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. 2000 Dec;20(23):8667–8675. doi: 10.1128/mcb.20.23.8667-8675.2000

Regulation of the Functional Interaction between Cyclin D1 and the Estrogen Receptor

Justin Lamb 1, Mohamed H Ladha 1, Christine McMahon 1, Robert L Sutherland 2, Mark E Ewen 1,*
PMCID: PMC86475  PMID: 11073968

Abstract

We report that the functional interaction between cyclin D1 and the estrogen receptor (ER) is regulated by a signal transduction pathway involving the second messenger, cyclic AMP (cAMP). The cell-permeable cAMP analogue 8-bromo-cAMP caused a concentration-dependent enhancement of cyclin D1-ER complex formation, as judged both by coimmunoprecipitation and mammalian two-hybrid analysis. This effect was paralleled by increases in ligand-independent ER-mediated transcription from an estrogen response element containing reporter construct. These effects of 8-bromo-cAMP were antagonized by a specific protein kinase A (PKA) inhibitor, indicating that the signaling pathway involved was PKA dependent. Further, we show that culture of MCF-7 cells on a cellular substratum of murine preadipocytes also enhanced the functional interaction between cyclin D1 and ER in a PKA-dependent manner. These findings demonstrate a collaboration between cAMP signaling and cyclin D1 in the ligand-independent activation of ER-mediated transcription in mammary epithelial cells and show that the functional associations of cyclin D1 are regulated as a function of cellular context.

Cyclin D1 is well recognized as a critical mitogen-regulated cell cycle control element which, in association with a catalytic subunit, cyclin-dependent kinase 4 (cdk4) or cdk6, effects the initial inactivating phosphorylation of the retinoblastoma protein, pRb, and thereby promotes proliferation (67, 78). Consistent with this biochemical function, cyclin D1 is demonstrably oncogenic in a variety of tissues (28).

The cyclin D1 gene is amplified in approximately 30% of human breast adenocarcinomas, and the protein is reportedly overexpressed in 60 to 80% of all cases (5, 8, 13, 23, 24, 48, 55, 79). Paradoxically, these tumors are characterized by low proliferation indices (55) and are thereby discriminated from cancers of this tissue associated with pRb inactivation (35). Indeed, there is no apparent relationship between cdk4 activity and cyclin D1 expression in breast cancer cell lines (75). Consistent with these findings, there has been one report that ectopic expression of cyclin D1 in mammary carcinoma cell lines can actually inhibit proliferation (29). Taken together, these observations suggest that cyclin D1 possesses functions independent of, or in addition to, participation in pRb-mediated promotion of cell cycle progression during mammary carcinogenesis.

Cyclin D1 also plays a specific and indispensable part in normal mammary gland biology. Mice nullizygous for the cyclin D1 gene exhibit, among surprisingly few defects, a dramatic impairment of lobuloalveologenesis associated with pregnancy (68). Further, in vitro models of this developmental process reveal a marked induction of cyclin D1 in the absence of corresponding increases in associated kinase activity toward the formation of milk-secreting structures (52). Thus, cyclin D1 appears to possess an exceptional function in the mammary epithelium, involved in both the normal development and malignant transformation of this tissue.

An intimation of what this exceptional function of cyclin D1 might be is provided by the demonstration that cyclin D1 can bind to, and stimulate transcription mediated by, the estrogen receptor (ER) in both a cdk- and ligand-independent manner (52, 82). cdk-independent functions of cyclin D1 are not now unprecedented (6, 33). Since the majority of cyclin D1-overexpressing mammary tumors also express ER (7, 32, 63) and since activation of ER-dependent transcription is reported to closely parallel cyclin D1 induction during the terminal differentiation of normal mammary epithelial cells in vitro (52), it is tempting to speculate that ER and cyclin D1 operate together during organo- and carcinogenesis of the breast.

If ER is indeed an alternative, functionally relevant partner for cyclin D1 in the mammary gland, it would seem reasonable to suppose that the interaction between these two proteins be regulated. Here we report that 8-bromo-cyclic AMP (8b-cAMP) acts synergistically with cyclin D1 to enhance ligand-independent transcription from an estrogen response element (ERE) reporter in mammary epithelial cells. As a corollary to these findings, we show that 8b-cAMP can significantly and specifically enhance the in vivo association between cyclin D1 and ER in a protein kinase A (PKA)-dependent manner. Finally, we demonstrate that culture of breast epithelial cells on a cellular substratum of murine preadipocytes mimics the effects of 8b-cAMP treatment by enhancing the functional interaction between cyclin D1 and ER in a PKA-dependent manner. These findings demonstrate a collaboration between cAMP signaling and cyclin D1 in the ligand-independent activation of ER-mediated transcription in mammary epithelial cells and show that the functional associations of cyclin D1 are regulated as a function of cellular context. These observations suggest a model in which stromal-epithelial communication directs the function of cyclin D1 to effect specific aspects of mammary gland organo- and carcinogenesis.

