Osteoblast-like cells from estrogen receptor alpha knockout mice have deficient responses to mechanical strain.

INTRODUCTION: In vivo, bones' osteogenic response to mechanical loading involves proliferation of surface osteoblasts. This response is replicated in vitro and involves ERK-mediated activation of the estrogen receptor (ER) alpha and upregulation of estrogen response element activity. This proliferative response can be blocked by selective estrogen receptor modulators and increased by transfection of additional ERalpha. MATERIALS AND METHODS: We have now investigated the mechanisms of ER involvement in osteoblast-like cells' early responses to strain by comparing the responses of primary cultures of these cells derived from homozygous ERalpha knockout (ERKO) mice (ERalpha-/-) with those from their wildtype (ERalpha+/+) and heterozygous (ERalpha+/-) littermates and from ER/beta knockout (BERKO) mice (ERbeta+/+, ERbeta+/-, and ERbeta-/-). RESULTS: Whereas ERalpha+/+, ERalpha+/-, ERbeta+/+, and ERbeta-/- cells proliferate in response to a single 10-minute period of cyclic strain, ERalpha-/- cells do not. Transfection of fully functional, but not mutant, ERalpha rescues the proliferative response to strain in these cells. The strain-related response of ERalpha-/- cells is also deficient in that they show no increased activity of an AP-I driven reporter vector and no strain-related increases in NO production. Their strain-related increase in prostacyclin production is retained. They proliferate in response to fibroblast growth factor-2 but not insulin-like growth factor (IGF)-I or IGF-II, showing the importance of ERalpha in the IGF axis and the ability of ERalpha-/- cells to proliferate normally in response to a mitogenic stimulus that does not require functional ERalpha. CONCLUSIONS: These data indicate ERalpha's obligatory involvement in a number of early responses to mechanical strain in osteoblast-like cells, including those that result in proliferation. They support the hypothesis that reduction in ERalpha expression or activity after estrogen withdrawal results in a less osteogenic response to loading. This could be important in the etiology of postmenopausal osteoporosis.     

Mechanical strain activates estrogen response elements in bone cells

The involvement of the estrogen receptor in the early responses of bone cells to mechanical strain was investigated by subjecting subconfluent monolayer cultures of ROS.SMER #14 cells (ROS 17/2.8 cells stably transfected with additional ER alpha) to 17 beta-estradiol or a single short period of dynamic mechanical strain (600 cycles, 1 Hz). The basal proliferation rate of ROS.SMER #14 cells was similar to ROS 17/2.8 cells, whose proliferative responsiveness to strain and estrogen is similar to that of primary cultures of rat long bone-derived osteoblasts. At peak strains of 3400 mu epsilon, strain-related proliferation in ROS.SMER #14 cells was 1.4 times that of ROS 17/2.8 cells. At 10(-8) mol/L, 17 beta-estradiol-related proliferation was nearly twice greater. The ROS.SMER #14 cells were transiently transfected with an estrogen-responsive reporter, 2ERE-pS2-CAT, containing two consensus estrogen response elements (ERE) linked to a chloroamphenicol acetyl transferase gene. Strain increased normalized ERE-CAT activity threefold and estradiol (10(-8) mol/L) sixfold. Both strain-related and estradiol-related increases in proliferation and ERE-CAT activity were blocked by the estrogen antagonist ICI 182,780 (10(-6) mol/L). These data show that strain as well as estrogen stimulates increased proliferation in ROS 17/2.8 cells and increased ER alpha-related ERE activity in ROS cells transfected with ER alpha. Proliferation is greater in the cells with more estrogen receptors. Both strain- and estrogen-related proliferation and ERE activity are blocked by the estrogen antagonist ICI 182,780. This indicates that ROS cells' early responses to mechanical strain involve ER alpha and estrogen-responsive genes.     

In vivo bone metastases, osteoclastogenic ability, and phenotypic characterization of human breast cancer cells

