Pancreatic Beta-Cell Proliferation Induced by Estradiol-17ß is Foxo1 Dependent.

Estradiol-17ß (E2) and the Foxo1 transcription factor have each been implicated in the regulation of ß-cell proliferation. Interaction between Foxo1and estrogen receptor alpha (ERα), effecting cell cycle, has been demonstrated in breast cancer cells, but has not been studied thus far in ß-cells. Using human islets and the INS1-E ß-cell line, this study investigated the contribution of Foxo1 to E2-mediated ß-cell replication. Foxo1 expression was knocked down in INS1-E cells using siRNA and Foxo1 activity was inhibited in human islets with a specific Foxo1 inhibitor (AS1842856). Cells were treated with E2 and the ERα agonist PPT and evaluated for proliferation by 3[H]-thymidine incorporation and for transcriptional activity through the estrogen response element by the luciferase assay. As Foxo1 activity is regulated by post-translational modifications, the effect of E2 on phosphorylation was also assessed. In INS1-E cells, knock down of Foxo1 abrogated the proliferative response to E2 and PPT. In human islets, inhibition of Foxo1 abrogated E2-mediated proliferation and attenuated the response to PPT. Foxo1 knock down and inhibition reduced activity through the estrogen response element by 25% (p<0.05) and 50% (p<0.01) respectively, in INS1-E cells. E2 increased Foxo1 phosphorylation in a time dependent manner in INS1-E and human islets (p<0.01, p<0.05, respectively). These findings suggest that Foxo1 is involved in E2-mediated proliferation in INS1-E cells and human islets. This may have implications vis-à-vis variations in circulating endogenous E2 concentrations in diabetes.

Rivaroxaban significantly inhibits the stimulatory effects of bone-modulating hormones: In vitro study of primary female osteoblasts.

BACKGROUND: Anticoagulant therapy is a mainstay of treatment subsequent to major orthopedic surgeries. Evidence linking anticoagulant therapy, osteoporosis, and delayed fracture healing is not conclusive. We have previously reported that rivaroxaban significantly inhibited cell growth and energy metabolism in a human osteoblastic cell line. This study analyzed the response of primary female osteoblast cells to rivaroxaban in combination with various bone-modulating hormones. METHODS: Bone samples were taken from both premenopausal (pre-Ob) and postmenopausal (post-Ob) women. Cells were isolated from each sample and cultured to sub-confluence. Each sample was then treated with Rivaroxaban (10 µg/ml) in combination with the following hormones or with the hormones alone for 24 hours: 30nM estradiol-17ß (E2), 390nM estrogen receptor α (ERα) agonist PPT, 420nM estrogen receptor ß (ERß) agonist DPN, 50nM parathyroid hormone (PTH), and 1nM of vitamin D analog JKF. RESULTS: No effects were observed after exposure to rivaroxaban alone. When pre-Ob and post-Ob cells were exposed to the bone-modulating hormones as a control experiment, DNA synthesis and creatine kinase (CK)-specific activity was significantly stimulated with a greater response in the pre-Ob cells. When the cells were exposed to rivaroxaban in combination with bone-modulating hormones, the increased DNA synthesis and CK-specific activity previously observed were completely attenuated. CONCLUSIONS: Rivaroxaban significantly inhibited the stimulatory effects of bone-modulating hormones in both pre-Ob and post-Ob primary human cell lines. This finding may have clinical relevance for patients at high risk of osteoporosis managed with rivaroxaban or other factor Xa inhibitors.

The response of cells derived from the supraspinatus tendon to estrogen and calciotropic hormone stimulations: in vitro study.

