Effects of gonadal and adrenal androgens in a novel androgen-responsive human osteoblastic cell line

While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR-6) that expresses a mature osteoblast phenotype and a physiological number (∼4,000/nucleus) of androgen receptors (AR). Treatment with 5α-dihydrotestosterone (5α-DHT) inhibited the proliferation of hFOB/AR-6 cells in a dose-dependent fashion, while it had no effect on the proliferation of hFOB cells, which express low levels of AR (<200/nucleus). In hFOB/AR-6 cells, co-treatment with the specific AR antagonist, hydroxyflutamide abolished 5α-DHT-induced growth inhibition. Steady-state levels of transforming growth factor-β₁ (TGF-β₁) and TGF-β-induced early gene (TIEG) mRNA decreased after treatment of hFOB/AR-6 cells with 5α-DHT, suggesting a role for the TGF-β₁-TIEG pathway in mediating 5α-DHT-induced growth inhibition of hFOB/AR-6 cells. In support of this, co-treatment of hFOB/AR-6 cells with TGF-β₁ (40 pg/ml) reversed the 5α-DHT-induced growth inhibition, whereas TGF-β₁ alone at this dose had no effect on hFOB/AR-6 cell proliferation. Furthermore, treatment of hFOB/AR-6 cells with 5α-DHT and testosterone (10⁻⁸ M) inhibited basal and 1,25-(OH)₂D₃-induced alkaline phosphatase (ALP) activity and type I collagen synthesis without affecting osteocalcin production. Thus, in this fetal osteoblast cell line expressing a physiological number of AR, androgens decrease proliferation and the expression of markers associated with osteoblast differentiation. These studies suggest that the potential anabolic effect of androgens on bone may not be mediated at the level of the mature osteoblast. J. Cell. Biochem. 71:96–108, 1998. © 1998 Wiley-Liss, Inc.     

Isoflavones regulate interleukin-6 and osteoprotegerin synthesis during osteoblast cell differentiation via an estrogen-receptor-dependent pathway

The hypothesis tested in this in vitro study was that the expression and production of dietary isoflavone-mediated osteoclastogenesis-regulatory cytokines, such as interleukin-6 (IL-6) and osteoprotegerin (OPG), are related to the different levels of estrogen receptors expressed in two hFOB osteoblastic cell lines. OPG mRNA expression was significantly increased in both hFOB1.19 and hFOB/ER9 cells treated with 17 beta-estradiol, genistein, or daidzein at 10(-8)M in comparison to vehicle (control) (P<0.05). In both cell lines, the release of IL-6 was suppressed, while OPG production was enhanced by isoflavone treatments (P<0.05). The increased expression of OPG and decreased IL-6 production by isoflavones were dose-dependent. Responses to isoflavones were much stronger in hFOB/ER9 cells, which express the estrogen receptor 20 times higher than those in hFOB1.19 cells. After adding the ER binding blocker, ICI-182,780, the effects of isoflavones on OPG and IL-6 production disappeared. In summary, the inhibition by dietary isoflavones of IL-6 production and the stimulation of OPG appear to be mediated, at least in part, via a genomic pathway operating through estrogen receptors and gene expression mechanisms.     

Estrogen and Progesterone Receptors in the Uterus of the Vervet Monkey

Abstract

Experimental conditions for the optimal measurement of estrogen (ER) and progesterone (PR) receptors in normal vervet monkey (Cercopithecus aethiops pygerythrus) uteri are described. The uteri of this primate were found to contain relatively high concentrations of both ER and PR. Levels of ER ranged from 151 to 822 femtomoles per mg protein (mean for group assayed is 327±165 femtomoles per mg protein). PR assays were performed on the same cytosols and the levels ranged from 444 to 2267 femtomoles per mg protein (mean of 1285±511 femtomoles per mg protein). Mean K_d values for the ER- and PR-ligand complexes were found to be 3.15±1.4x10^-10 M and 2.38±0.2x10^-9 M respectively, within the group analysed (n=21). The ratio of PR to ER varied between 1.1 and 13.1 with a mean of 4.5±2.4. Ligand specificity studies revealed that [³H]-17β-estradiol binding to the ER could only be inhibited by estrogens or estrogen analogues. The PR however exhibited an affinity for a wider range of ligand types. In low ionic strength buffers both ER and PR sedimented as ±8S type molecules in the presence or absence of 10mM sodium molybdate. Both receptors dissociated into smaller components, following a short exposure to 0.4 M KCl and subsequent centrifugation in a gradient containing 0.4 M KCl.     

