Repression of the Estrogen Receptor-α Transcriptional Activity by the Rho/Megakaryoblastic Leukemia 1 Signaling Pathway^*

Plasmids and Constructs

The ERE-tk-LUC, C3-LUC, and c-fos-LUC reporter genes as well as the internal control CMV-βgal, the pCR-ERα, pCR-ERα Δ79, and pCR-ERα CFs (Δ173) have been previously described (9). Expression plasmids encoding the negative and positive dominant Myc-tagged forms of RhoA (Q63L and T19N), Rac1 and Cdc42 (Q61L and T17N) are a gift from Prof. William Harris (University of California). The p3×flag-MKL1, p3×flag-MKL1 ΔN200 and p3×flag-MKL1 ΔC301, p3×flag-MKL1 ΔB, p3×flag-MKL1 ΔQ, and p3×flag-MKL1 ΔSAP expression vectors were kindly provided by Prof. R. Prywes (Colombia University) (17). The pTAL-LUC, pEGFP, and the pEGFP-β-actin plasmids were purchased from Clontech. Open reading frames from the MKL1-, MKL1 ΔN200-, and MKL1 ΔC301-expressing vectors as well as a modified open reading frame of the actin-expressing vector were generated by PCR and subcloned into pCR3.1 or pCDNA3.1 plasmid (Invitrogen) to obtain pCR MKL1, pCR MKL1 ΔN200, pCR MKL1 ΔC301, and pCDNA-actin G13R. The pR-CMV-CBP-HA was a gift from Prof. F. Gannon (Heidelberg, Germany). The pGEX2T, pGEX2T-ERα AB, and pGEX2T-ERα DF plasmids have been previously described (18). The pGEX3X-MKL N330 plasmid was constructed by subcloning the sequence coding the 330 N-terminal amino acids of MKL1, generated by PCR, into the pGEX3X plasmid (Amersham Biosciences). All generated constructs were sequenced.

Reagents and Antibodies

Cytochalasin D, the ROCK inhibitor Y-27632, and the Rho inhibitor C3 exoenzyme were purchased from Calbiochem. ICI_182,780 (ICI) was provided by TOCRIS Bioscience. The anti-GFP antibody (JL-8) and the anti-phospho-RNA polymerase II antibody (CTD4H8) were purchased from BD Biosciences and Upstate Biotechnologies, respectively. 17 β-estradiol (E₂), 4-hydroxytamoxifen (4-OHT), the anti-β-actin (AC-15) and the anti-FLAG M2 antibodies were purchased from Sigma-Aldrich. The anti-Myc tag antibody (9B11) was purchased from Cell Signaling. Antibodies raised against the HA epitope (HA probe), ERα (HC20), and MKL1 (MRTF-A and C-19) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture and Transfection

HeLa, HepG2, MCF-7, or MDA-MB231 cells or MDA-MB231 stably expressing ERα (19) were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 5% or 10% (MCF-7) fetal calf serum (Biowest) and antibiotics (Sigma-Aldrich) at 37 °C in 5% CO₂. Transfections were carried out using FuGENE 6^TM reagent according to manufacturer's instructions (Roche Applied Science). One day before transfection, cells were plated in 24-well plates at 50% confluence. 1 h prior transfection, the medium was replaced with phenol red-free Dulbecco's modified Eagle's medium-F12 (Sigma-Aldrich) containing 2.5% charcoal-stripped fetal calf serum (Biowest). Transfection was carried out with 100 ng of reporter gene, 100 ng of CMV-βGal internal control, and appropriate combinations of expression vectors. Plasmid mix was made up to 500 ng of total DNA per well with empty vector. Following an incubation overnight, cells were treated for 24 h with ligands or ethanol (vehicle control), and required drugs. The C3 exoenzyme (125 ng/ml) was added to the FuGENE/medium mix during the transfection step. Cells were then harvested, and luciferase and β-galactosidase assays were performed as previously described (18). Luciferase reporter gene activity was normalized to the β-galactosidase.

Protein Extracts and Western Blotting

To obtain whole cell extracts, cells were lysed in RIPA buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing a mixture of protease inhibitors (Roche Applied Science). Subcellular fractionation was performed as described in Current Protocols (20). Western blots were performed as previously depicted (9).

Pulldown Assays

To determine Rho activity in cell lines, the Rho Activation Assay Kit (Upstate) was used in accordance with manufacturer's instructions.

Reverse Transcription-PCR, ChIP, and Sequential ChIP Assays

Reverse transcription-PCR, ChIP, and sequential ChIP assays were performed as previously described (21). The primers against promoter regions are available upon request.

