Cy5 NHS ester Further proof is required as to

Further proof is required as to whether ER membrane receptors play a role in the maintenance of bone mass. It is also possible that other non-genomic pathways may be involved. Nevertheless, whatever the precise mechanism of action of estrogens may be, the imbalance between bone formation and resorption in postmenopausal women is restored by estrogen through inhibition of osteoclastogenesis resulting in a lower resorption rate and restoration of the balance between bone formation and bone loss. In mice, supraphysiological doses of estrogens may lead to bone formation, but this effect appears to be species-dependent. The high endogenous hormone concentrations in (B)ERKO mice [21], [22] may explain the lack of effect on bone after Cy5 NHS ester of one of the ER genes. SERMs or partial agonists also bind to ERs, but functional assays have shown that partial transactivation occurs and it is possible that selectivity for one of the receptors may exist. Tamoxifen, a triphenylethylene derivative and the first generation SERM (although the term was only introduced later), has shown surprisingly positive effects on bone [23], [24]. The metabolite 4-OH-tamoxifen is the active metabolite and has a two–three-fold higher binding affinity for ERβ (Ki=0.04nM) [19] than for ERα. Raloxifene, which has a benzothiophene chemophore, is structurally different from tamoxifen and does not need to be metabolized in order to bind to the ER. The conformation of SERM–ER complexes is different from that of complexes with natural estradiol [25]. It is probable that the conformation with each estrogen is different and therefore each estrogen may have unique properties [26]. In the raloxifene–ER complex, helix 12 does not lie over the binding pocket, as seen with the estradiol–ER complex, and this may prevent coactivators from binding to the ligand binding domain of the ER. Transactivation experiments have shown that raloxifene and 4-OH-tamoxifen have ERα agonistic and antagonistic activity, whereas they possess full antagonistic activity at the ERβ [7]. In bone, the effects of raloxifene and tamoxifen show similarities to pure ER agonists, whilst in other tissues no activation is found [24], [27]. As with estrogens, the final effect on bone is an inhibition of resorption, although the effects are less strong. The main distinctions from estradiol are differences in the binding to ERα and ERβ and the fact that ER responsive genes are less strongly induced by SERMs. The parent tibolone molecule does not bind to the ER, but estrogenic activity appears after metabolism to the two 3-OH-metabolites by 3α hydroxysteroid dehydrogenase (HSD) and 3β HSD/isomerase in the liver and intestine [17]. Due to the presence of a 3-OH-group in the A-ring of the steroid skeleton both metabolites bind and activate the ER, with a preference for the ERα [20]. In addition, a pool of inactively sulfated 3-OH-metabolites is found in the circulation. Conjugation occurs in the liver via sulfotransferases and this pool may be reactivated in the various tissues by sulfatases. De Gooyer et al. showed that tibolone and its metabolites inhibit sulfatase in a tissue-specific manner [28], inhibition occurring in breast and endometrial cells but not in bone cells. Therefore, in bone cells, the pool of sulfated metabolites may serve as a reservoir of estrogenic activity. Although the two estrogenic metabolites of tibolone have a lower intrinsic activity than estradiol, they are present in sufficient quantities in the circulation to generate a full estrogenic response. Tibolone and its metabolites are not aromatized by the enzyme aromatase [29]. The proposed metabolism of tibolone is depicted in Fig. 1. The third metabolite, the Δ4-isomer of tibolone, has progestogenic activity [20], [30], which is important for counteracting estrogenic activity in the endometrium, where it is also locally formed, and expressing androgenic properties in the brain. The receptor binding profile of the active tibolone metabolites is shown in Table 1 [20]. Tibolone does not possess the structural properties necessary to activate ERs; the positive signal found in ER transactivation assays must therefore be due to metabolism in the assay system. Since bone cells also possess progestogen receptors (PR) and androgen receptors (AR) [4], [5], [7], as well as ER, it was important to determine which hormonal activity was responsible for the positive effects of tibolone on bone. This has been investigated in experiments in which tibolone was combined with anti-hormones for ER, PR and AR and it was shown that only the ER was involved in these effects [30]. Thus, just like the SERMs, tibolone acts on bone via the ER [31].