TGF-B and Estrogen Regulate P27(Kip1) and Cyclin D(1) in Normal and Neoplastic Rat Pituitary Cells

Pituitary hyperplasia and tumor growth are regulated by various hormones and growth factors. Estrogen (E(2)) stimulates pituitary cell proliferation and prolactin (PRL) production. Estrogen also regulates transforming growth factor-B (TGF-B) effects in the pituitary. IGF-B in turn regulates various cell cycle proteins including p15 and p27(Kip1) (p27). To better understand the regulatory role of growth factors and hormones in the cell cycle we analyzed cyclin D(1), cyclin E, and p27 expression in normal and neoplastic rat pituitary cells. An in vitro analysis using cultured normal pituitary cells and GH(3) tumor cells and an in vivo analysis of estrogen-treated normal pituitary and implanted GH(3) cells were performed. Semiquantitative RT-PCR was used to analyze mRNA expression for cyclin D(1) cyclin E, and p27 in cultured pituitary cells and E(2)-treated pituitaries in vivo, Cyclin D(1) and p27 were localized in the nuclei of normal pituitary cells by immunocytochemistry (ICC). Very weak or absent immunostaining for cyclin D(1) and p27 was present in GH(3) cells. Both normal pituitary and GH(3) cells had strong nuclear staining for cyclin E. Normal pituitary had a 20-fold greater amount of cyclin D mRNA and a 3-fold greater amount of p27 mRNA compared to GH cells, whereas GH cells had slightly (1.5-fold) more cyclin E than normal pituitary cells. Treatment in vivo stimulated cell proliferation and decreased cyclin D(1) mRNA levels in normal pituitary. GH(3) tumor cells, implanted subcutaneously in the same animal, showed increased proliferation after E(2) treatment, but there was no change in cyclin B(1) mRNA in GH(3) cells. Cyclin E and p27 mRNA levels did not change significantly in normal pituitary or in GH(3) cells after E(2) treatment in vivo. Treatment of normal pituitary cells with 10(-9)M TGF-B1 for 3 d in vitro led to significant decreases in cyclin B(1) and p27 mRNAs (p < 0.05 ), whereas cyclin E levels were unchanged. These results indicate that cyclin B(1) and p27 mRNAs are present at significantly higher levels in normal pituitary compared to GH(3) cells, and that both E(2) and TGF-B1 can down-regulate cyclin B(1) 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 GH cells may explain the decreased regulatory control by E(2) in GH(3) tumor cells.