Rho GTPases and Actin Dynamics Control ERα Transactivation Potency

We previously demonstrated that the transcriptional activity of ERα depends upon the differentiation stage of the cell, with the highest activity detected in differentiated cells (9). De-differentiation of epithelial cells is correlated with significant changes in the activity of members of the Rho GTPases family such as RhoA, Rac1, and Cdc42 (11). The impact of the Rho/actin signaling pathway on ERα transactivation efficiency was analyzed by transient transfection experiments in HepG2 and HeLa cells, which are two epithelial-like cell lines exhibiting divergent phenotypes. HeLa cells, originating from a cervix carcinoma, present a poorly differentiated phenotype, do not express the calcium-dependent cell-cell adhesion molecule E-cadherin, and strongly express vimentin, an intermediate filament protein that expression is associated with increased invasive and metastatic potency. In contrast, the hepatocarcinoma HepG2 cell line appears more differentiated, producing a high level of E-cadherin but no vimentin (9). Moreover, Rho activity was much higher in HeLa than in HepG2 cell extracts, as indicated by glutathione S-transferase pulldown experiments using as a bait the Rho-binding domain of the human rhotekin protein, which has the ability to retain only active Rho (Fig. 1A). Although no signal was observed within the HepG2 extracts, we cannot exclude that some active Rho is present in these cells but at really low levels that could not be detected by the procedure. The transactivation efficiency of ERα on an ERE-tk-LUC reporter gene is also much higher in HepG2 than in HeLa cells following treatment with 10⁻⁸ m E₂ (Fig. 1B), corroborating our previous data (9, 10).

Constitutively active Myc-tagged forms of RhoA (Q63L), Rac1 (Q61L), or Cdc42 (Q61L), as well as their respective inactive forms (RhoA T19N, Rac1 T17N, and Cdc42 T17N), were then used to study the contribution of these Rho GTPases in transcriptional regulations mediated by ERα. In HepG2 cells, the transfection of increasing concentrations of plasmids encoding inactive RhoA, Rac1, and Cdc42 forms, which inhibit endogenous Rho GTPase activity, has little effect on ERα-mediated induction of the ERE-tk-LUC reporter. In contrast, the expression of these mutated Rho GTPases in HeLa cells increases 2- to 3-fold the transcriptional response of the reporter gene to ERα (Fig. 1B). Importantly, in HepG2 cells, although the transfection of low amounts of plasmids encoding the three constitutive Rho GTPase forms has little impact on ERα transactivation potency, the latter is severely reduced at higher amounts of plasmids. In contrast, in HeLa cells, the constitutively active forms have no obvious effect on the ERα-mediated stimulation of ERE-tk-LUC reporter, as expected (Fig. 1B). The use of the C3 exoenzyme, which is an inhibitor of the three Rho proteins (A, B, and C), restores the situation observed with the inactive form of RhoA (T19N) (Fig. 1B). Finally, we analyzed the expression level of ectopically expressed proteins in both cell lines through Western blots (Fig. 1C). These experiments showed no correlation between results from gene reporter assays and any variation in the amounts of ERα or Rho GTPase forms. Moreover, the increased amount of ERα detected within HepG2 cells transfected with the three constitutive Rho GTPase forms is inversely correlated with its activity. Altogether, these results indicate that ERα transcriptional efficiency is negatively regulated by active Rho GTPases.

Rho GTPases are mainly studied for their roles in regulating the actin cytoskeleton (22). Activation of RhoA induces the polymerization of actin, partly through its effector ROCK (23). We consequently evaluated the transcriptional activity of ERα in both cell lines following an inhibition of ROCK by the Y-27632 compound. In agreement with data presented before, the Y-27632 treatment enhances ERα transcriptional activity in HeLa cells only (Fig. 1B). Contribution of actin dynamics in ERα transcriptional regulation was then further evaluated by disrupting the intracellular levels of actin monomers (G-actin). For this purpose, cells were transfected with increasing concentrations of expression vectors encoding wild-type or unpolymerizable mutant (G13R) of β-actin, the overexpression of which enhances the G-actin level (24). When expressed in HeLa cells, both actin forms potentiate the induction of the ERα-dependent reporter gene. In contrast, ERα activity in HepG2 cells was not really modified in their presence (Fig. 1B). Western blots indicate that β-actin levels are not obviously affected by the transfections of both actin forms in both cell lines (Fig. 1C). In contrast, ERα expression level is slightly reduced by the overexpression of the wild-type β-actin form, while it remains active. Together, these results indicate that actin dynamics influence ERα activity. To substantiate this observation, we next used cytochalasin D, an actin-binding drug that mimics the coactivation of SRF by Rho (25). As expected, cytochalasin D strongly inhibits ERα transcriptional activity in HepG2 cells, thereby mimicking the situation observed upon the overexpression of constitutively active forms of Rho GTPases (Fig. 1B). Altogether, these results strongly suggest that Rho GTPases control ERα transcriptional activity by modulating actin dynamics.

