Regulation of Estrogen Receptor Activation of the Prolactin Enhancer/Promoter by Antagonistic Activation Function-2-Interacting Proteins

Fred Schaufele

Metabolic Research Unit University of California San Francisco, California 94143-0540


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional responses to estrogens are controlled by the cell- and gene-specific interactions of the nuclear estrogen receptor (ER) with cofactors and other transcription factors. The pituitary-specific PRL enhancer/promoter is regulated by estrogens only when it is bound by both ER and the pituitary-specific transcription factor, Pit-1. Cooperative ER/Pit-1 activation of the dormant PRL enhancer/promoter in pituitary progenitor cells requires the estrogen-dependent activation function-2 (AF-2) of ER, but is inhibited by one AF-2-interacting cofactor, RIP140. Here, the complex actions of RIP140 and other AF-2-interacting proteins at the PRL enhancer/promoter were shown to operate via ER itself. RIP140 inhibition of ER/Pit-1 activation in the absence of AF-1 and RIP140 inhibition of both ER{alpha} and ERß cooperative activation with Pit-1 suggested a conserved ER site for RIP140 action, possibly AF-2. Coexpression of other AF-2-interacting proteins, including the p160 factors, steroid receptor coactivator-1a (SRC-1a) and glucocorticoid receptor interacting protein-1 (GRIP1), had negligible effects on ER{alpha}/Pit-1 cooperative activation, but partially relieved RIP140 inhibition. Relief of RIP140 inhibition required the AF-2-binding, LXXLL motifs in SRC-1a and GRIP1. An ER AF-2 mutant that selectively blocked ER interaction with p160s, but not RIP140, still cooperated with Pit-1 and was inhibited by RIP140, but was not relieved by SRC-1a or GRIP1 expression. Thus, SRC-1a and GRIP1 binding to AF-2 counteracted the inhibition of ER/Pit-1 activation by another AF-2-interacting protein, RIP140. Complex, sometimes antagonistic, actions of different classes of AF-2-interacting proteins may play an important role in the cell- and gene-specific estrogen regulation of PRL and other genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogens regulate cellular response, differentiation, and proliferation in a variety of tissues. The postmenopausal reduction in estrogen synthesis is associated with significant declines in bone and cardiovascular health, which can be countered via estrogen replacement therapy. However, estrogen therapy is also associated with a higher estrogen-mediated risk of breast and uterine cancer. The development of estrogen analogs that mimic only the beneficial effects of estrogens in selected tissues would significantly improve hormone replacement therapy and breast cancer treatment. To do so, it will be necessary to understand the molecular nature of the tissue-specific estrogen responses.

Many estrogen responses are mediated by the estrogen receptor (ER), a DNA-binding transcription factor of the nuclear receptor superfamily that generally is more effective at promoting transcription when bound by estrogens (1, 2, 3, 4, 5, 6). ER activation is associated with the estrogen-dependent interaction of certain receptor-interacting proteins (RIPs) with activation function-2 (AF-2) (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), which is conserved in the carboxyl-terminal ligand-binding domain (LBD) of ER and most nuclear receptors (20). A separable amino-terminal domain of ER contains the more poorly conserved AF-1, which in most promoter contexts is dormant unless released by agonist binding to the LBD (2, 4, 21, 22). Ligand dependence of AF-1 may be related to relief of corepression at many nuclear receptors (23, 24, 25, 26, 27) or may be associated with the cobinding of the same receptor interacting coactivator to AF-1 and AF-2 (28, 29, 30).

AF-1 and AF-2 have been described mostly in studies of ER action at short, isolated DNA sequences to which ER binds. Estrogen regulation of natural genes is likely to use many of the same ER activities in conjunction with the activities provided by other promoter-binding factors. Synthesis of PRL declines with age in females and is strongly regulated by estrogens. Estrogen activation of pituitary-specific PRL gene transcription provides an excellent model in which to study cell-specific modulation of ER action by other promoter-binding factors.

ER regulates PRL gene transcription only if the PRL enhancer/promoter is bound by the pituitary-specific transcription factor Pit-1 (31, 32, 33, 34, 35). This cooperative activation by ER and Pit-1 explains why estrogen regulation of PRL gene transcription is pituitary specific. ER/Pit-1 cooperative activation requires AF-2 (35). However, RIP140, a 140-kDa cofactor that interacts with AF-2 of ER and a number of nuclear receptors in a ligand-dependent fashion (18), inhibits ER/Pit-1 activation (35). RIP140 may inhibit any of the PRL enhancer/promoter-binding proteins including ER. Understanding how RIP140 and other AF-2-interacting proteins modulate AF-2-dependent ER/Pit-1 cooperative activation will be crucial for understanding cell-specific estrogen regulation of gene transcription.

