (2.1) effects of gain- and loss-of-function of ASCLS on VSMC proliferation and, naturally derived, the (2.2) identification of ASCL3 target genes regulating VSMC proliferation. These specific aims will be addressed using immunochemistry and quantitative analysis of expression in vivo and in vitro in combination with growth factors of relevance in vascular remodeling as well as in the context of gain- and loss-of function through genetic and biochemical approaches. Development of this proposal will define ASCL3 as a novel bHLH TF with an active role in vascular proliferation and remodeling. This work is bound to transcend the field of vascular biology by shedding light on the general roles of this ubiquitous TF in cellular proliferation at large, with a potential to impact other fields where deregulated cell growth is at the roots of the involved biological processes as it is the case for cancer or developmental biology.