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Effects of the Phytoestrogen Genistein on the Circulating Soluble Receptor Activator of Nuclear Factor B Ligand-Osteoprotegerin System in Early Postmenopausal Women

Department of Biomedical Sciences (A.C.), Section of Pharmacology, University of Modena and Reggio Emilia, 41100 Modena, Italy; Department of Clinical and Experimental Medicine and Pharmacology (A.C., D.A., M.A.I., H.M., A.B., F.S.), Section of Pharmacology, and Departments of Internal Medicine (G.S., A.R., S.B., N.F.), Obstetrical and Gynecological Sciences (R.D., F.C.), and Biochemical Physiological and Nutritional Sciences (E.B.A., R.M.), School of Medicine, University of Messina, 98125 Messina, Italy

Address all correspondence and requests for reprints to: Professor Francesco Squadrito, M.D., Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, Azienda Ospedaliera Universitaria "G. Martino," Torre Biologica 5 degree Piano, Via Consolare Valeria, 98125 Messina, Italy. E-mail: francesco.squadrito{at}unime.it.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We investigated the serum levels of both receptor activator of nuclear factor B ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) in postmenopausal healthy women after a 1-yr therapy with genistein, (n = 30; 54 mg/d), hormone replacement therapy (n = 30; 1 mg/d 17ß-estradiol combined with norethisterone acetate) and placebo (n = 30). By comparison with placebo, the soluble RANKL (sRANKL)/OPG ratio was lower in the genistein group (-69 ± 7%; P < 0.01 vs. placebo 81 ± 24%) and in hormone replacement therapy-treated women (-11 ± 2%; P < 0.01 vs. placebo). A positive correlation (r = 0.63; P < 0.01) was found between 1-yr percentage change in sRANKL/OPG ratio and 1-yr change in urinary deoxypyridinoline, a bone resorption marker. A negative correlation was observed between 1-yr percentage change in sRANKL/OPG ratio and 1-yr change in femoral neck bone mineral density (r = -0.7; P < 0.01). Our findings suggest that the sRANKL-OPG system may mediate the beneficial effects of genistein on bone remodeling in postmenopausal women.

	Introduction

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GENISTEIN, AN ISOFLAVONE that is found in abundance in soybeans and their derivative food, structurally resembles 17ß-estradiol (1). There are animal studies showing that genistein prevents estrogen-deficiency bone loss (2, 3). Our randomized, double-blind clinical trial confirmed the positive effects of genistein treatment on bone loss induced by estrogen deficiency. A 1-yr genistein administration was associated with a normalization of elevated bone turnover: the phytoestrogen reduced bone resorption markers and enhanced bone neoformation parameters. Moreover this composite effect on bone turnover caused a significant increase in lumbar spine and femoral neck bone mineral density (BMD) without any significant adverse effect on the breast and uterus (4).

The mechanism by which genistein acts on bone is unknown. In vitro studies showed that the phytoestrogen modulates the osteoprotegerin (OPG)-receptor activator of nuclear factor B ligand (RANKL) system, which represents a major advance in bone physiology and plays a crucial role in the balance of bone remodeling (5, 6).

OPG and RANKL constitute a complex mediator system involved in the regulation of the resorption process in bone, which is probably responsible for the homeostatic mechanism of bone turnover. Alterations in this system could form the basis of some metabolic bone diseases, like osteoporosis and osteopetrosis, as suggested by recent experimental evidence (7, 8, 9, 10, 11, 12, 13).

In vivo and in vitro studies (9, 12, 13, 14) have shown that OPG, a member of the TNF receptor superfamily, is secreted by osteoblasts and is able to bind the RANKL, a product of the osteoblastic lineage cells: OPG neutralizes RANKL functions and negatively regulates osteoclast differentiation, activity, and survival.

Genistein and estrogen stimulate the expression of OPG in cultured bone cells (5, 15), therefore suggesting that the previously reported effects on bone of these two substances might be, at least in part, mediated by a modulation of this cytokine system.

Therefore, the present study was performed to evaluate the serum levels of OPG and soluble RANKL (sRANKL) in postmenopausal women with osteoporosis after a 1-yr genistein and hormone replacement therapy (HRT) to establish a possible relationship between changes in the serum levels of sRANKL/OPG and modifications in bone turnover markers and BMD.

	Subjects and Methods

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Subjects

We analyzed stored serum from our previous randomized, double-blind, HRT and placebo-controlled trial assessing the genistein role on bone loss in postmenopausal women (4).

The study subjects and procedures are described in detail elsewhere (4) and summarized briefly here.

