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Cell Growth & Differentiation Vol. 11, 99-110, February 2000
© 2000 American Association for Cancer Research


Articles

Inhibition of Mitogen-activated Protein Kinase and Phosphatidylinositol 3-Kinase Activity in MCF-7 Cells Prevents Estrogen-induced Mitogenesis1

Edward K. Lobenhofer, Gudrun Huper, J. Dirk Iglehart and Jeffrey R. Marks2

Department of Pathology [E. K. L., J. R. M.], Program in Cell and Molecular Biology [E. K. L., J. R. M.], and the Department of Surgery [G. H., J. D. I., J. R. M.], Duke University Medical Center, Durham, North Carolina 27710

Abstract

Estrogen acts to promote DNA synthesis in the MCF-7 human breast cancer cell line via its interaction with high levels of estrogen receptor. The primary mode of estrogen action has been considered to be through transcriptional activation of genes containing estrogen response elements, including the immediate early genes c-myc and fos. Recent reports have indicated that estrogen, acting through the estrogen receptor, is capable of inducing the mitogen-activated protein kinase (MAPK) cytoplasmic signaling cascade. In this study, specific small molecule inhibitors of MAPK and phosphatidylinositol 3-kinase activity were used to determine the influence of these cascades on estrogen-mediated mitogenesis. Phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, as well as inhibitors of MAPK kinase-1, PD098059 and U0126, decreased the fraction of cells entering DNA synthesis after treatment with 17ß-estradiol. These compounds did not inhibit expression of myc or fos. However, the drugs did prevent the accumulation of cyclin D1 and hyperphosphorylated retinoblastoma protein, indicating that the block occurred at, or prior to, this point in the cell cycle. Although these compounds were effective in preventing estrogen-mediated mitogenesis, the downstream kinases extracellular signal-regulated kinase 1, extracellular signal-regulated kinase 2, and protein kinase B were not activated over basal levels by estrogen treatment. These studies suggest that estrogen initiates mitogenesis by inducing the transcription of immediate early genes, but cytoplasmic signaling pathways play an important role in the control of subsequent events in the cell cycle.

Introduction

Estrogen can act as an effective mitogen in cells that express the ER3 both in cell culture and in vivo (reviewed in Ref. 1 ). E2, the most prevalent estrogen produced by the ovaries, bound to ER results in receptor translocation to the nucleus and dimerization. The ligand-bound ER dimer can interact with DNA at palindromic EREs, resulting in modulation of transcriptional activity. Antiestrogen treatment of cells that depend upon estrogen for their growth blocks this transcriptional activation and results in a G0-G1 cell cycle arrest (2) . A number of genes contain putative EREs including the immediate-early genes, c-myc and c-fos (3 , 4) . Therefore, it has been postulated that the mechanism by which estrogen mediates mitogenesis is via transcriptional activation of these and other key cell cycle regulatory genes.

After immediate-early gene synthesis, the cell cycle converges on the expression and activity of the cyclins and CDKs, particularly the D-type cyclins. Estrogen treatment of growth-arrested MCF-7 cells, a cancerous breast epithelial cell line that overexpresses ER, results in increased expression of cyclin D1 (5) . Concurrently, the activity of cyclin D1 binding partners, CDK4 and CDK6, increases (6 , 7) . Formation of active cyclin D1-CDK4 and cyclin D1-CDK6 holoenzymes serves two functions: (a) redistribution of CDK inhibitors (such as p27Kip1 and p21Cip1) away from cyclin E, facilitating the formation of active cyclin E-CDK2 complexes; and (b) phosphorylation of the Rb protein, which is also phosphorylated by cyclin E-CDK2 (8, 9, 10) .

The subcellular localization of ER and its role as a transcription factor led to the assumption that estrogen action occurs primarily in the nucleus. However, several recent reports have shown that elements of cytoplasmic signaling cascades may also be estrogen responsive. In cells expressing ER, the MAPK family members Erk1 and Erk2 are activated within 5 min in response to E2 treatment (11, 12, 13, 14) . Furthermore, cell membrane-impermeable estrogen (estradiol conjugated to BSA) stimulates MAPK activity (15) . A possible explanation for this phenomenon comes from several studies that have reported the existence of plasma membrane-associated ER (16, 17, 18, 19) . Activation of MAPK raises the possibility that some or all of the mitogenic activity of estrogen may be mediated through this pathway.

This theory was also supported by a study demonstrating that a MAPK inhibitor (PD098059) prevented estrogen-responsive proliferation in cardiac fibroblasts (20) . Furthermore, estrogen was able to induce mitogenesis in NIH-3T3 cells transiently transfected with transcriptionally inactive ER, presumably because of the ability of E2 to activate MAPK (21) . Cumulatively, these studies suggest that MAPK activation may be a requirement for estrogen-induced cell cycle progression.

Estrogen and its receptor may also interact with other cytoplasmic signaling components. In endometrial fibroblasts as well as selected malignant cell lines, E2 stimulates the activity of PKC (22) . Furthermore, in MCF-7 cells, synthesis of phosphatidylinositol and activation of phosphatidylinositol kinases can occur in response to E2 treatment (23) . ER itself can also act as a substrate for PKA. Serine-236 can be phosphorylated in vitro by PKA, which may be important in receptor dimerization (24) . Collectively, these reports suggest that cytoplasmic factors may be important intermediates in estrogen action.

In previous work, we found that MCF-7 cells grown in medium alone (no serum or added growth factors) proliferate in response to estrogen (25) . This system provides a model for studying the mitogenic effects of E2 in the absence of other exogenously added stimulatory molecules. In the current study, we have used a series of pharmacological compounds that specifically inhibit signaling intermediates that have been implicated in estrogenic action. Our results show that MAPK and PI3-K, but not PKA or PKC, activity are required for cell cycle progression in response to E2 stimulation. The cell cycle block caused by MAPK and PI3-K inhibitors occurs after the expression of immediate-early genes and prior to the induction of cyclin D1 expression, resulting in G1 arrest. Additional experiments show that E2 does not significantly activate MAPK or PKB, a downstream target of PI3-K, suggesting that either basal levels or delayed induction of these kinases are required for cell cycle progression in MCF-7 cells.

