Calciotrophic hormones and hyperglycemia modulate vitamin D receptor and 25 hydroxyy vitamin D 1-α hydroxylase mRNA expression in human vascular smooth muscle cells.

Estrogen receptors (ERα and ERß), the vitamin D receptor (VDR) and 25 hydroxyy vitamin D 1-α hydroxylase (1OHase) mRNA are expressed in vascular smooth muscle cells (VSMC). In these cells estrogenic hormones modulate cell proliferation as measured by DNA synthesis (DNA). In the present study we determined whether or not the calciotrophic hormones PTH 1-34 (PTH) and less- calcemic vitamin D analog QW as well as hyperglycemia can regulate DNA synthesis and CK. E2 had a bimodal effect on VSMC DNA synthesis, such that proliferation was inhibited at 30nM but stimulated at 0.3nM. PTH at 50nM increased, whereas QW at 10nM inhibited DNA synthesis. Hyperglycemia inhibited the effects on high E2, QW and PTH on DNA only. Both QW and PTH increased ERα mRNA expression, but only PTH increased ERß expression. Likewise, both PTH and QW stimulated VDR and 1OHase expression and activity. ERß, VDR and 1OHase expression and activity were inhibited by hyperglycemia, but ERα expression was unaffected by hyperglycemia. In conclusion, calcitrophic hormones modify VSMC growth and concomitantly affect ER expression in these cells as well as the endogenous VSMC vitamin D system elements, including VDR and 1OHase. Some of the later changes may likely participate in growth effects. Of importance in the observation is that several regulatory effects are deranged in the presence of hyperglycemia, particularly the PTH- and vitamin D-dependent up regulation of VDR and 1OHase in these cells. The implications of these effects require further studies. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

New vitamin D less-calcemic analog affect human bone cell line and cultured vascular smooth muscle cells similar to other less-calcemic analogs.

Primary cultures of human bone and vascular cells respond to vitamin D treatment by modulation of cell proliferation measured by DNA synthesis (DNA) and energy metabolism measured by creatine kinase specific activity (CK) via binding to vitamin D receptors (VDR) which are expressed in these cells. Vitamin D compounds also modulate the response to estradiol-17ß (E2) and the expression mRNAs of estrogen receptors (ERα and ERß), VDR, 25-hydroxy vitamin D3 1-α hydroxylase (1OHase) and lipoxygenases (12LO and 15LO). We now compared our newly synthesized analog: 1α,25-dihydroxy-9-methylene-19-norvitamin D3 JK152 (JK), on bone and vascular cells compared to other analogs. Human bone cell line SaOS2 respond to JK by increased DNA and stimulated CK dose-dependently, similar to the less-calcemic analogs CB 1093 (CB) and EB 1089 (EB). JK also up-regulated the response to E2 in terms of DNA and CK. JK inhibited DNA synthesis and increased CK in primary human vascular smooth muscle cells (VSMC) dose-dependently similar to EB and CB. JK up regulated the response to E2 in terms of CK with no effect on DNA. JK similar to CB and EB stimulated mRNA expression of VDR and ERα, 12LO and 15LO, with no effect on ERß and 1OHase mRNA expression in SaOS2 measured by real time PCR. Similar treatments of VSMC with JK, CB and EB stimulated 12LO and 15LO, VDR and ERα mRNA expression with no effect on ERß and 1OHase mRNA expression. The results presented here demonstrate that the new vitamin D less-calcemic analog JK is similar to other analogs in its effects on human cultured cells and therefore may be used in combined hormone replacement treatment (HRT) both in vitro and in vivo.

Effects of commonly used medications on bone tissue mineralisation in SaOS-2 human bone cell line: an in vitro study.

