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Molecular Endocrinology 18 (2): 291-302
Copyright © 2004 by The Endocrine Society

Potent Ligand-Independent Estrogen Receptor Activation by 3,3'-Diindolylmethane Is Mediated by Cross Talk between the Protein Kinase A and Mitogen-Activated Protein Kinase Signaling Pathways

Hoyee Leong, Jacques E. Riby, Gary L. Firestone and Leonard F. Bjeldanes

Departments of Nutritional Sciences and Toxicology (H.L., J.E.R., L.F.B.) and Molecular and Cell Biology (G.L.F.), University of California, Berkeley, California 94720

Address all correspondence and requests for reprints to: Leonard F. Bjeldanes, Department of Nutritional Sciences and Toxicology, 115 Morgan Hall, University of California-Berkeley, Berkeley, California 94720. E-mail: lfb{at}nature berkeley.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We investigated the mechanism of ligand-independent activation of the estrogen receptor (ER) by 3,3'-diindolylmethane (DIM), a promising anticancer agent derived from vegetables of the Brassica genus, in Ishikawa and HEC-1B human endometrial cancer cells. DIM stimulated the activity of an ER-responsive reporter by over 40-fold, equivalent to the maximum induction produced by estradiol (E2), whereas cotreatment of cells with the ER antagonist, ICI-182,780 (ICI), abolished the stimulatory effect of DIM. DIM also induced the expressions of the endogenous genes, TGF-{alpha}, alkaline phosphatase, and progesterone receptor similar to levels induced by E2. Induction of gene expression by DIM was inhibited by the protein synthesis inhibitor, cycloheximide. In addition, cotreatment of cells with the protein kinase A (PKA) inhibitor, H89, or the MAPK inhibitor, PD98059, reduced DIM activation of the ER by 75% and 50%, respectively. Simultaneous treatment of cells with both inhibitors completely abolished the effect of DIM. DIM stimulated MAPK activity and induced phosphorylation of the endogenous PKA target, cAMP response element binding protein (CREB), in a PKA-dependent manner. Expression of MCREB, a nonphosphorylatable CREB mutant, partially abolished activation of the ER by DIM. These results demonstrate that DIM is a mechanistically novel activator of the ER that requires PKA-dependent phosphorylation of CREB.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ESTROGEN RECEPTOR (ER) mediates a plethora of normal developmental and reproductive processes in human females and males, and it is associated with abnormal disease processes such as the initiation and promotion of cancers of the breast and other female reproductive organs (1, 2). The ER is a member of the steroid nuclear receptor superfamily of regulatory proteins that commonly function as ligand-regulated transcription factors. In the absence of estrogen, the ER is associated in a protein complex containing heat shock protein 90, which prevents the association of the receptor with DNA. Binding of the receptor to estrogen induces a conformational change that results in dissociation of the protein complex, and phosphorylation and binding of the receptor to its DNA-responsive element [estrogen response element (ERE)]. Association with the ERE promotes the recruitment of coactivators and interaction with basal transcription factors, resulting in the activation of target gene transcription (3). Studies with selective ER modulators show that the ER can mediate tissue-selective transcriptional effects that depend on structural variations in the ligand (4).

ER activity also can be stimulated in a ligand-independent manner by modulation of several kinase pathways including the MAPK and protein kinase A (PKA) pathways. Ligand-independent ER activators that function via the MAPK pathway include protein growth factors such as epidermal growth factor (EGF), TGF-{alpha}, IGF-I, and heregulin-2 (5, 6, 7, 8). Although the mechanisms by which TGF-{alpha} and heregulin-2 activate the ER remain unclear, EGF and IGF-I activate the ER by inducing MAPK-dependent phosphorylation of the ER S118 residue (9, 10). Activation of the PKA signaling pathway by 8-bromo-cAMP, a stable form of cAMP, and cholera toxin, an activator of adenylate cyclase, also enhances ER phosphorylation and activity. ER phosphorylation facilitates the interaction with coactivators and basal transcription factors, which leads to ER-mediated transcriptional activation (5, 11, 12). Furthermore, both PKA and MAPK pathways mediate phosphorylation and recruitment of the steroid receptor coactivator (SRC) family of coactivators (13, 14). In addition, the phosphatase inhibitor, okadaic acid, derived from toxic marine algae, also activates the ER presumably by inhibition of dephosphorylation of the activated ER or associated coactivators (15). Finally, the neurotransmitter, dopamine, can activate the ER in a PKA-dependent, ER ligand-independent mechanism in mammary tumor cells (15, 16). Thus, MAPK-mediated ER activation is induced by growth factor-stimulated membrane bound receptors, whereas PKA-mediated ER activation is induced by natural products and drugs that affect cellular cAMP levels.

Conceivably, the regulation of PKA and/or MAPK may be linked to the control of reproductive cell growth and steroid-responsive functions by different classes of anticancer agents. 3,3'-Diindolylmethane (DIM) is the major digestive product of indole-3-carbinol present in food plants of the Brassica genus, including broccoli and Brussels sprouts, and a promising cancer protective agent. Exposure of MCF-7 human breast cancer and Ishikawa human endometrial cancer cells to DIM reduced proliferation by 40% and 60%, respectively (16, 17). Additionally, oral administration of low doses of DIM inhibited the growth of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats by greater than 75% (18).

Although the chemoprotective effects of DIM in estrogen-responsive tissues are well established, we have previously observed that DIM can activate the ER (16, 17) under cell culture conditions with low estradiol concentrations. DIM induced the transcriptional activation of both estrogen-responsive endogenous genes and transfected reporter constructs and promoted the binding of the ER to its consensus DNA-responsive sequence in MCF-7 human breast cancer cells (16). Additionally, DIM induced the expression of TGF-{alpha} via ER activation in Ishikawa human endometrial cancer cells (17). DIM activation of the ER occurred in a ligand-independent and PKA-dependent manner (16, 17). The estrogenic activity of DIM has been confirmed in vivo in trout (19).