MATERIALS AND METHODS

Cell culture and reagents.

MCF-7 human mammary epithelial carcinoma cells, murine NIH 3T3 fibroblasts, and murine 3T3-L1 preadipocytes (26) were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. The clonal MCF-7 derivative cell line containing stably integrated cyclin D1 cDNA under the control of a zinc-inducible metallothionein promoter, designated D1.13 (57), was maintained in DMEM supplemented with 10% FBS, antibiotics, and 2 μg of insulin per ml. Cyclin D1 expression was induced in these cells by treatment with zinc sulfate at a final concentration of 70 μM. Where estrogen-free conditions were required, cells were cultured in phenol red-free DMEM containing 5% charcoal-dextran stripped FBS (HyClone) and antibiotics. 17β-Estradiol and 8b-cAMP were from Sigma. H-89 was from Calbiochem.

Plasmids and transfection.

Plasmid expression vectors for carboxy-terminal hemagglutinin (HA)-tagged human cyclin D1 (pRc/CMV-cyclin D1-HA), an amino-terminal GAL4 DNA-binding domain cyclin D1 fusion protein (pCMX-GAL4-cyclin D1), human ERα (pcDNA3.1-hER), human p27 (pCMV5-p27), human cdk4 (pCMV-cdk4), the GAL4 DNA-binding domain (pCMX-GAL4), the VP16 activation domain (pCMX-VP16), and β-galactosidase (pCMV-β-gal) have been described previously (20, 46, 52, 56, 76). The estrogen response element and GAL4-binding-site luciferase reporter constructs, p(ERE)2-tk-luc and pGAL4-TATA-luc, have also been described elsewhere (36, 53). The plasmid directing the expression of an amino-terminal VP16 activation domain ERα fusion protein (pCMX-VP16-ER) was constructed as follows. An in-frame EcoRI site was introduced immediately upstream of the human ERα start codon in pcDNA3.1-FLAG-ER (46) by site-directed mutagenesis (Mutagene kit; Bio-Rad) using the oligonucleotide 5′-GTGGAGGGTCATGGTCATCGAATTCTTGTCATCGTCGTCCTT-3′. The EcoRI-BamHI insert from the resulting plasmid was ligated into pCMX-VP16 linearized with EcoRI and BamHI. Transfections were made with Superfect reagent (Qiagen) as specified by the manufacturer. The total amount of plasmid transfected was kept constant within a given experiment by the addition of empty vector (pRc/CMV).

Immunoprecipitation and Western blotting.

Whole-cell lysates were prepared in EBC200 (50 mM Tris-HCl [pH 8], 200 mM NaCl, 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 0.1 mM sodium orthovanadate) and clarified by centrifugation. Lysates were subjected to immunoprecipitation with mouse monoclonal anti-ER antibodies (AER314; NeoMarkers) plus rabbit anti-mouse immunoglobulins (Sigma), rabbit polyclonal anti-cdk4 (C-22; Santa Cruz) or anti-p27 (C-19; Santa Cruz) antibodies, and protein A-Sepharose beads. Immune complexes were washed four times with NETN (20 mM Tris-HCl [pH 8], 100 mM NaCl, 1 mM EDTA, 0.5% NP-40), boiled in Laemmli buffer, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes and visualized by using antibodies against the HA epitope (12CA5), ER (AER314), cdk4 (C-22), p27 (C-19), or cyclin D1 (Ab-3; NeoMarkers), appropriate peroxidase-conjugated secondary antibodies (Amersham), and enhanced chemiluminescence detection (Amersham).

Transcriptional activation assays.

Cells were transfected with p(ERE)2-tk-luc or pGAL4-TATA-luc and pCMV-β-gal and the specified expression plasmids. Various amounts of 8b-cAMP or 17β-estradiol were added 24 h later, and incubation was continued for a further 24 h. For coculture experiments, transfected MCF-7 cells were trypsinized and replated on confluent cultures of NIH 3T3 fibroblasts or 3T3-L1 preadipocytes or on culture plastic. Where indicated, 8b-cAMP was added 24 h later and incubation was continued for a further 24 h. All ER transactivation assays were conducted under estrogen-free conditions. Luciferase and β-galactosidase activities were measured as described previously (17), and luciferase activities were corrected with respect to the corresponding β-galactosidase internal control.

RESULTS

The physical interaction between cyclin D1 and ER is regulated.

