TGF-β and estrogen regulate P27Kip1 and cyclin D1 in normal and neoplastic rat pituitary cells

Pituitary hyperplasia and tumor growth are regulated by various hormones and growth factors. Estrogen (E2) stimulates pituitary cell proliferation and prolactin (PRL) production. Estrogen also regulates transforming growth factor-β (TGF-β) effects in the pituitary. TGF-β in turn regulates various cell cycle proteins including p15 and p27Kip1 (p27). To better understand the regulatory role of growth factors and hormones in the cell cycle we analyzed cyclin D1, cyclin E, and p27 expression in normal and neoplastic rat pituitary cells. An in vitro analysis using cultured normal pituitary cells and GH3 tumor cells and an in vivo analysis of estrogen-treated normal pituitary and implanted GH3 cells were performed. Semiquantitative RT-PCR was used to analyze mRNA expression for cyclin D1, cyclin E, and p27 in cultured pituitary cells and E2-treated pituitaries in vivo. Cyclin D1 and p27 were localized in the nuclei of normal pituitary cells by immunocytochemistry (ICC). Very weak or absent immunostaining for cyclin D1 and p27 was present in GH3 cells. Both normal pituitary and GH3 cells had strong nuclear staining for cyclin E. Normal pituitary had a 20-fold greater amount of cyclin D1 mRNA and a 3-fold greater amount of p27 mRNA compared to GH3 cells, whereas GH3 cells had slightly (1.5-fold) more cyclin E than normal pituitary cells. E2 treatment in vivo stimulated cell proliferation and decreased cyclin D1 mRNA levels in normal pituitary. GH3 tumor cells, implanted subcutaneously in the same animal, showed increased proliferation after E2 treatment, but there was no change in cyclin D1 mRNA in GH3 cells. Cyclin E and p27 mRNA levels did not change significantly in normal pituitary or in GH3 cells after E2 treatment in vivo. Treatment of normal pituitary cells with 10−9M TGF-β1 for 3 d in vitro led to significant decreases in cyclin D1 and p27 mRNAs (p < 0.05), whereas cyclin E levels were unchanged. These results indicate that cyclin D1 and p27 mRNAs are present at significantly higher levels in normal pituitary compared to GH3 cells, and that both E2 and TGF-β1 can downregulate cyclin D1 mRNA levels in normal pituitary cells, suggesting that these factors regulate G1 to S phase transition in pituitary cells. The lower levels of specific cell cycle regulators in GH3 cells may explain the decreased regulatory control by E2 in GH3 tumor cells.