In this study, we identify the signaling pathways involved in ligand-independent activation of the ER by DIM in human endometrial cancer cells. We found that DIM-induced activation of the ER involves activation and cross talk between the PKA and MAPK signal transduction pathways. We establish that DIM is an efficient and mechanistically novel ER activator that functions through a mechanism that requires phosphorylation of CREB [cAMP response element (CRE) binding protein].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER Transcriptional Activation by DIM
To establish ligand-independent activation of the ER, we examined the ability of DIM to activate ER-mediated transcription in the absence of estradiol (E2). Ishikawa cells were transfected with the 4ERE-Luc reporter plasmid and treated for 24 h with the indicated concentrations of DIM or E2 for comparison. E2 activated transcription of the reporter in a concentration-dependent manner with a 50-fold maximum activation at 100 nM (Fig. 1AGo). Concentrations greater than 100 nM E2 did not result in further stimulation of the reporter. Cotreatment of cells with ICI, the ER antagonist, blocked the inducing activity of E2, consistent with the normal function of the ER in these cells (data not shown). DIM also increased transcriptional activity in a concentration-dependent manner (Fig. 1BGo). Maximum stimulation, similar to that of E2, was reached at 30 µM DIM and blocked by cotreatment with ICI, confirming that DIM activates the ER. In combination with increasing levels of E2, 30 µM DIM further enhanced reporter activation in an additive manner (Fig. 1AGo). A maximum reporter activation of 80-fold was reached in combination with 1 nM or greater concentrations of E2.



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  		Fig. 1. ER Activation by E2 and DIM in Ishikawa Cells

Ishikawa cells were transfected with 0.05 µg 4ERE-Luc and treated with vehicle control (DMSO) or the indicated concentrations of E2 in the presence or absence of DIM (A) or DIM (B) in the presence or absence of 0.1 µM ICI for 24 h. Luciferase activity was determined and normalized by protein content. The reported values are expressed as fold induction relative to DMSO-treated controls and represent the mean + SD of triplicate measurements from three independent experiments.

 
To demonstrate that the ER-inducing effects of DIM were not cell specific, reporter activation was examined in HEC-1B cells cotransfected with the expression vector for the human ER-{alpha}. E2 and DIM elicited similar inducing effects under these conditions (Fig. 2Go). Altogether, these results indicate that DIM is a potent and efficient inducer of ER-{alpha} transcriptional activation.



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  		Fig. 2. ER Activation by E2 and DIM in HEC-1B Cells

HEC-1B cells were cotransfected with 0.05 µg 4ERE-Luc and 0.05 µg WT ER-{alpha} expression vectors and treated with vehicle control (DMSO), 10 nM E2, or the indicated concentrations of DIM for 24 h. Luciferase activity was determined and normalized by protein content. The reported values are expressed as fold induction relative to DMSO-treated controls and represent the mean + SD of triplicate measurements from three independent experiments.

 
RT-PCR analyses established that DIM also induces the expression of several endogenous ER-responsive genes. DIM (30 µM) induced the expressions of TGF-{alpha}, alkaline phosphatase (AP), and progesterone receptor (PR) similar to levels induced by 10 nM E2 (Fig. 3Go). In combination with this high concentration of E2, DIM had minimal additional effect on further increasing gene expression. Unlike E2, however, the effects of DIM were indirect, as cotreatment of cells with the protein synthesis inhibitor, cycloheximide (50 µM), blocked the inducing effects of DIM but not of E2 on expression, as we showed previously for TGF-{alpha} (17). These results suggest that DIM requires action of an unknown intermediary factor for induction of ER-responsive gene expression.



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  		Fig. 3. Induction of ER-Responsive Gene Expression by E2 and DIM in Ishikawa Cells

Total RNA was isolated from Ishikawa cells treated with vehicle control (DMSO), 50 µM cycloheximide (CHX), 30 µM DIM in the presence or absence of CHX, or 10 nM E2 in the presence or absence of CHX or DIM for 6 h. For cotreatments, DIM and E2 were preceded by 1 h treatment with CHX. Total RNA was reverse transcribed and PCR-amplified to detect the expression of TGF-{alpha} (A), AP (B), or PR (C) along with GADPH as an internal control. PCR products were resolved and visualized on 1.8% agarose gels with ethidium bromide. Band intensities were quantitated and expression level was normalized to GADPH. The reported values are expressed as fold induction relative to DMSO-treated controls and represent the mean ± SD of measurements from three independent experiments.

 
Role of PKA and MAPK in DIM Activation of the ER
To assess whether phosphorylation plays a key role in DIM activation of the ER, we examined the effect of kinase inhibitors on reporter activation. Ishikawa cells were treated with a series protein kinase inhibitors before treatment with 30 µM DIM or 10 nM E2. Inhibitors of calmodulin-dependent kinase (KN62), PI3K (wortmannin), PKA (H89), protein kinase C (bisindolylmaleimide), MAPK (PD98059), and general tyrosine kinases (tyrophostin 25) had little or no effect on ER transcriptional activation by E2 (Fig. 4AGo). Treatment with PD98059 or tyrophostin 25, however, inhibited DIM-induced transcriptional activation by 50%. The most significant inhibitory effect was observed with H89. This PKA inhibitor reduced DIM activation of the reporter by 75%, even though E2-induced activation of the ER was fully functional. Combined treatment with H89 and PD98059 completely abolished the stimulatory effect of DIM (Fig. 4BGo). In contrast, KN62, wortmannin, and bisindolylmaleimide had no inhibitory effect on ER transcriptional activation by DIM. Our results confirm that E2 and DIM function by distinctly different mechanisms to activate the ER, and establish that activation of the ER by DIM is mediated solely by the PKA and MAPK signaling pathways.