PURPOSE: The most frequent complications after rotator cuff repair (RCR) are non-healing and re-tear. Age and gender are both proven risk factors for faulty RCR. This study analyzed the effects of female sex steroids and calciotropic hormones on tendon-derived cell characteristics. METHODS: Tendon-derived cells from rat supraspinatus were treated with estradiol-17ß (E2); soy isoflavones (daidzein, genistein, biochainin A); raloxifene and estrogen receptors α and ß agonists and antagonists; and less-calcemic vitamin-D analog, parathyroid hormone, and vehicle control for 24 h. Cell proliferation and mRNA expression of estrogen receptor α and ß, vitamin-D receptor (VDR), scleraxis, and collagen-1 were assessed. RESULTS: E2, Biochainin A, raloxifene, and vitamin-D significantly increased tendon-derived cell proliferation. Estrogen receptor α antagonists neutralized tendon-derived cells response to estradiol 17-ß; however, estrogen receptor ß antagonists did not have an effect. Scleraxis expression decreased following estradiol 17-ß and vitamin-D treatments. Vitamin-D significantly reduced collagen-1 expression, while estradiol 17-ß had no effect. Vitamin-D and estradiol 17-ß upregulated VDR expression. CONCLUSIONS: Significant tendon-derived cell proliferation can be achieved with commonly prescribed female sex and calciotropic hormones. However, collagen-1 expression remained constant or decreased following the administration of these hormones. Female sex steroids and vitamin-D promoted tendon-derived cell proliferation via estrogen receptor α and VDR, not estrogen receptor ß. Amplified cell proliferation was not associated with increased scleraxis and collagen-1 expression. These results have important implications to the properties of healing tendon and possible pharmaceutical therapies for patients with torn RC. Further research is warranted to expose the underling mechanisms of these effects.

Estrogens and Hyperglycemic Modulation of mRNAs Expressions Involved in Bone Metabolism: An Overshadowed Association?

Human bone cell line (SaOS2) express different mRNAs involved in bone biology and physiology such as estrogen receptor α (ERα), estrogen receptor ß (ERß), vitamin D receptor (VDR), 1α, 25 hydroxy vitamin D(3) hydroxylase (1OHase) as well as 12 and 15 lipoxygenases (12LO and 15LO). These mRNAs are modulated by estrogenic compounds. Since the skeletal protective effects of estrogens are not discernible in diabetic women, we tested whether the expression of the parameters measured here, and their modulations by estrogens, in SaOS2 cells grown in growth medium containing high glucose (HG; 9.0g/L; 44mM) compared to normal glucose (NG; 4.5g/L; 22mM). HG significantly increased DNA synthesis (DNA) and creatine kinase specific activity (CK) in SaOS2 cells. Stimulations of DNA but not of CK by E(2), by 4, 4', 4"-[4-propyl-(1H)-pyrazol-1, 3, 5- triyl] tris-phenol (PPT; ERα specific agonist), or by 2, 3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERß specific agonist), were abolished by HG. HG Itself up regulated the expression of mRNA of 12LO and 15LO and up regulated to much less extent ERß and VDR, but had no effect on the expression of mRNA of ERα and 1OHase. The different hormonal treatments modulated the expressions of 12LO and 15LO mRNAs which was reduced in HG, whereas the induction of their products 12 and 15HETE was only slightly affected by HG. The exact mechanism of HG effects on bone cell responses is yet to be investigated and its relationship to human bone physiology is not yet clear.

Estrogens and hyperglycemic modulation of mRNAs expressions involved in bone metabolism: an overshadowed association?

Human bone cell line (SaOS2) express different mRNAs involved in bone biology and physiology such as estrogen receptor α (ERα), estrogen receptor ß (ERß), vitamin D receptor (VDR), 1α, 25 hydroxy vitamin D(3) hydroxylase (1OHase) as well as 12 and 15 lipoxygenases (12LO and 15LO). These mRNAs are modulated by estrogenic compounds. Since the skeletal protective effects of estrogens are not discernible in diabetic women, we tested whether the expression of the parameters measured here and their modulations by estrogens, in SaOS2 cells grown in growth medium containing high glucose (HG; 9.0 g/L; 44 mM) compared to normal glucose (NG; 4.5 g/L; 22 mM). High Glucose (HG) significantly increased DNA synthesis and creatine kinase (CK) specific activity in SaOS2 cells. Stimulations of DNA but not of CK by E(2), by 4, 4', 4''-[4-propyl-(1H)-pyrazol-1, 3, 5- triyl] tris-phenol (PPT, ERα specific agonist), or by 2, 3-bis (4-hydroxyphenyl)-propionitrile (DPN, ERß specific agonist), were abolished by HG. HG itself upregulated the expression of mRNA of 12LO and 15LO and upregulated to much less extent of ERß and VDR, but had no effect on the expression of mRNA of ERα and 1OHase. The different hormonal treatments modulated the expressions of 12LO and 15LO mRNAs which was reduced in HG, whereas the induction of their products 12HETE and 15HETE was only slightly affected by HG. The exact mechanism of HG effects on bone cell responses is yet to be investigated and its relationship to human bone physiology is not yet clear.