We analysed the effects of commonly used medications on human osteoblastic cell activity in vitro, specifically proliferation and tissue mineralisation. A list of medications was retrieved from the records of patients aged > 65 years filed in the database of the largest health maintenance organisation in our country (> two million members). Proliferation and mineralisation assays were performed on the following drugs: rosuvastatin (statin), metformin (antidiabetic), metoprolol (ß-blocker), citalopram (selective serotonin reuptake inhibitor [SSRI]), and omeprazole (proton pump inhibitor (PPI)). All tested drugs significantly stimulated DNA synthesis to varying degrees, with rosuvastatin 5 µg/ml being the most effective among them (mean 225% (SD 20)), compared with metformin 10 µg/ml (185% (SD 10)), metoprolol 0.25 µg/ml (190% (SD 20)), citalopram 0.05 µg/ml (150% (sd 10)) and omeprazole 0.001 µg/ml (145% (SD 5)). Metformin and metoprolol (to a small extent) and rosuvastatin (to a much higher extent) inhibited cell mineralisation (85% (SD 5)). Our results indicate the need to evaluate the medications prescribed to patients in terms of their potential action on osteoblasts. Appropriate evaluation and prophylactic treatment (when necessary) might lower the incidence and costs associated with potential medication-induced osteoporosis.

Age-dependent responsiveness of human female bone cells to vitamin D analog and PTH.

Vitamin D less-calcemic analog JKF 1624 F2-2 (JKF) and PTH 1-34 stimulate in human female cultured osteoblasts (Ob) DNA synthesis (DNA), creatine kinase specific activity (CK), 1α, 25 vitamin D hydroxylase mRNA (1OHase) expression and 1,25(OH)2D3 (1,25) production, estrogen receptors (ER) mRNA expression and intracellular and membranal estrogen binding. In the present study, cultured Ob from different ages were subjected to hormonal stimulations and analyzed for different parameters. We found: 1) ERα expression is higher and ERß expression is lower in pre-meno - pausal Ob (prOb), with similar intracellular and membranal binding. 2) JKF and PTH up-regulated ERα and JKF downregulated ERß in both Ob, while PTH stimulated it in post- (poOb) and inhibited it in prOb. 3) There is higher expression of 1OHase mRNA in prOb, but 1,25 production is similar. Both parameters were hormonally stimulated to higher extent in prOb. 4) Ob express 12 and 15 lipoxygenase (LO) mRNA and produce 12- and 15-hydroxyeicosatetraenoic acid (H). 12LO expression is higher and 15LO is lower in prOb, while 12H is higher in prOb and 15H is similar in both. JKF inhibited 12LO expression in prOb and stimulated in poOb, whereas PTH stimulated it to higher extent in prOb. JKF stimulated and PTH inhibited 15LO expression in both; 12 and 15H were stimulated by both hormones in both Ob. 5. PTH and JKF stimulated DNA and CK in both Ob. In conclusion Ob demonstrate some age-dependent response to calciotrophic hormones, but the mechanism and beneficial outcome for human is unclear.

Vitamin D less-calcemic analog modulates the expression of estrogen receptors, vitamin D receptor and 1α-hydroxylase 25-hydroxy vitamin D in human thyroid cancer cell lines.

Estrogen receptors (ERs) are expressed in various "non-reproductive" cancer cell types. Some cancer types express 1α-hydroxylase 25-hydroxy vitamin D (1OHase) whose product, 1,25(OH)2D3 can retard cancer cell proliferation. Thyroid carcinoma cell growth is apparently promoted by estrogens, but whether or not this interaction is modified by vitamin D metabolites/analogs is presently unknown. Here we assessed the effect of a less calcemic vitamin D analog [JK 1624 F2-2 (JKF)] in three human thyroid cancer cell lines: ARO (anaplastic carcinoma), NPA (papillary carcinoma) and MRO (follicular carcinoma). (1) All cell lines expressed both ERα and ERß, vitamin D receptor (VDR) and 1OHase mRNA quantified by Real Time PCR. There was a general abundance of ERß over ERα expression, such that the ratio of ERß to ERα mRNA was >1000:1, 228:1 and 7.7:1 in ARO, MRO and NPA cells, respectively. (2) JKF up regulated ERß expression in ARO (by 110±15%) and MRO (by 280±10%) but down regulated ERß in NPA cells (by 40±15%). The expression of VDR was up regulated by JKF in NPA (21±6%), down regulated in ARO (-24±7%) and not affected in MRO. (3) All three human thyroid cancer cell lines were found to express 1OHase, which was up regulated by JKF in MRO (350±25%) and NPA (35±8%) but down regulated in ARO (-20±5%). This is the first report to describe direct regulation of VDR and 1OHase expression by a vitamin D analog in human thyroid cancer cells. A functional role for the vitamin D system in human thyroid cancer is suggested by the finding that the vitamin D analog can affect ERs expression which is in turn involved in estrogen-induced cell growth in an ER-type specific manner in these cells.

