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Molecular Endocrinology, doi:10.1210/me.2007-0080
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Molecular Endocrinology 21 (11): 2672-2686
Copyright © 2007 by The Endocrine Society

Farnesyl Pyrophosphate Is a Novel Transcriptional Activator for a Subset of Nuclear Hormone Receptors

Sharmistha Das, Matthieu Schapira, Marjana Tomic-Canic, Ritu Goyanka, Timothy Cardozo and Herbert H. Samuels

Department of Pharmacology (S.D., R.G., T.C., H.H.S.), New York University School of Medicine, New York, New York 10016; Structural Genomics Consortium (M.S.), Banting Building, University of Toronto, Toronto, Ontario, Canada M5G 1L5; and Department of Dermatology (M.T.-C.), Weill Medical College of the Cornell University, New York, New York 10021

Address all correspondence and requests for reprints to: Herbert Samuels, Departments of Pharmacology and Medicine, New York University School of Medicine, 550 First Avenue, Room MSB-424, New York, New York 10016. E-mail: herbert.samuels{at}med.nyu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In silico docking of a chemical library with the ligand-binding domain of thyroid hormone nuclear receptor-ß (TRß) suggested that farnesyl pyrophosphate (FPP), a key intermediate in cholesterol synthesis and protein farnesylation, might function as an agonist. Surprisingly, addition of FPP to cells activated TR as well as the classical steroid hormone receptors but not peroxisome proliferative-activating receptors, farnesoid X receptor, liver X receptor, or several orphan nuclear receptors the ligands of which are unknown. FPP enhanced receptor-coactivator binding in vitro and in vivo, and elevation of FPP levels in cells by squalene synthetase or farnesyl transferase inhibitors leads to activation. The FPP effect was blocked by selective receptor antagonists, and in silico docking with 143 nuclear receptor ligand-binding domain structures revealed that FPP only docked with the agonist conformation of those receptors activated by FPP. Our results suggest that certain nuclear receptors maintain a common structural feature that may reflect an action of FPP on an ancient nuclear receptor or that FPP could function as a ligand for one of the many orphan nuclear receptors the ligands of which have not yet been identified. This finding also has potential interesting implications that may, in part, explain the pleotropic effects of statins as well as certain actions of farnesylation inhibitors in cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR HORMONE RECEPTORS are transcription factors that play an important role in growth, development, metabolic regulation, and, in certain organisms, metamorphosis (1). These receptors were first identified as ligand-dependent transcription factors for known hormones such as glucocorticoids, mineralocorticoids, estrogens, progestins, and the thyroid hormones (see www.nursa.org as well as Refs 2, 3, 4, 5, 6, 7, 8, 9 for a detailed description of the nuclear receptor family). These receptors have a highly conserved DNA-binding domain (DBD) of approximately 70 amino acids and a less conserved C-terminal ligand-binding domain (LBD) of about 220 amino acids (1, 10). Other members of the receptor gene family were cloned using the DNA sequence of the conserved DBD to screen cDNA libraries. Ligands have been identified for a number of these receptors; lipids for the peroxisome proliferative-activating receptors (PPARs), oxysterols for liver X receptors (LXRs), bile acids for farnesoid X receptor (FXR), all-trans retinoic acid for the retinoic acid receptors (RARs), and 9-cis retinoic acid for the retinoid X receptors (RXRs) as well as the RARs, and the xenobiotic receptors, constitutive androstane receptor and pregnane X receptor (for review see Refs. 2, 3, 4, 5, 6, 7, 8, 9). A number of members of the nuclear receptor family are considered as orphan receptors that regulate transcription without ligand, or for which the ligand(s) have not yet been identified (6, 11).

The crystal structure of many of the LBDs of the nuclear receptor family have been solved as aporeceptors as well as bound to agonists and antagonists (10, 12). The LBD generally consists of 12 {alpha}-helical regions. Agonists bind to the central cavity of the LBD and reorient helix 12 (H12) to contribute to the formation of a hydrophobic groove on the surface of the receptor, which acts as a docking site for coactivator proteins facilitating transcriptional activation (10, 13, 14). Studies with antagonists indicate that they bind into the central cavity of the LBD but tend to protrude out of the LBD to prevent H12 from contributing to the coactivator-binding site (10). Certain orphan receptors such as hepatocyte nuclear factor (HNF)4{alpha}, HNF4{gamma}, and retinoid-related orphan nuclear receptors (RORs) are constitutively active, and their LBD region exists in an agonist-like conformation (15, 16, 17, 18).

Interestingly, the crystal structures of the LBDs of orphan receptors such as HNF4{alpha}, HNF4{gamma}, RORß, or RXR or the insect ortholog of RXR (ultraspiricle), which were expressed and purified from Escherichia coli, indicate that the central binding cavity contains a bacterial lipid(s) (15, 17, 19, 20, 21, 22, 23). In addition, all-trans retinoic acid has been shown to bind RORß and reduce its constitutive activity (16), and hLRH-1 (liver receptor homolog-1), hSF1 (steroidogenic factor-1), and mSF1 (but not mLRH-1) cocrystallize with a phospholipid(s) in their binding pocket (24, 25, 26, 27). Interestingly mLRH-1 does not associate with phospholipids. Whether such lipids act as physiological ligands for these and other orphan receptors or whether they fortuitously become incorporated in the hydrophobic cavity of the LBD during expression in bacteria requires more definitive functional studies.

An analysis of the evolution of the nuclear hormone receptor family suggests that the receptor family first functioned as transcription factors and that the utilization of ligands to regulate transcription evolved later (28, 29). Because ancient nuclear hormone receptors may have evolved as ligand-independent transcriptional regulators, it is likely that they were modulated by postsynthetic modification events such as phosphorylation. Alternatively, ancient nuclear hormone receptors may have modulated genes involved in essential metabolic pathways and, thus, have been regulated by the levels of metabolic intermediates. Thus, the initial ligand may have been an important metabolic intermediate and during evolution the LBD resulted in the acquisition of new ligand specificities.