The effects of direct factor Xa inhibitor (Rivaroxaban) on the human osteoblastic cell line SaOS2.

Thromboprophylaxis reduces the risk of surgery-related deep vein thrombosis, but anticoagulants were associated with systemic osteoporosis, a known risk factor for poor fracture healing. Rivaroxaban (XARELTO(®)) is a novel anticoagulant with specific ability to inhibit factor Xa, a serine endopeptidase, which plays a key role in coagulation. This study investigated the direct effects of rivaroxaban on bone biology using an in vitro cell culture model from the human female osteoblastic cell line SaOS2. Cells at subconfluence were treated for 24 hr with different concentrations of rivaroxaban and analyzed for DNA synthesis and creatine kinase- and alkaline phosphatase-specific activities, and were treated 21 days for analyzing mineralization. Rivaroxaban (0.01-50 µg/ml) dose-dependently inhibited up to 60% DNA synthesis of the cells. Creatine kinase-specific activity was also inhibited dose-dependently to a similar extent by the same concentrations. Alkaline phosphatase-specific activity was dose-dependently inhibited but only up to 30%. Cell mineralization was unaffected by 10 µg/ml rivaroxaban. This model demonstrated a significant rivaroxaban-induced reduction in osteoblastic cell growth and energy metabolism, and slight inhibition of the osteoblastic marker, alkaline phosphatase, while osteoblastic mineralization was unaffected. These findings might indicate that rivaroxaban inhibits the first stage of bone formation but does not affect later stages (i.e., bone mineralization).

Lipoxygenase metabolites are mediators of PTH-dependent human osteoblast growth.

PTH-induced osteoblast proliferation may contribute to its anabolic effects in bone. Since PTH-dependent osteoblast-like cell (Ob) growth is mediated via protein kinase C (PKC) and MAP kinase-kinase (MEK) and since lipoxygenase (LO) products activate PKC in a number of cell types, we assessed the expression of LO pathways in primary human cultured Ob. Ob from pre- or post-menopausal women were cultured and were treated with PTH and assayed for the expression of 12-LO and both type I and type II 15-LO mRNA and for the release their enzymatic products, 12- and 15-hydroxyeicosatetraenoic acid (HETE). Cells were also treated with PTH for stimulation DNA synthesis. First, Ob express platelet type- 12-LO and both type I and type II 15-LO mRNA and release their enzymatic products, 12- and 15-hydroxyeicosatetraenoic acid (HETE). Second, in female Ob, PTH induced a rapid increase in 12-HETE (50 fold increase) and 15-HETE (80 fold increase) and increased the expression of 12-LO mRNA but not of the two isoforms of 15-LO. PTH as well as 12 and 15-HETE stimulated DNA synthesis in Ob. The LO inhibitor baicalein inhibited PTH-stimulated DNA synthesis, which was reversed in the presence of either 12- or 15-HETE. A PKC inhibitor (bisindolylmaleimide I) as well as a MEK inhibitor (PD 98059) completely inhibited the stimulation of DNA synthesis by PTH, 12-HETE and the combination of PTH and 12-HETE. In contrast, 15-HETE-induced DNA synthesis was not abolished by these inhibitors. Further, 15-HETE partially restored the stimulatory effect of PTH on DNA synthesis in cells treated with PKC or MEK inhibitors. Finally, PTH- induced ERK1/2 phosphorylation, was blocked by a MEK inhibitor. These results demonstrate a novel mechanism of PTH-induced human bone cell proliferation operating through LO enzymes.