Mouse bone marrow cells cultured with human breast cancer MCF-7 cell-conditioned media showed osteoclastogenesis with an increment of bone resorption, although conditioned media from an adriamycin-selected MCF-7 clone (MCF-7ADR) had no effect. Consistently, MCF-7 cells induced 5-fold more in vivo experimental osteolytic bone metastases, with no soft tissue lesions, compared to MCF-7ADR cells. Paracrine factors stimulating (interleukin (IL)-6, IL-1beta, tumor necrosis factor-alpha (TNF-alpha)) or inhibiting (IL-12, IL-18, granulocyte macrophage-colony stimulating factor (GM-CSF)) osteoclastogenesis were significantly increased in MCF-7ADR relative to MCF-7 cells, suggesting that the inhibitory cytokines could selectively overwhelm the effects of the stimulatory ones. Treatment of osteoblast primary cultures with MCF-7-conditioned medium induced a selective upregulation of IL-6 expression, suggesting an indirect stimulation of osteoclastogenesis via the osteoblasts. MCF-7 and MCF-7ADR showed no difference in proliferation rate. However, a higher ability to migrate and invade gelatin and matrigel was observed in MCF-7ADR. Enhanced invasiveness might result from increased metalloproteinase (MMP) activity and cytoskeleton rearrangement. MCF-7ADR cells expressed higher levels of c-Src, focal adhesion kinase (FAK), and protein tyrosine kinase 2 (PYK2) involved in cell adhesion and motility. MCF-7 and MCF-7ADR expressed high and faint levels of functional estrogen receptor alpha (ERalpha), respectively. MCF-7ADR also showed significantly higher levels of the protein kinase C (PKC) alpha and beta2 and a selective activation of PKC compared to MCF-7, where the most abundant isoforms were beta1 and delta. Heat shock protein 27 (Hsp27) was more abundant in MCF-7 cells, but failed to translocate to the nucleus in response to heat shock. In conclusion, we have demonstrated that despite the fact that MCF-7ADR cells showed a more invasive phenotype relative to MCF-7, they have low potential to induce osteolytic bone lesions and stimulate osteoclastogenesis and osteoclast activity. Therefore, we believe that reduced aggressiveness of breast carcinomas could correlate with a greater osteolytic activity featuring their bone metastases.     

Breast cancer cells release factors that induced apoptosis in human bone marrow stromal cells.

Breast cancer is associated frequently with skeletal metastases, which cause significant morbidity. The main mechanism is an increase in osteoclast-mediated bone resorption. We postulated that osteoblasts could be other essential target cells and previously showed that conditioned medium (CM) of breast cancer cells (BCCs) inhibits the proliferation of osteoblast-like cells. In this study, we investigated the effects of BCC-secreted products on osteoprogenitor cells using a clonal fetal human bone marrow stromal preosteoblastic cell line (FHSO-6) that expresses alkaline phosphatase (ALP) activity, type I collagen (COLI), and increased osteocalcin (OC) and osteopontin under treatment with dexamethasone (Dex), 1,25-dihydroxyvitamin D [1,25(OH)2D], or recombinant human bone morphogenetic protein 2 (rhBMP-2). Treatment with MCF-7 CM inhibited FHSO-6 cell survival in a dose-dependent and irreversible manner. Morphological investigation indicated that MCF-7 CM increased both apoptotic and necrotic cell number. MCF-7 CM increased caspases activity and a broad inhibitor of caspase activity (benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone [z-VAD-fmk]) partly reversed the CM-induced inhibition of FHSO-6 cell survival. Western blot analyses revealed an increased bax/bcl-2 ratio in MCF-7 CM-treated FHSO-6 cells. MCF-7 cells exhibit FasLigand as membrane-bound protein and as a soluble cytokine in the CM. Deprivation of MCF-7 CM from active FasLigand by saturation with a soluble Fas molecule suppressed the induction of FHSO-6 apoptosis, whereas fibroblast CM, which did not contain FasLigand, only weakly modified FHSO-6 cell survival because of increased cell necrosis. These data indicate that FasLigand secreted by BCCs induces apoptosis and necrosis of human preosteoblastic stromal cells through caspase cascade modulated by the bax and bcl-2 protein level. The induction of apoptosis in human bone marrow stromal cells by BCCs may contribute to the inappropriately low osteoblast reaction and bone formation during tumor-induced osteolysis in bone metastases.     

Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-beta 1.

The biologic effects of recombinant human bone morphogenetic protein-2b (BMP-2b = BMP-4) were studied and compared with transforming growth factor-beta 1 (TGF-beta 1) in fetal rat osteoblast-like (ROB) cells. Similar to the effects of TGF-beta 1, BMP-2b stimulated DNA and collagen synthesis as well as protein accumulation. Unlike TGF-beta 1, which inhibited alkaline phosphatase activity, BMP-2b enhanced enzyme activity eight-to ninefold over the control level. The present study demonstrates direct actions of BMP-2b on bone-associated cells to stimulate osteogenic phenotypes in vitro and provides a cellular mechanism for the induction of bone formation by BMP-2b in vivo.     