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  		Fig. 4. Effects of Kinase Inhibitors on DIM and E2 Activation of the ER

Ishikawa cells were transfected with 0.05 µg 4ERE-Luc and pretreated with 10 µM KN62, 10 µM H89, 1 µM bisindolylmaleimide (Bis), 100 nM wortmannin (Wort), 10 µM tyrophostin 25 (Ty25), or 50 µM PD98059 (PD) (A) or cotreated with PD98059 and H89 (B) for 30 min. Cells were then treated with vehicle control (DMSO), 30 µM DIM, or 10 nM E2 for 24 h. Luciferase activity was determined and normalized by protein content. The reported values are expressed as fold induction relative to DMSO-treated controls and represent the mean + SD of triplicate measurements of three independent experiments.

 
Activation of MAPK
MAPK activation was determined by examining the induction of MAPK-mediated phosphorylation of ERK1 and ERK2. Protein was extracted from Ishikawa cells after 30 min exposure to 30 µM DIM, 10 µM forskolin (an activator of PKA), or 10 nM E2 and subjected to Western analysis using an antibody that recognizes phosphorylated ERK1 and ERK2 (phospho-ERK1 and ERK2). Forskolin and DIM primarily induced ERK1, and to a lesser extent, ERK2 phosphorylation in Ishikawa cells (Fig. 5AGo). E2 also induced ERK1 phosphorylation, but did not induce phosphorylation of ERK2. The compounds tested had no effect on total ERK protein levels (data not shown). These results confirm that DIM activates the MAPK pathway and reveal a further difference in the effects of DIM and E2 in endometrial cells.



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  		Fig. 5. Effects of DIM, E2, and Forskolin on MAPK Activation and cAMP Production

A, Ishikawa cells were treated with vehicle control (DMSO), 30 µM DIM, 10 nM E2, or 10 µM forskolin (Forsk) for 30 min. Levels of activated MAPK were determined by Western blot analysis using a phospho-MAPK-specific antibody. Band intensities for phospho-ERK1 (pERK1) and phospho-ERK2 (pERK2) were quantified and expressed as arbitrary units. Equal sample loading was confirmed by Coomassie blue staining of the Western blot membrane. B, Ishikawa cells were treated for 2 h with 30 µM DIM, 10 nM E2, or 10 µM forskolin (Forsk) for 2 h. Cell lysates were analyzed by EIA for cAMP levels. The reported values are expressed as picomoles per milliliter cell lysate and represent the mean + SD of triplicate measurements from three independent experiments.

 
PKA Activity
PKA activation is stimulated by the production of cAMP through activation of adenylate cyclase. Thus, we were interested in whether DIM increased cAMP production. Ishikawa cells were treated for 2 h with 30 µM DIM, 10 nM E2, or 10 µM forskolin and lysed for determination of cAMP by enzyme immunoassay (EIA). Forskolin increased cAMP levels by 8-fold over controls, whereas DIM and E2 had no effect (Fig. 5BGo). Direct measurement of PKA activity using alternate substrates indicated no increase in the activity of this kinase on treatment with DIM (data not shown). These observations indicate that DIM does not stimulate cAMP production, nor does DIM induce an increase in PKA activity against model substrates.

Effect of ER S118 Mutation on Transcriptional Activation by DIM
To determine whether DIM activates the ER similar to other ligand-independent activators that function through MAPK, the effect of the ER activation function (AF)-1 S118A mutant was examined. HEC-1B cells were cotransfected with the reporter and wild-type (WT) or S118A mutant ER. Expression of the mutant had no effect on cells treated with either 10 nM E2 or 10 µM DIM (Fig. 6Go). These results indicate a mechanistic difference between DIM and factors such as EGF that activate the ER by inducing MAPK-dependent phosphorylation of the S118 residue.



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  		Fig. 6. Effect of S118 Mutation on ER Activation

HEC-1B cells were cotransfected with 0.05 µg 4ERE-Luc and WT or S118A ER-{alpha} expression plasmids. Cells were treated for 24 h with DMSO vehicle control, 30 µM DIM, or 10 nM E2. Luciferase activity was determined and normalized by protein amount. The reported values are expressed as fold induction relative to DMSO-treated controls and represent the mean + SD of triplicate measurements from three independent experiments.

 
Involvement of CREB in ER Transcriptional Activity
The strong inhibitory effect of H89 on DIM activation of the ER suggests that DIM may regulate the activity of a downstream target of PKA. CREB is a transcription factor that can be activated by PKA-dependent phosphorylation (20). To functionally test the interaction of CREB and the ER in our system, the human CREB expression plasmid and the 4ERE-Luc reporter were expressed in HEC-1B cells with or without cotransfection of the ER expression plasmid. CREB expression had no effect on activity of the ERE-promoter in the absence of ER, whereas CREB activated the promoter in the presence of ER, demonstrating that the effect of CREB is ER dependent (Fig. 7Go). Expression of MCREB, a nonphosphorylatable S133A dominant-negative CREB mutant (20), had no effect in the presence of ER, suggesting S133 phosphorylation of CREB is required for ER activation.



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  		Fig. 7. Interaction between ER and CREB

HEC-1B cells were transfected with 0.05 µg 4ERE-Luc and 25 ng CREB or 25 ng MCREB in the presence or absence of 0.05 µg WT ER. Luciferase activity was determined and normalized by protein amount. The reported values are expressed as fold induction relative to DMSO-treated controls and represent the mean + SD of triplicate measurements from three independent experiments.