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 ERß 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-17ß (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 ERß 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.

Anti-thyroid cancer properties of a novel isoflavone derivative, 7-(O)-carboxymethyl daidzein conjugated to N-t-Boc-hexylenediamine in vitro and in vivo.

The incidence of thyroid cancer is up to 3 folds higher in women than in men, suggesting that estrogenic effects may be involved in the pathogenesis of this malignancy. Here, we explore whether or not human thyroid cancer cell growth can be curbed by a novel isoflavone derivative generated in our laboratory, the N-t-Boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc). With the exception of the follicular cancer cell line WRO, estrogen receptor (ER)α mRNA was only marginally expressed in cell lines derived from papillary (NPA), follicular (MRO), anaplastic thyroid carcinoma (ARO) such that the expression of estrogen receptor (ER) ßmRNA was more abundant than that of ERα mRNA in these cell types. Estradiol-17ß (E2; 0.03-300nmol/l) per se increased proliferation in all four cell-types. The ERß-specific agonist DPN increased [(3)H]-thymidine incorporation in all four thyroid cancer cell lines, whereas the ERα-specific agonist PPT increased growth only in NPA and WRO. By contrast, cD-tboc, derived from the weak estrogen daidzein, did not cause cell growth and dose-dependently diminished cell growth in all four cell lines via apoptosis and not necrosis, as detected by the release of histone-DNA fragments. The cytotoxic growth inhibitory effect of cD-tboc in these cells was modulated by E2 and the general caspase inhibitor Z-VAD-FMK, and the magnitude of this salvage was cell type-and dose-dependent. When nude mice carrying ARO thyroid xenografts were treated with cD-tboc, tumor volume decreased significantly, and no apparent toxicity was observed. These results suggest that cD-tboc may be a promising agent for therapy of thyroid carcinoma either alone or in combination with existing cytotoxic drugs.

The effects of estrogen receptors α- and ß-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line.

In cultured human osteoblasts estradiol-17ß (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERß-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERß-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERß mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERß pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.

DT56a (Femarelle), contrary to estradiol-17ß, is effective in human derived female osteoblasts in hyperglycemic condition.

We have reported previously, that female-derived cultured osteoblasts (hObs) responded to DT56a (Femarelle) measured by the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness and (3)[H] thymidine incorporation into DNA (DNA synthesis). Since the skeletal protective effects of estrogens are not discernable in hyperglycemic diabetic women, we sought to analyze the effect of estrogenic compounds on CK and DNA synthesis in hObs when grown in high glucose concentration (HG). Cells were grown either in normal glucose (NG) (4.5g/L; 22mM) or HG (9.0g/L; 44mM) for 7 days. HG increased constitutive CK but, the response of CK activity and DNA synthesis to estradiol-17ß (E(2)) treatment was reduced. In contrary, DT56a was found to be active (as measured by CK activity and DNA synthesis) in both NG and HG. HG decreases the hormonal responsiveness and might block important effects of estrogenic compounds, most likely contributing to their decreased skeletal preserving properties in hyperglycemic women. In hObs from post-menopausal women grown in HG, ERs mRNA expressions were unchanged. On the other hand, in hObs from pre-menopausal women HG increased ERs mRNA expressions. Since DT56a unlike E(2) is active in HG environment as well as in normal glucose, it may be an effective bone restoring agent in diabetic post-menopausal women.

The effects of native and synthetic estrogenic compounds as well as vitamin D less-calcemic analogs on adipocytes content in rat bone marrow.