Results

Cytoplasmic Signaling Inhibitors Prevent Estrogen-induced Mitogenesis.
The breast cancer epithelial cell line MCF-7 expresses high levels of ER{alpha}. When these cells are serum-starved in phenol red-free media for 24 h, the largest fraction of cells accumulates and arrests early in the G1 stage of the cell cycle. Treatment of starved cells with estrogen alone (2 x 10-8 M E2) stimulated cell cycle progression and resulted in a 3–4-fold increase in the number of cells undergoing DNA synthesis, a rate that is comparable or greater than the mitogenic effect of EGF (Fig. 1A)Citation .



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Fig. 1. Effect of cytoplasmic signaling inhibitors on E2-induced mitogenesis. A, percentage of MCF-7 cells in S phase after treatment with 2 x 10-8 M E2 or 10 ng/ml EGF for 24 h in the presence or absence of 50 µM PD098059 (PD), 25 µM U0126 (U), 5 µM LY294002 (LY), 100 nM wortmannin (W), 2 µM ICI 182,780 (ICI), 750 nM bisindolylmaleimide I (BisI), or 500 nM H-89. Compounds such as ICI 182,780, which inhibit MCF-7 cells by generating a potent cell cycle block, will exhibit lower rates of DNA synthesis than the asynchronous serum-starved control. The data represent an average of results obtained from three experiments, each performed in triplicate. Bars, SD. B, estrogen-stimulated MCF-7 cells were treated with variable concentrations of PD, LY, and U for 24 h, and the rate of DNA synthesis was measured. Bars, SD.

 
Several signaling cascades have been implicated in E2 function. To assess the importance of these pathways on E2-induced mitogenesis, we examined changes in the rate of DNA synthesis as a result of exposure to specific inhibitors. Serum-starved cells were stimulated with E2 in the presence of inhibitors of MEK, the upstream activator of MAPKs (50 µM PD098059 or 25 µM U0126), PI3-K (5 µM LY294002 or 100 nM wortmannin), PKA (500 nM H-89), PKC (750 nM bisindolylmaleimide I), or with an ER antagonist (2 µM ICI 182,780). These concentrations were chosen based upon published values demonstrated to inhibit each of the kinases effectively without causing nonspecific toxicity (26, 27, 28, 29) . Cell cycle distribution was measured by flow cytometry after 24 h of treatment (results from three representative experiments are averaged in Fig. 1ACitation ). The antiestrogen, as expected, effectively blocked E2- induced DNA synthesis, reducing the percentage of cells in S phase to below the levels in starved cells. Neither the PKA or PKC inhibitors (H-89 and BisI) had any significant effect. However, the MEK1 inhibitor, PD098059, reduced the rate of DNA synthesis by >50%, whereas the MEK1/MEK2 inhibitor, U0126 (U), decreased the rate of proliferation to below the level observed in starved cells. Inhibition of the PI3-K cascade with LY294002 was almost as effective as the ICI antiestrogen in blocking E2-induced S phase. However, wortmannin was not as potent an inhibitor. These data suggest that the activities of at least two cytoplasmic signaling molecules (MEK1 and PI3-K) are critical for the mitogenic effect of estrogen.

To compare the effects of these inhibitors on another ligand that is mitogenic in MCF-7 cells, we repeated the experiments on this breast cancer cell line stimulated with EGF (30) . As reported previously (31) , the ICI antiestrogen inhibited EGF-induced mitogenesis. Furthermore, EGF-stimulated DNA synthesis was inhibited by the same compounds to virtually the same degree as was seen with E2 treatment (Fig. 1A)Citation .

To determine the efficacy of these drugs in inhibiting estrogen-induced proliferation, we measured S-phase progression in the presence of a range of inhibitor concentrations (1–50 µM). At the lowest concentration for each drug, minimal effects, if any, were observed (Fig. 1B)Citation . However, in the presence of 5 µM LY294002, the percentage of cells in S phase was maintained at or near basal levels, a finding that is supported by the published IC50 (1.4 µM) of LY294002 for abolishment of PI3-K activity (26) . Similar kinetics of inhibition were noted with the MEK1/MEK2 inhibitor, U0126. PD098059 only weakly inhibits the activation of MEK2 and has a much greater IC50 for the inhibition of MEK1 as compared with U0126 (29) . Therefore, it was not surprising to note that PD098059 did not have as pronounced an inhibitory effect on estrogen-induced mitogenesis.

Effect of Estrogen on the Activation of the MAPK Cascade.
Inhibition of the PI3-K and MAPK cascades prevented efficient estrogen-induced cell cycle entry in MCF-7 cells. Several recent reports have demonstrated that the MAPK pathway is stimulated by estrogen in an ER-dependent fashion (12 , 13 , 15 , 21 , 32) . The dual specificity kinases, MEK1 and MEK2, phosphorylate a threonine and a tyrosine residue in the regulatory sites of Erk1 and Erk2, resulting in the activation of these MAPKs (reviewed in Ref. 33 ). We performed a series of experiments to determine whether Erk1 and/or Erk2 were activated by E2 under our conditions (Fig. 2A)Citation . The dually phosphorylated forms of Erk1 and Erk2 were specifically recognized on the immunoblot using a phosphospecific monoclonal antibody, whereas total Erk1 and Erk2 was detected using a separate antibody after stripping and reprobing the same blot. In this case, we measured activation 5 min after treatment with either EGF, a known inducer of MAPK activity, or E2. Compared with the untreated control (starved cells), 10 ng/ml EGF induced a dramatic increase in phosphorylated Erk1 and Erk2. Under the same conditions, 2 x 10-8 M E2 had little or no effect on the levels of activated Erk1 or Erk2 (Fig. 2A)Citation . A number of repetitions of this experiment failed to show more than a 1.5-fold induction at any time point (from 1 to 20 min after treatment) or at any cell density. Neither ICI 182,780 nor LY294002 had any effect on active MAPK levels in E2-stimulated cells (Fig. 2A)Citation . Although estrogen failed to activate MAPK, the MAPK inhibitors (PD098059 and U0126) effectively reduced the basal levels of phospho-Erk1 and -Erk2 (Fig. 2A)Citation .