In the present study, it was observed that RIP140 inhibition of ER/Pit-1 PRL enhancer/promoter activation operated completely via ER. Within ER, AF-1 was not required for RIP140 inhibition. RIP140 inhibition of the PRL enhancer/promoter activated by Pit-1 and either of the {alpha}- or ß-isoforms of ER suggested a conserved site for RIP140 action, possibly AF-2. Relief of RIP140 inhibition by the coexpression of other AF-2-interacting factors supported AF-2 as the target of RIP140 action. Disrupting interaction of those counteracting factors with AF-2 blocked their ability to relieve RIP140 inhibition. Thus, AF-2 dependent promoter- and cell-specific ER regulation of the PRL enhancer arises from the cooperative and antagonistic interactions of Pit-1, ER, and AF-2-interacting proteins docking directly to ER AF-2.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
p160 Coactivator Regulation of ER/Pit-1 Activation
We previously studied ER regulation of the rat PRL enhancer/promoter attached to the luciferase gene in transiently transfected mouse pituitary progenitor GHFT1–5 cells (34, 35). GHFT1–5 cells are ideal for studying cell-specific PRL enhancer/promoter activation because they are derived from an embryonic pituitary transformed immediately after the onset of low level Pit-1 expression (36) but before commitment to the PRL, GH, or TSH cell types that derive from the Pit-1-containing progenitor (37).

In GHFT1–5 cells, PRL enhancer/promoter activity was low unless an expression vector for the cDNA of Pit-1 was cotransfected (34, 35) (Fig. 1AGo). Expression of the cDNA for the {alpha}-isoform of human ER (ER{alpha}) had a marginal effect on PRL enhancer/promoter activity. In contrast, activation was substantial when ER{alpha} and Pit-1 were coexpressed, averaging 25.1 ± 5.1-fold activation over the uninduced PRL enhancer/promoter.



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Figure 1. RIP140 Is a Dose-Dependent Inhibitor of the Cooperative Activation of the 3-kb Rat PRL Enhancer/Promoter by Human ER and Rat Pit-1 Tagged with the FLAG Epitope in Mouse GHFT1–5 Pituitary Progenitor Cells

A, ER/Pit-1 cooperative activation was not affected by the expression of SRC-1a, GRIP1, TIF1{alpha}, or CBP, but was inhibited by RIP140 coexpression. B, Titration of RIP140 inhibition by cotransfection with 1, 3, or 10 µg RIP140 expression vector. C, Representative Western blot of an experiment in B, probed for ectopically expressed human ER (blotted with human-specific anti-ER antibody) or FLAG-Pit-1 expression (blotted with anti-FLAG antibody) demonstrated that RIP140 inhibited PRL promoter activity, not Pit-1 or ER expression. The FLAG epitope-tagged Pit-1 was used in all subsequent experiments and behaves exactly as native Pit-1 in cooperative activation with nuclear receptors (35 ). Three independent experiments were normalized to the activity of the ER/Pit-1-coactivated PRL enhancer/promoter (100%), and results are plotted as the mean ± SD in A and B.

 
ER/Pit-1 cooperative activation of the PRL enhancer/promoter was blocked by a point mutation in AF-2 of ER (35). This suggested that one or more of the known AF-2-interacting proteins may act as coactivators of cooperative ER/Pit-1 activation. Expression vectors encoding the cDNAs for the AF-2-interacting proteins, steroid receptor coactivator-1a (SRC-1a) (also called NCoA-1) (10), glucocorticoid receptor interacting protein (GRIP1, also called TIF2 or NCoA-2) (15), TIF1{alpha} (13), and RIP140 (18) were individually cotransfected along with the ER{alpha} and Pit-1 expression vectors and the PRL enhancer/promoter reporter into GHFT1–5 cells (Fig. 1AGo). PRL enhancer/promoter activities in the presence of ER, Pit-1, and SRC-1a, GRIP1, or TIF1{alpha} were 128 ± 29%, 129 ± 31%, and 129 ± 13%, respectively, as active as PRL enhancer/promoter activity in the absence of coactivator. Similarly, the coactivator CREB-binding protein (CBP) (38), which also participates in nuclear receptor action (39), did not significantly alter ER/Pit-1 activation of the PRL enhancer/promoter (120 ± 14%).

RIP140 Is a Dose-Dependent Inhibitor of ER/Pit-1 Synergy
Expression of RIP140, which, like SRC-1a, GRIP1, and TIF1{alpha}, binds AF-2 in a ligand-dependent fashion, strongly reduced ER/Pit-1 cooperative activation of the PRL enhancer/promoter (35) (Fig. 1AGo). RIP140 inhibition was dose dependent; transfection of 1–10 µg of the RIP140 expression vector caused a gradual decrease in ER/Pit-1 cooperative activation (Fig. 1BGo). ER/Pit-1 activation was effectively abolished to the level of Pit-1 activation by transfecting 10 µg of the RIP140 expression vector. In contrast, cotransfection of 10 µg of the RIP140 expression vector had no effect on C/EBP{alpha} activation of the PRL enhancer/promoter (35). Transfection of lower amounts of the same RIP140 expression vector was previously reported to enhance ER{alpha} activation at a minimal promoter containing an estrogen response element (ERE) (18), but in the current studies had no activating or inhibitory effect on ER{alpha}/Pit-1 cooperative activation of the PRL promoter (data not shown).

The levels of ER and Pit-1 expressed were determined by Western blots of nuclear extracts of the same transfected cells (Fig. 1CGo). Ectopically expressed human ER{alpha} and Pit-1 (with the FLAG epitope fused to the Pit-1 amino-terminus) were detected with antibodies specific for human ER and the FLAG epitope. Mouse GHFT1–5 cells contain some endogenous ER (35) that were not detected by the anti-human ER antibodies used here. The nuclear levels of ectopically expressed ER{alpha} or Pit-1 were not changed by the coexpression of Pit-1 or ER (Fig. 1CGo) by RIP140 (Fig. 1CGo) or by SRC-1a, GRIP1, or TIF1{alpha} expression (not shown). Similarly, endogenous ER and Pit-1 levels were not changed (35). This showed that ER/Pit-1 activation and its regulation by RIP140 were not artifacts of effects on the expression vectors themselves. Thus, the AF-2-interacting protein RIP140 directly inhibited estrogen-dependent, cooperative activation by ER and Pit-1.

