IGF-I and its binding proteins in vascular smooth muscle cells

描述(申请人提供)：在动脉粥样硬化病变的发病机制中，血管平滑肌细胞(SMC)在动脉内膜中异常聚集起关键作用。局部产生的胰岛素样生长因子(IGFS)是血管内膜SMC积聚的重要调节因子。IGF刺激SMC迁移、增殖、分化和存活。IGFS的这些不同作用是通过IGF-I受体(IGF-IR)介导的，IGF-IR是一种跨膜酪氨酸激酶。同样的配体如何激活相同的IGF-IR导致这些不同的生物反应还不是很清楚，但这是理解IGF信号系统在动脉粥样硬化病变发展中作用的分子基础的关键。最近，我们和其他人发现了几种由SMC合成和分泌的高亲和力的IGF结合蛋白(IGFBPs)。我们的研究表明，这些IGFBPs是对IGF刺激的特定细胞反应的重要决定因素，而IGFBP-5是这一范式中的关键角色。IGFBP-5与胰岛素样生长因子结合，调节胰岛素样生长因子的作用。IGFBP-5还通过不依赖配体的机制刺激SMC迁移。我们最近的研究表明，IGFBP-5定位于SMC核，核中的IGFBP-5很可能来自于分泌的蛋白。此外，保守的IGFBP-5 N结构域具有转录激活活性，不受IGF结合的影响。该提案的总体目标是进一步阐明IGFBP-5核转位途径，并确定其在调节SMC迁移、增殖、分化和凋亡中的作用。第一个目标将确定作为关键的IGFBP-5伙伴并介导IGFBP-5内化和核转位的膜和细胞质蛋白。第二个目的是研究IGFBP-5的功能意义，特别是它的核靶向性和配体结合。我们将在IGFBP-5 siRNA敲除的SMC和IGFBP-5缺失细胞中表达IGFBP-5，用于体外研究。在SMC和IGFBP-5基因敲除小鼠中定向过表达原生或突变IGFBP-5的转基因小鼠将用于体内研究。第三个目的是确定IGFBP-5反式激活活性的调控机制(S)，并鉴定IGFBP-5的靶基因。这些研究将使我们更好地了解IGFS、IGF-IR和IGFBPs之间的分子相互作用，并为SMC迁移、增殖、分化和凋亡的调控以及IGFBP-5作用的分子机制模型提供新的信息。我们相信，阐明IGF和IGFBP-5在SMC中的作用机制将具有重要的应用，包括开发未来可能纠正或绕过动脉粥样硬化和相关并发症的治疗策略。

DESCRIPTION (provided by applicant): Abnormal vascular smooth muscle cell (SMC) accumulation in the intima plays a key role in the pathogenesis of atherosclerotic lesions. Locally produced insulin-like growth factors (IGFs) are important regulators of intimal SMC accumulation. IGFs stimulate SMC migration, proliferation, differentiation, and survival. These diverse actions of IGFs are mediated through the IGF-I receptor (IGF-IR), a transmembrane tyrosine kinase. How activation of the same IGF-IR by the same ligands leads to these diverse biological responses is not well understood, but is key to understanding the molecular basis of the role of the IGF signaling system in development of atherosclerotic lesions. Recently, we and others have identified several high-affinity IGF-binding proteins (IGFBPs) that are synthesized and secreted by SMCs. Our studies indicate that these IGFBPs are important determinants of specific cellular responses to IGF stimulation, and that a key player in this paradigm is IGFBP-5. IGFBP-5 binds to IGF and modulates IGF actions. IGFBP-5 also stimulates SMC migration through a ligand-independent mechanism. Our recent studies reveal that IGFBP-5 is localized in the SMC nucleus and that nuclear IGFBP-5 is likely to be derived from the secreted protein. Moreover, the conserved IGFBP-5 N-domain possesses transcriptional activation activity which is not affected by IGF binding. The overall goal of this proposal is to further elucidate the IGFBP-5 nuclear translocation pathway and to determine its role in regulating SMC migration, proliferation, differentiation, and apoptosis. The first aim will determine the membrane and cytoplasmic proteins that act as key IGFBP-5 partners and mediate IGFBP-5 internalization and nuclear translocation. The second aim will investigate the functional significance of IGFBP-5, with special focus on its nuclear targeting and ligand binding. Native and mutant IGFBP-5 will be expressed in IGFBP-5 siRNA knockdown SMCs and in IGFBP-5 null cells for in vitro studies. Transgenic mice with targeted overexpression of native or mutant IGFBP-5 in SMCs and IGFBP-5 knock-out mice will be used for in vivo studies. The third aim will determine the regulatory mechanism(s) of the transactivation activity of IGFBP-5 and to identify IGFBP-5 target genes. The proposed studies will lead us towards a better understanding of the molecular interactions between IGFs, IGF-IR and IGFBPs and provide novel information on the regulation of SMC migration, proliferation, differentiation, and apoptosis, as well as, a model of the molecular mechanisms of IGFBP-5 actions. It is our belief that elucidating the mechanisms of IGF and IGFBP-5 actions in SMCs will have important applications, including the development of future therapeutic strategies that may correct or circumvent atherosclerosis and related complications.