In vitro effects of chemotherapeutic agents on human osteoblast-like cells

Osteopenia is a complicating problem that may occur during and after treatment for childhood malignancy. Clinical studies suggest that chemotherapeutic agents directly affect osteoblasts in vivo. Since combinations of agents are used for treatment, we individually investigated the chemosensitivity of human osteoblast-like cells to 11 of the chemotherapeutic agents used. The relative chemosensitivity of osteoblast-like cells representing different stages of cell differentiation was also examined. Cell numbers were evaluated following culture of an established human osteoblast-like cell line (MG63) for 3 days with clinically relevant concentrations of the chemotherapeutic agents. The chemosensitivity of MG63 cells was compared to that of a human osteoprogenitor cell line (HCC1) and primary osteoblast-like (HOB) cells derived from pediatric bone. Cell numbers were reduced by all agents in all cell types, although there was a varied response between agents at equimolar concentrations. In MG63 cells the lowest concentration of agent significantly reducing cell numbers varied between agents, for example, methotrexate (10(-7) M), vincristine (10(-9) M), and etoposide (10(-7) M) (all P <0.01). The less differentiated osteoblast phenotypes were significantly more chemosensitive at equimolar concentrations of methotrexate, vincristine, asparaginase, and dexamethasone than more differentiated phenotypes (all P <0.01). Furthermore, four agents significantly increased alkaline phosphatase (AP) activity in HOB cells. We conclude that individual chemotherapeutic agents added to osteoblast cell cultures reduce cell numbers, with osteoblast precursor cells being preferentially depleted. These results suggest that most of the agents may contribute to osteopenia in childhood malignancy by direct effects on cell numbers.     

Simvastatin induces estrogen receptor-alpha (ER-α) in murine bone marrow stromal cells

Simvastatin has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which simvastatin exerts its effects is still unclear. We previously reported that simvastatin promotes bone morphogenetic protein 2 (BMP-2) expression, induces osteoblastic differentiation, and inhibits adipocytic differentiation in mouse bone marrow stromal cells (BMSCs), and that this occurs, at least in part, via a BMP-2-dependent pathway. The aim of this study was to investigate further the mechanisms by which simvastatin stimulates osteogenesis in mouse BMSCs. To determine whether simvastatin-mediated osteogenesis was dependent on BMP-2, mouse BMSCs were treated with nonimmune normal mouse IgG or BMP-2 neutralizing antibodies combined with different concentrations of simvastatin. Surprisingly, the stimulatory effect of simvastatin on alkaline phosphatase (ALP) activity was not completely blocked by neutralizing BMP-2 monoclonal antibody treatment. Interestingly, we found that estrogen receptor-alpha (ER-α) protein levels increased after mouse BMSCs were treated with simvastatin for 72 h in a concentration-dependent manner. Moreover, the stimulatory effect of simvastatin on ALP activity in BMSCs was blocked by the estrogen receptor agonist ICI 182,780, and cotreatment with 17-β-estradiol and simvastatin increased ALP activities by two-to threefold in the BMSCs compared with treatment with simvastatin alone. These results suggest that simvastatin-induced in vitro osteogenesis in mouse BMSCs is mediated, at least in part, by induction of ER-α and not by BMP-2 alone. These results provide new insight into the mechanisms of simvastatin-induced bone formation in BMSCs.     

Nitric oxide and prostaglandin E2 production in response to ultra-high molecular weight polyethylene particles depends on osteoblast maturation state

BACKGROUND: Recent studies have shown that osteoblast-like cells respond directly to ultra-high molecular weight polyethylene particles in culture, suggesting that they may be involved in aseptic loosening of endoprostheses. We tested the hypothesis that the state of cell maturation plays a role in the response of osteogenic cells to ultra-high molecular weight polyethylene particles. METHODS: MG63 cells (immature osteoblast-like cells), OCT-1 cells (mature secretory osteoblast-like cells), and MLO-Y4 cells (osteocyte-like cells) were treated for twenty-four hours with commercial ultra-high molecular weight polyethylene particles with an average diameter of 1 mm. The effect of particle treatment on cell proliferation was assessed by measuring the number of cells, whereas the effects on differentiation and local factor production were assessed by measuring the production of osteocalcin, prostaglandin E2, and nitric oxide. The effect of particles on apoptosis was also evaluated. RESULTS: The addition of ultra-high molecular weight polyethylene particles increased the number of MG63 cells, did not affect the number of OCT-1 cells, and led to a decrease in the number of MLO-Y4 cells. The observed changes in cell number were not due to programmed cell death, as no more than 3% of the cells in cultures treated with the highest concentration of particles were undergoing apoptosis. Osteocalcin production was not affected by the addition of particles. Prostaglandin E2 production was increased in all three types of cultures, but the effect was greatest in OCT-1 cell cultures, as was the absolute amount of prostaglandin E2 produced. Nitric oxide production was unaffected in MG63 cell cultures, but it was stimulated in OCT-1 and MLO-Y4 cell cultures. CONCLUSIONS: The results of the present study support the hypothesis that osteoblast cell maturation state plays an important role in the response to ultra-high molecular weight polyethylene particles and that the terminally differentiated osteocyte may be involved in the bone response to wear debris in vivo.     

