A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.     

Responsiveness to phytoestrogens in primary human osteoblasts is modulated differentially by a "less-calcemic" analog of 1,25 dihydroxyvitamin D(3): JK 1624F(2)-2 (JKF)

We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and raloxifene (Ral), and male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. We also found that pre-treatment with the less-calcemic analog of Vitamin D, JK 1624 F(2)-2 (JKF), upregulated the response of CK to E(2) and Ral. In this study, we analyzed the response of human bone cells from pre- and post-menopausal females and males, to phytoestrogens. Bone cells derived from pre-menopausal women showed greater stimulation of CK than cells from post-menopausal women, after treatment with E(2) (30 nM), daidzein (D, 3000 nM), genistein (G, 3000 nM) and Ral (3000 nM); whereas the responses to biochainin A (BA 3000 nM), quecertin (Qu 3000 nM) or the carboxy derivative of G (cG 300 nM) were not age-dependent. Male-derived cells did not respond to phytoestrogens. When cells derived from female bones at both age groups were pre-treated with JKF, by daily addition of 1nM, for 3 days, there was an upregulation of the response to E(2), Ral, G and D but not to BA or Qu. Nuclear binding of (3)[H] E(2) was characteristic of the different phytoestrogens, with increase of the specific binding after pre-treatment with JKF. In contrast, the membranal binding of E(2)-Ov-Eu, which was specific for the estrogenic compounds except Ral, was inhibited by pre-treatment with JKF except for ICI 161480 (ICI). Male bone cells did not bind E(2)-Ov-Eu but bound T-BSA-Eu; this binding was abolished by pre-treatment with JKF. Pre-treatment with JKF increased mRNA for ERalpha and decreased mRNA for ERbeta in bone cells from both age groups of females and from males, all of which expressed both ERs, with a ratio of ERalpha to ERbeta of 121:1 in pre- and 78:1 in post-menopausal and 105:1 in male bone cells. This study raises the possibility of combined Vitamin D analog and phytoestrogen for hormonal replacement therapy to prevent post-menopausal osteoporosis, which is a subject of ongoing research in animal models.     

Mutual interaction of special phytoestrogenic compounds, their synthetic carboxy-derivatives and the less-calcemic vitamin D analog activities in human derived female cultured osteoblasts

Cultured female-derived human bone cells (hObs) responded by different parameters to different phytoestrogenic and vitamin D compounds. Pre- and post-menopausal hObs express ERα and ERβ mRNA with higher abundance of ERα. Pre-treatment with the less-calcemic vitamin D analog JKF 1624F(2)-2 (JKF) upregulated responsiveness to estrogens via modulation of ERs expression. These estrogenic compounds induce the expression and activity of 25 hydroxy-vitamin D(3)-1α hydroxylase (1OHase). We now analyzed the effects of carboxy-genistein (cG), carboxy-biocainin A (cBA) and carboxy-daidzein (cD), of BA, D or G and of licorice derived compounds glabridin (Glb) and glabrene (Gla) and estradiol-17β (E(2)) on DNA synthesis, creatine kinase specific activity (CK), intracellular and membranal E(2) binding and their modulations by JKF in hObs. We also analyzed modulation by phytoestrogenic compounds of 1OHase mRNA expression and activity. We showed that: (1) all compounds stimulated DNA synthesis and CK. (2) JKF and all estrogenic compounds modulated ERα and ERβ mRNA expression. (3) Pre-treatment with JKF increased DNA synthesis and CK responses only to E(2), D, G and Gla. (4) JKF increased the intracellular competitive binding only of E(2), D and G. (5) JKF abolished the membranal binding of all protein-bound estrogens. (6) JKF and all estrogenic compounds except the protein-bound ones up-regulated 1OHase expression and activity. In conclusion phytoestrogens and their analogs increase DNA synthesis and CK, and lead to increased production of 1,25(OH)(2)D(3) in hObs, while pre-treatment with JKF modulates the effect of estrogenic compounds via regulation of ERs mRNA expression in a yet unclear mechanism.     