Since the functional associations between cyclin D1 and cdk4 or cdk6 are tightly regulated processes (38, 44, 47), we wondered if the cyclin D1-ER interaction was similarly controlled. This possibility was tested by cotransfecting MCF-7 cells with plasmids directing the expression of human ERα and HA-tagged human cyclin D1. We discovered that treatment of these cells with the cell-permeable cAMP analogue 8b-cAMP resulted in a marked, concentration-dependent increase in the amount of cyclin D1-HA coimmunoprecipitated with ER (Fig. 1A). The total level of cyclin D1-HA was unaffected by 8b-cAMP (Fig. 1A). Increases in the amount of cyclin D1-HA associated with ER were apparent by 3 h, although 16 h of treatment was required for the maximum effect (Fig. 1B). Mammalian two-hybrid analyses also revealed an enhancement of the cyclin D1-ER interaction in the presence of 8b-cAMP (see Fig. 5B). The ability of 8b-cAMP to enhance the association between cyclin D1 and ER was unaffected by the presence or absence of added estrogen (data not shown).

FIG. 1.

FIG. 1

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The physical interaction between cyclin D1 and ER is enhanced by 8b-cAMP in a concentration- and PKA-dependent manner. MCF-7 cells were transfected with pRc/CMV-cyclin D1-HA (6.9 μg) and pcDNA3.1-hER (5.8 μg). (A) At 24 h later, various amounts of 8b-cAMP or vehicle control were added. Incubation was continued for a further 24 h. Whole-cell lysates were prepared, and cyclin D1-ER complex formation was analyzed by immunoprecipitation with ER antibodies (AER314) followed by Western blotting for cyclin D1-HA using 12CA5 antibody against the HA epitope (middle panel). The relative amounts of ER precipitated (top panel) and cyclin D1-HA present in the whole-cell lysates (WCL; bottom panel) were assessed by Western blotting with the same anti-ER and anti-HA antibodies. (B) 8b-cAMP (100 μM) or vehicle control was added at the indicated times before cell lysis. The total posttransfection incubation time was 48 h. Lysates were analyzed as described for panel A. (C) At 24 h after transfection, various amounts of H-89 or vehicle control were added, immediately followed by 8b-cAMP (100 μM) or vehicle control. Incubation was continued for a further 24 h. Lysates were analyzed as described for panel A.

FIG. 5.

FIG. 5

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Coculture with murine preadipocytes, but not fibroblasts, enhances the physical interaction between cyclin D1 and ER. (A) MCF-7 cells were transfected as described for Fig. 1 before being trypsinized and replated on confluent cultures of NIH 3T3 fibroblasts or 3T3-L1 preadipocytes (lanes 3 and 7, respectively) or culture plastic (lanes 2 and 6). At 24 h later, whole-cell lysates (WCL) were prepared from each. Whole-cell lysates were also prepared from NIH 3T3 or 3T3-L1 cells cultured in isolation (lanes 1 and 5, respectively). For mixing experiments, lysates of transfected MCF-7 cells grown on plastic were combined with those from NIH 3T3 or 3T3-L1 cells cultured in isolation and rocked for 60 min at 4°C (lanes 4 and 8, respectively). Cyclin D1-ER complex formation was assessed in all lysates by immunoprecipitation with ER antibodies (AER314) followed by Western blotting for cyclin D1-HA using 12CA5 antibody against the HA epitope (middle panel). The relative amounts of ER precipitated (top panel), and cyclin D1-HA present in the whole-cell lysates (bottom panel) were assessed by Western blotting with the same anti-ER and anti-HA antibodies. (B) Mammalian two-hybrid analysis. MCF-7 cells were transfected with pGAL4-TATA-luc, pCMV-β-gal, pCMX-GAL4 or pCMX-GAL4-cyclin D1, and pCMX-VP16 or pCMX-VP16-ER (each at 0.25 μg) before being trypsinized and replated on confluent cultures of NIH 3T3 fibroblasts, 3T3-L1 preadipocytes, or culture plastic. At 24 h later, 8b-cAMP was added to a final concentration of 100 μM as indicated, and incubation was continued for a further 24 h. Luciferase and β-galactosidase activities were determined, and normalized fold activations were calculated relative to the corrected luciferase activity resulting from expression of GAL4-cyclin D1 and VP16 under each culture condition. Results are means and standard deviations from three independent experiments each performed in triplicate. (C) Cocultures were prepared as described for panel A, except that H-89 (50 nM) was added as soon as the cells had become attached (lanes 3 and 5), immediately followed by 8b-cAMP (100 μM) where indicated (lane 2). At 24 h later, whole-cell lysates were prepared and analyzed as described for panel A. (D) MCF-7 cells were transfected with pRc/CMV-cyclin D1-HA (6.9 μg) and pCMV-cdk4 or pcDNA3.1-hER (each at 2.9 μg) before being trypsinized and replated on confluent cultures of NIH 3T3 fibroblasts or 3T3-L1 preadipocytes. At 24 h later, whole-cell lysates were prepared, and complex formation between cyclin D1-HA and cdk4 or ER was assessed by immunoprecipitation with antibodies against cdk4 (C-22; lanes 1 and 3) or ER (AER314; lanes 2 and 4) followed by Western blotting with the same antibodies or the 12CA5 antibody against the HA epitope of cyclin D1-HA. The relative amounts of cyclin D1-HA present in the whole-cell lysates (bottom panel) were assessed by Western blotting with the same anti-HA antibodies.