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Fig. 2. Effect of E2 stimulation on MAPK phosphorylation status. A, the phosphorylation status of Erk1 and Erk2 was assessed using a phosphospecific antibody and immunoblot analysis on lysates from cells treated with 10 ng/ml EGF or 2 x 10-8 M E2 in the presence or absence of 5 µM LY294002 (LY), 50 µM PD098059 (PD), 2 µM ICI 182,780 (ICI), or 25 µM U0126 (U). Levels of total Erk1 and Erk2 were detected on the same blot to control for loading variations. B, phosphorylated and total levels of Erk1 and Erk2 were assessed using the technique described above for cells treated with 2 x 10-8 M E2 in the presence of 1, 5, 10, 25, or 50 µM PD098059 or U0126.

 
The ability of U0126 and PD098059 to inhibit basal levels of activated MAPKs as well as cell cycle progression prompted us to examine whether a correlation exists between these two activities. Using the same antibodies described above, MCF-7 cells were stimulated with estrogen for 5 min in the presence of variable concentrations of PD098059 or U0126 (Fig. 2B)Citation . Estrogen had no effect on Erk1 or Erk2 levels, but increasing concentrations of these compounds inhibited phosphorylation of these kinases. A clear reduction in phospho-Erk1 and -Erk2 was noted with as little as 1 µM U0126 compared with starved or E2-treated cells. Peak inhibition was achieved between 5 and 10 µM. The PD098059 compound also reduced the levels of active Erk1/Erk2 with maximum inhibition reached at ~10 µM. Even at the highest concentration tested (50 µM), PD098059 was unable to completely inhibit phospho-Erk1/Erk2 levels. These data are consistent with relative S-phase inhibition observed with these two compounds and suggest that maintenance of basal levels of phospho-Erk1/Erk2 may be important in E2-mediated mitogenesis.

Effect of Estrogen on the Activation of the PI3-K Cascade.
PKB is a primary downstream target of PI3-K activation and has been implicated in cyclin D1 protein stabilization (34) . Treatment with estrogen had little, if any, effect on phosphorylated (active) PKB levels compared with stimulation with 30 ng/ml IGF-I, a known activator of PI3-K in MCF-7 cells (Fig. 3ACitation ; Ref. 35 ). Neither PD098059, U0126, nor ICI 182,780 affected the phosphorylation of PKB; however, the PI3-K drug, LY294002, was a potent inhibitor of PKB activation in response to IGF.



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Fig. 3. Effect of E2 stimulation on PKB phosphorylation status. A, phosphorylation of PKB was determined by Western analysis using a phosphospecific antibody on MCF-7 cell extracts from cells stimulated with 30 ng/ml IGF-I in the presence or absence of 5 µM LY294002 (LY) or with 2 x 10-8 M E2 in the presence or absence of 5 µM LY, 50 µM PD098059 (PD), 2 µM ICI 182,780 (ICI), or 25 µM U0126. The detection of total levels of PKB on the same blot controlled for loading discrepancies. B, phosphorylated and total levels of PKB were assessed using the technique described above for cells treated with 2 x 10-8 M E2 in the presence of 1, 5, 10, 25, or 50 µM LY294002.

 
The effect of LY294002 on the basal levels of phosphorylated PKB was also investigated. Only at the highest concentration (25 µM) was PKB significantly inhibited relative to starved or E2-treated cells (Fig. 3B)Citation . However, near-maximal inhibition of S-phase was achieved using 5 µM (Fig. 1B)Citation . As measured by phosphorylation of PKB, inhibition of the basal activity of PI3-K does not appear to correlate with suppression of E2-induced growth.

Toxicity and Reversibility of PI3-K and MAPK Inhibitor Molecules.
We next examined toxicity and reversibility of the pharmacological inhibitors of MAPK, PI3-K, and ER. Cell viability was measured by dye exclusion after 24 h of drug treatment in the presence of 2 x 10-8 M E2 (Fig. 4)Citation . A slight decline in viability was observed in cultures treated with the MAPK inhibitors PD098059 and U0126 and with the estrogen antagonist ICI 182,780. We observed no increase in the apoptotic fraction with these drugs, as measured by flow cytometry (data not shown). Next, we measured the reversibility of the growth-inhibitory effects of each drug by a washout experiment. MCF-7 cells were treated with the small molecule inhibitors for 24 h in the absence of any mitogen prior to washing three times with PBS and stimulating with E2 (Fig. 4)Citation . Cell cycle analysis showed that cells treated with LY294002 and PD098059 could respond mitogenically to estrogen stimulation after the drugs had been removed. However, inhibition by U0126 and ICI 182,780 could not be reversed in this time frame. Because this did not appear to be related to toxicity, it appears that these drugs exert a long-lasting effect on their targets. The ICI antiestrogen acts by inducing ER degradation; therefore, a long-lasting effect on ER response is not surprising (36) .