Estrogen regulation of human osteoblast function is determined by the stage of differentiation and the estrogen receptor isoform

Although osteoblasts have been shown to respond to estrogens and express both isoforms of the estrogen receptor (ER alpha and ER beta), the role each isoform plays in osteoblast cell function and differentiation is unknown. The two ER isoforms are known to differentially regulate estrogen-inducible promoter-reporter gene constructs, but their individual effects on endogenous gene expression in osteoblasts have not been reported. We compared the effects of 17 beta-estradiol (E) and tamoxifen (TAM) on gene expression and matrix formation during the differentiation of human osteoblast cell lines stably expressing either ER alpha (hFOB/ER alpha 9) or ER beta (hFOB/ER beta 6). Expression of the appropriate ER isoform in these cells was confirmed by northern and western blotting and the responses to E in the hFOB/ER beta 6 line were abolished by an ER beta-specific inhibitor. The data demonstrate that (1) in both the hFOB/ER cell lines, certain responses to E or TAM (including alkaline phosphatase, IL-6 and IL-11 production) are more pronounced at the late mineralization stage of differentiation compared to earlier stages, (2) E exerted a greater regulation of bone nodule formation and matrix protein/cytokine production in the ER alpha cells than in ER beta cells, and (3) the regulated expression of select genes differed between the ER alpha and ER beta cells. TAM had no effect on nodule formation in either cell line and was a less potent regulator of gene/protein expression than E. Thus, both the ER isoform and the stage of differentiation appear to influence the response of osteoblast cells to E and TAM.     

Estrogen mitogenic action. I. Demonstration of estrogen-dependent MTW9/PL2 carcinogen-induced rat mammary tumor cell growth in serum-supplemented culture and technical implications

Summary The MTW9/PL cell line was established by our laboratory in culture from the carcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a Wistar-Furth (W/Fu) rat. This tumor formed estrogen, androgen, and progesterone responsive tumors in W/Fu rats (Sirbasku, D. A., Cancer Res. 38:1154–1165; 1978). It was later used to derive the MTW9/PL2 cell population which was also estrogen-responsive in vivo (Danielpour, D., et al., In Vitro Cell. Dev. Biol. 24∶42–52; 1988). In the study presented here, we describe serum-supplemented culture conditions in which the MTW9/PL2 cells demonstrate≥80-fold steroid hormone growth responses. All sera used were steroid hormone-depleted by charcoal-dextran treatment at 34°C. The studies were done with horse serum as well as serum from other mammalian species. The growth of the MTW9/PL2 cells was biphasic in response to hormone-depleted serum. Concentrations of ≤5% (v/v) promoted optimum growth. Above this concentration, serum was inhibitory. Concentrations ≥40% (v/v) inhibited growth altogether. Addition of 1.0×10⁻¹³−1.0×10⁻⁸ M 17β-estradiol (E₂) reversed the inhibition completely. At 1.0×10⁻⁸ M, estrone, estriol and diethylstilbestrol promoted growth as well as E₂. Testosterone and dihydrotestosterone promoted growth only at ≥10⁻⁷ M. Progesterone was effective only at≥10⁻⁶ M. Cortisol was ineffective. Labeled-hormone-binding analysis and Western immunoblotting documented that MTW9/PL2 cells had estrogen and progesterone receptors but not androgen or cortisol receptors. Estrogen treatment of MTW9/PL2 cells induced a concentration and time dependent increase in progesterone receptors. We conclude (1) the MTW9/PL2 population is the first highly steroid hormone-responsive rat mammary tumor cell line to be established in culture from a carcinogen-induced tumor, and (2) sera from a number of species including horse, rat and human contain an inhibitor which mediates estrogen sensitive MTW9/PL2 cell growth in culture.     

