COCULTURING POSTERIOR PITUITARY AND GH3 CELLS - DRAMATIC STIMULATION OF PROLACTIN GENE-EXPRESSION

Recent evidence suggests that the posterior pituitary (PP), also called the neurointermediate lobe, regulates PRL release. We previously reported that cocultures of anterior pituitary and PP cells resulted in a 2- to 3-fold increase in PRL content and release. For this study we chose GH3 cells (a somatomammotroph tumor cell line) to determine whether coculturing GH3 with PP cells: 1) stimulates the release and cell content of PRL as compared with GH; 2) increases GH3 cell proliferation; and 3) affects PRL messenger RNA (mRNA) levels. Exp 1. GH3 cells (25,000 cells per well; 25K) were cocultured with PP cells (OK, 12.5K, or 25K) from male rats in serum-free media for 1, 2, 4, and 7 days; hormones were measured by RIA. Coculturing resulted in 5- to 10-fold increases in both media and cell PRL that were linear with time and dependent on the number of PP cells. In contrast, media GH increased only 1.5- to 2-fold, and GH cell content reduced by 75%. Exp 2. GH3, PP, and GH3 + PP Cells were cultured for 1, 2, and 4 days and then incubated with [H-3]thymidine for 5 h. The incorporation of [H-3]thymidine in GH3 cells remained constant over time and showed a small, early increase in cocultures. In contrast, incubation of PP cells alone resulted in a 50- to 60-fold rise in [H-3]thymidine incorporation from days 1-4 in culture. Exp 3. Cytoplasmic mRNA was determined by slot blot hybridization with ''P-labeled complementary DNA probes for PRL and GH. After coculturing 25K GH3 cells with 12.5K and 25K PP cells for 4 days, PRL mRNA levels increased 15- and 30-fold, respectively, whereas GH mRNA levels rose less than 2-fold. Neither PRL nor GH mRNA were detected in PP cells. Conclusions: 1) coculturing GH3 with PP cells dramatically stimulates PRL gene expression, synthesis, and release; 2) this response is specific for PRL, has little effect on GH, and is not due to increased GH3 cell proliferation; and 3) We speculate that a subpopulation of intermediate lobe cells, possibly with a proliferative capacity, is responsible for inducing these effects.