ACTION OF PTH-RELATED PROTEIN ON THE GUT

在该项目中提出的总体假设是胃肠（GI）肽甲状旁腺相关蛋白（PTHrP）在肠道中起重要的调节作用，并且其失调通过磷脂酰肌醇-3-激酶（PI 3 K）途径的异常调节而促成疾病。我们表明PTHrP增加体内结肠癌细胞异种移植物的生长。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水平与促侵袭整合素表达以及这些细胞在体外的迁移和侵袭潜力直接相关。我们发现PI 3 K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)通过询问PTHrP上调PI 3 K是否通过一个或多个信号通路发生，将确定PTHrP上调PI 3 K的机制。 PI 3 K p85 α x亚基与活化的PTHrP受体之间的相互作用，和/或继发于PTHrP介导的整联蛋白α 6 β 4的上调。在将PTHrP外源性添加到细胞培养物中后，以及在通过稳定转染工程化以过表达PTHrP的细胞中，测量PI 3 K活性。整合素在PI 3 K活化中的作用将使用siRNA下调其表达来确定。(2)将研究由PI 3 K调节的下游效应物的作用，包括Akt、PTEN和小GTP结合蛋白Rac和Cdc 42。这一目的将通过使用诸如转染的技术来实现。 显性阴性突变体，通过蛋白质免疫印迹测定蛋白质水平， 免疫细胞化学和直接活性测量。(3)PTHrP上调促侵袭性整合素表达的途径将使用北方印迹分析来检测整合素mRNA水平的变化，使用脉冲追踪分析来检测整合素合成和转换速率的变化，使用FACS分析来检测PTHrP过表达的结肠癌细胞与对照结肠癌细胞中整合素细胞内动员的变化。这些研究将使我们能够确定PTHrP促进结肠癌细胞侵袭和转移的机制，并最终允许开发旨在控制这一过程的新治疗策略。

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.