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Fig. 4. Reversibility of cytoplasmic signaling inhibitors and their effect on cell viability. Serum-starved MCF-7 cells were treated with 50 µM PD098059 (PD), 5 µM LY294002 (LY), 2 µM ICI 182,780 (ICI), or 25 µM U0126 (U) for 24 h. Cells were then washed three times with starvation media prior to stimulation with 2 x 10-8 M E2 in the absence of any inhibitory compounds for 24 h. The rate of DNA synthesis was then measured by propidium iodide staining and analysis by FACS. Inset, after 24 h of serum starvation, MCF-7 cells were stimulated with 2 x 10-8 M E2 in the presence or absence of the pharmacological compounds listed above (using the same dosage). Twenty-four h after treatment, cell viability was assessed by staining with erythrosin B and determining the percentage of viable cells. Bars, SD.

 
Inhibitor Effects on ER-mediated Transcription.
The most well-understood mode of estrogen action is the transcriptional activation of genes containing EREs. Among the genes containing putative EREs are several key cell cycle regulatory genes, including the immediate-early genes c-myc and c-fos (3 , 4) . If the PI3-K and MAPK inhibitors affected transcriptional regulation by the ER, they could stop cell cycle progression by preventing the induction of these genes. To determine whether the effects of signaling inhibitors on E2-stimulated mitogenesis were attributable to interference with ER-mediated transcriptional activity, MCF-7 cells were transfected with a luciferase gene under the control of either a naturally occurring (vitellogenin, TK-ERE) or artificial E2-responsive promoter (3X-ERE), and cell extracts were harvested 24 h later. In the absence of drugs, E2 induced a 5–6-fold increase in luciferase activity as opposed to the near basal levels seen in the presence of the ER antagonist ICI 182,780 (Fig. 5A)Citation . Blocking PKA or PKC activation had no impact on ER-mediated transcription, whereas the MEK1/2 inhibitor U0126 was as effective as the ICI compound at preventing ER-mediated transcription. Transcription declined ~50% in the presence of the MEK1 inhibitor PD098059 and with the PI3-K inhibitor LY294002.



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Fig. 5. Effect of cytoplasmic inhibitors on ER-dependent transcription. A, MCF-7 cells were transfected with a luciferase construct containing either an artificial (3X-ERE) or a naturally occurring (TK-ERE) estrogen-responsive promoter. Samples were harvested after 2, 6, 9, or 24 h of stimulation with 2 x 10-8 M E2 in the presence or absence of 5 µM LY294002 (LY), 50 µM PD098059 (PD), 25 µM U0126 )U), 2 µM ICI 182,780 (ICI), 750 nM bisindolylmaleimide I (BisI), or 500 nM H-89, cells were lysed, and luciferase activity was assessed. The 2-h interval was not sufficient to generate detectable levels of luciferase activity; however, the 6- and 9-h time points displayed similar kinetics of induction and inhibition, as the 24-h results show. Corrections for differences in protein concentration and transfection efficiencies were made using detected levels of a cytomegalovirus-luciferase construct from a different species and are given for at least three experiments in triplicate; bars, SD. B, MCF-7 cells were transfected with a human progesterone receptor B expression construct in addition to a progesterone-responsive luciferase construct. Twenty-four h after treatment with 10-7 M progesterone and inhibitory compounds, cells were lysed, and luciferase activity was measured and corrected as detailed above. Bars, SD. C, cells were incubated with inhibitors in the presence or absence of 2 x 10-8 M E2 for the times indicated prior to lysis. Western analysis was used to detect levels of ER (after 2, 6, and 9 h of stimulation). This blot was reprobed to quantitate actin levels to control for loading differences. LY, LY294002; W, wortmannin; PD, PD098059; U, 0126; ICI, ICI 182,780.

 
To examine the impact of cytoplasmic signaling cascades on earlier transcriptional events, this experiment was repeated with cell extracts harvested at shorter intervals after estrogen addition. Samples taken after 6 or 9 h displayed the same kinetics of induction and inhibition as the 24-h time point (data not shown). Attempts at detecting even earlier effects (2 h) using this reporter gene assay did not yield interpretable results because E2 alone failed to induce detectable luciferase activity.

The specificity of drug effects on ER-mediated transcription was examined by studying their impact on the transcriptional activity of another steroid hormone, progesterone. MCF-7 cells were transiently transfected with a human progesterone receptor expression vector and a luciferase gene with a progesterone-responsive promoter. Addition of 10-7 M progesterone for 24 h stimulated a 5-fold increase in luciferase activity, an activity that was not inhibited in the presence of LY294002 and ICI 182,780 (Fig. 5B)Citation . Both MAPK inhibitors prevented maximal induction by ~50%. Therefore, these cytoplasmic inhibitors have pleiotropic effects on nuclear hormone-mediated transcription.

The level of the ER itself may be decreasing in response to the inhibitory compounds, which could account for the decline in transcriptional activity. Therefore, we measured the levels of ER by immunoblotting over the course of a 24-h treatment regimen (Fig. 5C)Citation . ER is known to decline in response to E2 treatment (37 , 38) ; we observed this phenomenon with ER protein levels reduced 3-fold after 9 h of estrogen stimulation compared with basal levels in starved cells. Estrogen stimulation in the presence of LY294002, wortmannin, or PD098059 did not affect ER protein levels compared with E2 alone. Furthermore, in the absence of estrogen, these drugs caused no change in the steady-state levels of ER found in serum starved cells. Consistent with published data, ICI 182,780 reduced ER levels in the presence or absence of steroid hormone (36) . Unlike PD098059, treatment with U0126 decreased ER levels with kinetics similar to those observed with ICI 182,780. The diminishment of ER in the presence of U0126 and with ICI 182,780 is consistent with the dramatic inhibition of ER-mediated transcription at later time points.

