Cell-Cell Adhesion and Regulation of Pituitary Prolactin

Beverly C. Delidow， P.I.。本提案的目的是通过细胞间接触来研究垂体激素基因催乳素的调节。初步数据显示，当大鼠235-1垂体肿瘤细胞经历更高水平的细胞-细胞粘附时，催乳素（PRL）基因表达增加。相反，当细胞暴露于糖皮质激素时，PRL水平降低，这降低了细胞-细胞粘附分子和细胞再聚集的水平。所涉及的细胞粘附分子是钙依赖性细胞粘附蛋白的钙粘蛋白-连环蛋白系统。这些蛋白参与形成稳定的细胞-细胞接触和细胞内信号传导。在暴露于反义寡核苷酸降低了df -连环蛋白水平的细胞中，催乳素水平显著下降。这些观察结果导致了由钙粘蛋白介导的细胞-细胞接触在PRL基因表达中起调节作用的假设。在完成提出的工作后，假设将通过以下方式进行检验：1。数据强烈暗示235-1细胞含有钙粘蛋白样蛋白，但其身份尚不清楚。这些分子的鉴定是必不可少的，因为细胞表面钙粘蛋白与邻近细胞上的类似分子相互作用，以启动稳定的细胞-细胞粘附。也有必要表征表达的钙粘蛋白，以便它们的功能可能被特异性地干扰。RNA和蛋白质分析将使用现有的钙粘蛋白探针或抗体进行，以确定哪些钙粘蛋白相关基因在235-1细胞中表达。2. 为了确定钙粘蛋白在PRL基因调控中的作用，将使用Tet-on系统（一种将诱导基因稳定导入哺乳动物细胞的新方法）生成含有钙粘蛋白抑制片段诱导基因的235-1细胞亚系。当钙粘蛋白片段基因被诱导时，细胞应该会失去细胞间的粘附，从而可以详细研究这种丧失对PRL基因表达的影响。3. 最后，糖皮质激素处理235-1细胞导致连环蛋白表达减少；我们将研究这种减少的机制。连环蛋白在其他细胞中受到多种水平的调控。调控的潜在目标是RNA水平、蛋白质水平和蛋白质修饰。所有这些可能性都将被审查。从这些研究中获得的信息将为细胞-细胞粘附分子在分化细胞类型中的调节作用提供越来越多的证据。此外，新的数据将产生关于乳营养细胞的调节，多用途的内分泌细胞在繁殖，渗透调节和许多脊椎动物的行为中发挥作用。

IBN(MCB)-981027 Beverly C. Delidow, P.I. The goal of this proposal is to examine the regulation of a pituitary hormone gene, prolactin, by cell-cell contacts. Preliminary data show that prolactin (PRL) gene expression increases when rat 235-1 pituitary tumor cells experience greater levels of cell-cell adhesion. In contrast, PRL levels decrease when the cells are exposed to glucocorticoids, which decrease both the levels of cell- cell adhesion molecules and cell reaggregation. The cell adhesion molecules involved are the cadherin-catenin system of calcium-dependent cell adhesion proteins. These proteins participate in both forming stable cell-cell contacts and in intracellular signaling. Prolactin levels decrease profoundly in cells in which the DF-catenin levels have been decreased by exposure to antisense oligo-nucleotides. These observations led to the hypothesis that cell-cell contacts mediated by cadherins serve a regulatory function in PRL gene expression. In completion of the proposed work, the hypothesis will be tested in the following ways: 1. The data strongly imply that 235-1 cells contain cadherin-like proteins, but their identity is not known. The identification of these molecules is essential because the cell surface cadherin interacts with a like molecule on a neighboring cell to initiate stable cell-cell adhesions. It is also necessary characterize the cadherin(s ) expressed so that their function may be perturbed specifically. Both RNA and protein analyses will be carried out using existing cadherin probes or antibodies to determine which cadherin-related genes are expressed in 235-1 cells. 2. To determine the role of cadherin proteins in PRL gene regulation, the Tet-on system, a new means of stably introducing inducible genes into mammalian cells, will be used to generate sublines of 235-1 cells that contain an inducible gene for the inhibitory fragment of cadherin. When the cadherin fragment gene is induced, the cells shoul d lose cell-cell adhesion, allowing a detailed investigation of effect of that loss on PRL gene expression. 3. Finally, glucocorticoid treatment of 235-1 cells leads to a decrease in the expression of catenins; the mechanism of this decrease will be examined. Catenins are regulated at multiple levels in other cells. The potential targets for regulation are RNA levels, protein levels and protein modification. All of these possibilities will be examined. The information gained from these studies will add to the growing evidence for a regulatory role of cell-cell adhesion molecules in differentiated cell types. In addition, novel data will be generated on the regulation of lactotrophs, versatile endocrine cells with roles in reproduction, osmoregulation and behavior across many vertebrate species.