Structure-function relationships of the raloxifene-estrogen receptor-alpha complex for regulating transforming growth factor-alpha expression in breast cancer cells

Abstract

Amino acid Asp-351 in the ligand binding domain of estrogen receptor alpha (ERalpha) plays an important role in regulating the estrogen-like activity of selective estrogen receptor modulator-ERalpha complexes. 4-Hydroxytamoxifen is a full agonist at a transforming growth factor alpha target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with the wild-type ERalpha. In contrast, raloxifene (Ral), which is also a selective estrogen receptor modulator, is a complete antiestrogen in this system. Because D351G ERalpha allosterically silences activation function-1 activity in the 4-hydroxytamoxifen-ERalpha complex with the complete loss of estrogen-like activity, we examined the converse interaction of amino acid 351 and the piperidine ring of the antiestrogen side chain of raloxifene to enhance estrogen-like action. MDA-MB-231 cells were either transiently or stably transfected with Asp-351 (the wild type), D351E, D351Y, or D351F ERalpha expression vectors. Profound differences in the agonist and antagonist actions of Ralcenter dotERalpha complexes were noted only in stable transfectants. The agonist activity of the Ralcenter dotERalpha complex was enhanced with D351E and D351Y ERalpha, but raloxifene lost its agonist activity with D351F ERalpha. The distance between the piperidine nitrogen of raloxifene and the negative charge of amino acid 351 was critical for estrogen-like actions. The role of the piperidine ring in neutralizing Asp-351 was addressed using compound R1h, a raloxifene derivative replacing the nitrogen on its piperidine ring with a carbon to form cyclohexane. The derivative was a potent agonist with wild type ERalpha. These results support the concept that the side chain of raloxifene shields and neutralizes the Asp-351 to produce an antiestrogenic ERalpha complex. Alteration of either the side chain or its relationship with the negative charge at amino acid 351 controls the estrogen-like action at activating function 2b of the selective estrogen receptor modulator ERalpha complex.

Fig. 1. Structures of raloxifene and the derivative R1h used in structure-function studies

Compound R1h is a raloxifene derivative that has a cyclohexane ring instead of a piperidine ring.

Fig. 2. Comparison of agonist activities of 4-OHT and raloxifene in transient and stable transfection systems

MDA-MB-231 cells were transiently or stably transfected with wild type (A) or D351Y ERα (B) expression vector as described under “Experimental Procedures.” Transient transfection results are shown in solid bars (left y axis). Stable transfection data are presented in open bars (right y axis). The cells were treated with ethanol vehicle (EtoH), 1 nm E₂, 1 µm 4-OHT, or 1 µm Ral for 24 h.

Fig. 3. Characterization of mutated ERα stably expressed in MDA-MB-231 cells

A, the ERα expression levels in these stable transfectants were shown by Western blot analysis. B, binding affinities (K_d) for E₂ were calculated from saturation binding assay data using Prism 3.0 and are presented as the mean ± S.E. The saturation binding assays for ERα stable transfectants were done at least four times. C, competition binding assay results for Ral and R1h are shown. Each experiment was repeated at least five times.

Fig. 4. Determination of agonist or antagonist activities of raloxifene in stable transfectants by measuring induction of TGFα or pS2 mRNA

A, cells expressing wild type (Asp-351) or D351E ERα were treated with ethanol vehicle (control) or increasing concentrations of Ral (0.01, 0.1,1, 10, 100, or 1000 nM) for 24 h. TGFα mRNAlevels were measured by Northern blot analysis. β-actin was used as RNA-loading control. Quantitative results (TGFα/β-actin) from three independent Northern blots were presented as the mean ±S.D. B, TGFα mRNA levels were measured and compared in cells expressing D351Y and D351F ERα cells by Northern blot analysis. C, the cells were treated with 10 nM E₂ or in combination of increasing concentrations of Ral (0.01, 0.1, 1, 10, 100, or 1000 nM) for 24 h. TGFα mRNA levels were measured by Northern blot analysis. D, expression of pS2 and TGFα induced by E₂ or Ral were compared. The concentration of E₂ and Ral were 10 µM and 1 µm, respectively. Equal RNA loading for Northern blot (N.B) was ensured by measuring β-actin mRNA levels (data not shown). The data in C are D are a representative of three experiments. RT, reverse transcription.