 
To further determine the effect of CREB on ER transcriptional activity, Ishikawa cells were cotransfected with the 4ERE-Luc reporter and the indicated amounts of CREB. Addition of increasing amounts of CREB significantly enhanced ER transcriptional activation in all control and treatment groups (Fig. 8AGo). Furthermore, expression of CREB produced similar additive effects in cells treated with 30 µM DIM or 10 µM forskolin. However, all three levels of CREB expression in cells treated with 10 nM E2, produced synergistic activation of the reporter gene. These observations demonstrate that CREB can enhance the activation of the ER in Ishikawa cells. In addition, the observed E2/CREB synergy is consistent with an E2-mediated gene activation process that does not normally involve endogenous CREB. In contrast, the additive effect of CREB on gene activation by DIM and forskolin is consistent with the involvement of endogenous CREB in ER activation by these two compounds.



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  		Fig. 8. Enhancement of ER Activation by CREB

Ishikawa cells were cotransfected with 0.05 µg 4ERE-Luc and the indicated amounts of expression vectors for CREB (A) or MCREB (B). After treatment of cells for 24 h with 30 µM DIM, 10 nM E2, or 10 µM forskolin (Forsk), luciferase activity was determined and normalized by protein amount. The reported values are expressed as fold induction relative to DMSO-treated controls and represent the mean + SD of triplicate measurements from three independent experiments.

 
To further assess the role of CREB in ER transcriptional activation by DIM, cells were cotransfected with the reporter and the indicated amounts of MCREB. The results show a clear MCREB concentration-dependent decrease in DIM-induced ER activity of up to 50% (Fig. 8BGo). Expression of MCREB did not reduce ER activation by E2 or forskolin. The results demonstrate, however, that CREB activation contributes to DIM activation of the ER.

Induction of CREB Phosphorylation
We next determined the effect of DIM on CREB activation by examining the induction of CREB phosphorylation. After treatment with 30 µM DIM for the indicated times, total cellular protein was isolated from Ishikawa cells and examined by Western analysis using an antibody that recognizes CREB phosphorylated at the S133 residue (phospho-CREB). Induction of phospho-CREB was found after 30 min and was sustained for 3 h after exposure before loss of induction (Fig. 9AGo). Little or no change in CREB levels was observed after treatment with DIM. The results indicate that DIM induces a transient increase in levels of S133-phospho-CREB.



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  		Fig. 9. Induction of CREB Phosphorylation by DIM

A, Ishikawa cells were treated with vehicle control (DMSO) or 30 µM DIM for the indicated times. B, Ishikawa cells were pretreated for 30 min with 50 µM PD98059 or 10 µM H89 before 1 h treatment with vehicle control (DMSO) or 30 µM DIM. The levels of phosphorylated CREB (pCREB) and CREB were determined by Western blot analysis using antibodies to CREB and pCREB. pCREB levels were normalized to total CREB and expressed as fold induction relative to DMSO-treated controls. Equal sample loading was confirmed by Coomassie blue staining of the Western blot membrane.

 
Phosphorylation of CREB may be induced by several kinases including PKA, MAPK, calmodulin-dependent kinase, and protein kinase C (20). We observed that only the inhibition of PKA and MAPK activity resulted in decreased ER activation by DIM. Thus, we examined the effect of H89 and PD98059 on CREB phosphorylation. Pretreatment of cells with H89 resulted in a complete inhibition of DIM-induced S133-phospho-CREB levels, whereas pretreatment with PD98059 had no inhibitory effect (Fig. 9BGo). Similar to ER activation, the PKA signaling pathway plays an important role in DIM-mediated CREB activation. These results confirm that the DIM-induced increase in CREB phosphorylation is mediated by the PKA pathway.

CREB phosphorylation was further confirmed by subjecting Ishikawa nuclear extracts to gel mobility shift analysis. Using a radiolabeled CRE-containing oligonucleotide probe, the interaction of CREB with its DNA-responsive element, the CRE, was assessed. The analysis indicated that proteins in nuclear extracts from control and treated cells bound to CRE (Fig. 10Go). An anti-CREB antibody, which recognizes both CREB and phospho-CREB, supershifted the protein/DNA complexes from treated and control cells, indicating that CREB binds constitutively to the CRE (data not shown). The anti-phospho-CREB antibody caused a supershift when protein was derived from forskolin, E2, or DIM-treated cells with much less effect on protein derived from control cells. These observations confirm the stimulatory effect of DIM on CREB phosphorylation similar to the effects of E2 and forskolin.



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  		Fig. 10. Interaction of Phospo-CREB with the CRE

Ishikawa cells were treated with vehicle control, 10 µM forskolin (Forsk), 10 nM E2, or the indicated concentrations of DIM for 1 h. Nuclear extracts were obtained and subjected to gel shift analysis using a labeled CRE probe. Bands were super shifted by an antibody specific for phospho-CREB. Arrows indicate locations of the shifted (SB) and super shifted bands (SSB).

 
Effects on Protein Phosphatase Activity
We determined whether DIM exhibited effects similar to okadaic acid by examining whether DIM inhibited protein phosphatase activity. Okadaic acid, an inhibitor of protein phosphatases 1A and 2A (PP1A and PP2A), activates the ER in a ligand-independent manner (15). Ishikawa cells were treated with 30 µM DIM, 10 µM forskolin, or 5 µM okadaic acid. Protein phosphatase activity was determined by monitoring the generation of free phosphate after the incubation of cell lysates with a phosphopeptide substrate. Unlike okadaic acid, neither forskolin nor DIM exhibited inhibitory effects on protein phosphatase activity compared with control (Fig. 11Go). These results indicate that DIM activation of the ER does not involve inhibition of protein phosphatase activity.



