Control of the Membrane Sex Hormone-Binding Globulin-Receptor (SHBG-R) in MCF-7 Cells: Effect of Locally Produced SHBG

Abstract

The interaction between plasma sex hormone-binding globulin (SHBG) and its receptor (SHBG-R) inhibits estradiol-induced proliferation of MCF-7 cells (human estrogen-dependent breast cancer) through cAMP and PKA. Thus, SHBG can modulate estradiol action in breast cancer, but the implications of this require a more detailed knowledge of the SHBG-R. To this end, we have transfected MCF-7 cells with an expression vector carrying the human SHBG cDNA (S-MCF-7) and studied the effects of this on both SHBG-R binding and cell proliferation. Control cells were parental MCF-7 (P-MCF-7) and MCF-7 cells transfected with the β-galactosidase gene (B-MCF-7). Transfections were mediated by lipofectin followed by selection of transfected cells with G418. The amounts of SHBG in culture medium were evaluated by IRMA assay, with only S-MCF-7 cells shown to secrete SHBG; SHBG-R levels were evaluated by tracer binding technique. In P-MCF-7 and B-MCF-7 cells, SHBG-R was detectable as a two-binding site receptor, but no binding of SHBG was observed in S-MCF-7 cells. Proliferation of cells treated with estradiol was evaluated by [³H]thymidine incorporation in the three cell lines and in cells pretreated with SHBG (1 nM) purified from human serum or with conditioned medium from S-MCF-7 cells (medium S). In all three lines, cell proliferation increased after estradiol treatment. Preincubation with purified SHBG was effective in reducing estrogen-induced cell proliferation to basal levels in P-MCF-7 and B-MCF-7 but not in S-MCF-7 cells. The estradiol effect was also inhibited in P-MCF-7 cells treated with medium S. In conclusion, 1) SHBG inhibits estradiol-induced proliferation in cells containing a functional SHBG-R, whereas it has no detectable effect in cells in which the SHBG-R is either absent or not available to bind SHBG; and 2) S-MCF-7 cells are insensitive to SHBG (locally produced or exogenous) because their SHBG-R is occupied by SHBG.

Introduction

Sex hormone-binding globulin (SHBG or SBP) is a glycoprotein in human plasma that binds both androgens and estradiol with high affinity.1 The main biologic function of SHBG is the plasma transport of sex steroids, but descriptions of a specific membrane receptor for the protein (SHBG-R) in several human tissues2., 3., 4., 5., 6. and cultured cells7., 8., 9. have suggested other potential functions at the cellular level. Evidence that SHBG-R is present mainly on membranes of estrogen/androgen-dependent tissues10 and the observations that SHBG-R can be detected in the normal premenopausal endometrium11 and endometrial adenocarcinoma12 suggest that SHBG may influence the way sex steroids act in hormone-dependent neoplasms. In particular, data obtained in estrogen-dependent breast cancer suggest that the receptor-mediated action of SHBG may modulate estradiol effects in neoplastic cells.

Estrogen-dependent MCF-7 breast cancer cells express the SHBG-R, and the interaction of SHBG with the receptor does not modify the amount of estradiol entering cells.7 It does, however, cause a significant increase in the amount of estradiol “sequestered” at the cell membrane, which in turn appears to increase cAMP accumulation in these cells.13 More importantly, SHBG inhibits the estradiol-induced proliferation of MCF-7 cells, and this action is mediated by cAMP and PKA.14 SHBG-R is present in about 70% of breast cancer tissue samples positive for both estrogen and progesterone receptors, whereas only a small percentage of steroid hormone receptor-negative samples contain SHBG-R; SHBG-R-positive samples are characterized by a lower proliferation rate than negative samples.15

Taken together, these data suggest that SHBG and SHBG-R represent a possible “anti-estrogenic pathway” in estrogen-dependent breast cancer that could modulate estradiol action. Therefore, we have manipulated this system by transfecting MCF-7 cells with a human SHBG expression vector to determine how exogenous or locally produced SHBG might affect both SHBG-R levels and estrogen-induced cell proliferation.

MCF-7 cells were transfected with an expression vector carrying a human SHBG cDNA, the Neo resistance gene, and the CMV enhancer/promoter sequence.16 Transfection was by lipofectin17 followed by selection of transfected cells (S-MCF-7) with G418 (1.0–1.25 mg/mL). Control cells were parental MCF-7 cells (P-MCF-7) and MCF-7 cells transfected with the the same expression vector containing β-galactosidase cDNA (B-MCF-7).

The secretion of SHBG by stable transfectants was evaluated by double-antibody IRMA kit providing a specific measurement of SHBG concentrations (Orion Diagnostica, Finland). Medium SHBG concentrations were 0 nM for P-MCF-7 cells, 3.0–5.7 nM for S-MCF-7 cells, and 0 nM for B-MCF-7 cells, respectively.

SHBG-R was evaluated in all the three transfected cell lines with a binding assay using ¹²⁵I-SHBG (0.5–60 pM) as the labeled tracer in the absence or presence of 60 nM radioinert SHBG. Binding data from Scatchard plots showed a two-binding site receptor, as previously described,7 in both P-MCF-7 and B-MCF-7, whereas no significant binding was observed in S-MCF-7 cells. The observed B_max and K_d values were the following: -P-MCF-7 cells: B_max1, 6.8 fmol/10⁶ cells; K_d1 = 2.1 × 10⁻¹² M; B_max2 = 77 fmol/10⁶ cells; K_d2 = 8.3 × 10⁻¹⁰ M; -S-MCF-7 cells: undetectable; -B-CF-7 cells: B_max1 = 7.3 × 10⁶ cells; K_d1 = 2.1 × 10⁻¹² M; B_max2 = 76 fmol/10⁶ cells; K_d2 = 5.0 × 10⁻¹⁰ M.

The present study does not offer any explanation about the two binding sites for SHBG. It is probable that the difference in affinity of the two sites, being only about 50-fold, reflects a different functional state of a single binding site, although further studies are needed to answer this question definitively.

When proliferation of S-MCF-7, P-MCF-7, and B-MCF-7 cells was measured by [³H]thymidine incorporation, treatment with 1 nM estradiol produced a significant increase in the growth of all the three lines (Figure 1 ). In contrast, preincubation with 1 nM exogenous SHBG (40 min) very substantially reduced (p < 0.01) the estradiol-induced cell proliferation in P-MCF-7 and B-MCF-7 cells but to a much lesser extent in S-MCF-7 cells (Figure 2 ). Furthermore, the estradiol-induced proliferation of P-MCF-7 cells was completely inhibited by preincubation of cells with conditioned medium obtained from S-MCF-7 cells but not from P-MCF-7 and B-MCF-7 cells (Figure 3 ).