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

描述(由申请人提供)：在发育过程中参与细胞命运决定和调控细胞增殖、分化、凋亡和迁移的许多级联因子(Tf)受基本-螺旋-环-螺旋类(BHLH)中的转录因子(Tf)调控，包括bHLHachaete-scute家族中的那些转录因子，该家族从无脊椎动物到人类高度保守。虽然哺乳动物的ASCL1已经成为神经生物学中广泛研究的对象，但关于ASCL2到ASCL5家族的其他成员，特别是普遍表达的ASCL3的作用的信息却很少。我们的初步数据显示，ASCL3、ASCL2和ASCL1在血管平滑肌细胞(VSMC)中表达。聚焦于ASCL3，我们发现它的表达在血管紧张素II刺激增殖时增加，在较小程度上PDGF-BB的表达增加，而在Notch1激活或BMP2处理抑制生长的情况下表达降低。此外，ASCL3的稳定过表达在体外可促进VSMC的增殖，而在体内血管病变的新生内膜中ASCL3的表达明显上调。这些数据表明，ASCL3对VSMC的增殖有积极的促进作用，在病理性血管重塑的发展中具有潜在的作用，并将其定义为“血管调节”因子。这些观察结果导致了我们的中心假设，即ASCL3是一种促进生长的bHLH转录因子，调节VSMC的增殖。 为了解决这一假设的影响和预测，我们建议进行以下工作： 具体目标1：通过(1.1)体外分析ASCL3在不同刺激下的表达，重点关注血管病理中的相关因素，以将其与(1.2)体内血管重塑中ASCLS表达的分析相关联，来表征与VSMC增殖相关的ASCL3表达谱，以及 具体目的2：通过(2.1)ASCLS对VSMC增殖的获得和功能丧失的影响以及(2.2)调控VSMC增殖的ASCL3靶基因的鉴定，分析ASCL3对体外培养的VSMC增殖的影响。 这些特定的目标将通过体内和体外表达的免疫化学和定量分析结合与血管重塑相关的生长因子以及通过遗传和生化方法获得和丧失功能的背景来解决。这项提议的发展将把ASCL3定义为一种新的bHLHTF，在血管增殖和重塑中发挥积极作用。这项工作必将超越血管生物学领域，揭示这种普遍存在的转铁蛋白在细胞增殖中的一般作用，并有可能影响其他领域，在这些领域，放松调控的细胞生长是相关生物学过程的根源，就像癌症或发育生物学的情况一样。

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.