Many of the known cellular functions of cAMP and its analogues are mediated by PKA. To test the participation of PKA in the regulation of the association between cyclin D1 and ER, MCF-7 cells were transfected as described above and treated with 8b-cAMP (100 μM). These cells were then exposed to a range of concentrations of the highly specific PKA inhibitor H-89, and cyclin D1-ER complex formation was analyzed by coimmunoprecipitation. Treatment with H-89 at 50 nM and above completely abolished the effects of 8b-cAMP (Fig. 1C). Given that the reported Ki of this compound for PKA is 48 nM (11), these data suggest that the influence of 8b-cAMP on the interaction between cyclin D1 and ER is indeed mediated by PKA. Consistent with this observation, a number of other agents that stimulate this pathway—cholera toxin (1 μg/ml), forskolin (50 μM), and isobutylmethyxanthine (1 mM)—also enhanced the cyclin D1-ER interaction (data not shown).

Treatment with 8b-cAMP reveals an interaction between endogenous ER and cyclin D1.

The ability of 8b-cAMP treatment to enhance the physical interaction between ectopically expressed cyclin D1 and ER was such that we investigated the possibility that 8b-cAMP might also stimulate an interaction between the endogenous proteins. The clonal MCF-7 derivative cell line D1.13 expresses both endogenous ER and cyclin D1 but also exhibits modest zinc-inducible expression of exogenous cyclin D1 from a stably integrated metallothionein promoter construct (57). We reasoned that this cell system would allow us to assess the ability of 8b-cAMP to promote the formation of complexes between endogenous ER and cyclin D1 when its expression was induced to an extent comparable to the increases seen during mammary epithelial cell differentiation in vitro (52) and pregnancy-induced mammary gland development in vivo (42). Treatment of D1.13 cells with zinc sulfate did indeed increase cyclin D1 levels by about twofold relative to a cdk4 loading control (Fig. 2A), much as reported elsewhere (57). Anti-ER immunoprecipitation followed by Western blotting with cyclin D1 antibodies revealed that this modest increase was sufficient to induce a detectable interaction between endogenous ER and cyclin D1 when these D1.13 cells were treated with 8b-cAMP before being subjected to zinc induction (Fig. 2B). This finding suggests that activation of a PKA-dependent signaling pathway can promote a physical interaction between ER and cyclin D1 even when both proteins are expressed at physiological levels.

FIG. 2.

FIG. 2

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Treatment with 8b-cAMP reveals a physical interaction between endogenous ER and cyclin D1. (A) D1.13 cells were treated with zinc sulfate (70 μM) or vehicle control, and incubation was continued for a further 18 h. Whole-cell lysates (WCL) were prepared and analyzed by Western blotting with anti-cyclin D1 (Ab-3) and anti-cdk4 (C-22) antibodies. (B) D1.13 cells were treated with 8b-cAMP (100 μM) followed 6 h later by zinc sulfate (70 μM) or vehicle control. Incubation was continued for a further 18 h before whole-cell lysates were prepared, and cyclin D1-ER complex formation was analyzed by immunoprecipitation with ER antibodies (AER314) followed by Western blotting for cyclin D1 (Ab-3). The relative amounts of ER precipitated (top panel) and cyclin D1 present in the lysates (bottom panel) were assessed by Western blotting with the same anti-ER and anti-cyclin D1 antibodies.

The effect of 8b-cAMP is specific for the cyclin D1-ER interaction.

Next, we determined if 8b-cAMP could affect the association between cyclin D1 and two of its other protein partners, cdk4 and p27, and whether p27 would promote the assembly of cyclin D1 with ER, as it does for cyclin D1 with cdk4 (10, 38). MCF-7 cells were transfected with plasmids directing the expression of ER, p27, or cdk4, and cyclin D1-HA. These cells were then treated with 8b-cAMP (100 μM), and the effect on each protein-protein interaction was assessed by coimmunoprecipitation with antibodies specific for ER, cdk4, or p27. As is clear from Fig. 3, while the interaction between cyclin D1 and ER was significantly enhanced by 8b-cAMP treatment (lanes 7 and 8), cyclin D1-p27 complex formation was unaffected (lanes 5 and 6). Similarly, when cyclin D1-HA, cdk4, and p27 expression plasmids were cotransfected, 8b-cAMP had no influence on the amount of cyclin D1-HA immunoprecipitated with cdk4 (lanes 3 and 4).

FIG. 3.