ER Is Required for RIP140 Inhibition
RIP140 may inhibit the PRL enhancer/promoter by acting directly at the ER. Conversely, RIP140 may inhibit other factors, including Pit-1, that bind to other PRL enhancer/promoter sites (40, 41, 42, 43, 44, 45, 46). We determined that RIP140 did not affect PRL enhancer/promoter activity in the absence of ER (Fig. 2Go). Because GHFT1–5 cells contain endogenous ER (35), we conducted these experiments under conditions in which endogenous ER was selectively inactivated by incubating the transfected cells with 10-6 M ICI 164,384. ICI 164,384 is an inactive estrogen mimic that antagonizes estrogen action by competitive binding to the ligand-binding pocket of ER. A concentration of 10-6 M ICI 164,384 was sufficient to completely block ER/Pit-1 cooperative activation of the PRL enhancer/promoter (data not shown).



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Figure 2. RIP140 Does Not Inhibit PRL Enhancer/Promoter Activity in the Absence of Active ER

Incubation of GHFT1–5 cells with the estrogen antagonist ICI 164,384 showed that the observed Pit-1 activation of the PRL enhancer/promoter was not due to the cooperative action of expressed Pit-1 with endogenous GHFT1–5 cell ER. Three independent experiments were normalized to the activity of the Pit-1-activated PRL enhancer/promoter (100%), and the results are plotted as the mean ± SD.

 
In the presence of 10-6 M ICI 164,384, the PRL enhancer/promoter was still activated 3.26 ± 0.43-fold by the expression of Pit-1. This showed that Pit-1 activated PRL enhancer/promoter action in the absence of active ER. With ER inactivated by ICI 164,384, RIP140 coexpression did not inhibit Pit-1 activation of the PRL enhancer/promoter (Fig. 2Go). Thus, RIP140 inhibition of PRL enhancer/promoter specifically required the presence of active ER.

RIP140 Directly Inhibits ER Action
We showed above that ER is an essential participant in RIP140 inhibition of ER/Pit-1 cooperative activation at the PRL enhancer/promoter. To determine whether ER itself could be the site of RIP140 action, we studied ER action in isolation from all PRL enhancer/promoter elements. The well characterized vitellogenin ERE was appended to a minimal promoter containing the TATA box of the herpes simplex thymidine kinase gene (ERE-TATA). Like the PRL ERE, the vitellogenin ERE operates through what is referred to as the classical ER pathway (47) (see Fig. 4Go). The vitellogenin ERE can also replace the PRL ERE to support ER cooperative activation with Pit-1 (33).



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Figure 4. Both Human ER{alpha} (A) and Rat ERß (B) Activation of the PRL Enhancer/Promoter Are Pit-1 Dependent and Require AF-2

In GHFT1–5 cells grown in medium reduced in estrogens, the selective AF-2 antagonists/AF-1 agonists tamoxifen and raloxifene did not support Pit-1 cooperative activation with either ER isoform. Three independent experiments were normalized to the activity of the Pit-1-activated PRL enhancer/promoter (1-fold), and the results are plotted as the mean ± SD.

 
When transfected into GHFT1–5 cells grown in culture medium deficient in estrogens, the vitellogenin ERE-TATA promoter was activated, on the average, 33.6 ± 8.4-fold by the addition of 10-8 M estradiol (E2; Fig. 3Go). E2 activation was completely inhibited by 10-6 M ICI 164,384 (ICI+E2; Fig. 3Go). Therefore, E2 activation of the ERE-TATA promoter operated via endogenous ER{alpha} and/or ERß, both of which are detected by Western blots of nuclear extracts of GHFT1–5 cells (35) (our unpublished data).



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Figure 3. RIP140 Directly Represses ER Action in the Absence of Other PRL Enhancer/Promoter-Binding Factors

GHFT1–5 cells were grown in medium reduced in estrogens. RIP140 inhibited estrogen (E2) activation of the vitellogenin ERE controlling the activation of the minimal herpes simplex thymdine kinase TATA box. Estrogen (E2) activation of this promoter was marginally (1.3-fold) affected by the coexpression of 0.2 (shown) or 1.0 or 5.0 µg (not shown) human ER expression vector. ICI 164,384 antagonized estrogen activation (ICI + E2). Three independent experiments were normalized to the activity of the E2-activated ERE-TATA promoter (100%), and the results are plotted as the mean ± SD.

 
Cotransfection of 0.2 (Fig. 3Go) to 5 µg (data not shown) of ER{alpha} expression vector only modestly enhanced E2 activation of the ERE-TATA promoter 1.3-fold, on the average. In contrast, E2 induction of the PRL enhancer/promoter depended on the ectopic expression of ER{alpha} along with Pit-1 (Fig. 4AGo); maximum levels of ER/Pit-1 cooperative activation of the PRL enhancer/promoter were not reached even at 10 µg of the same ER{alpha} expression vector (35). Thus, E2 activation was saturated at much lower levels of ER at the ERE-TATA promoter than at the PRL enhancer/promoter. The differential response of different promoters to cellular ER levels may represent an overlooked component of the cell- and promoter-specific actions of ER.

