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

描述（申请人提供）：内膜内异常的血管平滑肌细胞（SMC）积聚在动脉粥样硬化病变的发病机制中起关键作用。局部产生的胰岛素样生长因子（IGFs）是内膜SMC积累的重要调节因子。IGFs刺激SMC迁移、增殖、分化和存活。igf的这些不同作用是通过IGF-I受体（IGF-IR），一种跨膜酪氨酸激酶介导的。相同的配体如何激活相同的IGF- ir导致这些不同的生物反应尚不清楚，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，我们和其他人已经确定了几种高亲和力的igf结合蛋白（igfbp），它们是由SMCs合成和分泌的。我们的研究表明，这些igfbp是特定细胞对IGF刺激反应的重要决定因素，而IGFBP-5在这一范式中起着关键作用。IGFBP-5结合IGF并调节IGF的作用。IGFBP-5也通过与配体无关的机制刺激SMC迁移。我们最近的研究表明IGFBP-5定位于SMC核中，核中的IGFBP-5可能来源于分泌的蛋白。此外，保守的IGFBP-5 n结构域具有不受IGF结合影响的转录激活活性。本研究的总体目标是进一步阐明IGFBP-5核易位途径，并确定其在调节SMC迁移、增殖、分化和凋亡中的作用。第一个目标是确定作为IGFBP-5关键伙伴并介导IGFBP-5内化和核易位的膜和细胞质蛋白。第二个目标将研究IGFBP-5的功能意义，特别关注其核靶向和配体结合。原生和突变的IGFBP-5将在IGFBP-5 siRNA敲除的SMCs和IGFBP-5空细胞中表达，用于体外研究。在SMCs中靶向过表达原生或突变IGFBP-5的转基因小鼠和IGFBP-5敲除小鼠将用于体内研究。第三个目标是确定IGFBP-5反活化活性的调控机制，并确定IGFBP-5的靶基因。这些研究将使我们更好地理解igf、IGF-IR和igfbp之间的分子相互作用，并为SMC迁移、增殖、分化和凋亡的调控提供新的信息，以及IGFBP-5作用的分子机制模型。我们相信，阐明IGF和IGFBP-5在SMCs中的作用机制将具有重要的应用价值，包括开发未来的治疗策略，以纠正或规避动脉粥样硬化及相关并发症。

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.