Inhibitor Effects on Estrogen-induced Gene Transcription.
The kinase inhibitors did exert a suppressive effect on estrogen-mediated transcription measured by reporter gene assay. These assays were performed between 6 and 24 h of drug treatment, long after the signaling cascades or the ER may transduce any immediate effects on the cell cycle. Decreased ER transcription may be a secondary effect of the arrest rather than a direct effect of kinase inhibitors. Expression of the immediate-early genes occurs rapidly after E2 treatment; therefore, we measured the expression of these genes directly by Northern blotting. We observed maximal expression of the two putative estrogen-responsive immediate-early genes, c-myc and c-fos, within 1 h of E2 treatment (Fig. 6)Citation . None of the signal transduction inhibitors (bisindolylmaleimide I, H-89, LY294002, and PD098059) had an effect on the kinetics or magnitude of estrogen-induced myc and fos expression (Fig. 6Citation and data not shown). Estrogen induction of myc and fos protein was also not inhibited by these compounds (data not shown). The ICI antiestrogen completely inhibited the induction of both c-myc and c-fos mRNA (and protein) as expected (Fig. 6Citation and data not shown). Despite decreasing the steady-state levels of ER, U0126 had no effect on the induction of myc and fos in response to estrogen treatment. Therefore, although the compounds did affect estrogen transcription measured in the transient assays, they had no effect on immediate-early gene synthesis. These results suggest that induction of these genes is directly dependent upon the hormone receptor’s nuclear action. Furthermore, inhibition of estrogen transcription in the reporter gene assay at later times is not correlated with effects on the transcription of these endogenous genes.



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Fig. 6. Effect of cytoplasmic signaling inhibitors on E2-induced immediate-early gene RNA levels. Representative Northern blot analysis of c-myc and c-fos RNA expression in MCF-7 cells treated with estrogen in the presence or absence of 5 µM LY294002 (LY), 100 mM wortmannin (W), 50 µM PD098059 (PD), 25 µM U0126 (U), or 2 µM ICI 182,780 (ICI; from a separate Northern blot) for the times (in hours) indicated for each lane. The starved lane displays the amount of c-myc or c-fos RNA present at time 0. Levels of the 28S ribosomal subunit were used as an internal control to correct for loading inconsistencies.

 
Timing of the Mitogenic Block.
It is readily understandable how the ICI antiestrogen blocks cell cycle progression via inhibition of immediate-early gene expression (39 , 40) . However, the cytoplasmic signaling inhibitors did not prevent the immediate early genes from being induced, raising the question of at what stage of the cell cycle the block is imposed. We next investigated the timing of this inhibition relative to the mitogenic stimulus. Estrogen was added at time 0, and the inhibitors (PD098059 or LY294002) were added at varying times after the mitogenic stimulus. At 24 h, all cultures were harvested, and the percentage of cells in S phase was determined. We have shown previously that starved MCF-7 cells begin to enter S phase ~12 h after the addition of estrogen, with peak S phase values reached at 24 h (25) . Effective inhibition of cell cycle progression was observed when LY294002 was added up to 6 h after exposure to estrogen (Fig. 7)Citation . By 12 h, the LY compound was no longer an effective inhibitor. In parallel experiments using the MEK1 inhibitor (PD098059), we observed a gradual increase in S-phase progression with delayed addition of the compound after E2 stimulation. Although the demarcation in PD098059 activity was not as clearly defined, the profile from both compounds suggested that inhibition of events early in G1 is important for the activity of these drugs in preventing S-phase progression.



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Fig. 7. Mitogenic effects of cytoplasmic signaling inhibitor addition after E2 stimulation. Starved cultures of MCF-7 cells were treated with 2 x 10-8 M E2 at time 0 and then treated with either 50 µM PD098059 (PD) or 5 µM LY294002 (LY) at 2, 6, 12, 15, or 18 h. At 24 h, cell cultures were harvested, and the fraction of cells in S phase was measured by flow cytometry. Bars, SD.

 
Cyclin D1 Expression and Rb Phosphorylation in Response to Estrogen and Cytoplasmic Signaling Inhibitors.
Increased cyclin D1 expression is considered to be a delayed-early event occurring after immediate-early genes are induced. Accumulation of cyclin D1 protein after E2 stimulation is a critical event in G1 progression (41) . In addition, overexpression of cyclin D1 can reverse cell cycle arrest in ER-positive cells accomplished by antiestrogen treatment (42) . These findings, coupled with our data showing that proliferation is inhibited by LY294002 only up to 6 h after E2 stimulation, suggested to us that a delayed early event was being specifically inhibited. Therefore, we examined cyclin D1 expression and its downstream effects, CDK2 activity and subsequent Rb phosphorylation. Estrogen treatment of serum-starved MCF-7 cells resulted in a 2–3-fold increase in cyclin D1 protein levels by 6 h (Fig. 8A)Citation . This modest induction is consistent with other reports and with the 3–4-fold increase in S phase that we routinely observed under these conditions (41) . As expected, cyclin D1 accumulation resulted in the activation of CDK2 and Rb hyperphosphorylation (Fig. 8, B and CCitation ; Ref. 43 ). The pharmacological compounds demonstrated a good correlation between antimitogenic activity and inhibition of the Rb pathway. The ICI antiestrogen, as reported previously (44) , and the MAPK inhibitor U0126 both prevented cyclin D1 induction and activation of CDK2 and led to decreased levels of hyperphosphorylated retinoblastoma protein. These findings are consistent with the effects of ICI and U0126 on mitogenesis. Of the PI3-K inhibitors, wortmannin was not as potent at preventing the accumulation of cyclin D1 protein as LY294002, a finding that is consistent with the relative S-phase inhibition observed with these compounds. The MEK1 inhibitor PD098059 also inhibited cyclin D1 levels; however, the effect appeared to be more transitory as cyclin D1 began to accumulate at later time points (Fig. 8ACitation and data not shown). This was also reflected by the incomplete inhibition of CDK2 activation, Rb hyperphosphorylation, and cell cycle progression. From these experiments, we place the critical antimitogenic effects of the PI3-K and MAPK inhibitors at or before the level of cyclin D1 induction.



