Distinct signaling pathways mediate stimulation of cell cycle progression and prevention of apoptotic cell death by estrogen in rat pituitary tumor PR1 cells

Abstract

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17beta-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones.

Figure 1.

Effects of estrogen and the pure antiestrogen ICI 182,780 on PR1 cell proliferation. (A) PR1 cells were maintained in estrogen-free medium (C) for 4 d, before replating and stimulation with the indicated concentrations of E2 or ICI 182,780 (ICI). At each indicated time, cells were collected and their number was evaluated by a colorimetric assay. (B) For [³H]TdR incorporation assays, cells were incubated as described above for the last 24 h before collection with radioactive thymidine and then processed as described under MATERIALS AND METHODS. The data shown (± SD) are representative of at least three, separate experiments carried out with six or eight replicate determinations.

Figure 2.

Effects of estrogen on ERE-mediated reporter gene transcription, DNA synthesis rate, and endogenous gene expression in PR1 cells. (A) Cells were maintained in estrogen-free medium (C) for 4 d, before transfection with the ERE-tk-luc reporter gene, followed by stimulation for 24 h with the indicated molar concentrations of either E2 or ICI 182,780 (ICI) and assay of luciferase activity in whole cell extracts, carried out as described in the text. (B) Intracellular prolactin (PRL) and cyclin D3 (D3) protein content was evaluated in whole cell lysates by Western blotting analysis, after 48-h stimulation carried out as described above. ERK1/2 assessment in the same blot or sample have been used for normalization, and the results are reported (ERKs). (C) Pulse labeling with [³H]TdR for 1 h was used to assess the effects of E2 and ICI treatment for 48 h on DNA synthesis rate. FCS, cells were maintained in medium containing 10% fetal bovine serum throughout the experiment. The data shown (± SD) are representative of multiple determinations carried out in replicate.

Figure 3.

Distinct responses of PR1 cells to progressive estrogen receptor blockade with the antiestrogen ICI 182,780. (A) Effects of 48-h blockade of estrogen signaling with the indicated concentrations of ICI 182,780 on DNA synthesis rate (full circles), measured by pulse-labeling with radioactive thymidine, cell growth (full squares), measured by labeling with radioactive thymidine for 24 h, cdc-2 cyclin-dependent kinase activity (open lozenges), ERE-tk-luc activity (open triangle). Each point is the mean ± SD of at least two determinations carried out in triplicate. (B) Whole cell lysates were prepared for evaluating by Western blotting intracellular prolactin (PRL) and cyclin D3 (D3) protein content after 48-h treatment of the cells as described above. ERK1/2 assessment in the same blot or sample have been used for normalization and the results are reported (ERKs). FCS, cells were maintained in medium containing 10% fetal bovine serum throughout the experiment. The data shown (± SD) are representative of multiple determinations carried out in replicate.

Figure 4.

Analysis of PR1 cells viability after estrogen deprival and restimulation or antiestrogen treatment. (A) Representative cytofluorimetric profile, cells growing in medium containing 10% FCS medium were analyzed before (top) or after treatment with ICI 182,780 (10^-6 M; bottom). M1 indicate the pre-G₁ fraction of dead cells. (B) Kinetics of induction of cell death by ICI 182,780 treatment of PR1 cells. (C) Kinetics of induction of PR1 cell death upon culture in estrogen-free medium (C), without or with ICI 182,780 (ICI), and reversal by stimulation with E2. (D) Cells were maintained in estrogen-free medium (C) for 2 d, before treatment with E2 10^-9 M or ICI 10^-8 M for 24 h and then were analyzed for Annexin V/BOBO-1 double staining by fluorescence microscopy. FCS, cells were maintained in medium containing 10% fetal bovine serum throughout the experiment. The data shown (± SD) are representative of multiple determinations carried out in replicate.

Figure 5.

Caspase-3 expression level and activity in PR1 cells after estrogen blockade with ICI 182,780. (A) Expression levels of caspase 3 mRNA in cells treated as indicated with either E2 or ICI 182,780 (ICI) were assessed by a multiple RNase protection assay. The signals corresponding to the mRNA of the housekeeping genes L32 and GAPDH are also shown for normalization. Cells were maintained in whole medium containing 10% fetal calf serum (FCS) or phenol red-free medium containing charcoal-stripped serum (DCC) throughout the experiment. (B) Cells were treated with 10^-6 M ICI 182,780 for the indicated times before evaluation of caspase-3 enzyme activity in cell extracts by a colorimetric assay, as described under MATERIALS AND METHODS. Where indicated, cells were maintained in DCC medium without (C) or with (E2) 10^-9 M 17β-estradiol for 48 h before analysis. The data shown (± SD) are representative of multiple determinations carried out in replicate.

Figure 6.

Effects of MEK and c-Src inhibition on estrogen-induced stimulation evaluated on different PR1 cells parameters: DNA synthesis rate, endogenous gene expression, cell proliferation, and prevention of cell death. PR1 cells cultured in estrogen-free medium (C) were treated with the indicated concentration of E2 in the presence of 2 μM c-Src kinase inhibitor PP-1 or of 30 μM MEK inhibitor U0126 (dissolved in dimethyl sulfoxide). (A) DNA synthesis rate was assessed after 48 h of treatment by pulse-labeling with radioactive thymidine. (B) Prolactin (PRL) and cyclin D3 (D3) expression in the cells were evaluated by Western blotting after normalization for ERK protein expression in the same blot or sample. (C) Extent of cell death in the cultures evaluated by cytofluorimetric analysis. (D) Growth of the cultures assessed by cell labeling with radioactive thymidine for 24 h or by direct cell count (top and bottom, respectively). The data shown (± SD) are representative of multiple replicate determinations.