We previously used in silico docking of a virtual library of compounds into the putative antagonist structure of RAR{alpha} and thyroid hormone receptor-{alpha} (TR{alpha}) and identified novel receptor antagonists (30, 31, 32). Compounds were also docked to the agonist structure of TRß (by M. Schapira) and surprisingly farnesyl pyrophosphate (FPP) was identified as a putative ligand for this receptor. Indeed, our functional and in vitro binding studies indicate that FPP can act as an agonist for TR and recruit coactivators to the TR-LBD. Remarkably, this agonist effect of FPP is not limited to TR but FPP also activates RAR{alpha}, RXR{alpha}, glucocorticoid receptor (GR), estrogen receptor (ER), and progesterone receptor (PR). In contrast, FPP does not activate LXR{alpha}, farnesoid X receptor (FXR){alpha}, PPAR{gamma}, ecdysone receptor, ultraspiricle, as well as a variety of studied orphan receptors. Molecular modeling and in silico docking of FPP into the solved crystal agonist structures of these receptors indicate that FPP efficiently docks only to those receptor LBDs that are functionally activated by FPP. The ability to activate many nuclear receptors suggests that FPP docks to a structure that may have been conserved in evolution and that FPP may have functioned as a ligand for an ancient nuclear receptor and/or for one or more orphan receptors that we have not examined. This finding also has potential interesting implications that may explain, in part, the pleotropic effects of statins as well as certain actions of farnesylation inhibitors in cells.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Transcriptional Activation of Nuclear Receptors by FPP
To identify novel ligands for nuclear receptors, we extended our in silico docking studies of virtual libraries (30, 32, 33) to the agonist-bound structure of TR. These studies indicated that FPP might act as a ligand for TR. To explore this possibility, we expressed chicken (c)TR{alpha} in HeLa cells along with a TR-responsive chloramphenicol acetyltransferase (CAT) reporter gene and incubated the cells with various concentrations of FPP (Fig. 1Go). Cells incubated with thyroid hormone (T3) served as a control. Surprisingly, addition of FPP to the medium resulted in transcriptional activation by TR. There was no effect of FPP with the CAT reporter gene alone, and FPP activation was dependent on expression of TR (data not shown). In contrast with FPP, incubation with farnesol had no effect, indicating the importance of the pyrophosphate moiety in receptor activation. Interestingly, the maximal extent of stimulation by FPP was generally about half that found with concentrations of T3 that lead to a maximal response. In some, but not all experiments, incubation with FPP + T3 resulted in stimulation found with FPP alone, suggesting that FPP might act as a partial agonist/antagonist for TR. cTR{alpha}(L398R) contains a mutation in H12. Although this mutation does not affect T3 binding, it interferes with the formation of a productive coactivator docking site and is not a functionally active receptor (34). FPP did not activate cTR{alpha}(L398R), indicating the importance of H12 in receptor activation by FPP (data not shown). An antagonist that we developed (D4) against TR (30) blocked cTR{alpha} activation by FPP, suggesting that FPP activates through binding to the LBD of the receptor (Fig. 1Go). Similar results for FPP were found with rat (r)TRß1 (data not shown). We also examined a number of other factors in the cholesterol synthetic pathway or derivatives of FPP that were without effect (not illustrated). These include, geranyl-pyrophosphate (PP), geranylgeranyl-PP, squalene, lanosterol, and cholesterol.


Figure 1
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  		Fig. 1. Transcriptional Activation of TR{alpha} by FPP

Hela cells were transfected with a vector expressing cTR{alpha} and a CAT reporter gene ({Delta}MTV-IR-CAT). The cells were then incubated with the concentrations of T3, FPP, farnesol, and the TR antagonist D4 as indicated in the figure. The cells were harvested and analyzed for CAT activity 40 h later. The results represent the mean ± SD of three experiments.

 
The FPP synthetic pathway (Fig. 2Go) involves the formation of mevalonic acid by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is then converted to geranyl-PP, a precursor of FPP. FPP has two fates: it is converted to squalene, a precursor of cholesterol, and is involved in the farnesylation of a variety of proteins including small GTPases such as Ras. The conversion of FPP to squalene is blocked by the squalene synthetase inhibitor zaragozic acid A (ZGA) (also known as squalestatin) (35), which can elevate the levels of FPP in cells (36). If FPP (and not a metabolite) is directly involved in activation of TR, Fig. 2Go predicts that elevation of FPP by ZGA or by inhibitors of farnesylation would lead to TR activation. Blocking the production of mevalonic acid by an HMG-CoA inhibitor would be expected to inhibit activation of TR by ZGA, and activation would be restored by addition of mevalonic acid or FPP, which bypasses the HMG-CoA inhibition.


Figure 2
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  		Fig. 2. Pathway of Cholesterol Synthesis and Protein Farnesylation and Prenylation

The figure depicts the pivotal role of FPP in the synthesis of cholesterol and farnesylation of proteins and the levels of inhibition of the pathways by mevastatin, ZGA, and farnesyltransferase inhibitors (FTI).

 
Figure 3Go shows such an experiment. HeLa cells transfected with vectors expressing cTR{alpha} and a TR-CAT reporter gene were incubated with ZGA, mevastatin (an HMG-CoA inhibitor), mevastatin + ZGA, mevastatin + ZGA + mevalonic acid, and mevastatin + ZGA + FPP. Our results indicate that activation of cTR{alpha} by ZGA is blocked by mevastatin and the activation is restored by addition of mevalonic acid or FPP. In addition, activation of TR also occurred when the cells were incubated with two different inhibitors of farnesylation (FTI277 and B589) that would be expected to increase the levels of FPP in cells (Fig. 3Go). The response to ZGA was dependent on expression of TR, and ZGA did not increase CAT activity when cells were only transfected with the TR-CAT reporter gene (data not shown).


Figure 3
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  		Fig. 3. TR{alpha} Is Activated by Factors that Elevate FPP Levels in Cells

Hela cells were transfected with a vector expressing cTR{alpha} and a CAT reporter gene ({Delta}MTV-IR-CAT). The cells were then incubated with the indicated concentrations of T3, FPP, ZGA, mevastatin, mevalonic acid (MVA), and the farnesyl transferase inhibitors FTI277 and B589. The cells were harvested 40 h later and analyzed for CAT activity. The results represent the mean ± SD of three experiments.