Cotransfection of the RIP140 expression vector reduced E2 activation of the vitellogenin ERE-TATA promoter (Fig. 3Go). In the presence of 0.2 µg of cotransfected ER, RIP140 reduced E2-dependent ERE-TATA promoter activity to 60.8 ± 7.6%. Therefore, RIP140 inhibition was not limited to the PRL enhancer/promoter and even occurred at a minimal promoter at which the effects of promoter binding factors other than ER were minimized. RIP140 is likely to directly inhibit ER action at a variety of EREs in a variety of enhancer/promoter contexts.

AF-2 Dominates ER/Pit-1 Activation of the PRL Enhancer/Promoter
The strong inhibition of ER/Pit-1 cooperative activation of the PRL enhancer/promoter by RIP140 might reflect RIP140 inhibition via ER AF-2 to which RIP140 is known to bind. Conversely, RIP140 might target a site in ER other than or in addition to AF-2. A second ER activation function, AF-1, is located in a domain separable from the LBD in which AF-2 resides. Some AF-2-interacting proteins have been observed to interact also with AF-1 of some nuclear receptors, including ER (28, 29). In the context of the full-length ER, AF-1 is dormant until released by ligand binding to the LBD. To study which domains of ER were required for RIP140 inhibition, we initially distinguished the relative contributions of AF-1 and AF-2 to ER/Pit-1 activation of the PRL enhancer/promoter pharmacologically.

GHFT1–5 cells were grown in medium containing low levels of estrogens and then transfected with ER{alpha} and/or Pit-1 expression vectors. Incubation with 10-8 M E2 resulted in significant activation of the PRL enhancer/promoter by ER and Pit-1 (Fig. 4AGo). E2 had little effect on the PRL enhancer/promoter activated only by Pit-1 expression (Fig. 4AGo). The small, but reproducible, activation observed with ER/Pit-1 expression in the absence of any ligand was due to residual estrogens in the medium, as it was blocked by 10-6 M ICI 164,384, which prevents both AF-1 and AF-2 activities.

Other ligands, including tamoxifen and raloxifene, that bind the E2-binding pocket of ER, commonly activate AF-1 (22, 28, 47, 48) without allowing the ER LBD to adopt the E2-dependent conformation in which AF-2 is available for coactivator binding (49). Tamoxifen and raloxifene are thereby partial agonists at promoters in which AF-1 is sufficient for activation. Incubation of the transfected cells with 10-6 M tamoxifen or 10-6 M raloxifene did not activate PRL enhancer/promoter activity. This suggested that ER/Pit-1 activation of the PRL enhancer/promoter is relatively AF-2 driven, with little or no contribution of AF-1 in the absence of AF-2.

AF-1 Is Not Required for RIP140 Inhibition of ER/Pit-1 Cooperative Activation
The lack of ER/Pit-1 activation in the presence of tamoxifen and raloxifene suggested that active AF-1 alone was insufficient for estrogen regulation of the PRL enhancer/promoter. However, AF-2 was also inhibited by these ligands. To examine whether any portion of ER/Pit-1 cooperative activation was affected by AF-1, we studied the ability of an ER deleted of its entire amino-terminal domain, including AF-1 (ER-{Delta}N), to activate the PRL enhancer/promoter. ER-{Delta}N was as effective as full-length ER in its ability to cooperate with Pit-1 and activate the PRL enhancer/promoter (Fig. 5Go). Thus, AF-1 makes no contribution to ER/Pit-1 activation. Moreover, RIP140 also inhibited ER-{Delta}N/Pit-1 activation as efficiently as ER/Pit-1 activation (Fig. 5Go). Therefore, despite the contribution of some AF-2-interacting proteins to AF-1/AF-2 cooperation (28, 29), RIP140 inhibition is complete in the absence of AF-1.



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Figure 5. RIP140 Inhibition of the PRL Enhancer/Promoter Requires Only the ER{alpha} DNA-Binding Domain and LBD

Deletion of the amino-terminal 179 amino acids of ER{alpha}, including all of AF-1 to the DNA-binding domain (ER-{Delta}N), did not affect ER/Pit-1 cooperative activation. RIP140 still inhibited Pit-1 cooperative activation with ER-{Delta}N. Three independent experiments were normalized to the activity of the Pit-1-activated PRL enhancer/promoter (1-fold), and the results are plotted as the mean ± SD.

 
PRL Enhancer/Promoter Activation via the Classical ER Pathway
The recently described ß-isoform of ER (50) is present in pituitaries (51) and pituitary cell lines (S. Gong and F. Schaufele, unpublished data), but its contribution to PRL enhancer/promoter activation is unknown. PRL synthesis in ER{alpha} knockout mice is reduced, but not eliminated (52), suggesting a role for ER{alpha} in PRL gene regulation that might be partially compensated by pituitary ERß. Like ER{alpha} (Fig. 4AGo), ERß activation of the PRL enhancer/promoter in GHFT1–5 cells grown in E2-deficient medium depended upon Pit-1 and E2 (Fig. 4BGo).