A daidzein-daunomycin conjugate improves the therapeutic response in an animal model of ovarian carcinoma.

The use of daunomycin against neoplasms is limited due to its severe cardiotoxicity. The cytotoxicity of daunomycin can be minimized by linking it to an affinity tag. Since ovarian cancer cells are sensitive to isoflavone action, we synthesized a daidzein daunomycin conjugate. In MLS human ovarian cancer cells, the conjugate was shown to have a larger cytotoxic effect than daunomycin per se at a low concentration. The conjugate was then tested in vivo in mice carrying MLS xenografts. Tumour growth in the groups of conjugate and daunomycin was inhibited by >50% as compared to vehicle treated mice. In contrast to daunomycin treated mice, no weight reduction or death was seen in mice treated with the conjugate. In vivo imaging of the fluorescence signal generated by daunomycin indicated uptake of both conjugate and daunomycin by the tumour. Tumour fluorescence was, however, higher in the conjugate treated mice than in the daunomycin treated mice, thus suggesting specific delivery of the drug to the tumour. Histological examination of myocardial tissue indicated that only the daunomycin, but not conjugate treated mice showed cardiac damage. These results indicate that targeting of daunomycin via carboxymethyldaidzein retains daunomycin's cytotoxic effects while averting its toxicity in an ovarian xenograft.

A sorafenib-sparing effect in the treatment of thyroid carcinoma cells attained by co-treatment with a novel isoflavone derivative and 1,25 dihydroxyvitamin D3.

BACKGROUND: Sorafenib improves progression-free survival in patients with progressive radioactive iodine-refractory differentiated thyroid carcinoma, but causes severe side effects. Estrogens may accelerate thyroid carcinoma cell growth. Our group recently reported that isoflavone derivative 7-(O)-carboxymethyl daidzein conjugated to N-t-boc-hexylenediamine (cD-tboc), a novel anti-estrogenic compound, retards the growth of both thyroid carcinoma cell lines and cultured human carcinoma cells. Vitamin D receptor (VDR) is expressed in malignant cells and responds to 1,25 dihydroxyvitamin D3 (1.25D) by decreased proliferative activity in vitro. The purpose of this study was to examine the effects of vitamin D metabolites (VDM) on the expression of estrogen receptors (ERs), VDR, and 1OHase mRNA, and to evaluate the inhibitory effect of low doses of sorafenib in combination with cDtboc and VDM on cell proliferation in cultured human papillary thyroid carcinoma (PTC). METHODS: In 19 cultured PTC specimens and 19 normal thyroid specimens, harvested during thyroidectomies from the same patients, expression levels of ERα, ERß, VDR, and 1 alpha-hydroxylase (1OHase) mRNA (by quantitative real-time PCR) were determined at baseline and after treatment with VMD. Cell proliferation was determined by measurement of 3[H] thymidine incorporation after treatment with sorafenib alone, sorafenib with added 1.25D or cD-tboc, and sorafenib with both 1.25D and cD-tboc added. RESULTS: 1,25D increased mRNA expression of all tested genes in the malignant and normal thyroid cells, while the ERα mRNA of the normal cells was unaffected. 1.25D dose-dependently inhibited cell proliferation in the malignant cells. The inhibitory effect of sorafenib on cell proliferation in the malignant cells was amplified after the addition of cDtboc and 1.25D, such that the maximal inhibition was not only greater, but also had been attained at a 10-fold lower concentration of sorafenib (20 µg/ml). This inhibition was similar to that of the generally used concentration of sorafenib (200 µg/ml) alone. CONCLUSIONS: The demonstration that low concentrations of cDtboc and 1.25D markedly amplify the inhibitory effect of sorafenib on the growth of human PTC supports the use of a 10-fold lower concentration of sorafenib. The findings may promote a new combination treatment for progressive radioactive iodine-refractory PTC.

Vitamin D modulation of the activity of estrogenic compounds in bone cells in vitro and in vivo.