FIG. 3

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The effect of 8b-cAMP is specific for the cyclin D1-ER interaction. MCF-7 cells were transfected with pRc/CMV-cyclin D1-HA (6.9 μg) and pCMV-cdk4, pCMV5-p27, pcDNA3.1-hER, or pRc/CMV (each at 2.9 μg) in the combinations indicated. At 24 h later, 8b-cAMP (100 μM) or vehicle control was added and incubation was continued for a further 24 h. Whole-cell lysates were prepared, and the formation of complexes between cyclin D1-HA, cdk4, p27, and ER was assessed by immunoprecipitation with antibodies against cdk4 (C-22; lanes 1 to 4), p27 (C-19; lanes 5 and 6), or ER (AER314; lanes 7 to 10) followed by Western blotting with the same antibodies or the 12CA5 antibody against the HA epitope of cyclin D1-HA.

Interestingly, the cyclin D1-cdk4 interaction was undetectable in the absence of transfected p27 plasmid (Fig. 3, compare lanes 1 and 2 with lanes 3 and 4). This observation is consistent with the reported function of p27 as an assembly factor for the cyclin D1-cdk4 complex (10, 38). However, cotransfection of p27 plasmid with ER and cyclin D1-HA expression vectors did not enhance the physical association between cyclin D1 and ER (compare lanes 7 and 9). If anything, ectopic expression of p27 reduced the 8b-cAMP-mediated increase in cyclin D1-ER complex formation (compare lanes 8 and 10), presumably by sequestering the cyclin in a ternary complex with cdk4. Further, we note that neither endogenous or exogenous p27 nor cdk4 is found together with the cyclin D1 coimmunoprecipitated with ER (lanes 7 to 10 and data not shown). These observations suggest that the complexes cyclin D1 forms with ER are distinct from those formed with cdk4 and p27.

Together, these data reveal that the physical interaction between cyclin D1 and ER is regulated differently from that between cyclin D1 and cdk4 and that the influence of 8b-cAMP is restricted to promotion of cyclin D1-ER complex formation.

Cyclin D1 and 8b-cAMP act synergistically to stimulate ligand-independent activation of ER.

Since cyclin D1 can stimulate the transcriptional function of ER in the absence of ligand (52, 82), we tested whether 8b-cAMP-mediated enhancement of the physical interaction between these two proteins would affect ER-dependent transcription. MCF-7 cells were transfected with cyclin D1-HA plasmid or vector control, together with an ER expression plasmid and an ERE-luciferase reporter construct. The addition of 8b-cAMP in the absence of cyclin D1 plasmid resulted in a modest increase in ligand-independent ER-mediated transactivation, as did the expression of cyclin D1 in the absence of 8b-cAMP, much as reported elsewhere (2, 16, 52, 82). However, 8b-cAMP exhibited a marked, concentration-dependent synergy with cyclin D1 in the activation of ER (Fig. 4). The extent of this activation not only was greater than that reported to date (52, 82) but also approximated that seen with 17β-estradiol, a well-recognized ER ligand.

FIG. 4.

FIG. 4

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Cyclin D1 and 8b-cAMP act synergistically to stimulate ligand-independent ER-mediated transcription. MCF-7 cells, cultured under estrogen-free conditions, were transfected with p(ERE)2-tk-luc (0.1 μg), pcDNA3.1-hER (0.1 μg), and pCMV-β-gal (0.25 μg), together with either pRc/CMV-cyclin D1-HA or pRc/CMV (each at 0.1 μg). At 24 h later, various amounts of 8b-cAMP, vehicle control, or 17β-estradiol (10 nM) were added, and incubation was continued for a further 24 h. Luciferase and β-galactosidase activities were determined, and normalized fold activations were calculated relative to the corrected luciferase activity in the absence of cyclin D1, 8b-cAMP, or estradiol. Results are means and standard deviations from three independent experiments each performed in triplicate.

A strict correlation between the amount of cyclin D1 coprecipitated with ER and the magnitude of the cyclin D1-dependent ER activation as a function of 8b-cAMP was observed (Fig. 1A and 4). This suggests that the extent of ER-mediated transcription from an ERE-containing promoter is, at least in this context, determined by the amount of the cyclin subunit bound to the receptor.

Taken together, these data indicate that the functional interaction between cyclin D1 and ER is subject to regulation by a specific, PKA-dependent pathway. These data also reveal that the extent of ER-mediated transcription activated by cyclin D1 can be greatly in excess of that previously thought, but only when given the appropriate signal.

Stromal-epithelial communication affects the functional interaction between cyclin D1 and ER.

The interactions of epithelial parenchyma and mesenchymal stroma have long been appreciated as critical to determination of the structure and normal function of the mammary gland (for reviews, see references 31, 59, and 62). The importance of the cellular microenvironment to ductal and alveolar morphogenesis, epithelial-cell differentiation, and tissue-specific or hormone-dependent gene expression can also be demonstrated in vitro (1, 4). For example, primary mouse mammary epithelial cells can undergo functional differentiation to form ductal and alveolar-type structures and can recapitulate the ability to synthesize milk proteins in response to lactogenic hormones exhibited in situ when cocultured with murine adipocytes or preadipocytes (40, 80). Because cAMP has been implicated in the transduction of intercellular signals (19, 54, 70) and since cyclin D1-mediated activation of ER-dependent transcription has been associated with extracellular matrix-dependent terminal differentiation of normal mammary epithelial cells in vitro (52), we used a preadipocyte coculture system to study the influence of stromal-epithelial communication on the cyclin D1-ER interaction and the possible involvement of PKA.