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Fig. 8. Effects of cytoplasmic signaling inhibitors on cyclin D1 expression, CDK2 activity, and Rb phosphorylation. Cells were preincubated with 2 x 10-8 M E2 in the presence or absence of 5 µM LY294002 (LY), 100 nM wortmannin (W), 50 µM PD098059 (PD), 25 µM U0126 (U), or 2 µM ICI 182,780 (ICI). A, after 6 h, the cells were lysed, and relative amounts of cyclin D1 and actin expression was detected by immunoblotting. B, after 24 h of treatment, cells were lysed, and CDK2 was immunoprecipitated; the activation status of this kinase was assayed by determining its ability to phosphorylate histone H1. C, Rb phosphorylation was assessed using Western analysis on cell lysates that were extracted after 24 h of stimulation.

 
Discussion

Estrogen, a nuclear hormone, exerts a mitogenic effect on MCF-7 cells without the addition of any other growth factors, which facilitates the study of this pathway in isolation. A great deal of work on estrogen-induced growth has led to several conclusions: (a) the response depends upon the presence of functional estrogen receptor; (b) immediate-early genes are transcriptionally activated; and (c) cyclin D1 is an important intermediate in inducing this cell cycle cascade (reviewed in Ref. 45 ). If one assumes that the primary mode of estrogen action is via its receptor acting as a ligand-inducible transcription factor, mitogenesis could be explained by the direct transcriptional activation of immediate-early genes that contain functional EREs. The recent reports of estrogen activation of cytoplasmic signaling components, particularly the MAPK pathway, complicate this relatively simple paradigm (11 , 13 , 15 , 22, 23, 24 , 46 , 47) . It is in this context that the current studies were performed.

We found that disruption of the PI3-K and MAPK cascades by specific inhibitors (PD098059, which inhibits MEK1; U0126, which inhibits MEK1 and MEK2; and LY294002 and wortmannin, which inhibit PI3-K) prevented or reduced the ability of MCF-7 cells to enter S phase in response to estrogen. These drugs arrested cells in G1 and did not induce any significant cytotoxicity. We also demonstrated that E2 does not measurably activate either PKB or MAPK under conditions that result in a mitogenic response. We did observe variable effects on estrogen-mediated transcription assayed on artificial EREs; however, the immediate-early genes c-myc and c-fos were both induced in the presence of these drugs. Both the PI3-K and MAPK inhibitors prevented cyclin D1 accumulation, which correlated with an absence of CDK2 activity and Rb phosphorylation. We concluded that inhibition of estrogen-mediated mitogenesis by these drugs was likely attributable to this repression of cyclin D1 expression, CDK2 activation, and Rb phosphorylation.

This study raises several issues regarding the mechanism of estrogen action and mitogenesis in general. The lack of any reproducible induction of MAPK activity, as measured by Erk1 and Erk2 phosphorylation, contradicts several published reports (13 , 15 , 21 , 32 , 47) . In light of these reports, we examined this parameter many times using different time points, cell densities, and variable concentrations of charcoal-stripped serum. On occasion, we did observe a small increase (<1.5 fold) in Erk1 and Erk2 phosphorylation between 2 and 5 min after estrogen stimulation. Published accounts of this activation have also shown a relatively modest induction (between 1.5- and 3-fold) over control values (13 , 47) . In all reported cases and in our experiments, the magnitude of activated MAPK resulting from a cell surface signal such as EGF far exceeds any induction attributable to estrogen (32) . Furthermore, estrogen can overcome a cell cycle block induced by Simvastatin, an HMG-CoA reductase inhibitor, without detectable MAPK activation (48) . It is also clear from our experiments that estrogen is mitogenic in the absence of measurable MAPK activation, which raises the issue of the physiological relevance of the response when it is observed.

MAPK and PI3-K inhibitors were shown to inhibit ER-mediated transcription, assayed using a gene reporter system. However, these inhibitors do not prevent the expression of immediate-early genes in response to estrogen. At least one plausible explanation can reconcile this apparent discrepancy. Maximal expression of c-myc and c-fos occurs after 1 h of stimulation, whereas luciferase activity required 6 h for a measurable response. Therefore, effects on ER transcription may not be manifest until after the rapid immediate-early response. How critical this delayed inhibition of ER activity is in mediating the growth arrest remains to be determined.

Estrogen stimulates the production and secretion of growth factors that can function in an autocrine fashion (49) . For example, protein levels of fibroblast growth factor-1 increase in response to E2 treatment in MCF-7 cells and may act synergistically with the hormone to generate a greater proliferative response (50 , 51) . Fibroblast growth factor-2 is also up-regulated in response to E2 stimulation of human endothelial cells and is required for the MAPK activation that is observed 3 h after exposure to estrogen (52) . Continuous incubation in the presence of MEK inhibitors would prevent the activation of MAPK by growth factors acting in an autocrine loop. Therefore, the cell cycle block observed with PD098059 and U0126 may be a result of inhibiting autocrine cascades necessary to realize the mitogenic potential of estrogen. Our finding that an antiproliferative response can still be achieved when adding the inhibitory drugs up to 6 h after hormone stimulation further substantiates this theory.