Vitamin D analogs modulate different organs, including modulation of energy metabolism, through the induction of creatine kinase (CK) activity. Skeletal organs from vitamin D-depleted rats showed lower constituent CK than those from vitamin D-replete rats. Moreover, estradiol-17beta (E2) or dihydrotestosterone (DHT), which increased CK in organs from intact female or male rats, respectively, stimulated much less CK in vitamin D-depleted rats. Treatment of intact female rats with noncalcemic vitamin D analogs significantly upregulated E2- and DHT-induced CKresponse. These analogs upregulated the CK response to selective estrogen receptor modulators (SERMs) in organs from intact or ovariectomized (Ovx) female rats but abolished SERMs' inhibitory effect on E2-induced CK. These analogs significantly increased estradiol receptor alpha (ERalpha) protein in skeletal organs as well as histomorphological and biochemical changes due to this treatment followed by E2 or DHT. The analogs alone markedly altered the growth plate and the trabeculae and increased trabecular bone volume (%TB V) and trabecular width. The addition of E2 or DHT to this treatment restored all parameters as well as increased %TBV and cell proliferation. Treatment of Ovx female rats with JK 1624 F2-2 (JKF) decreased growth-plate width and increased %TB V, whereas QW1624 F2-2 (QW) restored growth-plate width and %TB V. Treatment of E2 with JKF restored %TBV and growth-plate width, whereas E2 with QW restored all parameters, including cortical width. There was also upregulation of the response of CK to E2 in both combined treatments. Our human-derived osteoblast (hObs)-like cell cultures respond to estrogenic compounds, and pretreating them with JKF upregulated the CK response to E2, raloxifene (Ral), and some phytoestrogens. ERalpha and ERbeta proteins, as well as mRNA, were modulated by CB 1093 (CB) and JKF. JKF increased specific nuclear E2 binding in female hObs but inhibited specific membranal E2 binding. hObs express 25 hydroxyvitamin D3-1alpha hydroxylase (1-OHase)-mRNA and its biological activity, which are both modulated by parathyroid hormone (PTH) and estrogenic compounds. Our results demonstrate mutual interaction between vitamin D and estrogenic compounds. We therefore conclude that combined treatment with less-calcemic analogs of vitamin D and estrogenic compounds might be superior for treatment of bone damage caused by ovariectomy in female rats, with possible application for postmenopausal osteoporosis.

Synthesis and evaluation of the antiproliferative activities of derivatives of carboxyalkyl isoflavones linked to N-t-Boc-hexylenediamine.

The isoflavones biochanin A ( 1a), genistein ( 1b), and daidzein ( 4) at concentrations >20 microM inhibit cell growth of various cancer cell lines. To enhance the antiproliferative activities of these compounds, we synthesized three analogs, 2-[3-carboxy-(6-tert-butoxycarbonylamino)-hexylamino-propyl]-7,5-dihydroxy-4'-methoxyisoflavone ( 3a), 2-[3-[N-[6-(tert-butoxycarbonyl)-aminohexyl]]-caboxamidopropyl]-5,7,4'-trihydroxyisoflavone ( 3b), and 5-{2-[3-(4-hydroxy-phenyl)-4-oxo-4 H-chromen-7-yloxy]-acetylamino}-pentyl)-carbamic acid tert-butyl ester ( 6). When cancer cells expressing predominantly estrogen receptor mRNA of the beta- relative to alpha-subtype were treated with 3a, 3b, or 6, DNA synthesis was inhibited in a dose-dependent manner, ranging from 15 to 3000 nmol/L, with little inhibitory effect in normal vascular smooth muscle cells. Compound 6 was the most potent one, and its antiproliferative effect in cancer cells was modulated by estrogen and by the apoptosis inhibitor Z-VADFK. When tested in vivo, compound 6 decreased tumor volume of ovarian xenografts by 50%, with no apparent toxicity. Compound 6 may be a promising agent for therapy of cancer either alone or in combination with chemotherapeutic agents.

25 hydroxy-vitamin D(3)-1alpha hydroxylase expression and activity in cultured human osteoblasts and their modulation by parathyroid hormone, estrogenic compounds and dihydrotestosterone.

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.

DT56a (Femarelle): a natural selective estrogen receptor modulator (SERM).