All 90 women were 47–57 yr of age and with at least 12 months of menopause at baseline and in good general health without any clinical or laboratory evidence of confounding systemic diseases, such as cardiovascular, hepatic, or renal disorders. Other exclusion criteria were smoking habit of more than two cigarettes per day, previous treatment with any drug that could affect the skeleton in the preceding year, BMD at femoral neck greater than 0.795 g/cm². This BMD value corresponds to a T score of -1 SD.

Treatments

After a 4-wk stabilization on the standard fat-reduced diet, offering 30% energy from fat, less than 10% of energy from saturated fatty acid, and a cholesterol intake less than 300 mg/d, participants to the study were randomly assigned to receive continuous HRT (n = 30; 1 mg/d 17ß-estradiol combined with norethisterone acetate; Activelle, Nordisk), the phytoestrogen genistein (n = 30; 54 mg/d; Laboratory Plants, Messina, Italy), or placebo (n = 30). We used the above-mentioned diet for the stabilization period to ensure the same energy intake to all the subjects and avoid any interference on lipid profile.

Measurements

We measured the anteroposterior lumbar spine and femoral neck BMD and markers of bone formation (bone-specific alkaline phosphatase; osteocalcin-bone Gla protein) and resorption (pyridinoline and deoxypyridinoline) at baseline and after a 1-yr treatment (4).

OPG and sRANKL serum concentrations were analyzed blinded to any clinical information. OPG was measured with a commercially available ELISA kit according to the protocol of the manufacturer (Immundiagnostik, Bensheim, Germany). A monoclonal IgG antibody was used as capture antibody and biotin-labeled polyclonal antihuman OPG antibody was used as detection antibody. This assay detects monomeric, dimeric and ligand-bound forms of OPG [intraassay coefficient of variation (CV), 7%; interassay CV, 9%; lower detection limit: 0.14 pmol/liter]. Serum levels of sRANKL were measured by an ELISA system (Immundiagnostik) that detects free sRANKL but not sRANKL complexed to OPG (intraassay CV, 5%; interassay CV, 7%; lower detection limit: 0.08 pmol/liter).

Statistics

Data are given as means ± SEM. The significance of difference was assessed by ANOVA followed by post hoc evaluation. The relationship between sRANKL/OPG percentage changes from baseline and percentage changes in bone turnover markers and BMD was detected by simple regression analysis. P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS, Inc. for Windows release 6.0 (SPSS Inc., Chicago, IL).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Our previous published results (4) indicated that no statistically significant difference was observed in the baseline characteristics among the three groups. Moreover, in our previous study, we observed that daily administration of genistein and HRT significantly reduced in a similar manner the urinary excretion of pyridinium cross-links at 12 months. Placebo administration did not modify these markers (4). Genistein treatment significantly increased the markers of bone formation; by contrast, HRT treatment caused a reduction in bone-specific alkaline phosphatase and bone Gla protein, whereas placebo administration had no effects on the serum levels of these factors (4). Our previous data also showed that genistein and HRT significantly augmented BMD in all skeletal regions when compared with placebo (4).

Table 1 shows basal serum OPG and sRANKL levels as well as sRANKL/OPG ratio in the three experimental groups. No statistically significant difference was observed among the groups.

View larger version (11K): [in this window] [in a new window]   FIG. 1. Mean (±SEM) changes from baseline in serum OPG (A), sRANKL levels (B), and sRANKL/OPG ratio (C) after a 1-yr treatment with placebo (white bar), genistein (gray bar), and HRT (black bar). #, P < 0.05 vs. placebo; *, P < 0.05 vs. genistein.

By comparison with placebo, sRANKL/OPG ratio was lower in the genistein group (-69 ± 7%; P < 0.01 vs. placebo 81 ± 24%) and in HRT-treated women (-11 ± 2%, P < 0.01 vs. placebo) (Fig. 1C). A negative correlation (r = -0.7; P < 0.01) was found in the genistein group between 1-yr percentage change in sRANKL/OPG ratio and 1-yr percentage change in femoral neck BMD (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIG. 2. Relationship between 1-yr percentage change in sRANKL/OPG and 1-yr percentage change in femoral neck BMD and bone resorption marker (bottom) in the genistein group. DPYR, Deoxypyridinoline.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The OPG-RANKL-RANK system plays an important role in the regulation of bone turnover (16, 17, 18). Abnormalities of this system have been implicated in the pathogenesis of postmenopausal osteoporosis (7). However, the results have been conflicting: in one study (19), women with osteoporosis have been shown to have higher circulating levels of OPG than controls, whereas another study (20) has shown no difference between serum OPG levels in osteoporotic in comparison with healthy postmenopausal women.