Treatment with non-hypercalcemic analogs of 1,25-dihydroxyvitamin D3 increases responsiveness to 17beta-estradiol, dihydrotestosterone or raloxifene in primary human osteoblasts

Pretreatment with 1 nM 1,25-dihydroxyvitamin D(3) (1,25), or non-hypercalcemic Vitamin D analogs, upregulated the response of creatine kinase (CK) to 17beta-estradiol (30 nM E(2)), raloxifene (3000 nM RAL) or dihydrotestosterone (300 nM DHT) in primary human bone cells. Previously, we reported that these osteoblast-like cells responded to gonadal steroids in a sex specific manner. Bone cells derived from pre-menopausal women showed greater stimulation of CK specific activity by E(2) than bone cells from post-menopausal women; in male-derived cells no age related difference was found. In this study, we treated cells derived from female or male bones, at different ages, with the side chain modified analogs of Vitamin D: CB 1093 (CB), EB 1089 (EB), MC 1288 (MC) and the demonstrably non-calcemic hybrid analog JK 1624 F2-2 (JKF), by daily addition of 1 nM, for 3 days. On day 4, cells were incubated with sex steroids for 4h and cell extracts were prepared. Pretreatment with JKF or CB significantly upregulated the response to 30 nM E(2) in all female-derived cells and to 300 nM DHT in mature male-derived cells. In cells from older males, only JKF caused augmentation of DHT action. Bone cells from pre- or post-menopausal females responded to 3000 nM RAL by increased CK activity to the same extent as to 30 nM E(2); however, RAL and E(2), when applied together, resulted in mutual annihilation of their agonist activities. Vitamin D analogs prevented the antagonistic effect of RAL in the presence of E(2), possibly due to increased numbers of ERs. Both estrogen receptors, alpha (ERalpha) and beta (ERbeta), were expressed in male- as well as in female-derived cells. However, only in female-derived cells were ERalpha and ERbeta upregulated by pretreatment with Vitamin D analogs. This study raises the possibility of testing combined Vitamin D analog and estrogen replacement treatment for post-menopausal women to prevent osteoporosis.     

Modulation of the response to estradiol-17beta of rat vascular tissues by a non calcemic vitamin D analog

Estradiol-17beta (E(2)) increases creatine kinase (CK) specific activity in aorta (Ao) and left ventricle of the heart (Lv) from rat females. In the present study, we analyzed the effects of pretreatment with the non calcemic analog of vitamin D, JK 1624 F2-2 (JKF) on the response to E(2) (either 0.5 or 5 microg/rat) of Ao and Lv from prepubertal female rats. JKF did not affect CK in either organ. However, pretreatment with JKF (0.1 ng/g body weight for 1 or 2 weeks) increased the CK response to E(2) (0.5 microg/rat) by 50 +/- 10% in Ao and by 150 +/- 12% in Lv. The CK response to 5 microg/rat of E(2) in intact female rats, was increased by 118 +/- 15% and 99 +/- 11% in the Ao and by 89 +/- 6% and 112 +/- 13% in the Lv, in animals treated daily with JKF for 1 or 2 weeks, respectively, before administration of E(2). JKF also increased the response to 500 microg/rat raloxifene (Ral) by 47 +/- 8% in Ao and by 56 +/- 12% in Lv. Preliminary experiments showed that JKF treatment induced a approximately 50% increase in estradiol receptor ERalpha in both organs. The results indicate that the vitamin D analog JKF upregulates the response and sensitivity of vascular tissues to E(2), in association with increased expression of their ERalpha. These results should prompt examination of the possibility that the effects estrogen therapy in postmenopausal women can be augmented by vitamin D or its analogs.     

Less calcemic Vitamin D analogs enhance creatine kinase specific activity and modulate responsiveness to gonadal steroids in the vasculature