Rho/Actin Regulation of ERα Transactivation Potency Involves ERα AF1 Box 1 Activity

In epithelial cells, the attenuation of ERα activity observed following the loss of E-cadherin-mediated cell contacts partly results from a silencing of the ERα AF1 box 1 activity (10). We therefore evaluated the respective contribution exerted by ERα AFs toward the transcriptional activity of the receptor, upon changes of the Rho/actin pathway activity. Cell permissiveness to either ERα AFs was determined by comparing the transcriptional activity of ERα with those of ERα Δ79 (deletion of AF1 box 1) and ERα CF (additional deletion of AF1 box 2/3). Importantly, the ERE-tk promoter mainly used in our transient transfection experiments exhibits no intrinsic preference for a specific AF (9). In HepG2 cells, the main region involved in ERα transcriptional activity is the AF1 box 1 (ERα Δ79 versus ERα, Fig. 2B), the remaining activity depending upon the AF1 box 2/3, as expected (10). In contrast, the AF2 (ERα CF) represents ∼60% of the whole ERα transcriptional potency in HeLa cells. The AF1 box 2/3 (ERα Δ79 versus ERα CF) is responsible for the remaining 40% of the activity (Fig. 2B).

The overexpression of the constitutively active form of RhoA (Q63L) specifically abolishes the activity of ERα AF1 box 1 in HepG2 cells (ERα versus ERα Δ79) without significantly affecting the activities of the other AFs. In contrast, in HeLa cells, RhoA Q63L does not change the relative contribution exerted by the different AFs, as indicated by the similar activity promoted by the ERα, ERα Δ79, and ERα CF proteins (Fig. 2B). Thus, in the presence of a constitutively active form of RhoA, the transcriptional activity of ERα is similar in both cell types. As expected, the overexpression of the T19N-inactive form of RhoA had no effect on the transcriptional properties of the different ERα proteins in an AF1 box 1 permissive context such as HepG2 cells (Fig. 2B). As a consequence, the overexpression of this mutant in HeLa cells would have been expected to allow an activity of the AF1 box 1, but this is not the case (Fig. 2B). Similar results were observed following the overexpression of dominant positive and negative forms of Rac1 and Cdc42 (data not shown). To confirm the involvement of ERα AF1 in the regulation of ERα transactivation potency by the Rho/Actin pathway, we then evaluated the relative contribution of the different AFs toward ERα activity following the inhibition of the RhoA effector ROCK by the Y-27632. Importantly, while this treatment had no effect in HepG2 cells, it allowed HeLa cells to become permissive to an AF1 box 1 activity, as revealed by the higher transcriptional activity of full-length ERα versus ERα Δ79 (Fig. 2B). Importantly, this treatment did not affect the contribution of AF1 box 2/3 in HeLa cells, indicating that pathways initiated by RhoA were targeting the AF1 box 1 activity. Corroborating this conclusion, the overexpression of unpolymerizable β-actin (G13R) activated the AF1 box 1 activity in HeLa cells, as revealed by the different transcriptional potency of full-length ERα and ERα Δ79 proteins. In contrast, cytochalasin D totally abolished the activity of the AF1 box 1 in HepG2 cells and dictated a strict AF2 permissiveness in HeLa cells. Finally, as assessed by Western blots (Fig. 2C), although some ERα variants exhibited different expression levels, they were not affected upon the specific treatments that modify ERα activity in both cell lines.

These data demonstrate that Rho/actin axis controls ERα transcriptional activity, at least through the modulation of the respective contribution of each AFs toward the activity of the receptor. Notably, the activation of RhoA and subsequent actin polymerization inhibited the activity of the AF1 box 1 of ERα.