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  		Fig. 11. Effects of DIM, Forskolin, and Okadaic Acid on Protein Phosphatase Activity

Ishikawa cells were treated for 30 min with DMSO vehicle control, 30 µM DIM, 10 µM forskolin (Forsk), or 5 µM okadaic acid (OA). Protein phosphatase activity was determined on cell lysates. The reported values are expressed as picomoles of phosphate per minute per microgram of protein and represent the mean + SD of triplicate samples.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our results show that DIM strongly induces activation of the ER in Ishikawa and ER transfected HEC-1B human endometrial cancer cells. We demonstrate that DIM induces the expression of classical ER-responsive endogenous genes including TGF-{alpha}, AP, and PR. We have previously demonstrated that activations of both AP activity and TGF-{alpha} expression were abolished by cotreatment with ICI (17). Together, these results indicate that DIM can activate the promoters of a variety of ER-dependent genes. Furthermore, we previously demonstrated that such activation is independent of DIM binding to the ER in the MCF-7 and Ishikawa cells (Ref. 16 ; and data not shown). Our studies indicate that ligand-independent activation of the ER by DIM is mediated by the PKA and MAPK signaling pathways (Fig. 12Go). Inhibition of PKA and MAPK activity resulted in a 75% and 50% decrease in DIM activity, respectively. Simultaneous inhibition of both signaling pathways completely abolished DIM activity, indicating that DIM activation of the ER is mediated solely through a combination of PKA and MAPK signaling. Although DIM did not activate PKA activity nor increase cAMP levels, DIM induced the phosphorylation of CREB. The induction of CREB phosphorylation by DIM was completely abolished by a PKA inhibitor. Furthermore, expression of a nonphosphorylatable mutant CREB, MCREB, blocked DIM activation of the ER by over 50%. Taken together, these observations strongly indicate that activation of the ER by DIM is partially mediated by PKA-dependent phosphorylation of CREB and possibly other coactivators. We also demonstrated that DIM induced MAPK activity, suggesting that DIM may induce the MAPK-dependent phosphorylation and activation of the ER or another ER-associated protein involved in ER ligand-independent activation. In contrast, our previous studies demonstrated that inhibition of PKA activity was sufficient to abolish the ER-dependent induction of endogenous TGF-{alpha} expression by DIM (17). Whether the requirement of MAPK activity for ER activation is unique to a transfected ER-responsive reporter or reflects a promoter-specific effect requires further investigation.



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  		Fig. 12. Proposed Mode of ER Ligand-Independent Activation by DIM

DIM activation of the ER is mediated by PKA-dependent CREB phosphorylation without stimulation of PKA activity. We propose that DIM may sustain basal nuclear PKA activity, resulting in the phosphorylation of CREB and possibly other coactivators, such as SRC-1, and/or the ER. ER activation by DIM is also mediated by the MAPK signaling pathway. DIM stimulates MAPK activity and presumably induces MAPK-dependent phosphorylation of proteins involved in ER transcriptional activation or the ER in a S118-independent manner. Solid and dotted arrows indicate established and proposed mechanisms, respectively, by which DIM activates the ER.

 
We demonstrate here that DIM activation of the ER is indirect. Cotreatment with cycloheximide inhibited DIM but not E2 induction of ER-responsive gene expression. These results confirm that induction of gene expression by E2 is a primary response to ER activation and indicate that activation by DIM requires synthesis of an intermediary factor. This factor is not a general transcription activator, however, because we have demonstrated that DIM acts as an androgen receptor antagonist in prostate cancer cells (21). Hence, the effect of cycloheximide on DIM activity may result from several causes including, inhibition of de novo synthesis of a factor required for ER activation, loss of a coregulator with a high turnover rate, or from modified phosphorylation of the ER or coregulator resulting from activation of stress-activated protein kinase (22). Distinguishing these possibilities will be a focus of future studies.

The involvement of PKA in ER-mediated transcriptional activation is well documented (16, 23, 24, 25, 26, 27). Several studies have shown that activators of PKA enhance transcription of ER-regulated genes including PR, cathepsin D, and pS2 (9, 23, 24). Furthermore, 8-bromo-cAMP, an activator of PKA, enhanced the ligand-independent activation of the ER by cyclin D1 (27). These studies demonstrate that PKA mediates ligand-independent transcriptional activation of the ER. However, the observed effects of PKA are dependent on cell type and promoter context (25, 26).

Although the exact mechanisms are poorly understood, evidence shows that PKA mediates ligand-independent activation of steroid receptors by inducing receptor and/or receptor-associated coactivator phosphorylation. Activators of PKA, such as cholera toxin plus isobutyl methylxanthine (CT + IBMX), enhance phosphorylation of the ER (12, 26). CT and IBMX activate adenylate cyclase and inhibit the breakdown of cAMP, respectively. CT + IBMX induced a pattern of ER phosphorylation similar to that induced by E2 in COS-1 cells transfected with an ER expression vector (12). In transient transfection assays using an ER-responsive reporter in HeLa cells, CT + IBMX stimulated ER activity in cells cotransfected with an AF-1-truncated but not AF-2-truncated ER (29). Studies also indicate that PKA can regulate phosphorylation of SRC-1, a general coactivator for steroid receptors (13). SRC-1 interacts with CREB-binding protein (CBP), a general steroid receptor coactivator that associates with CREB. The interaction of these coactivators has been shown to synergistically increase ER and PR transcriptional activation (30, 31). 8-Bromo-cAMP was found to induce the phosphorylation of two residues on SRC-1 in COS-1 cells (13). Mutation of these residues resulted in a 50% loss of PR ligand-independent activation by 8-bromo-cAMP. The inhibition of PR activation was partially due to the loss of SRC-1 and CBP interaction, indicating that phosphorylation is necessary for coactivator interaction. Taken together, these results indicate that PKA mediates ER and coactivator phosphorylation and stimulates ER transcriptional activation in an AF-2-dependent manner. Our observations clearly indicate that DIM is unlike all PKA-dependent ER activating agents because DIM does not stimulate PKA activity.