Fig. 5. Agonist activity of compound R1h

A, the cells expressing the wild type ERα were treated with ethanol vehicle (Control) or increasing concentrations of R1h for 24 h. Top panel is a representative Northern blot. Bottom panel shows the quantitative results (mean ±S.E.) from three independent Northern blots. B, the cells expressing the wild type, D351E, D351Y, D351F, or D351G ERα were treated with either Control or 1 µm R1h for 24 h. The equal loading of total RNA was ensured by β-actin level. This experiment was repeated three times. C, the levels of pS2 mRNA were measured by Northern blot analysis in wild type ERα-expressing cells treated with control, 10 nM E2, 1 µm Ral, or 1 µm R1h for 24 h. D, stimulatory effect of R1h on MCF-7 cell growth was measured by DNA assay. The cells were treated for 5 days with vehicle control, 1 µM E2, 1 µm Ral, 1 µm R1h, 1 µm ICI 182,780, or a combination of E₂ and Ral, R1h and ICI 182,780, E₂ and ICI 182,780.

Fig. 6. Hormone-dependent interaction of ERα with SRC-1 in vitro

A, ³⁵S-labeled wild type or mutated ERα-translated in vitro was incubated with GST-ΔSRC-1 immobilized on glutathione-Sepharose in the presence of ethanol vehicle (Control), 1 nm E₂, or 1 µm Ral at 4 °C for 2 h. After extensive washing, the bound ER·SRC-1 complexes were eluted by glutathione and separated on a 7.5% SDS-PAGE. B, the pull-down assay was done with the wild type ERα and GST-ΔSRC-1 in the presence of Control, 1 nm E₂, or 1 µm R1h.

Fig. 7. Western blot analysis of the effects of different ligands on the wild type or mutated ERα protein levels

The cells were treated with ethanol vehicle (Control), 1 nm E₂, 1 µm Ral, or 1 µm R1h for 24 h. 25 µg of whole cell lysates were loaded and separated on a 7.5% SDS-PAGE. The levels of ERα were determined by Western blot analysis using anti-ERα antibody G20. Even loading was ensured by β-actin levels measured using anti-β-actin antibody. Each Western blot was repeated at least three times.

Fig. 8. Surface structures around amino acid 351 of Ral-bound LBDs of ERα

A structural model of dimeric human ERα bound to raloxifene was derived from the Protein Data Bank (code 1ERR) (19) by removing all water molecules with the exception of the ordered water-forming H-bond with the O₃ of raloxifene, adding hydrogens and minimizing in the consistent valance force field (CVFF) using Discover (Accelrys, San Diego, CA). Mutant receptors were constructed using Biopolymer (Accelrys) to replace Asp-351 with Gly, Glu, Phe, or Tyr and to obtain a minimum energy rotomer for the mutant side chain. The results were visualized using Insight II (Accelrys).

Fig. 9. Computer modeling of the LBDs of ERα occupied by Ral or R1h

A, relationships between the side chain of Asp-351 or Glu-351 with the piperidine of raloxifene. The nitrogen in the piperidine of raloxifene forms a H-bond with Asp-351 (left panel). When Asp-351 is replaced by a glutamic acid, the positions of both the piperidine and the side chain of Glu-351 changes (middle panel). As a result, the distance between the nitrogen in the piperidine of raloxifene and the side chain of Glu-351 is longer (3.5–5.0 Å) (right panel). Thus, the negative charge of Glu-351 is exposed on the surface. B, a comparison of Ral-Asp-351 versus R1h–Asp-351 (left panel), Ral-Tyr-351 versus R1h–Tyr-351 (right panel). Because the cyclohexane of R1h cannot form a hydrogen bond with Asp-351, the side chain of R1h assumes a more extended conformation. As a result, the negative charge of Asp-351 is exposed, and R1h–ERα is an agonist. When Asp-351 is replaced by a tyrosine, Tyr-351 partially occupies the space filled by the piperidine of raloxifene in wild type ERα. Thus, the piperidine of raloxifene is displaced and the side chain of raloxifene is allowed to assume an extended conformation. Ral-D351Y ERα is a partial agonist. However, the cyclohexane interacts with Tyr-351, and R1h–D351Y ERα does not display any agonist activity.