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

描述（申请人提供）：内膜中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着关键作用。局部产生的胰岛素样生长因子（IGF）是内膜 SMC 积累的重要调节因子。 IGF 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。相同配体激活相同 IGF-IR 如何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，我们和其他人鉴定了几种由 SMC 合成和分泌的高亲和力 IGF 结合蛋白 (IGFBP)。我们的研究表明，这些 IGFBP 是 IGF 刺激的特定细胞反应的重要决定因素，而该范例中的关键角色是 IGFBP-5。 IGFBP-5 与 IGF 结合并调节 IGF 作用。 IGFBP-5 还通过配体独立机制刺激 SMC 迁移。我们最近的研究表明，IGFBP-5 位于 SMC 细胞核中，并且核 IGFBP-5 很可能源自分泌蛋白。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。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。第一个目标是确定作为关键 IGFBP-5 伙伴并介导 IGFBP-5 内化和核转位的膜和细胞质蛋白。第二个目标将研究 IGFBP-5 的功能意义，特别关注其核靶向和配体结合。天然和突变型 IGFBP-5 将在 IGFBP-5 siRNA 敲低 SMC 和 IGFBP-5 无效细胞中表达，用于体外研究。在 SMC 中靶向过度表达天然或突变 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 反式激活活性的调控机制并鉴定 IGFBP-5 靶基因。拟议的研究将引导我们更好地了解 IGF、IGF-IR 和 IGFBP 之间的分子相互作用，并提供有关 SMC 迁移、增殖、分化和凋亡调节的新信息，以及 IGFBP-5 作用的分子机制模型。我们相信，阐明 IGF 和 IGFBP-5 在 SMC 中的作用机制将具有重要的应用，包括开发可能纠正或规避动脉粥样硬化和相关并发症的未来治疗策略。

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.