MCF-7 cells were transfected with expression plasmids encoding ER and HA-tagged cyclin D1, trypsinized, and replated on confluent cultures of mouse 3T3-L1 preadipocytes or NIH 3T3 fibroblasts. The physical interaction between cyclin D1 and ER in the epithelial (MCF-7) cells was then assessed by coimmunoprecipitation and Western blotting with an anti-HA antibody. This analysis revealed that culture on a cellular substratum of preadipocytes, but not fibroblasts, resulted in a marked enhancement of cyclin D1-ER complex formation (Fig. 5A, compare lanes 3 and 7). A fully differentiated adipocyte substratum also promoted cyclin D1-ER assembly (data not shown).

Although the absence of detectable ER in either mouse cell type and the use of an epitope-tagged cyclin D1 expression construct excluded the possibility that the effects observed were attributable to the participation of murine cyclin D1 and/or ER, this method could not discount the possibility that a protein present in either the fibroblasts or the preadipocytes could affect cyclin D1-ER complex formation once the cell mixtures had been lysed. Mixing lysates of 3T3-L1 and MCF-7 cells grown in isolation did not mimic the effect of coculture on cyclin D1-ER complex formation (Fig. 5A, compare lanes 7 and 8), but we decided, nonetheless, to use mammalian two-hybrid analyses to directly measure the interaction occurring within the epithelial cells.

MCF-7 cells were transfected with plasmids directing the expression of GAL4-cyclin D1 and VP16-ER hybrid proteins and a GAL4-luciferase reporter construct, trypsinized, and plated on confluent cultures of preadipocytes or fibroblasts or on cell culture plastic. The cyclin D1-ER interaction under each condition was then assessed by a luciferase assay (Fig. 5B). Consistent with coimmunoprecipitation data (Fig. 5A), culture on a cellular substratum of 3T3-L1 preadipocytes, but not NIH 3T3 fibroblasts or culture plastic, or following treatment with 8b-cAMP (Fig. 1A) resulted in a significant enhancement of cyclin D1-ER complex formation (Fig. 5B).

The magnitude of the cyclin D1-ER interaction induced by preadipocyte coculture was comparable to that associated with 8b-cAMP treatment (Fig. 5B and C). To establish whether the influence of this cellular microenvironment was also qualitatively similar to that of 8b-cAMP treatment in being PKA dependent, the effect of H-89 on cyclin D1-ER assembly induced by preadipocyte coculture was tested. As shown in Fig. 5C, treatment with the PKA inhibitor at a concentration sufficient to block the effect of 8b-cAMP (50 nM; compare lanes 2 and 3) could also at least partially suppress the increase in the cyclin D1-ER interaction induced by coculture with preadipocytes (compare lanes 4 and 5).

It was also of interest to determine whether, like 8b-cAMP treatment, preadipocyte coculture was without a discernible effect on the interaction between cyclin D1 and cdk4. MCF-7 cells were cotransfected with expression plasmids for cyclin D1-HA and ER or cdk4 before being plated on either fibroblast or preadipocyte cultures. Immunoprecipitation with antibodies specific for ER or cdk4 revealed that neither coculture condition resulted in a detectable cyclin D1-cdk4 complex (compare Fig. 3), although, as before, the preadipocyte substratum markedly enhanced the interaction between cyclin D1 and ER (Fig. 5A and C).

Together, these data suggest that the stromal-epithelial communication promoting cyclin D1-ER assembly is rather specific for this complex and is mediated by PKA. It is likely that 8b-cAMP acts by stimulating this pathway.

Given the correlation between the magnitudes of the cyclin D1-ER interaction and cyclin D1-dependent ER-mediated transcription (Fig. 1 and 4) we tested whether coculture with preadipocytes would also result in an increase in ER transactivation in the epithelial cells. As a direct parallel of our coimmunoprecipitation and mammalian two-hybrid data (Fig. 5), culture of MCF-7 cells on a preadipocyte substratum resulted in a significant enhancement of ERE-luciferase reporter activity to levels comparable to those induced by 8b-cAMP (Fig. 6). These data indicate that the transcriptional activation of ER by cyclin D1 is significantly greater when epithelial cells are cultured in a cellular microenvironment designed to mimic that of the mammary gland in vivo.

FIG. 6.