Estrogen regulation results from classical ER interactions with an ERE in the PRL enhancer. Estrogen regulation also may arise from ER interactions with other factors, including AP-1 (47), which is a potent regulator of the PRL promoter (44, 53). We used differences in the pharmacology of the estrogen response via the classical and AP-1 pathways to determine their respective contributions to Pit-1 cooperative activation with both ER{alpha} and ERß. In general, ER{alpha} and ERß activation via AP-1 are mechanistically distinct and distinguishable by differing agonist/antagonist profiles (48). At AP-1, tamoxifen acts as an agonist for both isoforms, and raloxifene selectively activates ERß. Both ER{alpha}/Pit-1 and ERß/Pit-1 activation of the PRL enhancer/promoter were insensitive to tamoxifen, raloxifene, and ICI 164,384 and were only activated by estradiol (Fig. 4Go). The lack of any agonist activity by either tamoxifen or raloxifene suggested that the AP-1 pathway did not contribute to estrogen regulation of Pit-1 cooperation with either ER isoform.

RIP140 Inhibits Pit-1 Cooperation with ERß
The similar pharmacology and similar Pit-1 dependence of ER{alpha} and ERß suggest that the two ER isoforms play similar roles in the estrogen regulation of the PRL gene. ER{alpha} and ERß are homologous over the DNA-binding domain and LBD (50), which are sufficient for RIP140 inhibition of ER{alpha}/Pit-1 cooperative action (Fig. 5Go). We, therefore, examined whether RIP140 would inhibit ERß cooperative activation with Pit-1.

Like ER{alpha}, the coexpression of RIP140 completely blocked ERß/Pit-1 cooperative activation (Fig. 6Go). RIP140 expression also did not change the relative abilities and inabilities of E2, tamoxifen, raloxifene, or ICI 164,384 to promote ER{alpha} or ERß cooperative activation with Pit-1 at the PRL enhancer/promoter (data not shown). Thus, ER{alpha} and ERß behave similarly with respect to RIP140 inhibition of ER/Pit-1 cooperativity at the PRL enhancer/promoter.



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Figure 6. ERß/Pit-1 Cooperative Activation of the PRL Enhancer Promoter Is Inhibited by RIP140

Four independent experiments were normalized to the activity of the Pit-1-activated PRL enhancer/promoter (1-fold), and the results are plotted as the mean ± SD.

 
AF-2 Interacting Coactivators Counteract RIP140 Inhibition
The similar effects of RIP140 on ER{alpha} and ERß action at the PRL enhancer/promoter most likely indicated that they possess a common target for RIP140 action. Both ER{alpha} and ERß contain AF-2, which is required for direct RIP140 binding to ER and other nuclear receptors. Within AF-2, the binding sites for some of the AF-2-interacting proteins overlap, albeit with AF-2 amino acid requirements specific for each interacting protein (8, 16, 54, 55, 56, 57). Consistent with binding site overlap, at least RIP140 and SRC-1a have been shown to compete for binding to PPAR{gamma} (58).

If AF-2 were a target for RIP140 inhibition, other AF-2-interacting proteins that did not inhibit ER/Pit-1 cooperative interaction (Fig. 1Go) should compete with RIP140 binding and reduce RIP140 inhibition of the PRL enhancer/promoter. Expression of SRC-1a, GRIP1, and TIF1{alpha} shifted the dose response of RIP140 inhibition to the right (Fig. 7AGo). Western blots showed that these effects were not due to changes in the expression of ER or Pit-1 (Fig. 7BGo). PRL enhancer/promoter activity progressively declined with higher RIP140 amounts in the presence of each coactivator (Fig. 7AGo). However, there were some differences in the coactivator responses.



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Figure 7. Other AF-2-Interacting Proteins Relieve RIP140 Inhibition of Human ER/Pit-1 Cooperative Activation of the PRL Enhancer/Promoter

A, Cotransfection of 10 µg of the expression vectors for the AF-2-interacting proteins SRC-1a, GRIP, or TIF1{alpha} partially relieved dose-dependent RIP140 inhibition, albeit with differing characteristics. Three independent experiments were normalized to the activity of the ER/Pit-1-coactivated PRL enhancer/promoter (100%), and the results are plotted as the mean ± SD. B, ER and Pit-1 expressions, detected by Western blot, were not affected by expression of the AF-2-interacting proteins.

 
Relief of RIP140 inhibition by TIF1{alpha} became less effective and was eventually eliminated at higher RIP140 expression levels. Intersection of the RIP140 dose-response curves in the presence and absence or TIF1{alpha} at high RIP140 levels would be expected if RIP140 competed for and occluded TIF1{alpha} binding to the same site. At high RIP140 levels, the RIP140 dose-response curve in the presence of GRIP1 also approached that in the absence of GRIP1, although it did not intersect it. The difference in the degree to which GRIP1 and TIF1{alpha} counteracted RIP140 inhibition may reflect relative differences in TIF1{alpha} and GRIP1 expression levels, interaction with AF-2, or ability to coactivate the PRL enhancer/promoter.

In contrast, the slope of the RIP140 dose-response curve in the presence of coexpressed SRC-1a diverged from the RIP140 dose-response curve. This would not be anticipated if the only effect of SRC-1a was to occlude AF-2 and prevent RIP140 from binding. This may suggest that the SRC-1a-containing complex was a strong coactivator of ER/Pit-1 action at the PRL enhancer/promoter or that the SRC-1a-containing complex was more stable to RIP140 competition than its GRIP1 or TIF1{alpha}-containing counterparts. An alternative possibility was that SRC-1a acted through a different site on the ER than did GRIP1 or TIF1{alpha}.

