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.