In our study we not only measured serum OPG and sRANKL, but we also evaluated the ratio between these two factors in an effort to better understand the link between the sRANKL-OPG system and bone loss in early postmenopausal women.

Our postmenopausal women treated with placebo had increased serum OPG and sRANKL and enhanced sRANKL/OPG ratio when compared with basal values. These women showed also a marked decrease in BMD and a high bone turnover rate. Therefore, these findings, taken together, suggest that in early postmenopause there is a tendency toward an enhanced osteoclastogenesis mediated, at least in part, by an enhancement in serum RANKL. However, OPG also increases in postmenopausal women, thus suggesting that the decoy receptor tries to oppose against the RANKL effects. This mechanism seems to be ineffective in fully counterbalancing the RANKL increased activity; in fact, the sRANKL/OPG ratio markedly rises.

Therefore, this increased ratio unmasks an ineffective OPG response to the enhanced osteoclastic bone resorption in these osteoporotic patients. In agreement with our hypothesis, it has been also shown that serum OPG concentrations are significantly higher in postmenopausal women with osteoporosis than in age-matched normal controls and that, within the osteoporotic group, OPG concentrations are higher in women with lower BMD (16).

We observed that genistein administration decreased sRANKL/OPG ratio after 12 months of treatment. This effect largely resulted from a decrease in serum sRANKL and, to a lesser extent, from an increase in serum OPG. Genistein therapy might regulate OPG levels acting via an estrogen-dependent mechanism, as showed by an in vitro study in which genistein enhanced OPG mRNA expression in osteoblastic cell line via a genomic pathway operating through estrogen receptors (21). However, this phytoestrogen binds to estrogen receptor (ER)ß with higher affinity than ER (22),and it has been demonstrated that ER, but not ERß, is involved in the regulation of the vertebral OPG-RANKL system (23). Thus, the effect on the sRANKL-OPG system might not be limited to an estrogen receptor interaction, but it might also be due to inhibition of topoisomerase II; in fact, genistein depresses RANKL mRNA expression and stimulates OPG mRNA in clonal osteogenic stromal ST2 cells (2).

In our patients, we observed that the 1-yr sRANKL/OPG ratio percentage change was either positively correlated with change in resorption markers or negatively correlated with change in femoral neck BMD. Therefore, the RANKL-OPG system may mediate at least in part some of the antiresorptive effects of genistein.

In contrast, no significant correlation between sRANKL/OPG ratio and BMD was found in our HRT group. This result further highlights the different action on bone exerted by these two active treatments. In our previous study, we observed a different effect on bone formation: genistein enhanced markers of osteoblast activity, whereas HRT reduced these markers. Also in this study, there are clear-cut differences.

In the HRT group, the sRANKL/OPG ratio was not significantly reduced. This event is likely a consequence of a decrease in serum OPG. However, this is in contrast with an in vitro study indicating that estrogen stimulates the expression of OPG in human osteoblasts (24) and mouse stromal cells (25) via transcriptional activation of ER; moreover, estrogen deficiency in ovariectomized rodents results in decreased OPG expression (26, 27) and increased RANKL production (27, 28) by cells of the bone and bone marrow microenvironment.

A possible explanation for this discrepancy might be that we used an association between estrogen and norethisterone acetate. This latter has a 19-nortestosterone derivative with a partial androgen effect (29). An in vitro study showed that progesterone had no effects on OPG mRNA and protein in normal human osteoblast-like cells (30); furthermore, androgens inhibit OPG mRNA levels and protein secretion by osteoblastic cells (31). In addition, in vivo testosterone decreases serum OPG levels in normal elderly men (32). Therefore, it may be speculated that the association of norethisterone acetate with estrogen attenuates the effect of estrogen on bone cells.

Our study has two limitations. The first one is that we measured OPG and sRANKL levels in the peripheral circulation, and it is unclear to what extent this reflects changes in the bone microenvironment. Moreover, OPG is produced mainly by bone cells and also by a number of other tissues (33); therefore, the serum changes may be underestimated because the nonskeletal sources may increase background noise. The second one is that the methodology for measuring OPG and RANKL in serum has not been fully validated.

In conclusion, our data suggest that the OPG-RANKL-RANK system plays a crucial role in the bone loss of the early postmenopause and indicate that the phytoestrogen genistein positively alters the sRANKL/OPG balance in such manner to improve bone turnover.

	Footnotes

 
Abbreviations: BMD, Bone mineral density; CV, coefficient of variation; ER, estrogen receptor; HRT, hormone replacement therapy; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor B ligand; sRANKL, soluble RANKL.

Received May 27, 2003.

Accepted October 9, 2003.

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