Modulation of connexin43 alters expression of osteoblastic differentiation markers

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43^–). hFOB/Cx43^– cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43^– cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43^– cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor 1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43^–. Osteopontin mRNA levels were increased in hFOB/Cx43^– relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43^– and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells. gap junction communication; alkaline phosphatase activity; osteopontin; osteocalcin; hFOB 1.19     

Characterization of estrogen and progesterone receptors in monkey endometrium: methodology and effects of estradiol and/or progesterone on endometrium of castrate monkeys.

Appropriate methods for repeated surgical collection of endometrial tissue from rhesus monkeys, and characterization of cytosol and nuclear estrogen (E₂) and progesterone (P) receptors (R) are described. Tissue collection was made in the mid-luteal phase at abdominal fundal hysterotomy. Functional status of the ovaries was determined by visual inspection and RIA of E₂ and P in serum. Receptor assay procedures were devised permitting the measurement of total cytosol and nuclear receptor concentration. Sucrose density gradients of labelled cytosol were made and a 4S saturable binding component for ³H P and for ³H E₂ were found. Equilibrium dissociation constants of ³H E₂ and ³H R5020 were 2.1×10^?10M and 3.6 × 10^?9M, respectively. These binding characteristics are similar to those found in human endometrium and suggest that these surrogate primates have extensive utility in investigation of factors influencing E₂R and PR concentrations in endometrial tissue during the menstrual cycle and implantation. Simulated menstrual cycle were produced in 20 castrate monkeys by sequential treatment with estradiol and progesterone in silastic capsules. RIA of E₂ and P, and gonadotropins in peripheral serum provided assuredness of the hormonal status of each monkey under treatment. Cytosol and nuclear receptors for E₂ and P were measured in the endometrium after different intervals of the treatment. E₂ receptor (E₂R) levels were not changed during the estrogen sequence, but were lowered by progesterone therapy in both cytosol and nuclear components. Progesterone receptor (PR) synthesis in cytosol was induced by exogenous estrogen. The total concentration of PR decreased with the uptake of P by the cell; meanwhile, the ratio of cytosol to nuclear P receptors declined. These data suggest that this sequential estrogen-progesterone regimen induces the changes in E₂R and PR patterns in the endometrium of ovariectomized monkeys which occurs due to ovarian cyclicity in the normal menstrual cycle.     

Nongenomic effects of an anti-idiotypic antibody as an estrogen mimetic in female human and rat osteoblasts

We investigated the early effects of the anti-idiotypic antibody (clone 1D₅), which recognized the estrogen receptor (ER), on cytosolic free calcium concentration ([Ca²⁺]i) and its long term effects on creatine kinase (CK) specific activity in female human and rat osteoblasts. These actions were compared to the known membrane and genomic effects of 17β estradiol (E₂). Like E₂, clone 1D₅ increased within 5 s [Ca²⁺]i in both cell types by two mechanisms: 1) Ca²⁺ influx through voltage-gated Ca²⁺ channels as shown by using EGTA, a chelator of extracellular Ca²⁺, and nifedipine, a Ca²⁺ channel blocker; 2) Ca^2+; mobilization from the endoplasmic reticulum as shown by using phospholipase C inhibitors, such as neomycin and U-73122, which involved a Pertussis toxin-sensitive G-protein. Clone 1D₅ and E₂ stimulated CK specific activity in human and rat osteoblasts with ten fold higher concentrations than those needed for the membrane effects (0.1 μg/ml and 10 pM, respectively). Both effects were gender-specific since testosterone and 5α-dihydotesterone were uneffective. Tamoxifen and Raloxifene, two estrogen nuclear antagonists, inhibited CK response to 1D₅ and E₂ and Ca²⁺ response to 1D₅, but not CA²⁺ response to E₂. By contrast, (Fab′)₂ dimer, a proteolytic fragment of 1D₅ with antagonist properties, inhibited both membrane and genomic effects of 1D₅ and E₂. In conclusion, these results imply that clone 1D₅ has an estrogen like activity both at the membrane and nuclear levels in female human and rat osteoblasts. 1D₅ must therefore interact with membrane binding sites, penetrate the cells, and reach the nuclear receptors by an as yet uncharacterized mechanism. J. Cell. Biochem. 65:53–66. © 1997 Wiley-Liss, Inc.     