 
FPP Activates a Variety of Nuclear Receptors
We next examined the effect of FPP on a wide variety of ligand-dependent nuclear receptors as well as orphan receptors. These studies were carried out with Gal4-LBD receptor chimeras to compare the effect of FPP using the same CAT reporter gene (pMC110) (Fig. 4Go). FPP activated mouse (m)ER{alpha}, human (h)GR, mRXR{alpha}, and hRAR{alpha} (Fig. 4AGo). FPP did not stimulate CAT activity when only the Gal4 DBD was expressed (data not shown). In addition, FPP did not to activate the hGR variant hGRß, mPPAR{gamma}, hFXR{alpha}, hVDR (vitamin D receptor), or mLXR{alpha} (Fig. 4BGo). The latter four receptors were expressed because we could activate them with their respective agonists (1 µM rosiglitazone, 20 µM farnesol, 1 µM 1,25-dihydroxyvitamin D3, or 10 µM 27-hydroxycholesterol) (Fig. 4BGo). Farnesol did not activate TR (Fig. 1Go), or ER, GR, RXR, or RAR (data not shown). Unlike TR, FPP appears to consistently act as a full-agonist for ER{alpha} and GR because the extent of activation by FPP was similar to that found for estradiol (E2) and dexamethasone (Dex), respectively (Fig. 4AGo). Studies of transcriptional activation by hGR, hER{alpha}, and mRXR{alpha} using mevastatin, mevalonic acid, and ZGA gave similar results as with cTR{alpha} in Fig. 3Go (data not shown). In addition, antagonists of GR (RU486) or ER{alpha} (ICI182,780) inhibited activation of these receptors by FPP (Fig. 5Go), similar to that found with the TR inhibitor D4. We also found that FPP could activate AR as well as PR but somewhat less efficiently compared with TR, RXR, GR, or ER (data not shown).


Figure 4
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  		Fig. 4. FPP Activates a Wide Variety of Nuclear Hormone Receptors

Hela cells were transfected with vectors expressing Gal4-DBD chimeras of the LBDs of (A) cTR{alpha}, mER{alpha}, hRAR{alpha}, mRXR{alpha}, hGR or (B) mPPAR{gamma}, hFXR{alpha}, hVDR, mLXR{alpha}, and a Gal4-CAT reporter gene (pMC110). For panel B, hGRß is a full-length protein and the reporter used was mouse mammary tumor virus-CAT. The cells were then incubated with the indicated concentrations of their respective cognate agonists [T3, E2, all-trans retinoic acid (ATRA) and 9-cis retinoic acid, (9-cis RA), Dex, rosiglitazone, farnesol, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), 27-hydroxycholesterol (27-OH-Chol)] or FPP at the indicated concentrations. The cells were harvested 40 h later and analyzed for CAT activity. The results represent the mean ± SD of three experiments.

 

Figure 5
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  		Fig. 5. Nuclear Hormone Receptor Antagonists Block Receptor Activation by FPP

Hela cells were transfected with a vectors expressing Gal4-DBD chimeras of the LBDs of cTR{alpha}, mER{alpha} or hGR, and pMC110. Cells were then incubated with the indicated concentrations of their respective cognate agonists, FPP, or FPP + the selective receptor antagonists: D4 for TR{alpha}, Ru486 for GR, or ICI182,780 (ICI) for ER{alpha} as indicated. The cells were harvested 40 h later and analyzed for CAT activity. The results represent the mean ± SD of three experiments.

 
We examined the effect of FPP or ZGA on a number of orphan receptors expressed as Gal4-LBD chimeras (mSHP, rHNF4{alpha}, hROR{alpha}, mLRH-1 and hLRH-1, mSF1, dFTZ-F1, hERR (estrogen-related receptor) {alpha}, hERRß, hERR{gamma}, mNur77, and the primordial ER-related receptors from Aplysia and from Octopus vulgaris) and found no activation (data not shown). In addition, FPP did not activate the ecdysone receptor or ultraspiracle, the insect homolog of RXR. The lack of activation of ultraspiricle by FPP is interesting in view of the fact (although somewhat controversial) that Juvenile hormone III and methyl farnesoate, which are derived from farnesol after dephosphorylation of FPP in insect cells (37), may function as high-affinity ligands for this receptor (38, 39). HNF4, ROR{alpha}, the ERRs, Nur77 (6, 23) and the Aplysia-ER and Octopus vulgaris-ER are constitutively active (26, 40). This activity was not affected by blocking the formation of FPP with mevastatin (data not shown), suggesting that endogenous FPP is not a ligand for these receptors.

FPP Incubation with Receptors Leads to Coactivator Recruitment in Vitro and in Vivo in Yeast
Our results suggest that FPP activates certain receptors such as TR, ER, and GR through direct binding to the receptor LBD. In contrast, FPP does not appear to activate PPAR{gamma}, LXR{alpha}, and VDR in cells. To assess whether these differences relate to coactivator recruitment by FPP, we examined the binding of reticulocyte lysate synthesized [35S]cTR{alpha}, [35S]hER{alpha}, [35S]hGR, [35S]mPPAR{gamma}, [35S]hLXR{alpha}, and [35S]hVDR with glutathione-S-transferase (GST)-nuclear receptor coactivator (NRC)(849–1153) in vitro. The 849- to 1153-amino acid region of NRC contains the LxxLL-1 receptor interaction domain that interacts with a wide variety of agonist-bound nuclear receptor LBDs (14, 41). Figure 6Go shows that incubation with either T3 or FPP enhances the interaction of TR{alpha} with GST-NRC. The binding of ER{alpha} and GR to GST-NRC was also enhanced by FPP. In contrast, FPP did not enhance the interaction of [35S]mPPAR{gamma}, [35S]hLXR{alpha}, or [35S]hVDR with GST-NRC whereas binding of these receptors to NRC was stimulated by their cognate ligands (Fig. 6Go). In keeping with previous findings (41), control studies with GST alone showed no effect of ligand receptor recruitment (data not shown). FPP was also without effect (data not shown).