Vitamin D receptors are widely expressed in the cardiovascular system, in which Vitamin D and its metabolites exert a variety of biological activities such as regulation of cellular proliferation and differentiation, cell calcium transients and cell energy metabolism in vitro. The latter is mediated through the control of the brain type creatine kinase specific activity (CK), which serves to provide a readily available reservoir for ATP generation under increased work-load. In the present study we undertook to assess the role of Vitamin D on energy metabolism in the rat heart and aorta in vivo by using CK, which is a key energy metabolizing enzyme and compare Vitamin D depleted and repleted animals. Vascular tissues from female or male Vitamin D-depleted rats showed 61-80% lower CK activity in the aorta (Ao) and left ventricle of the heart (Lv) than control, Vitamin D-replete rats. Moreover, neither estradiol-17beta (E2) nor dihydrotestosterone (DHT), which increases CK specific activity in Ao and Lv of intact female or male rats, respectively, were able to stimulate CK in Vitamin D-depleted rats. Treatment of intact female rats for 2 weeks or 2 months with the less-calcemic Vitamin D analogs JKF 1624F2-2 (JKF) or QW 1624F2-2 (QW) (Fig. 1), did not significantly affect CK specific activity. However, after pretreatment with these analogs, there was an up regulation of the E2-induced CK response in Ao and Lv. In intact female rats, all Vitamin D analogs also potentiated the in vivo CK response to the SERMs raloxifene (Ral) and tamoxifen (TAM) in Ao and Lv. However the inhibitory effect of Ral or TAM on E2-induced CK activity was lost after pretreatment with Vitamin D analogs. The non-calcemic analog CB 1093 (CB) induced a significant increase in estradiol receptor alpha (ERalpha) protein in both myocardial and aortic tissue from intact and from ovariectomized female rats. Collectively, these results indicate that Vitamin D analogs modulate cell energy homeostasis in vascular tissues through induction of CK and up regulation of the response and sensitivity of CK in vascular tissues to E2 and to SERMs, possibly through via an increase in ERalpha protein in female derived organs. These results corroborate our previous in vitro studies in human vascular cells and further suggest that the Vitamin D system plays an important physiological role in maintaining normal cell energy reservoir in the vasculature.     

A non-calcemic analog of 1 alpha,25 dihydroxy vitamin D(3) (JKF) upregulates the induction of creatine kinase B by 17 beta estradiol in osteoblast-like ROS 17/2.8 cells and in rat diaphysis.

We have reported that multiple treatments with so-called 'non-hypercalcemic' analogs of 1 alpha,25(OH)(2) vitamin D(3) (1,25(OH)(2)D(3)) stimulate the specific activity of creatine kinase BB (CK) in ROS 17/2.8 osteoblast-like cells, and that pretreatment with these analogs upregulates responsiveness and sensitivity to 17 beta estradiol (E(2)) for the induction of CK. However, since the analogs showed toxicity in vivo, we have now studied the action of a demonstrably non-calcemic hybrid analog of vitamin D in ROS 17/2.8 cells, and prepubertal rats. The analog JKF was designed to separate its calcemic activity from other biological activities by combining a calcemic-lowering 1-hydroxymethyl group with a potentiating C, D-ring side chain modification including 24 difluoronation. Treatment with 1 pM JKF alone significantly stimulated CK specific activity at 4 h by 30+/-10%. However after three daily pretreatments, JKF upregulated the extent of induction by 30 nM E(2) by 33% at 1 pM and by 97% at 1 nM; the E(2) dose needed for a significant stimulation of CK activity was lowered to 30 pM. The action of the SERMS tamoxifen, tamoxifen methiodide and raloxifene, at 3 microM, was also upregulated by three daily pretreatments with 1 nM JKF; unexpectedly, this pretreatment prevented the inhibition of E(2) stimulation by the SERMS. Upregulation of E(2) action by 1 nM JKF was inhibited by 1 nM ZK159222, an inhibitor of the nuclear action of 1,25(OH)(2)D(3). In vivo, three daily injections of 0.05 ng/g body weight of JKF augmented the response of prepubertal female rat diaphysis and epiphysis to E(2). Therefore, demonstrably non-calcemic analogs of 1,25(OH)(2)D(3) may have potential for use in combination with estrogens or SERMS in the prevention and/or treatment of metabolic bone diseases such as postmenopausal osteoporosis.     