Rapid action of estradiol in primate GnRH neurons: the role of estrogen receptor alpha and estrogen receptor beta

Estrogens play a pivotal role in the control of female reproductive function. Recent studies using primate GnRH neurons derived from embryonic nasal placode indicate that 17β-estradiol (E₂) causes a rapid stimulatory action. E₂ (1 nM) stimulates firing activity and intracellular calcium ([Ca²⁺]_i) oscillations of primate GnRH neurons within a few min. E₂ also stimulates GnRH release within 10 min. However, the classical estrogen receptors, ERα and ERβ, do not appear to play a role in E₂-induced [Ca²⁺]_i oscillations or GnRH release, as the estrogen receptor antagonist, ICI 182,780, failed to block these responses. Rather, this rapid E₂ action is, at least in part, mediated by a G-protein coupled receptor GPR30. In the present study we further investigate the role of ERα and ERβ in the rapid action of E₂ by knocking down cellular ERα and ERβ by transfection of GnRH neurons with specific siRNA for rhesus monkey ERα and ERβ. Results indicate that cellular knockdown of ERα and ERβ failed to block the E₂-induced changes in [Ca²⁺]_i oscillations. It is concluded that neither ERα nor ERβ is required for the rapid action of E₂ in primate GnRH neurons.     

Glucocorticoid evoked upregulation of RCAN1-1 in human leukemic CEM cells susceptible to apoptosis

Background

Estradiol (E₂) mediates various intracellular signaling cascades from the plasma membrane via several estrogen receptors (ERs). The pituitary is an estrogen-responsive tissue, and we have previously reported that E₂ can activate mitogen-activated protein kinases (MAPKs) such as ERK1/2 and JNK1/2/3 in the membrane ERα (mERα)-enriched GH₃/B₆/F₁₀ rat pituitary tumor cell line. Phytoestrogens are compounds found in plants and foods such as soybeans, alfalfa sprouts, and red grapes. They are structurally similar to E₂ and share a similar mechanism of action through their binding to ERs. Phytoestrogens bind to nuclear ERs with a much lower affinity and therefore are less potent in mediating genomic responses. However, little is known about their ability to act via mERs to mediate nongenomic effects.

Methods

To investigate the activation of different nongenomic pathways, and determine the involvement of mERα, we measured prolactin (PRL) release by radio-immunoassay, MAPK activations (ERK1/2 and JNK1/2/3) via a quantitative plate immunoassay, and intracellular [Ca²⁺] by Fura-2 fluorescence imaging in cells treated with E₂ or four different phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol).

Results

Coumesterol and daidzein increased PRL release similar to E₂ in GH₃/B₆/F₁₀ cells, while genistein and trans-resveratrol had no effect. All of these compounds except genistein activated ERK1/2 signaling at 1–10 picomolar concentrations; JNK 1/2/3 was activated by all compounds at a 100 nanomolar concentration. All compounds also caused rapid Ca²⁺ uptake, though in unique dose-dependent Ca²⁺ response patterns for several aspects of this response. A subclone of GH₃ cells expressing low levels of mERα (GH₃/B₆/D₉) did not respond to any phytoestrogen treatments for any of these responses, suggesting that these nongenomic effects were mediated via mERα.

Conclusion

Phytoestrogens were much more potent in mediating these nongenomic responses (activation of MAPKs, PRL release, and increased intracellular [Ca²⁺]) via mERα than was previously reported for genomic responses. The unique non-monotonic dose responses and variant signaling patterns caused by E₂ and all tested phytoestrogens suggest that complex and multiple signaling pathways or binding partners could be involved. By activating these different nongenomic signaling pathways, phytoestrogens could have significant physiological consequences for pituitary cell functions.     