Figure 6
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  		Fig. 6. FPP Recruits the NRC to TR{alpha}, ER{alpha}, and GR, But Not PPAR, LXR, or VDR in Vitro

cTR{alpha}, hER{alpha}, hGR, mPPAR{gamma}, hLXR{alpha}, and hVDR were labeled in vitro with [35S]L-methionine using a rabbit reticulocyte lysate system. The receptors were incubated with GST or GST-NRC in the presence and absence of the indicated ligands as described in Materials and Methods. The bound receptor was then analyzed by sodium dodecyl sulfate gel electrophoresis followed by autoradiography. Shown is the binding to GST-NRC. No effect of ligand or FPP was found with GST alone (data not shown). 27-OH-Chol, 27-Hydroxycholesterol; Ros, rosiglitazone.

 
We also examined the effect of FPP on stimulating the interaction of cTR{alpha}, hGR, or hER{alpha} with NRC in a yeast two-hybrid interaction assay (41). LexA-LBD fusions were expressed in yeast along with B42-NRC, which is conditionally expressed by galactose. Parallel studies were carried out with B42-NRC in which the three leucines in LxxLL-1 were changed to alanines. This NRC mutant does not interact with agonist-bound nuclear receptors (14, 41). The association of B42-NRC by agonist-bound receptor was assessed using pSH18–34, a LexA-LacZ reporter gene. Figure 7Go shows that addition of T3, FPP, or ZGA to the culture media enhances the interaction of LexA-cTR{alpha} with B42-NRC (although yeast synthesize ergosterol instead of cholesterol, the synthesis of ergosterol also requires the synthesis of squalene from FPP, which is blocked by ZGA). In contrast, no interaction occurred with the B42-NRC LxxLL-1 mutant, suggesting that FPP mediates a conformational change similar to that formed by T3-bound TR{alpha}. Studies were carried out with LexA-hGR and LexA-hER{alpha}, which gave similar results (not illustrated).


Figure 7
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  		Fig. 7. FPP Increases the Interaction of TR{alpha} with Coactivators in Yeast

A yeast interaction trap assay was carried out as described in Materials and Methods and in Ref. 41 . EGY48 yeast were transformed with vectors expressing a LexA-LBD of cTR{alpha}, wild-type (wt) NRC or an LxxLL NRC mutant (mutNRC), and the LexA-LacZ reporter (pSH18–34). The various yeast clones were grown in galactose/raffinose medium (to induce the NRCs) without or with T3, FPP, or ZGA (1 µM, 10 µM, and 100 µM, respectively). After incubation overnight at 30 C the yeast cultures were assayed for ß-galactosidase. T3, FPP, or ZGA stimulate an increase in ß-galactosidase activity in yeast expressing LexA-TR{alpha} + wtNRC whereas no activity was found with LexA-TR{alpha} alone or LexA-TR{alpha} + mutNRC.

 
FPP Binds to ER and TR in Vitro
The recruitment of coactivators to the TR, ER, and GR LBDs (Fig. 6Go) by FPP supports the notion that FPP acts as an agonist by binding directly to these receptor LBDs. To further document this, we examined the effect of FPP on binding of [3H]E2 to hER{alpha} and [125I]T3 to cTR{alpha}. GST-cTR{alpha} and GST-hER{alpha} were expressed in E. coli and purified with glutathione agarose. A similar study with hGR was not carried out because we found that GST-GR did not efficiently bind ligand. In fact, the coactivator recruitment study for GR in Fig. 6Go could only be performed if FPP or Dex was present in the reticulocyte lysates during GR synthesis, as described previously (42). Figure 8AGo shows that FPP inhibits the binding of [3H]E2 to GST-ER{alpha}, although with a much lower efficacy than E2 (~100-fold). In contrast with FPP, 20 µM farnesol did not inhibit [3H]E2 binding to GST-ER{alpha} (data not shown). Figure 8BGo also shows that [3H]FPP binds to hER{alpha} and that this binding is inhibited by unlabeled FPP. Similar studies with [125I]T3 and cTR{alpha} indicated that the affinity of FPP for cTR{alpha} was about 10-fold lower than that found for FPP and ER (Fig. 8CGo). This may explain, in part, the finding that FPP acts as partial agonist for TR in some experiments, whereas FPP acts as a full-agonist for ER{alpha}.


Figure 8
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  		Fig. 8. Binding of [3H]E2 and [3H]FPP to the ER-LBD

A, GST-hER{alpha} (150 ng) was incubated with 1 nM [3H]E2 alone along with the indicated concentrations of nonradioactive E2 or FPP. The samples were incubated for 30 min at 25 C and then 1 h at 4 C. The samples were then washed at 4 C three times and then analyzed for bound [3H]E2 using a liquid scintillation counter. GST alone served as a control, and no [3H]E2 binding to the GST control was found. The results were plotted as [3H]E2 bound. The amount of [3H]E2 bound without addition of E2 or FPP was considered as 100. The amounts of [3H]E2 bound with the different concentrations of E2 or FPP are plotted as a percent of [3H]E2 bound without addition of E2 or FPP. B, GST-hER{alpha} (150 ng) was incubated with [3H]FPP (0.5 µM) alone or with 10 µM nonradioactive FPP and analyzed as indicated in Materials and Methods. Binding of [3H]FPP to GST-hER{alpha} is inhibited by unlabeled FPP. [3H]FPP does not bind to the GST control. C, Inhibition of [125I]T3 binding to GST-cTR{alpha} by FPP and nonradioactive T3 was carried out as described for GST-hER{alpha} in panel A above except that 2 nM [125I]T3 was used.

 
ZGA Incubation Leads to Transcriptional Activation through Endogenous ER in MCF-7 Cells
The functional studies described above were carried out in HeLa cells using transiently expressed receptors. To assess whether FPP can activate through endogenous receptor levels, we examined the effect of FPP in MCF-7 cells using an ER-specific CAT reporter gene. Interestingly, addition of FPP to the medium did not lead to stimulation even when ER was overexpressed using an ER expression vector. In contrast with FPP, we found that incubation with ZGA resulted in stimulation of the ER-CAT reporter gene by endogenously expressed ER in MCF-7 cells, and this effect was similar to that found with E2 (Fig. 9AGo). The effect of ZGA was inhibited by the ER antagonist ICI182780. Similar to the results found for TR in HeLa cells (Fig. 3Go), stimulation by ZGA was inhibited by mevastastin, and this inhibition could be rescued by incubation with mevalonic acid (Fig. 9AGo). Incubation with farnesylation inhibitors also resulted in stimulation (data not shown). Although the results of Fig. 9AGo suggest that activation of endogenous ER can occur through elevation of FPP, addition of FPP to the medium does not lead to activation, suggesting that FPP does not enter MCF-7 cells. This conclusion is supported by the finding that FPP did not activate TR or GR when expressed in MCF-7 cells, but these receptors could be activated by ZGA (Fig. 9BGo). Why FPP does not enter MCF-7 cells is unclear. Actually, because FPP is a charged lipid, it is more surprising that the molecule enters HeLa cells.