MKL1 Links Rho/Actin Pathway to ERα Transcriptional Activity

RhoA and changes in actin dynamic are integral components of a T cell factor-independent pathway leading to the activation of SRF. One mediator of this pathway is the coactivator MKL1 (12, 13). The organization of MKL1 is shown in Fig. 3A. MKL1 was expressed in both cell lines (Fig. 3B), therefore, we sought to evaluate the influence of this protein on ERα transcriptional activity. For this purpose, flagged versions of wild-type or deletion mutants of MKL1 were overexpressed in the cells. The MKL1 ΔN200 protein was deleted from the N-terminal RPEL motifs (Fig. 3A), which are critical for actin binding, and acted as a constitutively active variant on SRF target genes such as c-fos. The MKL1 ΔC301 protein was deleted from the C-terminal transactivation domain and behaves as a dominant negative form on c-fos regulation (12, 17). The correct expression of these proteins, their cellular localization, and their appropriate behavior on a c-fos-LUC reporter gene were first established as shown in Fig. 3, C and D. The amount of the plasmids encoding MKL1 variants used was previously determined by a dose-response experiment (data not shown). Their impact on the ERE-tk-LUC reporter gene was then analyzed. Wild-type and mutated forms of MKL1 dramatically change the basal activity of the ERE-tk-LUC measured in the absence of ERα, paralleling variations observed on the c-fos-LUC reporter (Fig. 3D). The constitutively active mutant MKL1 ΔN200, and to a lesser extent MKL1, enhanced the basal activity of the ERE-tk-LUC, whereas the dominant negative form MKL1 ΔC301 had an opposite effect, notably in HeLa cells (Fig. 3D). Similar results were observed on other ERE-driven reporter genes as well as on ERE-less reporters (Fig. 3D and data not shown). Thus, besides exhibiting different amplitudes of their response to MKL1 variants, with the ERE-less pTAL reporter gene being the less sensitive, the responses of all reporters were similar, in the presence or absence of an ERE. Finally, the overexpression of MKL1 variants internally deleted of short basic (ΔB), glutamine-rich (ΔQ), or SAP (SAF-A/B, Acinus, and PIAS) (ΔSAP) regions, which are conserved within the myocardin-related transcription factor family (17), also modify the basal activity of the reporter genes in a cell- and promoter-dependent manner (Fig. 3E and data not shown).

To facilitate the analysis of the influence of MKL1 forms on ERα transcriptional activity and minimize the interpretation bias created by the variations in the basal transcriptional activity of the ERE-tk-LUC observed following MKL1 expression, ERα transactivation potency were expressed, for each MKL1 forms, as the -fold increase above reporter gene activity measured in the absence of receptor. The results of these assays clearly show that the overexpression of MKL1 and MKL1 ΔN200 dramatically reduced the transactivation efficiency of ERα in HepG2 cells (Fig. 3E), whereas the dominant negative MKL1 ΔC301 almost doubled the activity of ERα. The overexpression of MKL1 and MKL1 ΔN200 proteins has a similar impact on ERα transcriptional potency in HeLa cells, but to a lesser extent. Additionally, as in HepG2 cells, MKL1 ΔC301 also enhanced by 2-fold the transcriptional activity of ERα in HeLa cells (Fig. 3E). On the other hand, the internal deletions of MKL1 B, Q, or SAP regions influenced ERα activity in a cell-dependent manner (Fig. 3E).

The impact of MKL1 forms on cell permissiveness to either ERα AFs was then determined by comparing ERα, ERα Δ79, and ERα CF transcriptional activity. These experiments demonstrate that, in HepG2 cells, MKL1 and MKL1 ΔN200 dramatically reduced (MKL1) or even abolished (ΔN200) the activity of the AF1 box 1 activity. This is revealed by the decrease of full-length ERα activity to levels similar to those induced by ERα Δ79 (Fig. 4A). In HeLa cells, the reduction of ERα activity provoked by these two MKL1 proteins was apparently a consequence of an inhibition of the AF1 box 2/3, which generated a cell context permissive only to ERα AF2 (Fig. 4A). These results therefore parallel those observed following the overexpression of dominant positive RhoA (Q63L) and cytochalasin D treatments (Fig. 2B). Importantly, the stimulation of ERα activity observed following the overexpression of MKL1 ΔC301 in both cell lines seems to be a consequence of the de-repression of an AF1 box 1 activity in HeLa cells, and of the stimulation of its contribution toward ERα activity in HepG2 cells (compare ERα with ERα Δ79 in Fig. 4A). Lastly, the overexpression of MKL1 variants internally deleted of B, Q, or SAP regions abolished the AF1 box 1 activity of ERα in HepG2 cells (data not shown). In HeLa cells, these deleted forms of MKL1 had a similar impact on ERα AF1 box 2/3 activity as full-length MKL1 (data not shown).

