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 (0K, 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 [3H]thymidine for 5 h. The incorporation of [3H]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 [3H]thymidine incorporation from days 1-4 in culture. Exp 3. Cytoplasmic mRNA was determined by slot blot hybridization with 32P-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.
Corcia
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A
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202.
Steinmetz
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R
Liu
J W
JW
Ben-Jonathan
N
N
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D015870:Gene Expression; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism; D008297:Male; D009693:Nucleic Acid Hybridization; D010904:Pituitary Gland, Posterior / Q000502:physiology*; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011388:Prolactin; D013006:Growth Hormone; D004247:DNA