Figure 9
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  		Fig. 9. FPP Activates through Endogenous ER in MCF-7 Cells

A, MCF-7 cells were transfected with an ER-selective CAT reporter gene ({Delta}MTV-IR+3-CAT) using phenol red-free DMEM supplemented with hormone-depleted calf serum (10% vol/vol). Cells were then incubated with E2, ZGA, mevastatin, mevalonic acid (MVA), and ICI182,780 (ICI) as indicated. The cells were harvested 40 h later for CAT activity. B, MCF-7 cells were transfected with Gal4-DBD chimeras of the LBDs of cTR{alpha}, hGR, mPPAR{gamma}, hVDR, mLXR{alpha}, and the Gal4-CAT reporter gene, pMC110. Cells were then incubated with the indicated concentrations of cognate ligand, FPP, or ZGA and 40 h later were harvested for CAT activity. C, MCF-7 cells, or HeLa cells transiently expressing ER{alpha}, were incubated with 7.5 nM [3H]E2, 7.5 nM [3H]E2 + 7.5 µM unlabeled E2, or 7.5 nM [3H]E2 + 200 µM ZGA as described in Materials and Methods. After a 1.5-h incubation at 37 C, the cells were chilled to 4 C and washed three times with chilled isotonic saline, and the extent of [3H]E2 bound was determined by liquid scintillation counting. Each point represents an assay carried out in quadruplicate. 27-OH-Chol, 27-Hydoxycholesterol.

 
Elevation of FPP in HeLa cells does not activate mPPAR{gamma}, hLXR{alpha}, and hVDR (Fig. 4BGo). To assess whether this might be unique to HeLa cells, we used MCF-7 cells to determine whether elevation of endogenous FPP levels leads to activation of these receptors (Fig. 9BGo). Each of these receptors was activated by their cognate ligands but not by incubation of MCF-7 cells with ZGA, which activates TR, GR, and ER.

Our results suggest that elevation of FPP in cells can enhance transcriptional activation by endogenously expressed ER{alpha} in MCF-7 cells, and by ER{alpha} when transiently expressed in HeLa cells (Figs. 4Go, 5Go, and 9AGo). In addition, FPP inhibits the binding of [3H]E2 to the hER{alpha} LBD in vitro, and [3H]FPP binds to the hER{alpha} LBD in vitro as well (Fig. 8Go). These in vitro studies support the notion that elevation of FPP in cells leads to activation of ER{alpha} through binding to the receptor.

To provide further evidence for this, we carried out in vivo studies in MCF-7 cells, and in HeLa cells transiently expressing ER{alpha}, to further document that FPP acts in cells through binding to ER{alpha} (Fig. 9CGo). MCF-7 cells, or HeLa cells transiently expressing ER{alpha}, were first incubated with or without ZGA (200 µM) for 20 h to elevate intracellular FPP levels. The cells were then incubated with 7.5 nM [3H]E2 alone (a concentration expected to give ~80% maximal binding to ER) or with a 1000-fold molar excess of unlabeled E2, or with ZGA for 1.5 h. The cells were chilled, and then washed three times, after which the extent of whole cell binding of [3H]E2 was assessed. Figure 9CGo shows that incubation of unlabeled E2 or ZGA with HeLa cells or MCF-7 cells inhibits the binding of [3H]E2 to ER although unlabeled E2 was slightly more efficient than ZGA. HeLa cells which were not transfected to express ER{alpha} showed no effect of unlabeled E2 or ZGA on the extent of [3H]E2 binding, which was 10-fold lower than with cells expressing ER{alpha} and reflects nonspecific binding (not illustrated). Parallel studies of the extent of [3H]E2 binding to the nuclei of these cells showed a similar pattern as the whole-cell binding assay (not illustrated). The results of Fig. 9CGo supports the notion that the levels of FPP increased by ZGA in both cell types is sufficient to bind to ER as reflected by the inhibition binding of [3H]E2 to ER. These in vivo findings, along with the evidence that FPP binds to ER{alpha} in vitro, and that ZGA incubation enhances transcriptional activation of an ER-reporter gene in HeLa and MCF-7 cells, which is inhibited by an ER{alpha} antagonist, further supports the notion that elevation of FPP in cells acts through binding to nuclear receptors.

Taken together, our findings indicate that elevation of FPP in MCF-7 cells can lead to transcriptional activation of ER-responsive genes through endogenous ER. To further study this, we used microarray studies to compare the genes activated by E2 and FPP in MCF-7 cells. Cells were incubated with 100 nM E2 or 100 µM ZGA to elevate FPP levels. Cells that received dimethylsulfoxide vehicle served as controls. The microarray analysis indicated that E2 and ZGA modulated both distinct and common set of genes. Supplemental Fig. 10 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org) shows the results of two independent experiments in which ZGA (FPP inferred) and E2 stimulate (yellow) or repress (blue) the same set of genes in MCF-7 cells. In summary, E2 incubation resulted in an increase (at least 3-fold) of 672 mRNAs. ZGA resulted in an increase of 138 mRNAs (at least 3-fold). Of the 138 mRNAs increased by ZGA, 38 were common with those increased by E2. E2 resulted in a decrease (at least 3-fold) of 994 mRNAs whereas ZGA resulted in at least a 3-fold decrease in 575 mRNAs. Of these 575 mRNAs, 45 were also found to be reduced by E2 incubation.