Relief of RIP140 Inhibition Requires SRC-1a and GRIP1 Interaction with AF-2
Inhibition of ER/Pit-1 cooperative activation of the PRL enhancer/promoter by the AF-2-interacting protein RIP140 was therefore counteracted by the expression of AF-2-binding coactivators. To determine whether AF-2 itself was the site of action for either or both SRC-1a and GRIP1, mutations that disrupt SRC-1a and GRIP1 interaction with ER AF-2 were examined for their effect on the relief of RIP140 inhibition. SRC-1a and GRIP1 bind to AF-2 via a conserved sequence motif, LXXLL (59), present in multiple copies in SRC-1a and GRIP1.

Point mutants changing all four copies of LXXLL in SRC-1a or two of the three copies of LXXLL in GRIP1 to LXXAA block interaction of those coactivators with ER AF-2 (57, 59). SRC-1a or GRIP1 containing those same mutants were no longer capable of relieving RIP140 inhibition of the PRL enhancer/promoter (Fig. 8Go). Upon coexpression of RIP140 and mutant SRC-1a or GRIP1, PRL enhancer/promoter activity remained at 38.8 ± 18.3% or 34.4 ± 16.8% that of the ER/Pit-1-activated PRL enhancer/promoter. This was not statistically different (P > 0.10) from the 41.8 ± 13.0% activity upon coexpression of RIP140. In contrast, a statistically significant relief of RIP140 inhibition was observed with the coexpression of either wild-type SRC-1a (P = 0.01) or GRIP1 (P = 0.03) to levels that were not significantly different (P > 0.10) from those of the enhancer/promoter activity in the absence of RIP140 or coactivator. Thus, SRC-1a and GRIP1 relieve RIP140 inhibition through a target that binds the LXXLL motifs.



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Figure 8. Mutation of the LXXLL Boxes in SRC-1a and GRIP1 to LXXAA Eliminates SRC-1a and GRIP1 Relief of RIP140 Inhibition

A dose of RIP140 expression vector within the linear inhibitory range (5 µg) was transfected in these experiments. Ten micrograms of each wild-type (wt) or mutant coactivator were cotransfected. Four independent experiments were normalized to the activity of the ER/Pit-1-coactivated PRL enhancer/promoter (100%), and the results are plotted as the mean ± SD.

 
The suggestion that the LXXLL-binding site targeted by SRC-1a and GRIP1 was AF-2 in ER was confirmed by using a mouse ER containing the K366A point mutation that selectively disrupts binding to SRC-1a without affecting RIP140 binding (56). The K366A mutant ER remained capable of cooperating with Pit-1 to activate the PRL enhancer/promoter, albeit at a level 50.0 ± 9.8% that of the wild-type mouse ER (Fig. 9AGo). This activity was still significantly above (P < 0.05) the level of PRL enhancer/promoter activity that would have been obtained by the addition of the independent Pit-1 and ER K366A activation levels.



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Figure 9. The SRC-1a- and GRIP1-Binding Site in ER Is Required for SRC-1a and GRIP1 Relief of RIP140 Inhibition

A, The K366A mutant of the mouse ER{alpha}, which blocks SRC-1a binding without affecting RIP140 binding (56 ), cooperated with Pit-1 to activate the PRL enhancer/promoter. PRL enhancer/promoter activation by the K366A ER mutant was less than that of wild-type (wt) mouse ER under saturating and subsaturating ER conditions. B, PRL enhancer/promoter activation by Pit-1 and the K366A ER{alpha} mutant was inhibited by the cotransfection of 5 µg RIP140 expression vector; this was not relieved by the cotransfection of 10 µg SRC-1a or GRIP1 expression vector. Three independent experiments were normalized to the activity of the wild-type mouse ER/Pit-1-coactivated PRL enhancer/promoter (100%), and the results are plotted as the mean ± SD in A and B.

 
PRL enhancer/promoter activity cooperatively activated by ER K366A and Pit-1 was reproducibly reduced by RIP140 expression to a level 61.0 ± 17.9% of the activity in the absence of RIP140. In contrast to the wild-type mouse ER, RIP140 inhibition of cooperative activation of ER K366A with Pit-1 was not relieved by expression of SRC-1a and GRIP1 (Fig. 9BGo). In the presence of RIP140 and SRC-1a or GRIP1, ER-K366A/Pit-1-activated PRL enhancer/promoter activity was not statistically different from and even tended to be lower than the activity in the absence of coactivator. Thus, cooperative activation by Pit-1 and the K366A mutant mouse ER was still inhibited by RIP140, but was no longer relieved by SRC-1a or GRIP1 coexpression. Together, the results shown in Figs. 8Go and 9Go conclusively demonstrated that counteraction of RIP140 inhibition of ER/Pit-1 cooperative activation of the PRL enhancer/promoter by SRC-1 and GRIP1 required the interaction of both coactivators with ER AF-2.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Most natural estrogen-regulated promoters contain not only ER-binding sites, but also sites for other transcription factors. Indeed, estrogen regulation of many natural promoters is cell specific, suggesting that other factors affect ER action. Pituitary-specific estrogen regulation of the PRL enhancer/promoter results from ER dependence on the pituitary-specific PRL enhancer/promoter-binding factor, Pit-1 (31, 32, 33, 34, 35). Thus, at the PRL enhancer/promoter, Pit-1 may even be thought of as a sequence-specific DNA-binding coactivator for ER. To fully understand cell-specific estrogen regulation, it will be important to define how ER activities are used in the context of Pit-1 and other promoter-binding factors.