A selective estrogen receptor modulator (SERM) is defined as a substance with dissimilar effects on different tissues: agonist in some and antagonists in others. The natural compound DT56a (Femarelle) was shown to activate estrogen receptors in human cultured female derived osteoblasts. It was also shown to relieve menopausal symptoms and to increase bone mineral density with no effect on sex steroid hormone levels and on the endometrial thickness. DT56a, similarly to estradiol-17beta (E2), stimulated the specific activity of creatine kinase (CK) in skeletal and vascular tissues of female rats, as a marker of estrogen receptor (ER) activation. However, in the uterus, CK was activated only by E2 but not by DT56a. In order to prove that DT56a is a SERM, we examined the mutual interaction between DT56a and E2, at supra physiological doses, in different tissues in both intact and ovariectomized female rats, as well as in human cultured vascular and bone cells. Administration of DT56a or E2 stimulated CK in all tissues tested, but when given simultaneously, in intact immature female rats, DT56a completely abolished E2 stimulation of CK in all organs except in the diaphyseal bone where the inhibition was partial. In ovariectomized female rats, DT56a abolished E2's stimulation of CK in diaphyseal bone, thymus, uterus and pituitary but caused a partial inhibition in aorta, left ventricle and epiphyseal cartilage. In human bone cells E2 stimulation of CK, of alkaline phosphatase (AP) activity and of DNA synthesis was completely abolished by DT56a in post-menopausal cells and partially inhibited in pre-menopausal cells. In human vascular cells, inhibition of DNA synthesis by E2 was completely abolished by DT56a and E2-induced CK was partially inhibited by DT56a. The results support the finding that DT56a is a SERM; it stimulated different parameters similar to E2, but when given simultaneously, at supra physiological doses, inhibited these E2's effects. Further investigations regarding intra and extra cellular mechanism of action of DT56a are currently performed.

Interaction between the effects of the selective estrogen modulator femarelle and a vitamin D analog in human umbilical artery vascular smooth muscle cells.

To further investigate the interaction between vitamin D system and estrogen-mimetic compounds in the human vasculature we studied the effect of the "less- calcemic" analog of 1,25(OH)2D3 (1,25D); JK 1624F2-2 (JKF) in the presence of selective estrogen modulator femarelle (F), the phytoestrogen daidzein (D) and estradiol-17b (E2) on 3[H] thymidine incorporation (DNA synthesis) and creatine kinase specific activity (CK) in human umbilical artery vascular smooth muscle cells (VSMC). F, D and E2, stimulated DNA synthesis at low concentrations, and inhibited it at high concentrations. All estrogen-related compounds increased CK dose- dependently. Daily treatment with JKF (1nM for 3days) resulted in decreased DNA synthesis, increased CK and up- regulation of the stimulation of DNA synthesis by low estrogen-related hormones whereas D- and E2- mediated inhibition of cell proliferation was abolished by JKF. In contrast, inhibition of cell proliferation by F could not be blocked by JKF. JKF also up-regulated the stimulatory effects on CK by F, E2 and D. VSMC expressed Estrogen Receptor (ER)a and ERb mRNA at a relative ratio of 2.7:1.0, respectively. JKF pretreatment increased ERa (∼50%) and decreased ERb (∼25%) expression. E2 did not affect ERs whereas both D and F up-regulated ERb (∼100%) and ERa (∼50%). Additionally, JKF increased the intracellular competitive binding of F (from ∼70 to ∼310%), of D (from ∼60 to ∼250%) and of E2 from (from∼70 to ∼320%). F reciprocally modulated the vitamin D system by up-regulating VDR- and 25 hydroxyy vitamin D 1-a hydroxylase (1OHase) mRNA expression (∼120%). F also stimulated 1OHase activity as indicated by an increase in the production of 1, 25D (∼250%). A similar increase was elicited by D (∼90%) but not by E2. In conclusion, F has unique effects on human VSMC in that it can sustain inhibition of cell growth even in the presence of the vitamin D analog JKF. That JKF increases ER expression and F increased the endogenous production of 1, 25D and VDR expression offer new opportunities to modulate VSMC growth. Whether or not these mutual effects of F and JKF can be exploited to promote vascular health, particularly in estrogen-deficient states (e.g., menopause) is under investigation.