FIG. 6

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Coculture with preadipocytes enhances ligand-independent ER-mediated transcription induced by cyclin D1. MCF-7 cells, cultured under estrogen-free conditions, were transfected with p(ERE)2-tk-luc (0.1 μg), pcDNA3.1-hER (0.1 μg), and pCMV-β-gal (0.25 μg), together with either pRc/CMV-cyclin D1-HA or pRc/CMV (each at 0.1 μg) before being trypsinized and replated on confluent cultures of NIH 3T3 fibroblasts, 3T3-L1 preadipocytes, or culture plastic. At 24 h later, 8b-cAMP was added to a final concentration of 100 μM as indicated and incubation was continued for a further 24 h. Estrogen-free conditions were maintained throughout. Luciferase and β-galactosidase activities were determined, and normalized fold activations were calculated relative to the corrected luciferase activity in the absence of cyclin D1 under each culture condition. Results are means and standard deviations from three independent experiments each performed in triplicate.

DISCUSSION

Our data show that the physical interaction between cyclin D1 and ER is markedly and specifically enhanced, in a concentration-dependent manner, by the cAMP analogue 8b-cAMP. The sensitivity of this effect to a specific inhibitor of PKA and the fact that a number of agents known to activate adenylate cyclase mimic the influence of 8b-cAMP suggest that cyclin D1-ER complex formation is regulated by a PKA-dependent signaling pathway.

The functional consequence of activating this regulatory pathway is revealed by our demonstration that 8b-cAMP exhibits a strong, concentration-dependent synergy with cyclin D1 for ligand-independent activation of ER transcription. The level of transactivation approached that associated with estrogen treatment, indicating that maximal activation of ER can be achieved by cyclin D1 overexpression, provided that the appropriate cellular signals are present.

The candidacy of cAMP as an important second messenger in mammary gland biology is substantially supported by in vivo data. It has been known for many years that intracellular cAMP levels increase markedly in the mammary gland during pregnancy in the mouse (58), rat (60, 61), and guinea pig (41) and are persistently elevated in rat and human mammary tumors (12, 27, 34, 37, 51), as is PKA activity (21, 25, 43, 65). It is also now appreciated that cAMP-binding proteins—especially the RIα regulatory subunit of PKA (type I)—are overexpressed in a significant proportion of primary human breast tumors and that this feature has independent diagnostic and prognostic significance, being correlated with poor clinicopathology and outcome (49, 50).

Perhaps the most compelling evidence for an important role for cAMP in normal mammary development is provided by the observations that systemic administration of cholera toxin, an activator of adenylate cyclase, potently stimulates lobuloalveologenesis in the mouse and, together with estrogen and progesterone, causes mammary gland development closely resembling that seen during pregnancy (66, 69). Indeed, in a classical text of mammary gland biology, Daniel and Silberstein conclude that cAMP probably “serves as an intracellular effector of as yet unidentified mammogens” and highlight the importance of this “as yet unidentified, cAMP-mediated pathway” (14).

In vitro studies not only appear to complement these in vivo findings but also implicate ER in the cellular response to increases in cAMP levels, showing that both cholera toxin and 8b-cAMP can stimulate ligand-independent transcription from an ERE reporter (2) and of estrogen-responsive genes, LIV-1 and pS2 (16).

The means by which cAMP induces ER-mediated transactivation are unknown. However, given that (i) cyclin D1 can also stimulate ligand-independent activation of ER-mediated transcription (52, 82), (ii) increases in its levels during pregnancy (42, 71) and in tumors (5, 23, 24) broadly parallel those in the levels of intracellular cAMP and PKA activity, and (iii) artificial elevation of cAMP levels in the mammary gland (66, 69) brings about the developmental changes (i.e., alveologenesis) characteristically defective in the cyclin D1−/− mammary phenotype (68), we believe that the discovery that cyclin D1 and 8b-cAMP exhibit a marked synergy in ER activation highlights a cooperativity between cyclin D1 and cAMP signaling in mammary organo- and carcinogenesis.

The cooperativity between cyclin D1 and cAMP may simply reside in positive regulation of the physical interaction between cyclin D1 and ER. Consistent with this hypothesis, we found that the level of ER activity closely correlated with the amount of cyclin subunit bound to the receptor. Cyclin D1 can associate with P/CAF and SRC-1 and can recruit these transcriptional coactivators to unliganded ER (46, 81). Therefore, it is likely that increases in the cyclin D1 content and hence in the coactivator content of the ER transactivation complex account for the influence of cAMP and its analogues on ER-mediated transcription.

It remains to be determined how PKA-dependent signals promote the functional interaction between cyclin D1 and ER. Cyclin D1 has long been recognized as being subject to posttranslational modification (45), and both cyclin D1 and ER are reportedly phosphorylated, at multiple sites, by PKA (9, 39, 64). However, preliminary experiments indicate that cyclin D1 is the sole target of this pathway, although none of the published cyclin D1 phosphorylation sites appear important for the interaction (data not shown). An alternative model implicates an as yet unidentified inhibitor of cyclin D1-ER complex formation as the ultimate target of PKA.