Our results strongly suggest that cyclin D1 induction is a key intermediate in estrogen mitogenesis. Several reports have focused on the role of cyclin D1 activation in estrogen-mediated mitogenesis. MCF-7 cells overexpressing cyclin D1 are unable to proliferate in the presence of antiestrogens (53) . Planas-Silva and Weinberg (41) demonstrated that estrogen relieves a cell cycle block in tamoxifen arrested MCF-7 cells by increasing levels of cyclin D1 protein, which causes the redistribution of p21cip1/waf1 away from the cyclin E/CDK2 complexes, allowing Rb phosphorylation and cell cycle progression. Prall et al. (42) derived MCF-7 cell lines with inducible expression of c-myc or cyclin D1. Cell cycle arrest of these lines by the estrogen antagonist ICI 182,780 was reversed by expression of either myc or cyclin D1. In both cases, loss of the CDK inhibitor p21 from cyclin E/CDK2 complexes was noted. Interestingly, c-myc overexpression did not result in elevated cyclin D1 levels, and when cyclin D1 was induced, myc levels remained constant, implicating the existence of multiple pathways to remove p21. Our data indicate that c-myc induction is not sufficient to allow estrogen-mediated cell cycle progression in the face of diminished cyclin D1 expression.

Functional activity of ectopically expressed ER is not sufficient for the expression of cyclin D1 and subsequent proliferation in response to estrogen (54) . However, Castoria et al. (21) reported recently that fibroblasts transiently transfected with a transcriptionally inactive ER (lacking the DNA binding domain) exhibited E2-dependent cell cycle progression, presumably attributable to the ability of the receptor to activate the MAPK cascade. This report suggests that proliferation in response to estrogen may be a result of nonnuclear events rather than ER-mediated transcription. In contrast, in a host of other studies, ectopic expression of active ER (measured by transcriptional activity) is not sufficient to convert cells into E2 responders (55, 56, 57, 58, 59, 60, 61) . Our data indicate that E2-induced proliferation can occur in the absence of detectable MAPK activation. Although other studies have shown E2-induced MAPK activity, the link between MAPK and mitogenesis after E2 stimulation has not been established. Specific culture conditions, experimental conditions, and divergence of cells in long-term culture may explain the discrepant results.

The question raised by our data is how the inhibitors of cytoplasmic signaling prevent S phase although the targets of these inhibitors are apparently not activated. Repression of cyclin D1 may be the key to understanding these observations. The molecular mechanism by which the PI3-K cascade functions in cyclin D1 accumulation has begun to be characterized as a posttranscriptional phenomenon. In the absence of mitogen, cyclin D1 is targeted for ubiquitin-dependent degradation because of the presence of a phosphate group at threonine 286, a substrate for GSK-3ß (62 , 63) . Inactivation of GSK-3ß occurs as a result of PKB activation, a downstream target of the PI3-K cascade (Ref. 44 and reviewed in Ref. 64 ). Thus, the cell cycle block induced by LY294002 in the presence of E2 may be attributable to the inability of GSK-3ß to be inactivated by PKB, thereby preventing the accumulation of cyclin D1.

The role of MAPK in cyclin D1 accumulation is less well defined. Typically, activation of MAPK results in nuclear translocation and activation of immediate-early gene expression. Several studies, however, do suggest a more direct effect. Using a Chinese hamster fibroblast cell line, CCL39, Lavoie et al. (65) demonstrated that not only was MAPK activation required for expression of endogenous cyclin D1, but also sufficient for full induction of this protein in the absence of growth factors. One theory to explain this phenomenon is that activated MAPK phosphorylates PHAS-1, which in turn impacts mRNA cap recognition and translation via eIF-4E (66) . Therefore, inhibition of these cascades may prevent cyclin D1 accumulation at the level of mRNA utilization.

We propose that whereas estrogen may directly impact some components of cytoplasmic signaling cascades, the primary mode of action is within the nucleus. The finding that MAPK and PI3-K inhibitors prevent estrogen-induced mitogenesis indicates that cytoplasmic events are still required in this process. This underscores the importance of determining the molecular events required for the accumulation of cyclin D1 protein and the role of estrogen in autocrine loops, which may stimulate signaling cascades.

Materials and Methods

Materials.
ICI 182,780 was purchased from Tocris. E2, EGF, IGF-I, LY294002, wortmannin, bisindolylmaleimide I, and H-89 (dihydrochloride) were obtained from Calbiochem. PD098059 was obtained from New England Biolabs. A monoclonal antibody against diphosphorylated Erk1 and Erk2 was purchased from Sigma Chemical Co.; polyclonal antibodies against Erk2 (clone K-23), ER{alpha} (G-20), CDK2 (M-2), the monoclonal antibody recognizing cyclin D1 (HD11), and normal goat IgG were obtained from Santa Cruz Biotechnology. Actin (C4) monoclonal antibody and protein G-agarose beads were from Boehringer Mannheim, and the monoclonal antibody against Rb protein (G3–245) was purchased from PharMingen. Polyclonal antibodies against PKB and Ser-473-phosphorylated PKB were obtained from New England Biolabs. Donald P. McDonnell kindly provided the estrogen-responsive luciferase constructs (3X-ERE and TK-ERE), the progesterone-responsive luciferase construct (3X-PRE), and the human progesterone receptor expression plasmid.

Cell Culture.
MCF-7 cells were obtained from American Type Culture Collection and propagated in RPMI 1640 containing 10% (v/v) FBS (Life Technologies, Inc.). ER content was measured using the ERICA kit (Abbot Labs, Inc.) and quantitated using the CAS 200 Image Analyzer (Cell Analysis Systems, Inc.). MCF-7 cells express 170 fmol/mg of protein as compared with <10 fmol/mg of protein found in normal breast epithelial cells.

For experimental purposes, cells were seeded in six-well format at a density of 3 x 105 cells/well or in a 60-mm dish at a density of 5 x 105 cells. After 24 h, cells were washed one time with phenol red-free RPMI without FBS. Cells were then incubated for an additional 24 h in this medium to effect growth factor deprivation (67) .