In the present study, we demonstrate that ligand-independent activation of the ER by DIM is partially mediated by PKA-dependent phosphorylation of CREB. CREB contains a kinase-inducible domain, which contains several acidic residues as well as the S133 residue. Phosphorylation of S133 is required for the transcriptional activation of CREB. Phosphorylation of CREB results in transcriptional activation due to interaction with CBP, a transcriptional adapter that bridges phosphorylated CREB to basal transcriptional machinery (32). In addition, CBP interacts with the ER and enhances ER transcriptional activation (13, 30). CREB is proposed to indirectly interact with the ER as a secondary coactivator through CBP (33). Whether CREB is able to function independently or synergistically with other coactivators remains unclear and will be a focus of future studies to further detail the mechanism by which DIM activates the ER.

Although we established the role of CREB phosphorylation in ER activation, the mechanism by which DIM induces phosphorylation remains unclear. Exposure of cells to H89 completely abolished the inducing effect of DIM, indicating the central role of PKA in CREB phosphorylation. However, DIM did not induce PKA activity, unlike all other agents that induce ligand-independent activation of the ER via PKA. PKA is an enzymatically inactive tetramer composed of two catalytic subunits (C{alpha}/ß/{gamma}) bound to two regulatory subunits (RI{alpha}/ß, RII{alpha}/ß). PKA activation is induced by increased production of cAMP via activation of adenylate cyclase. cAMP binds to the regulatory subunits of PKA, and upon binding, induces the dissociation of two active catalytic subunits that translocate to the nucleus and phosphorylate target proteins (34). Kinase activity is inhibited by the interaction with protein kinase inhibitors that promote nuclear export of the catalytic subunits (35). We found that DIM had no effect on nuclear translocation of PKA catalytic subunits (data not shown). Thus, we propose that DIM may sustain the activity of catalytic subunits already in the nucleus by preventing their interaction with protein kinase inhibitor. Previous studies have demonstrated this phenomenon in ethanol-treated neuroblastoma cells (36). However, we propose that DIM may be the first agent shown to activate the ER by such a mechanism.

Whereas DIM failed to activate PKA, we demonstrated that DIM did induce MAPK activity. Several studies have demonstrated the mediation of ER ligand-independent activation by MAPK signaling (9, 10, 37, 38, 39, 40). Activators of MAPK, for example EGF, mimicked the effects of estrogen in the mouse uterus (37, 38, 39). These effects were abolished upon ICI administration or were nonexistent in ER-deficient mice (38, 40). Studies in COS-1 and HeLa cells have demonstrated that EGF mediates ER activation by phosphorylation of the ER on the S118 residue (9, 10). In Ishikawa cells, ER activation was induced by expression of constitutively active MAPK kinase kinase (MEKK1) (41). Ligand-independent activation of the ER by MAPK-activating agents appears to be dependent only on the ER AF-1 domain (9, 29). In the present study, DIM induced the phosphorylation of ERK1/2 in the Ishikawa and transfected HEC-1B cells. Although DIM-induced CREB phosphorylation was unaffected by the MAPK inhibitor, PD98059, whether DIM induces MAPK-mediated phosphorylation of the ER or ER-associated proteins remains to be determined. However, our results suggest that DIM activation of the ER does not require phosphorylation of the ER S118 residue, indicating a mechanistic difference between DIM and EGF.

DIM also shows mechanistic differences with dopamine, another ligand-independent activator of the ER. Although DIM and dopamine are G400-dependent and S118-independent ER activators, dopamine, by itself, was not active in Ishikawa cells (data not shown; and Refs. 42, 43). However, in combination with DIM, dopamine exhibited a synergistic activation of the ER (data now shown), further demonstrating mechanistic differences between the two compounds. Altogether, our studies clearly distinguish DIM from other ligand-independent ER activators.

Despite the apparent estrogenic effects, DIM paradoxically exerts potent cytostatic effects in vitro and in vivo (16, 17, 18). DIM shows estrogenic and antiproliferative activities at the same concentrations in Ishikawa cells (17). Thus, we propose that DIM simultaneously activates an antiproliferative pathway that counters the estrogenic effects. Both estrogenic and antiproliferative effects may be elicited through similar signaling pathways. We have previously demonstrated that PKA-dependent DIM induction of TGF-{alpha} gene expression may contribute to growth inhibition because TGF-{alpha} itself inhibits cell growth in Ishikawa cells (17). We have also found that the inhibition of PKA activity by H89 reduces the cytostastic activity of DIM in Ishikawa cells by 50% (data not shown). PKA activity is likely required for the expression and/or activity of additional cytostatic factors because the neutralization of TGF-{alpha} only partially reversed DIM’s antiproliferative effects (17). Altogether, our studies demonstrate that DIM is likely to have multiple effects that include ER activation. Overall, however, DIM is effective at controlling the growth of estrogen-dependent cancer cells. Given the association of endometrial cancer in women using tamoxifen for the control of breast cancer, the potent cytostatic effects of DIM in both breast and uterine tissues may prove significant. In light of such advantage over tamoxifen, the mechanism by which DIM exerts simultaneous estrogenic effects is important to determine given that DIM may also exert similar estrogenic effects in other tissues such as the brain, bone, and cardiovasculature. Such effects are beneficial and sought after in the search for improved selective ER modulators for the treatment of breast cancer. Whether DIM may exert estrogenic effects in these tissues by the same mechanism we have discovered in uterine cancer cells remains to be determined. Characterization of these effects will be informative because DIM already shows promise as a therapeutic agent for cancers of the female reproductive tract.