To confirm the role exerted by MKL1 in controlling the transactivation potency of ERα AF1 box 1, we used another system that relies on the response of the human complement C3 gene (C3) promoter to the agonist activity of 4-OHT. This response indeed depends exclusively upon ERα AF1 box 1 activity (9, 10, 26). Importantly, the overexpression of the dominant negative form of MKL1 (ΔC301) renders HeLa cells permissive to the AF-1 agonistic activity of 4-OHT (Fig. 4B). Correspondingly, the dominant positive MKL1 ΔN200 abolishes the C3 promoter response to 4-OHT in HepG2 cells (Fig. 4B). Interestingly, upon the overexpression of MKL1 ΔN200 in HeLa cells, ERα bound to 4-OHT represses the reporter gene activity. These results ultimately highlight the role of MKL1 in regulating ERα AF1 box 1 activity. Altogether, these results complete the demonstration that the Rho/actin/MKL1 signaling pathway plays a major role in modulating ERα transactivation efficiency through the modification of the respective activities of ERα AF subregions.

MKL1 Is Present on Estrogen-responsive Promoters in Vivo and Is a Potential ERα-interacting Protein

We next explored the hypothesis that MKL1 could regulate ERα activity in cells endogenously expressing the receptor. The ERα-positive breast carcinoma cell line MCF-7 was selected for this purpose. Firstly, the impact of transiently transfected forms of MKL1 on the transactivation efficiency of endogenous ERα was measured on the ERE-tk-LUC reporter. As shown in Fig. 5A, the constitutively active mutant MKL1 ΔN200 and MKL1, to a lesser extent, enhance the basal activity of the reporter gene measured in the absence of E₂. Importantly, the transcriptional activity of endogenous ERα is up- or down-regulated by dominant negative or dominant positive forms of MKL1, respectively. These results are in total agreement with those previously observed for transfected ERα in HepG2 and HeLa cells. As expected from their differentiated phenotype (E-cadherin-positive and vimentin-negative) (9), MCF-7 cells exhibited a cell context that was mainly permissive to the ERα AF1 box 1 activity (compare ERα with ERα Δ79 and ERα CF in Fig. 5B). The overexpression of MKL1 and MKL1 ΔN200 totally abolished this permissiveness, whereas the dominant negative form of MKL1 (ΔC301) enhanced it (Fig. 5B). These results clearly indicated that MCF-7 cells provide a similar context to HepG2 cells, in terms of ERα transactivation efficiency on an ERE-driven reporter gene. In contrast, the dedifferentiated ERα-negative breast cancer MDA-MB 231 cell line (MDA) was insensitive to AF1 box 1 activity, as HeLa cells (Fig. 5C). Importantly, the stable re-expression of ERα into these cells (MDA- ERα), which induced a more differentiated phenotype, restored an AF-1 box 1-sensitive cell context (Fig. 5C).

Having shown that MKL1 regulates ERα transcriptional activity, we next investigated whether MKL1 is present on endogenous estrogen-responsive promoters. We therefore performed ChIP experiments and sequential ChIP in MCF-7 cells to determine the (co)occupancy of several gene promoters with both ERα and MKL1. As shown in Fig. 5E, MKL1 was recruited to the promoter of all tested E₂-regulated genes (control reverse transcription-PCR within Fig. 5D), including pS2/TFF1 (trefoil factor 1), Wisp2 (WNT1-inducible signaling pathway protein 2), PR_A (progesterone receptor, A isoform), and SDF-1 (stromal cell derived factor-1) in the absence or presence of E₂. Furthermore, with the exception of PR_A, a co-recruitment of MKL1 and ERα may occur on the three other analyzed promoters, as revealed through sequential ChIP (Fig. 5E). However, the association of ERα and MKL1 on these three promoters seems to exhibit some differences respective to the treatment of the cells with E₂. For instance, MKL1 appears constitutively associated with pS2/TFF1 and Wisp2 promoters, in conjunction with ERα of which the recruitment is stimulated by E₂. In contrast, the recruitment of MKL1 on the SDF-1 promoter seems enhanced by E₂ and appears associated with ERα only in the presence of E₂. This reflects the strict E₂ dependence of ERα recruitment on this promoter. These results were partly confirmed on the MDA-ERα cells, which offer a different system to analyze the association of MKL1 with the promoter region E₂-regulated genes. However, differing from the MCF-7 cells, the recruitment of MKL1 on the pS2/TFF1 promoter appears to be enhanced by E₂. Among the tested genes, only SDF-1 was not expressed in these cells (Fig. 5D). Importantly, the lack of expression of SDF-1 in MDA-ERα cells is reflected by its inability to recruit MKL1 or ERα, likely illustrating a closed chromatin conformation (Fig. 5E). This provides an interesting additional control of the specificity of the signals observed. In conclusion, these data indicate that ERα and MKL1 can be co-recruited and are likely to cooperate on the promoter region of several E₂-regulated genes.