The proposed research will examine the role of Ca2+-dependent cadherin mediated cell-cell adhesion in the induction of prolactin (PRL) gene expression. There are three general aims: 1. Cadherin expression in GH3 cells. We have preliminary data for multiple cadherin expression in GH3 cells. This work will be extended in order to confirm our preliminary findings, as well as to generate full length cDNA clones and specific antibodies.
The specific aims are: 1. Cloning and sequencing of PCR- generated fragments of rat N-cadherin, E-cadherin, and P-cadherin from GHG3 cell cDNA in order to confirm their identity. 2. Use of these partial cDNA clones as probes to examine cadherin expression in GH3 cells by Northern blot. 3. Isolation of full length cDNA clones for those cadherins expressed in GH3 cells. 4. Use of sequence information to generate specific antibodies against rat expressed in GH3 cells. This will be done on a contractural basis. 5. Use of specific cadherin antibodies to examine cadherin protein expression and localization in GH3 cells and related pituitary tumor cell lines. II. Effects of experimentally induced disruption of cell-cell adhesion on PRL gene expression. We have reproducibly observed a close correlation between CaCl2-induced cell-cell adhesion and CaCl2-induced PRL gene expression in GH3 cells. We propose to utilize experimentally induced inhibition or disruption of cell-cell contacts for the purpose of examining the requirement of cell-cell adhesion in the CaCl2 induction of PRL gene expression. Cell-cell adhesion will be inhibited or disrupted in the following ways: 1. Introduction by stable transfection of an N-cadherin gene containing a large deletion of the extracellular domain. Based on previous work with this gene, it is expected to behave as a dominant negative mutant cadherin gene, which will disrupt the ability of endogenous cadherins to form cell-cell junctions. 2. Competitive inhibition of the homophilic binding site by introduction of peptides corresponding to the homophilic binding site of the extracellular domain. 3. Effectively increasing c=src activity, since c-src phosphorylates cadherins and associated proteins, and induces the disassembly of adherins junctions. III. Examination of cadherin expression and function in the pituitary gland. We propose examine of cadherin function in the normal pituitary gland. This section has two specific aims: 1. Expression of cadherins in adult and fetal rat pituitaries and; 2 Effect of a dominant negative cadherin mutation that is targeted to lactotrophs in transgenic mice on lactotrope abundance and position within pituitary, and on content of pituitary PRL. The proposed research represents the first examination of cadherin function in the pituitary gland, both in terms of its morphogenesis and of the differentiated function of lactotropes. These studies may provide the first demonstration of a role for cadherins in the expression of a specific hormone. Since cadherins have tumor suppressor activity, future work may also reveal a role for cadherins in the control of estrogen-induced proliferation of lactotropes.