Our demonstration that the functional interaction between cyclin D1 and ER is significantly enhanced in mammary epithelial cells when cultured on particular cellular substrata highlights the potential biological significance of a regulatory pathway controlling cyclin D1-ER assembly. Fibroblasts were unable to promote assembly, while preadipocytes or adipocytes, which approximate the normal supporting connective tissue in vivo, were markedly competent in this regard. The parallels between these findings and others in the literature are striking. Primary murine mammary epithelial cells form branching ductal and alveolar-type structures when cocultured with preadipocytes and adipocytes but not with fibroblasts (80). Also, such epithelial cells acquire a basal lamina, cellular polarity, and secretory organelles and synthesize milk proteins in response to lactogenic hormones only when provided with a microenvironment of adipocytes or preadipocytes (40, 80).

Since the specific cellular substrata required to support cytodifferentiation and secretory differentiation also promote cyclin D1-ER function and since cyclin D1-mediated activation of ER-dependent transcription has been associated with extracellular matrix-dependent differentiation of normal mammary epithelial cells (52), it is tempting to speculate that these observations are related. It is possible that the cellular microenvironment of the mammary gland “specifies” the cyclin D1-ER interaction and that this complex accounts for the exceptional, tissue-specific function of cyclin D1 (i.e., alveologenesis) revealed by the cyclin D1 nullizygous mouse (68).

A recent demonstration that cyclin E expressed in the mouse under the control of the natural cyclin D1 promoter (“cyclin E→D1 knock-in”) can rescue the mammary development defect associated with cyclin D1 nullizygosity (22) has cast doubt on the notion that cyclin D1 has any exceptional activity whose contribution is required for the complete development of a mammary gland. However, while the neurological and retinal defects of the cyclin D1−/− mouse appear to be fully reversed by knock-in cyclin E, only a subset (35%) of cyclin E→D1 females were able to nurse their pups normally (22). This discrepancy might imply that cyclin D1 does indeed exert a function unique to the mammary gland for which cyclin E cannot substitute. It is conceivable that cyclin D1 must contribute both a proliferative influence—which can be approximated by cyclin E—and a cdk-independent ER-mediated action for the full and reliable development of a functional mammary gland.

Disruption of the stromal environment has now been convincingly shown to promote the phenotypic conversion and malignant transformation of mammary epithelial cells (72). Conversely, suppression of the aberrant transduction of signals from the extracellular matrix can cause reversion of the malignant phenotype of human breast cancer cells (77). Whether substratum-directed promotion of cyclin D1-ER complex formation contributes to mammary tumorigenesis is currently unknown. However, it is an intriguing possibility that the nature of cyclin D1-mediated oncogenesis could be modified by stromal influences.

The effects of a preadipocyte substratum and 8b-cAMP could be independent or could operate through a common signaling pathway. The observations that enhancement of the cyclin D1-ER interaction afforded by coculture can be at least partially antagonized by a PKA inhibitor and that treatment with 8b-cAMP does not further enhance coculture-induced assembly (data not shown) would appear to argue for the latter possibility. Indications that cAMP signaling is integral to the transduction of stromal-epithelial communications (19, 54, 70) also support this notion, but the question remains open.

Lethal irradiation of the preadipocyte layer did not block the enhancement of cyclin D1-ER complex formation (data not shown) or the promotion of epithelial-cell differentiation in the coculture experiments described previously (40, 80). These findings suggest that the effects are mediated by direct cell-cell contacts or through the formation of a basement membrane. This has indeed been shown to be true for the functional differentiation of mammary epithelial cells, with the communication involving integrins, laminin (18, 73, 74), and β-1,4-galactosyltransferase (3, 30). Whether these elements also transduce signals that promote cyclin D1-ER complex formation or whether other participants in intercellular communication, such as the Wnt proteins (15), are involved has yet to be determined. Future work will seek to elucidate the signaling pathways from the cell surface, through PKA, to cyclin D1-ER assembly.

ACKNOWLEDGMENTS

We thank Joan Massague for the human p27 expression plasmid (pCMV5-p27), Bruce Spiegelman for 3T3-L1 cells, Ron Evans for pCMX-GAL4 and pCMX-VP16, and Donald McDonnell for pGAL4-TATA-luc.

This work was supported by grants from the Massachusetts Department of Public Health Breast Cancer Research Program (to J.L.), the National Health and Medical Research Council of Australia (to R.L.S.), the New South Wales Cancer Council (to R.L.S.), the National Institutes of Health (PO1 CA80111) (to M.E.E.), and Novartis Pharmaceutical Corp. (to M.E.E.). J.L. is a Suzanne Sheats Breast Cancer Research Fellow. M.E.E. is a Scholar for the Leukemia and Lymphoma Society of America.

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