ER contains at least two transcriptional activation functions, AF-1 and AF-2, which regulate basal promoter activity (3, 4). Deletion of AF-1 had no effect on ER/Pit-1 cooperative activation at the PRL enhancer/promoter (Fig. 5Go). Consistent with this, the thyroid hormone receptor, which contains no or poor AF-1 activity (60), also cooperates with Pit-1 to activate the pituitary-specific GH gene promoter (61). In contrast, null mutations within ER AF-2 (35) or blocking ER AF-2 activity with the selective AF-2 antagonists tamoxifen or raloxifene (Fig. 4Go) did not allow ER cooperative activation with Pit-1. Similarly, thyroid hormone receptor cooperative activation with Pit-1 required AF-2 (35). Thus, Pit-1 cooperative activation with at least two nuclear receptors seems to be relatively AF-2 driven. Selective use of AF-2 may be related to activities provided by Pit-1 that supercede, mimic, or, in ER, preclude the use of AF-1.

Part of AF-2 consists of a conserved sequence (20) that in all nuclear receptors forms an amphipathic {alpha}-helix on one side of a ligand-induced hydrophobic pocket. This pocket acts as a docking site (62) for certain ER-interacting proteins, including SRC-1a, GRIP1, TIF1{alpha}, and RIP140. In mammalian cells, at least SRC-1a, GRIP1, and RIP140 can potentiate the activities of artificial promoters in which an ERE is placed in close proximity to the transcription start (18, 56, 57, 59) and are thus bona fide ER coactivators. However, expression of GRIP1, SRC-1a, or TIF1{alpha} did not substantially affect ER/Pit-1 activation of the PRL enhancer/promoter (Fig. 1Go). Perhaps GRIP1, SRC-1a and TIF1{alpha} are already present in excess in GHFT1–5 cells, or Pit-1 cooperative activation with ER mutated such that it cannot bind SRC-1 (Fig. 9AGo) might indicate that other known (7, 8, 9, 11, 12, 14, 17, 19, 23) or unknown AF-2-interacting coactivators participate in ER action at the PRL enhancer/promoter.

The putative coactivator is not RIP140, which strongly inhibited ER/Pit-1 cooperative activation at the PRL enhancer/promoter (35) (Fig. 1Go). RIP140 directly repressed ER in GHFT1–5 cells (Fig. 3Go), but did not inhibit the PRL enhancer/promoter when ER was selectively inactivated (Fig. 2Go). Thus, RIP140 inhibition of PRL gene transcription operated via ER and not via any of the other transcription factors that bind to the PRL enhancer/promoter. Within ER, the target of RIP140 inhibition appeared to be common to both ER isoforms (Figs. 4Go and 6Go) and was not affected by the deletion of AF-1.

Competition by TIF1{alpha} for RIP140 binding to a common site in ER seemed to explain partial relief of RIP140 inhibition by TIF1{alpha} and probably GRIP1 and SRC-1a (Fig. 7Go). In support of direct competition at AF-2, the relief of RIP140 inhibition by both SRC-1a and GRIP1 was dependent on AF-2 (Figs. 8Go and 9Go). Similarly, RIP140 inhibition of ER/Pit-1 cooperative activation of the PRL enhancer/promoter in GHFT1–5 cells may arise from RIP140 competitive inhibition of the binding of the putative endogenous AF-2-interacting coactivator. The prevention of activation by mass action interference of the binding of a strong coactivator by the binding of a poorer coactivator, such as RIP140, is emerging as a mechanism of corepression that is distinct from the active mechanisms involving the recruitment of enzymatic activities. Mass action competition at AF-2 is, however, likely to be modified by the recruitment of activating or inhibitory complexes by the competing factors, which may explain the observed differences in coactivator relief of RIP140 inhibition (Fig. 7AGo).

Therefore, in pituitary cells, estrogen regulation of the PRL enhancer/promoter is a synthesis of mutual, complementary, and/or counteracting activities possessed by ER, its AF-2 interacting cofactors, and Pit-1. The estrogen responses of other promoters might similarly be regulated by the amount and binding preferences of competing AF-2-interacting proteins in a cell. This would also be influenced by the amount of ER and the differential sensitivity of some promoters to ER levels (Figs. 3Go and 4Go). Thus, the relative effects and amounts of ER, different AF-2-interacting proteins, and cooperating transcription factors will probably determine the level of cell-specific estrogen response at many natural promoters. The development of ER ligands that specifically affect any of those interactions may one day provide a means to selectively activate or inhibit estrogen responses in specific tissues.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transfection and Analysis
GHFT1–5 cells were grown in DMEM-H21 supplemented with 10% FCS (Figs. 1Go, 2Go, and 5Go–7). The agonist/antagonist response was determined in cells that had been grown for 24 h before transfection in medium lacking phenol red and containing 10% iron-supplemented newborn calf serum tested for reduced amount of estrogens (Figs. 3Go and 4Go). Transfected cells were grown in the estrogen-deficient medium and treated with 10-8 M estradiol, 10-6 M ICI 164,384, 10-6 M tamoxifen, 10-6 M raloxifene, and/or delivery vehicles 1 day after transfection, then collected the following day. Transfection was performed by electroporation using the conditions and buffers previously described (45).