We demonstrate in the present study that DIM activates the ER in a PKA- and MAPK-dependent manner. We establish that PKA-dependent activation of the ER is partially mediated by phosphorylation of CREB. The mechanism of ER activation via MAPK remains unclear but is likely to be mediated by phosphorylation of the ER or a coactivator. We demonstrate further that DIM is mechanistically unique from agents reported to mediate activation of the ER through the MAPK and PKA signaling pathways because DIM activation of the ER was dependent on the AF-2 domain and independent of induction of PKA activity. Finally, DIM does not inhibit protein phosphatase activity as does okadaic acid, which has also been reported to activate the ER (15). Thus, DIM is a novel ER ligand-independent activator that is dependent on both PKA and MAPK activities. These studies have identified a novel ligand-independent ER activation pathway that involves cross talk between the PKA and MAPK signal transduction pathways. Furthermore, this work identifies DIM as a new class of phytoestrogen available from the diet.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
DMEM, Opti-MEM, and Lipofectamine were purchased from Life Technologies (Gaithersburg, MD). Phenol red-free DMEM and fetal bovine serum (FBS) were purchased from Sigma Chemical Co. (St. Louis, MO). Lavendustin A, PD98059, KN62, and ICI were purchased from Tocris (Ellisville, MO). Anti-CREB, anti-phospho-CREB, anti-MAPK, and anti-phospho-MAPK antibodies were purchased from Upstate (Waltham, MA). DIM was prepared from indole-3-carbinol and purified as described (44, 45). All other reagents used were of highest commercial grade available.

The 4-ERE-luciferase reporter construct was a gift from Dr. David Shapiro (University of Illinois, Champaign, IL). The CREB and MCREB cDNAs were kindly provided by Dr. Richard Goodman (Oregon Health Science University, Portland, OR) and Dr. Jane Reusch (Veterans Affairs Medical Center, Denver, CO). WT ER-{alpha} (HEGO), AF-2 G400V mutant (HEO), and AF-1 S118A mutant (HE457) were purchased from Dr. Pierre Chambon (University of Strasbourg, Strasbourg, France).

Cell Culture
Ishikawa (ER positive) and HEC-1B (ER negative) human endometrial adenocarcinoma cells were obtained from B. A. Lessey (Chapel Hill, NC) and ATCC (Manassas, VA). Cells were grown in DMEM supplemented with 10% FBS, 3.0 g/liter glucose, and 3.7 g/liter sodium bicarbonate and maintained at 37 C in humidified air containing 5% CO2. For all experiments, cells were depleted of estrogen for 1–3 d in stripped media before treatment. Stripped media consisted of phenol red-free DMEM (Sigma) supplemented with 5% calf serum doubly stripped in dextran-coated charcoal (Life Technologies), 4.0 g/liter glucose, 3.7 g/liter sodium bicarbonate, and 2 mM glutamine. Treatments were administered by addition of 1 µl of the concentrated agent in dimthylsulfoxide (DMSO) per 1 ml of medium or addition of an equal volume of DMSO for control cells.

Transient Transfection and Reporter Assay
Ishikawa and HEC-1B cells were plated in 6.0-cm tissue culture plates and transfected with 0.05 µg, unless stated otherwise, of the indicated plasmids in Opti-MEM by the lipofection method using Lipofectamine. Subsequently, cells were treated with the indicated test compounds for 24 h. Cells were rinsed twice with PBS and harvested in 150 µl lysis buffer [25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 2.5% Triton X-100, 1 mM dithiothreitol (DTT)]. Protein extract (50 µl) in 180 µl assay buffer (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 15 mM KH2PO4, 2 mM ATP, 1 mM DTT) was combined with reaction buffer (0.25 mM luciferin, 25 mM glycylglycine) to determine luciferase activity using the Lumat LB9507 luminometer (EG&G Berthold, Wildbad, Germany). Total protein concentration was measured using Bradford reagent (Bio-Rad, Hercules, CA) and used to normalize luciferase activity.

RT-PCR Analysis
After the indicated treatments, total RNA was isolated from Ishikawa cells using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) as described previously (16). Subsequently, mRNA was purified from 50 µg of total RNA using the Oligotex mRNA Purification System (QIAGEN, Valencia, CA). Poly(A)+ RNA was reverse transcribed into cDNA using the Superscript Preamplification System (Life Technologies) at 42 C for 50 min with random oligomers in a 100 µl reaction mixture. The resultant cDNA (5 µl) was then subjected to PCR (94 C for 45 sec, 58 C for 45 sec, 72 C for 2 min) using AP (28 cycles), TGF-{alpha} (28 cycles), or PR (23 cycles) primers. Glyceraldehyde-3-phosphate dehydrogenase was also amplified as an internal control. Sequences of the primers, synthesized by Genosys (The Woodlands, TX), are as follows: placental AP, 5'-TCAGGAAAAGAGGAGGCTCA-3', 5'-TCTGAGTGGCTGTGACTTGG-3'; TGF-{alpha}, 5'-ATGGTCCCCTCGGCTGGACA-3', 5'-ACGAAGGTACCTTGGACGTC-3'; PR, 5'-CACGAGTTTGATGCCAGAGA-3', 5'-AGGGAGGAGAAAGTGGGTGT-3'; glyceraldehyde-3-phosphate dehydrogenase, 5'-ACCTGGTGCTCAGTGTAGCC-3', 5'-GTCAGTGGTGGACCTGACCT-3'. PCR products were electrophoresed on 1.8% agarose gels and visualized by ethidium bromide staining.