In Silico Docking of FPP with the LBDs of Nuclear Hormone Receptors
Our results with receptor antagonists suggest that FPP activates through binding to the receptor ligand binding pocket. To further explore this, we used a previously validated computational molecular docking algorithm (30, 31, 32, 43, 44, 45, 46) to assess the mode of interaction of FPP with the various nuclear receptors. One hundred forty-three crystallographic three-dimensional (3D) structural models of the LBDs of RARß and {gamma}, RXR{alpha} and ß, AR, ER{alpha} and ß, ERR{alpha} and {gamma}, FXR, GR, HNF4{gamma}, PPAR{alpha}, {gamma}, and {delta}, PR, pregnane X receptor, ROR{alpha} and {gamma}, SF1, TR{alpha} and ß, and VDR have been deposited in the Protein Structural Database (supplemental Table 1). Docking of FPP to each revealed that FPP docked consistently in the ligand-binding pocket of the agonist conformation of those receptors found to be activated by FPP in vitro. The common docked conformation placed the FPP pyrophosphate group at the opposite end of the ligand binding pocket from H12, and, for each receptor activated by FPP, the distal carbon tail of FPP makes hydrophobic contacts with H11–H12 loop and/or H12 (supplemental Fig. 11A), most commonly with the side chains of Ile-Met-Phe (supplemental Fig. 11B). In contrast, docking to the group of agonist structures representing the LBD of nuclear receptors not activated by FPP demonstrated either inability of FPP to dock into the ligand-binding pocket or docking into large ligand-binding pockets in conformations making no contact with the H11–H12 loop. The docking results, which were unbiased because they depend only on the 3D structures of FPP and the LBDs, correlate very well with the in vitro activation data. Detailed study of the interactions suggests that a few specific residues in helix H11–H12 are necessary but not sufficient to allow FPP to activate a particular nuclear receptor. Rather, the overall size and shape of the ligand-binding pocket, a feature that is more indirectly dictated by the nuclear receptor protein sequence, appears to limit which nuclear receptor may be activated by FPP.

Taken together, our findings provide compelling evidence that FPP can bind to the LBDs of a subset of nuclear hormone receptors and lead to activation by mediating a structural modification of the LBD involving coactivator recruitment through an H12-dependent mechanism. Thus, mutations of H12 that abrogate activation by cognate ligand agonists (e.g. cTR{alpha}(L398R) also abrogate activation by FFP. In addition, as examined for TR, ER{alpha}, and GR, activation by FPP is blocked by respective antagonist ligands for these receptors. Lastly, using an unbiased in silico docking approach, we found that FPP docks to the ligand-binding pocket of receptors that are activated by FPP but not to the receptors that are not activated by FPP.

Our findings may have implications for some of the biological effects observed with farnesyl transferase inhibitors (47), which interfere with the farnesylation of small GTPases such as members of the Ras family. Although such compounds act through inhibition of protein farnesylation, they would also be expected to increase the level of FPP in cells that might act through an orphan receptor or one of the receptors that we have shown to be activated by FPP in this study. Our results also explain an interesting observation reported by Doisneau-Sixou et al. (48), who found that incubation of farnesyl transferase inhibitors (e.g. FTI277) with MCF-7 cells leads to stimulation of several ER-responsive genes as well as recruitment of SRC-1 to ER{alpha} as assessed by immunoprecipitation assays. Because the farnesylation inhibitors did not directly bind to ER{alpha}, the results were interpreted to indicate that farnesylated or prenylated proteins act to somehow repress activation by ER{alpha}. Our results suggest, however, that the activation of ER{alpha} by farnesylation inhibitors reported by Doisneau-Sixou et al. (48) can be explained through elevation of FPP, which acts as an agonist for ER{alpha}.

Although the biological implications of our findings are not absolutely clear, they raise a number of interesting possibilities. First, some of the known pleotropic effects of statins, which appear independent of their effect on reducing cholesterol levels (49, 50), may be mediated, in part, through reduction in the activity of one or more constitutively acting orphan receptors that might use FPP as an agonist. The nitrogen-containing bisphosphonates are thought to act by inhibiting the enzyme involved in FPP synthesis (farnesyl diphosphate synthetase) (51). Thus, like the statins, some of the pleotropic effects of bisphosphonates may also occur, in part, by a reduction of FPP levels. Lastly, knockout of the squalene synthase gene in mice leads to defective neural tube closure (52), which is reminiscent of some of the neural defects seen in rodents receiving high doses of all-trans retinoic acid (53). In this regard, a squalene synthase inhibitor (TAK-475) (54) is in Phase III clinical trials for the treatment of low-density lipoprotein cholesterol. Results of this trial have not yet been reported (55) so it is unknown whether a patient group received the squalene synthase inhibitor without statins, which might lead to side effects related to nuclear receptor activation.

An interesting question is whether sufficient levels of FPP occur in certain tissues or pathological processes to mediate effects through nuclear receptors. A study of FPP levels in liver before and after ZGA administration indicates a basal level of approximately 5.4 nmol/g (wet weight) in control livers and about 110 nmol/g in livers from ZGA-infused rats (35). Assuming that the liver is approximately 75–80% water, the concentration of FPP in liver would be about 7 µM in the basal state and approximately 140 µM after ZGA administration. Figure 1Go indicates that addition of 5–10 µM FPP to cell culture media is sufficient to activate TR (the intracellular concentration FPP is likely lower than that in the medium). Because ER, and possibly GR, appears to have a higher affinity for FPP than TR, the levels of FPP in liver cells would be in the range to activate these receptors as well.

