The bone-protective effect of the phytoestrogen genistein is mediated via ERα-dependent mechanisms and strongly enhanced by physical activity

Abstract

Reduced estrogen levels occurring during menopause in women are accompanied by a variety of disorders, e.g. hot flushes, depressions, osteoporosis, increase in body weight and reduced movement drive. The phytoestrogen genistein (GEN) has been demonstrated to have a significant bone-protective potency. In order to study the ER subtype-specific effects of this phytoestrogen on bone in an animal model, ovariectomized (OVX) female Wistar rats were either treated with 17β-estradiol (E₂) (4 μg/kg/day), the ERα-specific agonist (ALPHA) 16α-LE₂ (10 μg/kg/day), the ERβ-specific agonist (BETA) 8β-VE₂ (100 μg/kg/day) or GEN (10 mg/kg/day) for 3 weeks. Vehicle-treated OVX animals served as controls. All animals had the opportunity of voluntary wheel running. Movement activity, changes of body weight and trabecular bone mineral density (BMD) in the tibia were analyzed. E₂ and ALPHA treatment, but not treatment with BETA, significantly increased the movement activity of OVX rats. Treatment with GEN resulted in a significant decrease of movement activity as compared to OVX animals. Bone mineral density in the trabecular area of the tibia and the expression of bone morphogenetic protein-2 (BMP-2) were significantly reduced in OVX- and BETA-treated rats as compared to rats substituted with E₂, ALPHA and GEN. The bone-protective effect of ALPHA was antagonized by co-treatment with the pure antiestrogen Faslodex (ICI). In order to distinguish hormone-dependent effects from those of exercise, we performed an additional experiment where the animals had no opportunity of wheel running. The results demonstrate that physically inactive rats have a stronger decrease of bone mineral density than physically active animals. Very surprisingly, our data demonstrate that GEN has no bone-protective activity in the absence of physical activity. In contrast, ALPHA and E₂ are bone-protective in the presence and absence of physical activity.

In conclusion, our data provide evidence that the effects of E₂ on body weight, movement drive and protection of bone mineral density are mediated via ERα, whereas activation of ERβ has only a limited effect. Our data also indicate that the bone-protective effects of GEN may be mediated via ERα-dependent mechanisms and that physical activity has a strong impact on the bone-protective potency of this phytoestrogen.

Introduction

Growth, differentiation and functioning of many typical tissues of the female reproductive tract like uterus, ovary and the mammary gland are influenced by estradiol. Estrogens also play an important role in bone maintenance [1], [2] and in the cardiovascular system where they have certain cardioprotective effects [3]. Reduced estrogen levels occurring in menopausal and postmenopausal women are accompanied by a variety of disorders, e.g. hot flushes, depressions, osteoporosis, increase in body weight and reduced movement drive. As a therapeutic strategy, hormone replacement therapy (HRT) has been used for decades as the “gold standard” to treat estrogen deficiency symptoms such as vasomotor disturbances and genito-urinary atrophy [4]. In addition, use of HRT prevents long-term development of postmenopausal osteoporosis [5], [6]. Recently, the data from the Million Women Study [7] have once again opened up the discussion about the benefit/risk ratio of HRT and alternative strategies for the treatment of menopausal and postmenopausal disorders. Besides the use of selective estrogen receptor modulators (SERMs) [8], [9] the use of ER subtype-specific ligands is discussed to be useful in HRT, too. Cloning of a second estrogen receptor in the rat prostate in 1996 led to the description of ERβ [10]. Thus, estrogen receptor signaling has become much more complex and multifaceted [11], [12], [13]. Even though estrogens bind with similar affinity to estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) [14], the ligand-binding domains (LBD) of these two transcription factors differ significantly from each other [15].

Unlike E₂, several synthetic ER ligands like Tamoxifen (Tam), Raloxifene (Ral), or other SERMs exhibit a distinct binding affinity to ERα and ERβ [16], [17], [18]. There are speculations that the molecular mechanisms involved in the tissue-selective action of theses substances include ER subtype-specific effects [19], [20]. Moreover, the effects of some phytoestrogens, compounds of plant origin with affinity to the ER, are discussed to be mediated via ER subtype-specific mechanisms [21].

The phytoestrogen GEN, for example, is supposed to function as an ERβ-selective partial agonist, which binds to the LBD of both ER isoforms with moderate affinity [15], [22], but preferentially to ERβ and in a manner similar to E₂[23]. These differences in binding activities suggest that phytoestrogens like GEN may selectively trigger ERβ-mediated transcriptional pathways [21].

In the context of different menopausal and postmenopausal disorders information on the tissue specific action of estrogens and phytoestrogens associated with specific estrogen receptor signaling is very limited. The identification of isotype-selective ER ligands provides an alternative tool to study the biological role of ERα and ERβ adding to the information generated by the analysis of ER knockout animals. The studies shown in the present paper have been performed with the novel highly selective steroidal ERβ agonist 8β-VE₂[24], and the highly selective ERα agonist 16α-LE₂[25], [26], which belong to the most potent and isotype-selective estrogens identified so far.

Therefore, the aim of this study was to investigate if observed impacts of the phytoestrogen GEN on menopausal disorders are based upon its property as a weak ERα or as a potent ERβ agonist.