Western Blot Analysis
Ishikawa cells were harvested in lysis buffer containing 10 mM Tris, 150 mM NaCl, 1% deoxycholate, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 1 mM sodium orthovanadate and protease inhibitors (10 µg/ml aprotinin, 5 µg/ml pepstatin, 10 µg/ml leupeptin, and 50 µg/ml phenylmethylsulfonylflouride). Total cellular protein (50 µg) was electrophoresed on 15% sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose membranes (Millipore, Bedford, MA), and subjected to Western blot analysis using anti-CREB, anti-phospho-CREB, anti-MAPK, and anti-phospho-MAPK antibodies (Upstate). Protein was detected using the Phototope-STAR Western Blot Detection Kit (New England Biolabs, Beverly, MA) and scanned and quantified using the National Institutes of Health Image Program. Blotted membranes were stained with Coomassie blue to verify equal protein loading.

cAMP Assay
For cAMP determination, cells were treated for 2 h with the indicated compounds and washed twice with PBS. cAMP levels were detected using the cAMP EIA EIA kit (Biomol, Plymouth Meeting, PA) according to the manufacturer’s instructions.

PKA Assay
After treatment with test compounds for 15–120 min, Ishikawa cells were harvested and homogenized in extraction buffer [25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM ß-mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin]. PKA activity was determined on cell homogenates using the SignaTECT cAMP-dependent protein kinase assay system (Promega, Madison, WI) according to the manufacturer’s instructions.

Nuclear Extraction and Gel Mobility Shift Assay
Nuclear extracts were obtained after treatment for 1 h with the indicated compounds. Cells were washed twice and incubated with hypotonic buffer [10 mM HEPES (pH 7.5)]. After 15 min of incubation, cells were harvested in buffer I [3 mM MgCl2, 1 mM DTT, 25 mM HEPES (pH 7.5)] and homogenized with a Teflon pestle. Homogenates were centrifuged for 4 min at 1000 x g at 4 C and the supernatant containing the cytosolic proteins was discarded. The nuclei were washed three times with buffer II [3 mM MgCl2, 1 mM DTT, 0.1 M KCl, 25 mM HEPES (pH 7.5)], and resuspended in buffer III [1 mM DTT, 0.4 M KCl, 25 mM HEPES (pH 7.5)]. Glycerol (10% vol/vol) was added after a 20-min incubation on ice.

The oligonucleotides containing the palindromic CRE consensus motif (5'-TGGCTGACGTCAGAGA-3', 5'-TCTCTGACGTCAGCCA-3') were annealed and 5' end-labeled with {gamma}32P-ATP. The labeled oligonucleotide probe was subsequently purified using a Sephadex G50 spin column, ethanol precipitated, and dissolved in Tris-EDTA buffer. Nuclear extract (12 µg) was combined with 100 ng poly(deoxyinosine-deoxycytidine) (Sigma) for 15 min in buffer containing 1 mM DTT, 10% glycerol, 1 mM EDTA, 160 mM KCl, and 25 mM HEPES. For supershift experiments, 1 µg of anti-CREB or anti-phospho-CREB antibody (Upstate) was added. An aliquot of the labeled probe containing 25,000 cpm was added to the mixture and incubated for an additional 15 min. The protein-DNA complexes were loaded with Ficoll loading buffer (0.25% bromophenol blue, 25% Ficoll type 400) onto prerun, nondenaturing 4% polyacrylamide gels in TAE [7 mM Tris, 33 mM sodium acetate, 10 mM EDTA (pH 8.0)].

Protein Phosphatase Activity
Cells were treated for 30 min with test compounds, washed twice with PBS, and lysed in extraction buffer containing 20 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.1% ß-mercaptoethanol, 2 µg/ml leupeptin, 10 µg/ml aprotinin, and 10% (vol/vol) glycerol. Cells were disrupted by sonication on ice (3 x 10 sec), and cell lysates were clarified by centrifugation for 1 h at 16,000 rpm. Protein phosphatase activity was determined on the resulting supernatant (5 µg) using the phosphatase assay system obtained from Promega. The phosphatase assay was performed in a PP2A-specific buffer [50 mM imidazole (pH 7.2), 0.2 mM EGTA, 0.02% ß-mercaptoethanol, 0.1 mg/ml BSA] with 100 µM phosphopeptide substrate [RRA(pT)VA] in a 50-µl reaction volume. After incubation for 30 min, generation of free phosphate was determined (OD 620 nM) using the molybdate-malachite green-phosphate complex assay as described by the manufacturer. PP2A-specific activity was reported as picomoles of phosphate generated per minute per microgram of protein.


   FOOTNOTES
 
This work was supported by the Department of Defense Army Breast Cancer Research Program, the NIH, and the National Institute of Environmental Health Sciences Center.

H.L. is currently at the Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637.

Abbreviations: AF, Activation function; AP, alkaline phosphatase; CBP, CREB-binding protein; CRE, cAMP response element; CREB, CRE binding protein; CT, cholera toxin; DIM, 3,3'-diindolylmethane; DMSO, dimethylsulfoxide; DTT, dithiothreitol; E2, estradiol; EGF, epidermal growth factor; EIA, enzyme immunoassay; ER, estrogen receptor; ERE, estrogen response element; IBMX, isobutyl methylxanthine; MCREB, a nonphosphorylatable CREB mutant; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PP, phostphatase (1A and 2A); PR, progesterone receptor; SRC-1, steroid receptor coactivator-1; WT, wild-type.

Received for publication May 27, 2003. Accepted for publication November 18, 2003.


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