Cell Cycle Analysis/Viability.
After 24 h of stimulation with 2 x 10-8 M E2 in the presence or absence of inhibitory drugs, cells were trypsinized and pelleted prior to washing once in PBS. Cell pellets were fixed in 70% ethanol (in PBS) overnight on ice. Cells were washed with PBS and resuspended in 500 µl of PBS supplemented with 100 µg of RNase A and 50 µg of propidium iodide. DNA content was determined on a per cell basis using flow cytometry. Cell viability was assessed on trypsinized cells by staining with erythrosin B and determining the percentage of viable cells (500 cells counted) by microscopic examination in three separate experiments.

Northern Blotting.
Total RNA was extracted from 5 x 105 cells using the Trizol reagent according to the manufacturer’s instructions (Life Technologies). Five µg of RNA were electrophoresed on a 1% agarose-2.2 M formaldehyde gel and then transferred onto a nylon membrane (ICN). Blots were cross-linked using UV irradiation and hybridized with a 32-P-labeled probe prepared by random priming (cDNA plasmid clones were purchased from American Type Culture Collection). Blots were washed for 10 min at 65°C with 2x SSC (150 mM NaCl and 15 mM Na3C6H3O7, pH 7.0) containing 1% SDS and then with 0.1x SSC/0.1% SDS prior to autoradiography at -80°C with intensifying screens. IMAGEQuant (Molecular Dynamics) was used to quantify the expression levels of the gene of interest, and loading inconsistencies were corrected using detected levels of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase.

Transient Transfections/Luciferase Assay.
For liposome delivery, cells were washed with serum-free medium (Opti-Mem I; Life Technologies). The firefly luciferase gene (Photinus pyralis), under the control of estrogen- or progesterone-responsive promoters (artificial or from the human vitellogenin gene) as well as a cytomegalovirus-driven luciferase construct (Renilla reniformis) were combined (2.5 µg/ml and 5 ng/ml, respectively) prior to transfection, following the standard protocol for Lipofectin (Life Technologies). (Because of low endogenous expression levels of progesterone receptor in MCF-7 cells, a human progesterone receptor B expression construct was also included in the transfection mix for experiments using progesterone). Cells were incubated with the liposome solution for 5 h, at which time the medium was aspirated and replaced with RPMI 1640 without phenol red, supplemented with 10% (v/v) charcoal-stripped FBS. Twenty-four h later, cells were treated with 2 x 10-8 M estrogen in fresh medium, either in the presence or absence of inhibitory drugs. Cells were lysed after 24 h of stimulation, and luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega) and a 20/20 dual-channel luminometer (Turner Designs).

Immunoblotting.
Cells were lysed in 2x SDS sample buffer [25 mM Tris-HCl (pH 6.8), 12.5% glycerol, 6% SDS, 0.045% bromphenol blue, 4% ß-mercaptoethanol, and 1 mM sodium orthovanadate]. Protein separation was achieved using 10% SDS-PAGE and electrotransferred to nitrocellulose membranes (Schleicher & Schuell). To prevent nonspecific interactions of the antibody, the blots were blocked for 1 h at room temperature with PBS containing 0.01% Tween 20 (PBS-T) and 5% nonfat dry milk. Membranes were incubated with primary antibodies in PBS-T milk, at recommended dilutions, overnight at 4°C. Antigen-antibody complexes were visualized by incubation with horseradish peroxidase-conjugated goat antirabbit or antimouse IgG (Jackson Laboratories) for 1 h at room temperature, followed by ECL detection (DuPont NEN Life Science). Quantitation of protein levels was performed as detailed above, with actin levels being used to correct for variable loading.

CDK2 Kinase Assay.
After 24 h of estrogen stimulation in the presence or absence of inhibitory compounds, cells were lysed in NP40 lysis buffer [50 mM Tris (pH 8), 5 mM EDTA, 150 mM NaCl, and 0.5% NP40] on ice for 30 min. Insoluble proteins were removed by centrifugation, and 500 µg of the remaining lysate were precleared with 1 µg of normal goat IgG and 20 µl of protein G-agarose beads. Precleared lysate was immunoprecipitated with an anti-CDK2 antibody and immunocomplexes were washed twice with lysis buffer and once with kinase buffer [50 mM Tris (pH 7.5), 10 mM MgCl2, and 1 mM DTT]. Five µg of histone H1, 1 µM dATP, and 5 µCi [{gamma}32P]dATP in 50 µl of kinase buffer were added to the beads prior to a 30-min incubation at 30°C. The reaction was terminated with the addition of 10x SDS sample buffer, and the samples were separated using 15% SDS-PAGE. The gel was dried and exposed to X-ray film.

Acknowledgments

We thank Donald P. McDonnell for the generous gift of the 3X-ERE, TK-ERE, 3X-PRE, and hPR-B constructs; Lenora Q. Blount for invaluable assistance in cell culturing; Carey J. Oliver for helpful discussions and comments; and Rita Nahta for critical review of the manuscript.

Footnotes

1 This work was supported by Predoctoral Training Grant DAMD17-97-1-7224 (to E. K. L.) from the Department of Defense and the Duke University Medical Center Specialized Programs of Research Excellence in Breast Cancer Grants PHS CA68438 and PHS CA73802. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, Duke University Medical Center, Box 3873, Durham, NC 27710. Phone: (919) 681-6133; Fax: (919) 681-6291; E-mail: marks003{at}mc.duke.edu Back

3 The abbreviations used are: ER, estrogen receptor; E2, 17ß-estradiol; ERE, estrogen response element; CDK, cyclin-dependent kinase; Rb, retinoblastoma; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; MEK, MAPK kinase; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PI3-K, phosphatidylinositol 3-kinase; EGF, epidermal growth factor; IGF, insulin-like growth factor; GSK, glycogen synthase kinase; FBS, fetal bovine serum. Back

Received for publication 6/17/99. Revision received 12/20/99. Accepted for publication 12/21/99.

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