BACKGROUND: We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17ß (E2) modulate bone morphology in rat female model. AIM: We now analyze the effects of phytoestrogens, E2, selective E2 re ceptor modulators, and the less-calcemic analogs of vitamin D: JKF1624F2-2 (JKF) or QW1624F2-2 (QW) on fat content in bone marrow (BM) from long bones in ovariectomized female rats (OVX). MATERIALS AND METHODS: OVX rats were injected with treatments known to affect bone formation, 5 days per week for 2.5 month for analysis of fat content in BM. RESULTS: In OVX young adults there is a decreased bone formation and a 10-fold increase in fat cells content in BM. Treatment with E2, raloxifene (Ral) or DT56a resulted in almost completely abolishment of fat cells content. Daidzein (D) decreased fat cells content by 80%, genistein (G) or biochainin A (BA) did not change fat cells content and carboxy BA (cBA) had a small but significant effect. JKF or QW did not affect fat cells content, whereas combined treatment of JKF or QW with E2 resulted in complete abolishment of fat cells content. These changes in fat cells content are inversely correlated with changes in bone formation. CONCLUSIONS: Our results demonstrate that adipogenesis induced by OVX is a reversible process which can be corrected by hormonal treatments. The awareness of a relationship between fat and bone at the marrow level might provide a better understanding of the pathophysiology of bone loss as well as a novel approach to diagnosis and treatment of 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.

Bone loss in ovariectomized rats: dominant role for estrogen but apparently not for FSH.

Estrogen deficiency as the sole factor underlying post-menopausal osteoporosis was challenged, in light of reports that both follicular stimulation hormone (FSH) receptor and FSHß knockout mice were resistant to bone loss, suggesting a detrimental role for FSH. We assessed whether lowering FSH levels by gonadotropin realizing (GnRH) analog decapeptyl in ovariectomized female rats (OVX) affects bone. Wistar-derived 25 days old OVX female rats were injected for 10 weeks with estradiol-17ß (E(2)), with GnRH analog (decapeptyl) or with both. FSH and luteinizing hormone (LH) serum levels were markedly increased in OVX rats, with smaller growth plates with disrupted architecture; heavy infiltration of bone marrow with numerous adipocytes and reduced thickness of cortical bone. In OVX rats treated with E(2), FSH, and LH levels were intermediate, the tibia was similar to that of intact rats, but there was reduced thickness of cortical bone. In decapeptyl treated OVX rats, FSH and LH levels were suppressed, the organization of growth plate and the trabecular bone were disrupted, and there were fewer proliferative and chondroblastic cells and a large adipocytes population in bone marrow, but an increased trabecular bone volume (TBV). In the E(2) + decapeptyl treatment, FSH and LH levels were suppressed, with partially restored growth plate architecture and improved TBV. In conclusion, E(2) deficiency is the dominant factor impairing bone loss in OVX and concomitant changes in FSH/LH levels achieved by decapeptyl have some modulating, though complex role in this setting. The role of high FSH levels in post-menopausal bone loss requires further investigation using combined sub-optimal doses of the different hormones.

The effects of peptides with estrogen-like activity on cell proliferation and energy metabolism in human derived vascular smooth muscle cells.

Hormone replacement therapy (HRT) for post-menopausal symptoms in diabetes is associated with increased risk of coronary heart disease and stroke. Therefore, there is a need for new HRT with no adverse effects on diabetic post-menopausal women. We developed peptides as potential estrogen mimetic compounds and now we evaluated the effects of the most efficacious peptide; hexapeptide estrogen-mimetic peptide 1 (EMP-1) (VSWFFE) in comparison to estrogen (E(2)) and peptides with weak activity A44 (KAWFFE) and A45 (KRAFFE) on modulation of cell proliferation of vascular smooth muscle cells (VSMC) growing in normal (ng) or high glucose (hg) concentrations. In ng EMP-1-like E(2) inhibited cell proliferation at high concentration, and stimulated at low concentration. EMP-1 did not affect E(2) stimulation of DNA, but inhibited E(2) inhibition of cell proliferation at high concentration. All effects by the combination of EMP-1 and E(2) were abolished at hg. A44-stimulated cell proliferation at all concentrations and A45 had no effect. When A44 was co-incubated with E(2) at both concentrations, DNA synthesis was stimulated, but abolished at hg. A45 abolished E(2) stimulation and inhibition of cell proliferation at both glucose concentrations. All peptides tested except A45-stimulated CK-specific activity at both glucose concentrations. In hg A44 stimulation of DNA was unaffected as well as its inhibition by EMP-1. EMP-1 and A44 similar to E(2)-stimulated MAPK activity in ng or hg, suggesting similar mechanism of action. The results presented here suggest that EMP-1 provided it acts similarly in vivo can replace E(2) for treatment of post-menopausal women in hyperglycemia due to diabetes.