Interstitial Cajal-like cells (ICLC) as steroid hormone sensors in human myometrium: immunocytochemical approach

Expression of estrogen (ER) and progesterone (PR) receptors was investigated in cultured human normal myometrial cells (non-pregnant uterus, fertile period). The ER and PR expression was studied by immunohistochemistry and immunofluorescence on either myocytes or interstitial Cajal-like cells (ICLC). Only those cells double immunostained for c-kit and steroid receptors were considered as ICLC. ER and/or PR immunoreactivity was localized in ICLC, primarily concentrated at the nucleus level, but it was also observed in the cell body (cytoplasm) and processes. Stronger immunopositive reaction in the ICLC nucleus for PR than for ER was noted. Under our experimental conditions, a clear positive repeatable reaction for steroid receptors could not be detected in myocytes. In conclusion, these data suggest that ICLC could be true hormonal 'sensors', possibly participating in the regulation of human myometrial contractions (via gap junctions with myocytes and/or by paracrine signaling).     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17β-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17β-estradiol (E₂). Comparative studies were conducted with E₂ and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E₂ and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E₂ and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [³H]E₂ under saturating conditions (10 nM E₂) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [³H]E₂ was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 μM α-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E₂ metabolism, and subsequent E₂ depletion. In conclusion, while TPA and E₂ effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD. © 1996 Wiley-Liss, Inc.     

Comparative effects of raloxifene, tamoxifen and estradiol on human osteoblasts in vitro: Estrogen receptor dependent or independent pathways of raloxifene

SERMs bind to both estrogen receptor (ER)α and β, resulting in tissue dependent estrogen agonist or antagonist responses. Both raloxifene and tamoxifen are most frequently used SERMs and exert estrogen agonistic effects on human bone tissues, but the details of their possible direct effects on human bone cells have remained largely unknown. In our present study, we examined the comparative effects of raloxifene, tamoxifen, and native estrogen, estradiol on human osteoblast cell line, hFOB in vitro. Both the cell numbers and the ratio of the cells in S phase fraction were significantly increased by the treatment of raloxifene or tamoxifen as well as estradiol treatments in hFOB. Gene profile patterns following treatment with raloxifene, tamoxifen, and estradiol demonstrated similar patterns in a microarray/hierarchal clustering analysis. We also examined the expression levels of these genes detected by this analysis using quantitative RT-PCR. MAF gene was induced by raloxifene treatment alone. GAS6 gene was induced by raloxifene and tamoxifen as well as estradiol. An estrogen receptor blocker, ICI 18, 286, inhibited an increase of GAS6 gene expression but not the levels of MAF gene mRNA expression. Results of our present study demonstrated that raloxifene exerted direct protective effects on human osteoblasts in both estrogen receptor dependent and independent manners.     

Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication

Gap junctional channelsfacilitate intercellular communication and in doing so maycontribute to cellular differentiation. To test this hypothesis, weexamined gap junction expression and function in atemperature-sensitive human fetal osteoblastic cell line (hFOB 1.19)that when cultured at 37°C proliferates rapidly but when culturedat 39.5°C proliferates slowly and displays increased alkalinephosphatase activity and osteocalcin synthesis. We found that hFOB 1.19 cells express abundant connexin 43 (Cx43) protein and mRNA. Incontrast, Cx45 mRNA was expressed to a lesser degree, and Cx26 and Cx32mRNA were not detected. Culturing hFOB 1.19 cells at 39.5°C,relative to 37°C, inhibited proliferation, increased Cx43 mRNA andprotein expression, and increased gap junctional intercellularcommunication (GJIC). Blocking GJIC with 18-glycyrrhetinic acid prevented the increase in alkaline phosphataseactivity resulting from culture at 39.5°C but did not affectosteocalcin levels. These results suggest that gap junction functionand expression parallel osteoblastic differentiation and contribute tothe expression of alkaline phosphatase activity, a marker for fullydifferentiated osteoblastic cells.