Transit of normal rat uterine stromal cells through G1 phase of the cell cycle requires progesterone-growth factor interactions

Understanding of cell cycle regulation in hormonally responsive cells lags behind studies in other systems because few models have been available to i dentify the role of steroid hormones and their receptors in this process; T his study investigates progesterone-dependent effects on the progression of normal uterine stromal cells through early G1 phase of the cell cycle. Qui escent rat uterine stromal cells were stimulated to reenter the cell cycle by adding serum-free medium containing medroxyprogesterone acetate (MPA) an d basic Fibroblast growth factor (FGF). [H-3]thymidine incorporation increa sed significantly (P = 0.025) in cells stimulated with both FGF alone and M PA plus FGF compared with the control cells. Moreover, cells stimulated wit h MPA plus FGF incorporated significantly more (P = 0.01) [H-3]thymidine th an cells treated with FGF alone, suggesting requisite interactions between progesterone and FGF for stromal cell entry into S phase. Flow cytometric a nalysis of stimulated stromal cells showed FGF alone and MPA plus FGF incre ased significantly (P = 0.002) the percentage of cells in S phase at 12 h. Incorporation of bromodeoxyuridine into stromal cell nuclei indicated that FGF alone and MPA plus FGF increased the percentage of cells entering S pha se at 18 and 24 h compared with the control cells. In addition, MPA plus FG F increased significantly (P = 0.001) the number of cells entering S phase at 24 h compared with FGF alone and sustained S phase entry compared with F GF alone, MPA alone, or the control cells. Stromal cells inhibited from G1 reentry by inhibition of mitosis showed accelerated entry into S phase in r esponse to MPA plus FGF compared with FGF alone. Cyclin D1 messenger RNA in creased in stromal cells treated with MPA plus FGF at 9, 12, and 15 h. Addi tion of RU 486 to cells stimulated with MPA plus FGF for 9 h reduced cyclin D1 messenger RNA accumulation by 40%. Western blot analysis of cyclin D1 i mmunoprecipitates indicated complex formation with both cyclin-dependent ki nase 4 (Cdk4) and cyclin dependent kinase 6 (Cdk6). Cyclin D1-Cdk complexes and kinase activity correlated temporally with increased cyclin D1 express ion in cells cultured with MPA plus FGF. Taken together, these results show that progesterone-FGF interactions increase cyclin D1 expression, correlat ing with accelerated stromal cell entry into S phase compared with cells tr eated with FGF alone. Morever, progesterone plus FGF sustains the timing of stimulation for transit of uterine stromal cells through G1 into S phase c ompared with FGF alone.