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

Vitamin D metabolites or its less-calcemic analogs (JKF or QW) are beneficial for bone biology. We analyzed whether or not 25(OH)D3 (25), 1,25(OH)2D3 (1,25), JKF or QW regulate lipooxygenase (LO) enzymes expression and their products hydroxyeicosatetraenoic acid (12 and 15 HETE) formation as well as reactive oxygen species (ROS) production in human bone cell lines (SaOS2 and hFOB) and primary cultured human bone cells (Obs) from males or females. All compounds except 25 increased LOs mRNA expression and HETE production in female or male Obs. ROS formation was induced by JKF and QW in both cell lines, and was inhibited by different inhibitors. Baicalein (baic) an inhibitor of 12 and 15 LO activity, inhibited partially ROS formation by JKF or QW in SaSO2 and hFOB. JKF-stimulated DNA synthesis in female Obs was inhibited by baic but unchanged by addition of HETE or HETE with baic. These results indicate that vitamin D increased oxidative stress in bone cells is in part via induction of LO expression and activity. This new feature of vitamin D is probably mediated by intracellular and/or membranal receptors and its potential hazard could lead to potential damage due to increased lipid oxidation.

The response of creatine kinase specific activity in rat pituitary to estrogenic compounds and vitamin d less-calcemic analogs.

We examined the response of rat female pituitary at different metabolic stages to treatments with estrogenic compounds and vitamin D analogs. Immature or ovariectomized (Ovx) female rats responded by increased creatine kinase specific activity (CK) to estradiol-17beta (E(2)), genistein (G), daidzein (D), biochainin A (BA), quecertin (Qu), carboxy- G (cG), carboxy- BA (cBA), and raloxifene (Ral). The response was inhibited when Ral was injected together with the estrogens. CK was increased when hormones were injected daily into Ovx rats for 4 different time periods. Pretreatment with the less-calcemic vitamin D analogs JK 1624 F(2)-2 (JKF) or QW 1624 F(2)-2 (QW) followed by estrogenic injection resulted in increased response and sensitivity to E(2) and loss of inhibition of E(2) by Ral. CK was also increased by feeding with E(2) or licorice or its components dose- and time- dependent in immature or Ovxrats. Diabetic female rats did not respond to increased doses of E(2). In conclusion, rat female pituitary is estrogens-responsive organ, suggesting to considerits response for HRT in postmenopausal women for both beneficial and hazardous aspects.

Daidzein but not other phytoestrogens preserves bone architecture in ovariectomized female rats in vivo.

Ovariectomy of immature female rats, results in significant decrease of trabecular bone volume and in cortical bone thickness. Previously, we found that estradiol-17beta (E(2)) restored bone structure of ovariectomized (Ovx) female rats to values obtained in intact sham-operated female rats. E(2) also selectively stimulated creatine kinase (CK) specific activity a hormonal-genomic activity marker. In the present study, we compared the effects of E(2) and the phytoestrogens: daidzein (D), biochainin A (BA), genistein (G), carboxy-derivative of BA (cBA), and the SERM raloxifene (Ral) in Ovx, on both histological changes of bones and CK, when administered in multiple daily injections for 2.5 months. Bone from Ovx rats, showed significant disrupted architecture of the growth plate, with fewer proliferative cells and less chondroblasts. The metaphysis underneath the growth plate, contained less trabeculae but a significant increased number of adipocytes in the bone marrow. D like E(2) and Ral but not G, BA, or cBA, restored the morphology of the tibiae, similar to that of control sham-operated animals; the bony trabeculeae observed in the primary spongiosa was thicker, with almost no adipocytes in bone marrow. Ovariectomy resulted also in reduced CK, which in both epiphysis and diaphysis was stimulated by all estrogenic compounds tested. In summary, only D stimulated skeletal tissues growth and differentiation as effectively as E(2) or Ral, suggesting that under our experimental conditions, D is more effective in reversing menopausal changes than any of the other isolated phytoestrogens which cannot be considered as one entity.