The finding that FPP activates a number of nuclear receptors, particularly those with small ligand-binding pockets, suggests that it binds to a structural feature(s) that has been conserved in evolution. This implies that FPP may have initially functioned as a ligand for an ancient steroid nuclear receptor that was involved in metabolic regulation. Nursa.org (Nuclear Receptor Signaling Atlas) lists 104 known receptor isoforms and orphan receptors. Therefore, in addition to being able to activate TR, ER, GR, and the retinoid receptors under certain conditions, FPP may function as an even higher affinity ligand for one of the many receptor isoforms or orphan receptors that we did not have the opportunity to study. In this context FPP might be considered as an orphan ligand in search of an orphan receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Chemicals
Plasmid vectors expressing full-length cTR{alpha}, rTRß, hER{alpha}, hERß, hPR, hGR, hVDR, and hAR were previously described as were vectors expressing Gal4-hRAR{alpha}, Gal4-mRXR{alpha}, Gal4-mER{alpha}, Gal4-hGR and LexA-cTR{alpha}, LexA-GR, and LexA-hER{alpha} (41, 56). Vectors expressing Gal4-mPPAR{gamma}, Gal4-hLXR{alpha}, and Gal4-rFXR{alpha} (57, 58) along with wild-type mPPAR{gamma} were obtained from Barry M. Forman (City of Hope, Duarte, CA). Wild-type hLXR{alpha} was also previously described (59). Ken-ichirou Morohashi (National Institute For Basic Biology, Japan) provided Gal4 vectors expressing the LBDs of mLRH-1, hERRß, rHNF4{alpha}, hROR{alpha}, dFTZ-F1, and mSF1 (60). Peder Norby (Molecular Genetics, Novo Nordisk AIS, Denmark) provided Gal4 vectors expressing hERR{alpha}, hERRß, and hERR{gamma} (61); Yoon-Kwang Lee and David Moore (Baylor College of Medicine, Houston, TX) provided Gal4 vectors expressing mSHP, rHNF4{alpha}, and mNur77 (17, 62). Johan Auwerx (Universite Louis Pasteur, France) provided Gal4-hLRH-1 (63); Patricia Willy (X-ceptor Therapeutics, San Diego, CA) also provided Gal4-hERR{alpha} (64); and Keith Baker, David Mangelsdorf, and Carl Thummel provided us with Gal4 vectors expressing the LBDs of ecdysone and ultraspiracle insect receptors (65). John Cidlowski (National Institute of Environmental Health Sciences, Research Triangle Park, NC) provided us with a full-length LBD variant isoform of GR (hGRß) that is not activated by glucocorticoid agonists (66). Joseph Thornton (Department of Biology, University of Oregon, Eugene, OR) provided us with vectors expressing the Gal4 DBD with the LBDs of primordial ER-related receptors from Aplysia and Octopus vulgaris (40, 67). All hormones, ligands, and chemicals used in this study were obtained from Sigma-Aldrich Corp. (St. Louis, MO) except for the farnesyl transferase inhibitors FTI277 and B589, which were obtained from Cayman Chemical Co. (Ann Arbor, MI) and 27-hydroxycholesterol, which was a gift of Dr. Norman Javitt, Division of Gastroenterology (NYU School of Medicine, New York, NY). The TR antagonist D4 has been previously described (30).

Transfections into Mammalian Cells
Transfections in HeLa cells or MCF-7 cells were performed in DMEM using calcium-phosphate coprecipitation as described earlier (41). The medium was supplemented to 10% with calf serum treated with AG1X8 resin and charcoal to remove hormones (41). The various receptor agonists and antagonists were used at the concentrations indicated in the text and/or figures. Typically 100 ng of CAT reporter plasmid was used per sample (4-cm2 well) unless otherwise indicated. Expression plasmid (200 ng) was used for full-length receptors whereas Gal4 vectors were used at 150 ng/transfection. The reporters used were {Delta}MTV-IR-CAT for TR, {Delta}MTV-IR+3-CAT for ER, and pMC110 for the Gal4-receptor chimeras (41, 56). All transfections were performed in duplicate or triplicate. CAT assays were performed 40 h after transfection as described elsewhere (41). Each study was performed in duplicate or triplicate at least three times, and the results are presented as mean ± SD of the experiments.

Yeast Interaction and ß-Galactosidase Assays
A yeast interaction trap assay was carried out as previously described (41). EGY48 yeast expressing a LexA-DBD fusion with the cTR{alpha}-LBD was transformed with pJG4–5 (conditionally regulated by galactose) expressing wild-type NRC containing the LxxLL receptor-interacting domain (amino acids 849-1153) or an LxxLL NRC mutant (mutNRC). pSH18–34 containing LexA binding sites that expresses LacZ was used as a reporter gene. Yeast clones were grown overnight at 30 C in galactose/raffinose medium containing T3, FPP, or ZGA (1 µM, 10 µM, and 100 µM, respectively) and then quantitated for ß-galactosidase in triplicate (41). The units of ß-galactosidase are expressed as (O.D. 420 nm x 1000)/(minutes of incubation) x O.D. 600 nm of yeast suspension. As indicated in the legend to Fig. 7Go, similar studies were carried out with LexA-hER{alpha} and LexA-hGR.

Effect of FPP on the Binding of Nuclear Hormone Receptors to a Receptor Coactivator in Vitro
A GST fusion with the receptor interacting region of the coactivator NRC [GST-NRC(849–1153)] was expressed in SG1117 E. coli by induction with isopropyl-ß-D-thiogalactopyranoside and then purified and immobilized on glutathione-agarose (14, 41). cTR{alpha}, hER{alpha}, hGR, mPPAR{gamma}, hLXR{alpha}, and hVDR were labeled with [35S]L-methionine by in vitro transcription/translation using rabbit reticulocyte lysates. We found that for 35S-labeled GR to bind NRC, ligand had to also be included during the reticulocyte translation as previously reported for agonists and GR (42). 35S-labeled receptors were mixed with 300 ng of GST or GST-NRC(849–1153) immobilized on beads. The samples were incubated at 25 C without or with cognate ligands [T3, E2, Dex, rosiglitazone, 27-hydroxycholesterol (27-OH-Chol), 1,25-dihydroxyvitaminD3 (1,25-(OH)2D3) (each as 1 µM) or 20 µM FPP] for 30 min followed by incubation at 4 C for 1 h in binding buffer (Tris-HCl 20 mM, pH 7.7, at 25 C; 2 mM MgCl2; 100 mM NaCl; 1 mM dithiothreitol; 1 µg/ml ovalbumin; 0.5 mM phenylmethylsulfonylfluoride; 0.25% Nonidet P40; and 0.25 µM ZnCl2). The samples were then washed three times with the same buffer at 4 C without or with the same concentration of ligand. This was particularly important for FPP because its affinity is lower than the other ligands and, thus, binding may be reduced during the washing procedure. The bound 35S-labeled receptors were analyzed by sodium dodecyl sulfate-gel electrophoresis followed by autoradiography.