25-hydroxyvitamin D3-1alpha-hydroxylase is expressed in human vascular smooth muscle cells and is upregulated by parathyroid hormone and estrogenic compounds.

BACKGROUND: 1,25(OH)2 vitamin D3 exerts multiple effects in human vascular smooth muscle cells (VSMCs). We therefore tested the possibility that VSMCs possess an endogenous 25-hydroxyvitamin D3-1alpha-hydroxylase system, the final enzyme in the biosynthetic pathway of 1,25(OH)2D3. METHODS AND RESULTS: We assessed the expression and activity of 25-hydroxyvitamin D3-1alpha-hydroxylase by real-time polymerase chain reaction and the conversion of 25(OH)D3 into 1,25(OH)2D3. First, 25-hydroxyvitamin D3-1alpha-hydroxylase mRNA was identified in cultured VSMCs by real-time polymerase chain reaction. Second, in cells treated daily (3 days) with parathyroid hormone (66 nmol/L), estradiol-17beta (30 nmol/L), raloxifene (3 micromol/L), and the phytoestrogens genistein (3 micromol/L), biochainin A (3 micromol/L), and 6-carboxy biochainin A (30 nmol/L), 25-hydroxyvitamin D3-1alpha-hydroxylase mRNA increased by 43+/-13%, (P<0.05) 7+/-24% (P=NS), 176+/-28% (P<0.01), 65+/-11% (P<0.05), 152+/-24% (P<0.01), and 71+/-9% (P<0.05), respectively. Third, production of 1,25(OH)2D3 from 25(OH)D3 was seen with a Km of 25 ng/mL and increased dose dependently after treatment with parathyroid hormone, genistein, and the phytosetrogen derivative 6-carboxy biochainin A. Estradiol-17beta and biochainin A also increased the generation of 1,25(OH)2D3 by 40+/-23% (P<0.05) and 55+/-13% (P<0.05), respectively. CONCLUSIONS: We provide here the first evidence for the expression of an enzymatically active 25(OH)D3-1alpha-hydroxylase system in human VSMCs, which can be upregulated by parathyroid hormone and estrogenic compounds. Because exogenous vitamin D inhibits VSMC proliferation, the role of this system as an autocrine mechanism to curb changes in VSMC proliferation and phenotype is a subject for future investigation.

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.

DT56a stimulates gender-specific human cultured bone cells in vitro.

DT56a found to have SERM-like properties is used for the treatment of menopausal symptoms and osteoporosis. In vivo experiments demonstrated that DT56a displayed selective estrogenic activity; it stimulated creatine kinase (CK) specific activity in the skeletal tissues but not on the uterus of ovariectomized rats. DT56a, when applied together with estradiol-17beta (E(2)), completely inhibited the E(2)-stimulated CK, as demonstrated by other SERMs. DT56a stimulated bone formation in a rat model as measured by histological and histomorphometrical parameters. In a clinical study, administration of DT56a (Femarelle) resulted in a considerable elevation of bone mineral density and relief of menopausal symptoms. The aim of the present study was to analyze the effects of DT56a in vitro on human-derived bone cultured osteoblasts (Ob), by measuring its effects, at different concentrations, on DNA synthesis, CK and alkaline phosphatase (ALP) specific activities as well as changes in intracellular [Ca(2+)](i) concentrations. DT56a stimulated CK and DNA synthesis in both pre- and post-menopausal female Ob with maximal effect at 100 ng/ml for both age groups. In addition, DT56a stimulated ALP in Ob from both pre- and post-menopausal women with maximal effect at lower dose of 50 ng/ml, with higher response of pre-menopausal cells. Raloxifene (Ral) inhibited all DT56a-stimulated changes in Ob from both age groups. DT56a, when given together with E(2), completely antagonized E(2)-stimulated effects demonstrating its nature as a phyto-SERM. DT56a also, dose dependency, stimulated the intracellular levels of [Ca(2+)](i) with maximal effect at 10 ng/ml. Male-derived Ob did not respond to DT56a in any parameter. In summary, DT56a stimulated sex-specifically female-derived Ob, indicating its unique nature compared to the compounds currently used for postmenopausal osteoporosis by being bone-forming and not only an anti-resorptive agent.