Less-calcemic vitamin D analogs enhance creatine kinase specific activity and modulate responsiveness to gonadal steroids in rat skeletal tissues.

Vitamin D metabolites and analogs exert a variety of biological activities, such as regulation of cellular proliferation, differentiation and energy metabolism, exerted through the brain type isozyme of creatine kinase (CK) specific activity, serving to provide ATP generation. In the present study we assess the role of vitamin D in induction of CK in rat epiphyseal cartilage (Ep) and diaphyseal bone (Di). Skeletal tissues from female or male vitamin D-depleted rats showed lower CK than in vitamin D-replete rats in both Ep and Di. Moreover, estradiol-17beta (E2) or dihydrotestosterone (DHT), which increased CK in Ep and Di of intact female or male rats, respectively, stimulated CK in vitamin D-depleted rats to a much lower extent. Treatment of intact female rats for 1, 2 or 8 weeks with the less-calcemic vitamin D analogs JKF 1624F2-2 (JKF) or QW 1624F2-2 (QW) and the non-calcemic analog CB 1093 (CB), slightly affected CK, although there was an up-regulation of the E2- and DHT-induced CK response in Ep and Di from these rats. In intact female rats, all vitamin D analogs potentiated CK response to the SERM raloxifene (Ral) and tamoxifen (TAM) in these organs but the inhibitory effect of Ral or TAM on E2-induced CK was lost after this pre-treatment. CB induced a significant increase in estradiol receptor alpha (ERalpha) protein in both Ep and Di from intact female rats. Collectively, these results indicate that vitamin D analogs modulate CK in skeletal tissues and up-regulate its response and sensitivity to E2 and to SERM in these tissues, possibly via an increase in ERalpha protein. These results corroborate our previous studies in human bone cells, and further suggest that the vitamin D system plays an important physiological role in maintaining normal cell energy reservoir in the skeleton.

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.

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.

The role of non-calcemic analogs of vitamin D in differentiation of cultured rat bone marrow into osteoblast-like cells: age and sex differences.

We have previously demonstrated that rat bone in vivo and rat bone cells in vitro, responded sex-specifically to gonadal steroids in stimulation of the specific activity of the BB isozyme of creatine kinase (CK), a marker for hormonal responsiveness. Pre-treatment with vitamin D analogs up-regulated the sex-specific responsiveness and sensitivity to gonadal steroids. We also found that mice cultured femoral bone marrow (BM) in the presence of dexamethasone (DEX) and 1,25(OH)2D3 (1,25D) or both differentiated into osteoblast-like cells (Obs), which acquired sex-specific responsiveness to gonadal steroids. This response was significantly augmented in the presence of both agents. In the present study, we examined the effect of age, sex and vitamin D non-hypercalcemic analogs on the differentiation of rat derived femoral BM into Obs. In female or male derived BM from intact but not gonadectomized rats DEX and DEX+1,25D increased the constitutive levels of CK. BM derived from old females showed lower stimulation of CK than BM originated from young females by estradiol (E2) or raloxifene (Ral) in the presence of both DEX and 1,25D. The non-hypercalcemic analogs of vitamin D: CB 1093 (CB), EB 1089 (EB) and MC 1288 (MC) were more effective than 1,25D in both age groups in stimulating CK in the absence of DEX. In the presence of DEX, there was a further increase in CK with the same differential effectiveness. BM from gonadectomized male or female rats lost the sex-specific response, responding to both E2 and dihydrotestosterone (DHT). BM derived from both intact and gonadectomized males and females, growing with DEX or DEX+1,25D showed increased specific activity of constitutive levels of alkaline phodphatase (AP). No significant stimulation of AP was seen in any BM by gonadal steroids. These findings suggest that manipulation of the hormonal milieu in early stages of differentiation sequence of Obs determines the subsequent selective responsiveness of the developing bone tissue to sex steroids. Also non-calcemic vitamin D analogs were more effective in this process than 1,25D and showed activity even in the absence of DEX and may be applied to the differentiation process for bone tissue engineering.