ACTION OF PTH-RELATED PROTEIN ON THE GUT

本项目的总体假设是胃肠道(Gl)肽甲状旁腺激素相关蛋白(PTHrP)在肠道中起着重要的调节作用，其失调通过对磷脂酰肌醇-3-激酶(PI3K)途径的异常调节而参与疾病的发生。我们发现PTHrP可促进体内异种结肠癌细胞的生长。PTHrP在转移性结肠癌细胞中的表达高于非转移性结肠癌细胞，并且PTHrP水平与体外侵袭整合素亲和表达以及这些细胞的迁移和侵袭潜能直接相关。我们发现PI3K通路介导了这些效应。本项目的重点是通过追求三个目标来了解PTHrP促进结肠癌细胞侵袭的分子机制。(1)PTHrP上调PI3K的机制(S)将通过询问这是否通过 PI3K p85和lt；x亚基与激活的PTHrP受体之间的相互作用，和/或次级于PTHrP介导的整合素a6p4的上调。PI3K活性将在外源PTHrP加入细胞培养后被检测，并在稳定转染过表达PTHrP的细胞中进行检测。整合素在PI3K激活中的作用将通过siRNAs下调其表达来确定。(2)将研究受PI3K调控的下游效应分子的作用，包括Akt、PTEN以及小的GTP结合蛋白Rac和CDc42。这一目标将通过以下技术来实现： 显性-负性突变体，蛋白质水平的免疫印迹测定和 免疫细胞化学和直接活性测量。(3)PTHrP上调侵袭前整合素表达的途径(S)将通过Northern印迹分析来检测整合素mRNA水平的变化，脉冲追逐分析来检测整合素合成和周转速度的变化，以及流式细胞仪分析来检测PTHrP过表达的结肠癌细胞内整合素动员的变化。这些研究将使我们能够确定甲状旁腺素rp促进结肠癌细胞侵袭和转移的机制(S)，并最终应该允许开发旨在控制这一过程的新的治疗策略。

The overall hypothesis addressed in this project is that the gastrointestinal (Gl) peptide parathyroid hormone-related protein (PTHrP) plays an important regulatory role in the gut, and its dysregulation contributes to disease via aberrant regulation of the phosphatidylinositol-3-kinase (PI3K) pathway. We show that PTHrP increases colon cancer cell xenograft growth in vivo. PTHrP expression is higher in metastatic vs. non-metastatic colon cancer cells, and there is a direct correlation of PTHrP levels with pro-invasive integrin expression and the migratory and invasive potential of these cells in vitro. We find that the PI3K pathway mediates these effects. The focus of this project is to understand the molecular mechanisms via which PTHrP promotes colon cancer cell invasion, by pursuing three Aims. (1) The mechanism(s) governing the upregulation of PI3K by PTHrP will be determined, by asking whether this occurs via an interaction between the PI3K p85<x subunit with an activated PTHrP receptor, and/or is secondary to the PTHrP-mediated upregulation of integrin a6p4. PI3K activity will be measured after exogenous addition of PTHrP to the cell culture, and in cells engineered by stable transfection to overexpress PTHrP. The role of integrins in PI3K activation will be determined using siRNAs to downregulate their expression. (2) The role of downstream effectors regulated by PI3K, including Akt, PTEN, and the small GTP-binding proteins Rac and Cdc42, will be investigated. This aim will be accomplished using such techniques as transfection with dominant-negative mutants, determination of the protein levels by Western immunoblotting and immunocytochemistry, and direct activity measurements. (3) The pathway(s) via which PTHrP upregulates pro-invasive integrin expression will be addressed using Northern blot analysis to detect changes in integrin mRNA levels, pulse-chase analysis to detect alterations in the rate of integrin synthesis and turnover, and FACS analysis to detect changes in integrin intracellular mobilization in PTHrP-overexpressing vs. control colon cancer cells. These studies will allow us to identify the mechanism(s) by which PTHrP promotes invasion and metastases of colon cancer cells, and should ultimately allow the development of new therapeutic strategies aimed at controlling this process.