Collected cells were lysed in reporter lysis buffer (Promega Corp., Madison, WI). Luciferase (Figs. 1Go, 2Go, and 4Go–7) and chloramphenicol acetyltransferase (Fig. 3Go) activities in these extracts were determined as previously described (34, 35, 45, 61). Data from multiple independent experiments were normalized to specific reference points (see figure legends for n and points), and the mean ± SD were determined for each.

For Western blots (Figs. 1Go and 7Go), the cell pellet posttreatment with reporter lysis buffer was resuspended in 50 mM Tris buffered to pH 7.8 with 2-[N-morpholino]ethanesulfonic acid, 1 mM dithiothreitol, and 0.1% Triton X-100; pelleted; resuspended in the same buffer; then pelleted again. The resulting crude nuclei were resuspended three times with 20 µl 20 mM HEPES (pH 7.9), 300 mM KCl, 200 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, and 15% glycerol, and the extracts were pooled. Equivalent amounts of extract protein (5–20 µg depending on the experiment) were loaded onto 10% SDS-polyacrylamide gels and probed with either the FLAG {alpha}M2 mouse monoclonal antibody (ICI-Kodak, Rochester, NY) or the human-specific ER HC-20 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After washing away the primary antibodies, horseradish peroxidase-linked antimouse IgG (Life Technologies, Gaithersburg, MD) or antirabbit IgG (Amersham, Arlington Heights, IL) secondary antibodies were incubated with the filters, washed, and detected with enhanced chemiluminescence reagents (Amersham).

Plasmids
Levels of 2.5 µg (Figs. 1Go, 2Go, and 4Go–7) and 2.0 µg (Figs. 8Go and 9Go) of the 3-kb PRL enhancer/promoter cloned in front of the luciferase gene (63) were transfected in each experiment shown. The plasmid containing the vitellogenin ERE inserted in front of the thymidine kinase TATA box controlling the bacterial chloramphenicol acetyltransferase gene was previously described (47); 1 µg of this reporter was transfected in Fig. 3Go.

Ten micrograms of the human ER{alpha} (64), 10 µg rat ERß (48), 5 µg FLAG-tagged rat Pit-1 (35), and 1–10 µg RIP140 (18) expression vectors were transfected into GHFT1–5 cells (Figs. 1Go, 2Go, and 4Go–7) as previously described (35). Expression vectors were under the control of the simian virus-40 (human ER{alpha}), cytomegalovirus (ERß, Pit-1), and elongation factor-1{alpha} (RIP140) promoters. A level of 0.2 µg human ER{alpha} expression vector was transfected in Fig. 3Go; 0.3–3 µg wild-type or K366A mutant of the mouse ER (56) were transfected in Fig. 9Go. Activation of the PRL enhancer/promoter saturated at 1 µg mouse ER expression vector, whereas saturation was not reached even with 15 µg human ER expression vector. This presumably reflected a much higher transcriptional activity in GHFT1–5 cells of the adenovirus major late promoter used to express the mouse ER.

Expression vectors containing CBP (38) under the control of the Rous sarcoma virus promoter (Fig. 1Go), wild-type SRC-1a (10) under the control of the cytomegalovirus promoter (Figs. 1Go and 7Go), wild-type SRC-1a and SRC-1a mutated in its four LXXLL motifs (L636A/L637A, L693A/L694A, L752A/L753A, and L1438A/L1439A; Figs. 8Go and 9Go), wild-type GRIP1 (15), GRIP1 mutated in its two LXXLL motifs (L693A/L694A and L748A/L749A; Figs. 1Go and 7Go–9) (57), and TIF1{alpha} (Figs. 1Go and 7Go) (13), all under the control of the simian virus-40 promoter, were previously described. For points in which a particular cDNA was not expressed in a matched experiment, the "empty" expression vector not containing the specific cDNA was cotransfected.


    ACKNOWLEDGMENTS
 
I thank Dr. Paul Webb for critical reading of the manuscript, Dr. Tom Scanlon for providing raloxifene, and the following for the indicated expression vectors: Drs. V. Cavaillès and M. Parker (RIP140, wild-type and K366A mutant of mouse ER, wild-type and LXXLL mutants of SRC-1a); Drs. S. Oñate, M.-J. Tsai, and B. O’Malley (SRC-1a); Drs. H. Hong and M. Stallcup (wild-type GRIP1); Carol Anderson and Dr. Peter Kushner (LXXLL mutant of GRIP1); Drs. B. LeDouarin and R. Losson (TIF1{alpha}); Drs. R. Kwok and R. Goodman (CBP); and K. Paech and Dr. P. Webb (ERß).


    FOOTNOTES
 
Address requests for reprints to: Dr. Fred Schaufele, Metabolic Research Unit, University of California, San Francisco, California 94143-0540. E-mail: freds{at}metabolic.ucsf.edu

This work was supported by Grant RPG-94–028-TBE from the American Cancer Society (to F.S.).

Received for publication September 30, 1998. Revision received March 15, 1999. Accepted for publication March 19, 1999.


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