Responsiveness to estradiol-17beta and to phytoestrogens in primary human osteoblasts is modulated differentially by high glucose concentration.

We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and to raloxifene (Ral), whereas 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. In cells derived from pre-menopausal women, E(2), G, D and Ral stimulated CK to higher extent compared to post-menopausal bone cells, whereas quecertin (Qu), carboxy-biochainin A (cBA) and carboxy-genistein (cG) stimulated CK in both age groups similarly, and biochainin A (BA) stimulated post-menopausal cells to a bit higher extent than pre-menopausal cells. Since the skeletal protective effects of estrogens are not discernable in diabetic women, we tested in this study, the effects of high glucose concentration in the growth medium, on the effects of estrogenic compounds on CK in human-derived bone cells (hObs). Female-derived hObs were grown either in normal (4.5 g/l; 22 mM, NG) or high glucose (9.0 g/l; 44 mM, HG) for 7 days. HG increased constitutive CK, but attenuated E(2)- and DHT-induced CK in female or male hObs, respectively. HG also inhibited genistein (G) and daidzein (D) stimulated CK in female hObs, but not the effects of biochainin A (BA), quecertin (Qu) or Ral. Intracellular, mainly nuclear binding of (3)[H]E(2) was characteristic of the different phytoestrogens in female hObs, was abolished by HG. Membranal binding of Eu-Ov-E(2), was displaced only by E(2)-Ov, ICI, cG-Ov or cD-Ov but decreased total binding of Eu-Ov-E(2) in both age groups and completely abolished the competition with E(2)-Ov or ICI in both age groups, but the competition with cD-Ov and cG-Ov was decreased only slightly but not statistically significant. HG also abolished Eu-BSA-T, which bound similarly male-derived hObs. All hObs expressed mRNA for ERalpha and ERbeta with higher abundance of ERalpha. HG increased mRNA for both ERs in female-derived hObs, but decreased mRNA for both ERs in male-derived hObs. Hence, human bone cells, which express specific nuclear and membranal binding sites for estrogenic compounds, are modulated by HG, leading to altered hormonal responsiveness, which might block important effects of estrogenic compounds, contributing probably to their decreased skeletal preserving properties under hyperglycemia.

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.

Vitamin D receptor expression is linked to potential markers of human thyroid papillary carcinoma.

Genes regulated cell-cell and cell-matrix adhesion and degradation of the extracellular matrix (ECM) have been screened as potential markers of malignant thyroid nodules. The mRNA expression levels of two of them, the ECM protein-1 (ECM1) and the type II transmembrane serine protease-4 (TMPRSS4), were shown to be an independent predictor of an existing thyroid carcinoma. The vitamin D receptor (VDR) is expressed in epithelial cells of the normal thyroid gland, as well as in malignant dividing cells, which respond to the active metabolite of vitamin D by decreased proliferative activity in vitro. We evaluated the relationship between mRNA gene expressions of TMPRSS4, ECM1 and VDR in 21 papillary thyroid carcinoma samples and compared it to 21 normal thyroid tissues from the same patients. Gene expression was considered as up- or down-regulated if it varied by more or less than 2-fold in the cancer tissue relative to the normal thyroid tissue (Ca/N) from the same patient. We found an overall significant adjusted correlation between the mRNA expression ratio (ExR) of VDR and that of ECM1 in Ca/N thyroid tissue (R=0.648, P<0.001). There was a high ExR of VDR between Ca/N thyroid tissue from the same patient (3.06±2.9), which also exhibited a high Ca/N ExR of ECM1 and/or of TMPRSS4 (>2, P=0.05).The finding that increased VDR expression in human thyroid cancer cells is often linked to increased ECM1 and/or TPMRSS4 expression warrants further investigation into the potential role of vitamin D analogs in thyroid carcinoma.