Osteocytes use estrogen receptor alpha to respond to strain but their ERalpha content is regulated by estrogen.

The role of mechanical strain and estrogen status in regulating ERalpha levels in bone cells was studied in female rats. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas habitual strain and artificial loading has only a small but positive effect, except on the ulna's medial surface, where artificial loading stimulates reversal of resorption to formation. INTRODUCTION: Osteoporosis is the most widespread failure of bones' ability to match their architectural strength to their habitual load bearing. In men and women, the severity of bone loss is associated with bioavailability of estrogen. This association could result from the estrogen receptor (ER) involvement in bone cells' adaptive response to loading. MATERIALS AND METHODS: In vivo semiquantitative analysis of the amount of ERalpha protein per osteocyte was performed in immuno-cytochemically stained sections from control and loaded rat ulna, as well as tibias of ovariectomy (OVX) and sham-operated female rats. In vitro, the effect of exogenous estrogen (10(-8) M) and mechanical strain (3400 microepsilon, 1 Hz, 600 cycles) on the expression of ERalpha mRNA levels was assessed in ROS 17/2.8 cells in monolayers using real-time PCR and ER promoter activity. ERalpha translocation in response to exogenous estrogen and mechanical strain was assessed in both ROS 17/2.8 and MLO-Y4 cells. RESULTS: More than 90 percent of tibial osteocytes express ERalpha, the level/osteocyte being higher in cortical than cancellous bone. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas in the ulna habitual strain and that caused by artificial loading had only a small but positive effect, except on the medial surface, where loading stimulates reversal of resorption to formation. In unstimulated osteocytes and osteoblasts in situ, and osteocyte-like and osteoblast-like cells in vitro, ERalpha is predominantly cytoplasmic. In vitro, both strain and estrogen stimulate transient ERalpha translocation to the nucleus and transient changes in ERalpha mRNA. Strain but not estrogen also induces discrete membrane localization of ERalpha. CONCLUSIONS: Bone cells' responses to both strain and estrogen involve ERalpha, but only estrogen regulates its cellular concentration. This is consistent with the hypothesis that bone loss associated with estrogen deficiency is a consequence of reduction in ERalpha number/activity associated with lower estrogen concentration reducing the effectiveness of bone cells' anabolic response to strain.     

Direct action of naturally occurring estrogen metabolites on human osteoblastic cells.

This article describes experiments that were performed to examine the direct action of estrogen metabolites on cultured human osteoblast cells. The human fetal osteoblastic cell line, hFOB/ER9, which expresses high levels of the estrogen receptor (ER) alpha, was used to examine the direct effects of 16alpha-hydroxyestrone (16alpha-OHE1) and 2-hydroxyestrone (2-OHE1) on osteoblast differentiation. The 16alpha-OHE1 caused a decrease in osteocalcin (OC) secretion to a maximum of 40% of control values (vehicle-treated cells) at 10(-7) M. Alkaline phosphatase (AP) activity was significantly induced at 10(-7) M 16alpha-OHE1 with greater than 500% of control at 10(-6) M 16alpha-OHE1. Finally, AP steady-state messenger RNA (mRNA) levels were increased within 24 h of 16alpha-OHE1 treatment. In contrast to 16alpha-OHE1, 2-OHE1 had no effects on the secretion of OC, AP activity, or AP gene expression. The 2-OHE1 also did not display any antiestrogen activity because treatment in combination with 17beta-estradiol (E2) and 16alpha-OHE1 had no significant effect on the reduction in OC secretion or induction of AP activity. Similar to E2, 16alpha-OHE1 stimulated the expression of an early response gene, a TGF-beta inducible early gene, designated TIEG, as early as 60 minutes after treatment, whereas treatment with 2-OHE1 displayed no effect. Support that the 16alpha-OHE1 regulation of these osteoblasts (OB) markers was mediated through the ER is shown by the fact that the estrogen antagonist ICI 182,780 abrogated these effects. These data suggest that is a potent estrogen agonist on human osteoblastic hOB/ER9 cells. In contrast, 2-OHE1 displayed no estrogenic or antiestrogenic activity in this human osteoblast cell model.     

Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs)

BACKGROUND: Bone morphogenic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway to evaluate the ability of certain BMPs to promote fracture-healing and spinal fusion. The optimal BMPs to be used in different clinical applications have not been elucidated, and a comprehensive evaluation of the relative osteogenic activity of different BMPs is lacking. METHODS: To identify the BMPs that may possess the most osteoinductive activity, we analyzed the osteogenic activity of BMPs in mesenchymal progenitor and osteoblastic cells. Recombinant adenoviruses expressing fourteen human BMPs (BMP-2 to BMP-15) were constructed to infect pluripotent mesenchymal progenitor C3H10T1/2 cells, preosteoblastic C2C12 cells, and osteoblastic TE-85 cells. Osteogenic activity was determined by measuring the induction of alkaline phosphatase, osteocalcin, and matrix mineralization upon BMP stimulation. RESULTS: BMP-2, 6, and 9 significantly induced alkaline phosphatase activity in pluripotential C3H10T1/2 cells, while BMP-2, 4, 6, 7, and 9 significantly induced alkaline phosphatase activity in preosteoblastic C2C12 cells. In TE-85 osteoblastic cells, most BMPs (except BMP-3 and 12) were able to induce alkaline phosphatase activity. The results of alkaline phosphatase histochemical staining assays were consistent with those of alkaline phosphatase colorimetric assays. Furthermore, BMP-2, 6, and 9 (as well as BMP-4 and, to a lesser extent, BMP-7) significantly induced osteocalcin expression in C3H10T1/2 cells. In C2C12 cells, osteocalcin expression was strongly induced by BMP-2, 4, 6, 7, and 9. Mineralized nodules were readily detected in C3H10T1/2 cells infected with BMP-2, 6, and 9 (and, to a lesser extent, those infected with BMP-4 and 7). CONCLUSIONS: A comprehensive analysis of the osteogenic activity of fourteen types of BMPs in osteoblastic progenitor cells was conducted. Our results suggest an osteogenic hierarchical model in which BMP-2, 6, and 9 may play an important role in inducing osteoblast differentiation of mesenchymal stem cells. In contrast, most BMPs are able to stimulate osteogenesis in mature osteoblasts.     

Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells.

Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP-2-induced matrix calcification was studied. BMP-2 enhances Pit-1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification. INTRODUCTION: Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP-2-induced matrix mineralization. MATERIALS AND METHODS: Confluent MC3T3-E1 osteoblast-like cells were exposed to BMP-2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit-1 and Pit-2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). RESULTS: BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport in MC3T3-E1 cells by increasing the V(max) of the transport system. This effect was preceded by an increase in mRNA encoding Pit-1 but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineralization induced by BMP-2 were blunted by a specific inhibitor of the c-Jun-N-terminal kinase (JNK) pathway. CONCLUSIONS: Results presented in this study indicate that, in addition to its well-known effect on several markers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1 cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP-2.     

17β-雌二醇对子宫内膜异位症患者在位子宫内膜间质细胞β-catenin mRNA和蛋白表达的影响

目的研究17β-雌二醇(17β-E2)对子宫内膜异位症(内异症)患者在位子宫内膜间质细胞β-catenin mRNA和蛋白表达的影响,探讨Wnt/β-catenin信号通路在介导雌激素促进内异症发生发展的作用。方法体外分离培养内异症患者在位子宫内膜间质细胞。用不同浓度17β-E2处理子宫内膜间质细胞48 h;此后选用10-10mol/L 17β-E2处理子宫内膜间质细胞12、24和48 h,逆转录聚合酶链反应(RT-PCR)和免疫印迹法(Western blotting)检测17β-E2处理前后子宫内膜间质细胞β-catenin mRNA和蛋白的表达水平。同法分析雌激素受体拮抗剂ICI182,780(10-6mol/L)对17β-E2促进β-catenin mRNA和蛋白表达的影响。免疫组织化学染色观察17β-E2作用后β-catenin在子宫内膜间质细胞中的定位。结果17β-E2能明显促进内异症患者在位子宫内膜间质细胞β-catenin mRNA和蛋白的表达,并呈剂量和时间依赖性,于10-10mol/L作用48 h最明显。雌激素受体拮抗剂ICI182,780能明显抑制17β-E2对子宫内膜间质细胞β-catenin mRNA和蛋白的表达。免疫组织化学染色发现17β-E2能促进β-catenin在子宫内膜间质细胞核内的表达。结论雌激素可能通过激活Wnt/β-catenin信号通路促进内异症在位子宫内膜的异位种植。