Vitamin D metabolites and analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cell line

Vitamin D metabolites and its less-calcemic analogs (vitamin D compounds) are beneficial for bone and modulate cell growth and energy metabolism. We now analyze whether 25(OH)D(3) (25D), 1,25(OH)(2)D(3) (1,25D), 24,25(OH)(2)D(3) (24,25D), JKF1624F(2)-2 (JKF) or QW1624F(2)-2 (QW) regulate lipooxygenase (LO) mRNA expression and its products; hydroxyl-eicosatetraenoic acid (12 and 15HETE) formation, as well as reactive oxygen species (ROS) production in human bone cell line (SaOS2) and their interplay with modulation of cell proliferation and energy metabolism. All compounds except 25D increased 12LO mRNA expression and modulated 12 and 15HETE production whereas ROS production was increased by all compounds, and inhibited by NADPH oxidase inhibitors diphenyleneiodonium (DPI) and N-acetylcysteine (NAc). Baicaleine (baic) the inhibitor of 12 and 15LO activity blocked only slightly the stimulation of DNA synthesis by all compounds, whereas DPI inhibited almost completely the stimulation of DNA and CK by all compounds. Treatments of cells with 12 or 15HETE increased DNA synthesis and CK that were only slightly inhibited by DPI. These results indicate that vitamin D compounds increased oxidative stress in osteoblasts in part via induction of LO expression and activity. The increased ROS production mediates partially elevated cell proliferation and energy metabolism, whereas the LO mediation is not essential. This new feature of vitamin D compounds is mediated by intracellular and/or membranal binding sites and its potential hazard could lead to damage due to increased lipid oxidation, although the transient mediation of ROS in cell proliferation is beneficial to bone growth in a yet unknown mechanism.     

Systemic treatments with the low-calcemic 1,25(OH)(2)D(3) analogs JKF or QW increase both the morphological and biochemical responses to estradiol-17beta in rat tibiae

We demonstrated previously that daily injection for 3 days of the less calcemic vitamin D analogs: JK 1624 F(2)-2 (JKF) and QW 1624F(2)-2 (QW) followed by estradiol-17beta (E(2)) in female rats upregulated creatine kinase-specific activity (CK) in skeletal tissues. In this study, we evaluated both histomorphological and biochemical changes due to a regime of 4 days treatment with JKF or QW, followed by injection of E(2) on day 5, repeated for 2.5 months. Ovariectomized female rats (Ovx) were injected 2 weeks after surgery, with JKF or QW at 0.2 ng/g BW followed by injections of E(2) (1 microg/rat) on day 5 of each week for 2.5 months. Rats were sacrificed 24 h after the last injection and bones were analyzed. JKF alone decreased growth plate width, increased % total bone volume (%TBV), with no change in cortical thickness. In contrast, QW restored growth plate width and %TBV with no change in cortical thickness. Combined with E(2), JKF restored %TBV and growth plate width but with no change in cortical thickness, while QW restored significantly all parameters including cortical thickness. Moreover, there was also an increase in the responsiveness of CK to E(2) in epiphyseal cartilage and diaphyseal bone but not in uterus. Thus, vitamin D less calcemic analogs increased responsiveness to E(2) morphologically as well as biochemically. We, therefore, conclude that combined treatment of less calcemic analogs vitamin D and E(2) might be superior for treatment of bone damage caused by ovariectomy in female rats and might be applied for post-menopausal osteoporosis.     

Estrogenic activity of glabridin and glabrene from licorice roots on human osteoblasts and prepubertal rat skeletal tissues

Data from both in vivo and in vitro experiments demonstrated that glabridin and glabrene are similar to estradiol-17β in their stimulation of the specific activity of creatine kinase, although at higher concentrations, but differ in their extent of action and interaction with other drugs. In pre-menopausal human bone cells, the response to estradiol-17β and glabridin (at higher concentration) was higher than in post-menopausal cells; whereas, glabrene (at higher concentration) was more effective in post-menopausal cells. At both ages, the response to estradiol-17β and glabridin was enhanced by pretreatment with the less-calcemic Vitamin D analog CB 1093 (CB) and the demonstrably non-calcemic analog JK 1624 F₂-2 (JKF). The response to glabrene was reduced by this pretreatment. Both glabridin and glabrene stimulated creatine kinase specific activity in diaphyseal bone and epiphyseal cartilage of prepubertal female rats. Daily feeding (3–14 days) of prepubertal female rats with glabridin, estradiol-17β or their combination, also stimulated creatine kinase specific activity. Glabridine, similarly to estradiol-17β, also stimulated creatine kinase specific activity in ovariectomized female rats. Raloxifene, in combination with glabridin or estradiol-17β, demonstrated the phenomenon of mutual annihilation of stimulation of creatine kinase specific activity in both epiphysis and diaphysis. Glabrene activity was not inhibited by raloxifene. Therefore, glabridin shows greater similarity to estradiol-17β and thus greater potential, with or without Vitamin D, to modulate bone disorders in post-menopausal women.     

Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells

Sex steroid hormone receptors play a central role in modulating telomerase activity, especially in cancer cells. However, information on the regulation of steroid hormone receptors and their distinct functions on telomerase activity within the mesenchymal stem cell are largely unavailable due to low telomerase activity in the cell. In this study, the effects of estrogen (E2) treatment and function of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) on telomerase activity were investigated in human mesenchymal stem cells (hMSCs). Telomerase activity and mRNA expression of the catalytic subunit of telomerase (hTERT) were upregulated by treatment of the cells with E2. The protein concentration of ERalpha was also increased by E2 treatment, and enhancement of ERalpha accumulation in the nucleus was clearly detected with immunocytochemistry. When ERalpha expression was reduced by siRNA transfection into hMSCs, the effect of E2 on the induction of hTERT expression and telomerase activity was diminished. In contrast, the transient overexpression of ERalpha increased the effect of E2 on the expression of hTERT mRNA. These findings indicate that the activation of hTERT expression and telomerase activity by E2 in hMSCs depends on ERalpha, but not on ERbeta.     

"Non-hypercalcemic" analogs of 1alpha,25 dihydroxy vitamin D augment the induction of creatine kinase B by estrogen and selective estrogen receptor modulators (SERMS) in osteoblast-like cells and rat skeletal organs

We have demonstrated previously that daily treatments for 3 days with the so-called "non-hypercalcemic" analogs of 1alpha,25 dihydroxy vitamin D in ROS 17/2.8 osteoblast-like cells, stimulate the specific activity of creatine kinase BB (CK), and that such treatment with these analogs followed by a single treatment with gonadal steroids, upregulates responsiveness and sensitivity to estradiol 17beta (E(2)) for the induction of CK. This study was designed to determine if these same "non-hypercalcemic" vitamin D analogs could upregulate in vivo the response to E(2) and whether substitution of selective estrogen receptor modulators (SERMS) for E(2) would result in the same upregulation. We found that one week or 2 weeks pretreatment of prepubertal rats with vitamin D analogs led to increased induction of CK by E(2) and by the SERMS tamoxifen, tamoxifen methiodide and raloxifene, in epiphysis and diaphysis of the femur but not in the uterus. However, in contrast to their antiestrogenic activity in the uterus, there was no inhibition of E(2) action by the SERMS in skeletal tissues. The induction of mRNA for ckb in ROS 17/2.8 cells by E(2) or SERMS was demonstrated only after vitamin D pretreatment; there was no inhibition of E(2) induction by SERMS. Antagonists of vitamin D dependent calcium transport (transcaltachia) did not inhibit stimulation by vitamin D analogs. These results support the involvement of a nuclear mechanism in the upregulation of induction of CK by E(2), which may be due, in part, to the ability of vitamin D to increase estrogen receptor(s).     

Vitamin D-interacting protein 205 (DRIP205) coactivation of estrogen receptor alpha (ERalpha) involves multiple domains of both proteins

Vitamin D-interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17beta-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE(3)), DRIP205 coactivates ERalpha-mediated transactivation. DRIP205Delta587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205Delta587-636 also coactivates ERalpha. Moreover, both wild-type and variant DRIP205 also colocalize with ERalpha in the nuclei of transfected cells. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ERalpha and in interactions with ERalpha. Coactivation of ERalpha by DRIP205 does not require NR boxes, and variants with deletion of N-terminal (amino acids 1-639) and C-terminal (amino acids 576-1566) significantly coactivate ERalpha. DRIP205 resembles p160 coactivators that also interact with multiple regions of ERalpha; however, unlike p160 coactivators, DRIP205 coactivation of ERalpha does not require NR boxes.     

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.