Growth and Function of Cultured Gastrointestinal Muscle

描述(申请人摘要)：本提案的目的是描述调节生长促进效应的信号通路。 胰岛素样生长因子-I和胰岛素样生长因子结合蛋白-5(IGFBP-5)的表达及其生长抑制作用 转化生长因子-β1和胰岛素样生长因子结合蛋白-β在人肠道平滑肌细胞上的表达。这个 实验方法利用正常人类肠道肌肉细胞在 培养中的生长，它概括了肌肉组织在 发展中的或发炎的肠子。此前的研究表明，这些水平 生长细胞产生的IGF-I和IGFBP-5在 增殖期和衰退期随着汇流的接近而下降，而 在细胞达到融合后，转化生长因子-β1和IGFBP-3的水平最高。 当前提案的具体目标所依据的假设是 生长因子和结合蛋白激活刺激和抑制 信号通路调节：(I)IGF-I受体的表达；(Ii) 生长因子和结合蛋白的产生以及它们的能力 促进或抑制生长；以及(Iii)细胞周期从G 1至S阶段。具体目的是：(1)描述监管的特征 胰岛素样生长因子-I、转化生长因子-β1、胰岛素样生长因子结合蛋白-5和胰岛素样生长因子结合蛋白-3对胰岛素样生长因子-I受体表达的影响 培养；(2)表征介导刺激的信号通路 胰岛素样生长因子-I和胰岛素样生长因子结合蛋白-5及转化生长因子-β1和胰岛素样生长因子结合蛋白-3的抑制作用 细胞生长和生长因子和结合蛋白的产生；以及(3) 识别调控细胞周期从G1期到S期的信号 相位。每个具体目标都有大量的初步数据支持。IGF-I 和IGFBP-5通过调节其生产的相互作用的途径相连， 促生长作用和抑制IGF-I受体表达的能力。这个 途径包括IGF-I激活p42/44 MAP和PI3-激酶 受体酪氨酸激酶--IGFBP-5受体激活p38 MAP激酶 丝氨酸激酶。转化生长因子-β1和胰岛素样生长因子结合蛋白-3激活转化生长因子-β1受体丝氨酸激酶 导致Smad2磷酸化和IGF-I受体抑制 磷酸化。IGF-I刺激细胞周期蛋白D1表达、CDK4活性和pRb 磷酸化，转化生长因子-β1刺激cdk抑制因子p15INK4b。这个 研究提供了对细胞机制的分析，这些细胞机制决定了 肌肉生长的开始和停止。

DESCRIPTION (Applicant's Abstract): The objective of this proposal is to characterize the signaling pathways that mediate the growth-promoting effects of IGF-I and IGF-binding protein-5 (IGFBP-5), and the growth-inhibiting effects of TGF-beta1 and IGFBP-on human intestinal smooth muscle cells. The experimental approach utilizes normal human intestinal muscle cell during growth in culture, which recapitulates the rapid growth of muscle tissue in the developing or inflamed intestine. Previous studies have shown that the levels of IGF-I and IGFBP-5 produced by growing cells are high during the proliferative phase and decline with the approach of confluence, whereas the levels of TGF-beta1 an IGFBP-3 are highest after the cells attain confluence. The hypotheses that underlie the Specific Aims of the current proposal are that the growth factors and binding proteins activate stimulatory and inhibitory signaling pathways that regulate: (i) the expression of IGF-I receptors; (ii) the production of growth factors and binding proteins, and their ability to promote or inhibit growth; and (iii) the progress of the cell cycle from the G 1 to the S phase. The Specific Aims are to: (1) characterize the regulation of IGF-I receptor expression by IGF-I, TGF-beta1 IGFBP-5, and IGFBP-3 during culture; (2) characterize the signaling pathways that mediate the stimulatory effects of IGF-I and IGFBP-5, and inhibitory effects ofTGF-B1 and IGFBP-3 on cell growth and the production of growth factors and binding proteins; and (3) identify the signals that regulate cell cycle progression from the G1 to S phase. Each Specific Aim is supported by substantial preliminary data. IGF-I and IGFBP-5 are linked by reciprocal pathways that regulate their production, growth-promoting effects, and ability to inhibit IGF-I receptor expression. The pathways include activation of p42/44 MAP kinase and PI 3-kinase by IGF-I receptor tyrosine kinase, an activation of p38 MAP kinase by IGFBP-5 receptor serine kinase. TGF-beta1 and IGFBP-3 activate TGF-beta1 receptor serine kinase leading to Smad2 phosphorylation and inhibition of IGF-I receptor phosphorylation. IGF-I stimulates cyclin Dl expression, cdk4 activity, and pRb phosphorylation, and TGF-beta1 stimulates the cdk inhibitor, p15INK4B. The studies provide an analysis of the cellular mechanisms that determine initiation and cessation of muscle growth.