Binding of E2 and FPP to the ER-LBD in Vitro
GST-hER{alpha} (150 ng) was incubated with 1 nM [3H]E2 (95 Ci/mmol) (PerkinElmer Corp., Wellesley, MA) alone along with the indicated concentrations of nonradioactive E2 or FPP. The samples were incubated for 30 min at 25 C and then for 1 h at 4 C in binding buffer (25 mM Tris, pH 7.8, at 25 C; 0.05% Triton X-100; 0.5 mM EDTA; 100 mM KCl; 10 mM 2-mercaptoethanol; 1 mM phenylmethylsulfonylfluoride). The samples were then washed three times in the same buffer at 4 C, and analyzed for bound [3H]E2 using a liquid scintillation counter. GST alone served as a control and no [3H]E2 binding to the GST control was found. The amount of [3H]E2 bound without addition of E2 or FPP was considered as 100. The [3H]E2 bound with the different concentrations of E2 or FPP are plotted as a percent of [3H]E2 bound without addition of E2 or FPP. A similar study was carried out with GST-cTR{alpha} and [125I]T3 (3000 Ci/mmol) (PerkinElmer). GST-hER{alpha} was also used to assess whether [3H]FPP (20.5 Ci/mmol) (Amersham Pharmacia Biotech, Piscataway, NY) binds to ER. GST-hER{alpha} (150 ng) or GST alone (150 ng) was incubated with 0.5 µM [3H]FPP alone or in the presence of 10 µM nonradioactive FPP using the conditions described above.

ZGA Incubation Inhibits the Binding of [3H]E2 to ER{alpha} in Vivo
MCF-7 cells or HeLa cells were plated in six spot multiwell plates (~750,000 cells per 9-cm2 well). The HeLa cells were then transiently transfected to express ER{alpha}. The HeLa cells and MCF-7 cells were then incubated twice for 1.5 h with DMEM-Phenol red free medium supplemented to 10% with resin-charcoal-treated bovine serum to remove E2 (41, 56). The medium was replaced a third time, and the cells were incubated without or with 200 µM ZGA for 20 h. The medium was then replaced with DMEM-Phenol red free medium without serum, and the cells were incubated with 7.5 nM [3H]E2 or 7.5 nM [3H]E2 + 7.5 µM unlabeled E2. ZGA was added to the cells that had received 200 µM ZGA for 20 h, and the cells were also incubated with 7.5 nM [3H]E2. After 1.5 h incubation at 37 C, the plates were chilled on ice and the attached cells were washed three times with chilled isotonic saline. The cells were then harvested and analyzed for the extent of [3H]E2 binding using a liquid scintillation counter. To study the extent of nuclear associated [3H]E2, cells were isolated with STM buffer (0.25 M sucrose; 10 mM Tris, pH 8, at 25 C; 1 mM MgCl2; 0.2% Triton X-100) and the nuclei prepared as previously described (30). All binding studies were carried out in quadruplicate.

Gene Expression Profiling and Microarray Data Analysis
Microarray analysis was performed at the NYU Cancer Institute Genomics Facility. MCF-7 cells were incubated in DMEM-Phenol red free medium supplemented to 10% with resin-charcoal-treated bovine serum to remove E2 (41, 56). Cells were then incubated with 100 nM E2 or 100 µM ZGA for 24 h. Cells that received only vehicle (dimethylsulfoxide) served as controls. Total RNA was prepared with Trizol reagent (Invitrogen, Carlsbad, CA), and the preparation of cRNA probes and hybridization to GeneChip HGU133A 2.0 arrays followed the recommendations of the manufacturer (Affymetrix). Raw GeneChip data were normalized at the probe level by Robust Multichip Average algorithm (68) and further filtered using GeneSpring 7.2 (Agilent Technologies, Palo Alto, CA). The differentially abundant mRNAs (between the different groups) were statistically filtered using an intersection of t test/one-way ANOVA results (with P value cut-off of 0.05) and Significance Analysis of Microarrays with false discovery rate set to 5% (69). Results are presented from two independent experiments and are shown as supplemental data Fig. 10.

In Silico Molecular Docking of FPP with the LBDs of Nuclear Hormone Receptors
Docking was performed as previously described (30, 31, 32, 43, 44, 45, 46) to assess the mode of interaction of FPP with 143 crystallographic 3D structural models of nuclear receptor LBDs (see supplemental Table 1). Grid potentials, including those for electrostatics, van der Waals, and hydrogen bonding, were generated from the full-atom Protein Data Bank models of the nuclear receptors. A full-atom flexible FPP ligand was then docked to these grid potentials using the Biased probability Monte Carlo procedure (70) and resulting conformations of the ligand within the grid potentials were ranked according to energy score. Calculation for each model took approximately 4 CPU h on a 1.67-gHz PowerPC G4 chip (~900,000 functional calls).


   ACKNOWLEDGMENTS
 
We thank the New York University Cancer Institute Genomics Facility for microarray data processing and analysis. We especially thank all the investigators who provided us with the plasmids and other reagents listed in Materials and Methods.


   FOOTNOTES
 
This work was supported by Grants DK16636 (to H.H.S.) and NR08029 (to M.T.-C.) from the National Institutes of Health and a grant from the Entertainment Industry Foundation (to H.H.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 31, 2007

Abbreviations: CAT, Chloramphenicol acetyltransferase; DBD, DNA-binding domain; E2, estradiol; ER, estrogen receptor; ERR, estrogen-related receptor; FPP, farnesyl pyrophosphate; FXR, farnesoid X receptor; GR, glucocorticoid receptor; H12, helix 12; HNF, hepatocyte nuclear factor; LBD, ligand-binding domain; LRH, liver receptor homolog; LXR, liver X receptor; NRC, nuclear receptor coactivator; PP, pyrophosphate; PPAR, peroxisome proliferative-activating receptor; PR, progesterone receptor; RAR, retinoic acid receptor; ROR, retinoid-related orphan nuclear receptor; RXR, retinoid X receptor; SF1, steroidogenic factor-1; TR, thyroid hormone receptor; VDR, vitamin D receptor; ZGA, zaragozic acid A.

Received for publication February 12, 2007. Accepted for publication July 25, 2007.


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 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   TRα  |  RARα  |  LXRα  |  FXRα  |  VDR  |  RXRα  |  ERα  |  GR
Coregulators:   ASC-2
Ligands:   all-trans-Retinoic acid  |  Calcitriol  |  Dexamethasone  |  17β-Estradiol  |  9-cis-Retinoic acid  |  Thyroid hormone  |  Mifepristone  |  Rosiglitazone  |  Fulvestrant



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