TE85 Cells Research Articles

Expression of a Structurally Unique Osteoclastic Protein-tyrosine Phosphatase Is Driven by an Alternative Intronic, Cell Type-specific Promoter

An osteoclastic protein-tyrosine phosphatase (PTP-oc), essential for osteoclast activity, shows sequence identity with the intracellular domain of GLEPP1, a renal receptor-like transmembrane PTP. PTP-oc has been assumed to be a truncated variant of GLEPP1, resulting from alternative splicing. However, the 5'-untranslated region sequence of PTP-oc mRNA contains 217 bp from an intron of GLEPP1. There are no splicing acceptor sites at the PTP-oc transcription site. The intronic sequence flanking the 5' end of the PTP-oc transcription start site contains potential promoter elements essential for transcriptional initiation. To test the hypothesis that the PTP-oc gene has an alternative, tissue-specific, intronic promoter, the promoter activity of a 1.3-kb PCR fragment covering the 5'-flanking region of the PTP-oc gene was measured. The putative PTP-oc promoter fragment showed strong promoter activity in U937 cells. Mutation of the putative TATA box within the PTP-oc promoter abolished 60-90% of its promoter activity. The PTP-oc promoter fragment showed strong promoter activity in cells that express PTP-oc (U937 cells and RAW264.7 cells) but not in cells that do not express the enzyme (skin fibroblasts, TE85 cells, and HEK293 cells). These findings strongly support the conclusion that the 1.3-kb intronic fragment contains the tissue-specific, PTP-oc proximal promoter. Deletion and functional analyses indicate that the proximal 5' sequence flanking the TATA box of the PTP-oc contains potential repressor elements. The removal of the putative repressor elements led to the apparent loss of tissue specificity. In summary, we conclude that an intronic promoter within the GLEPP1 gene drives the expression of the PTP-oc in a cell type-specific manner. This GLEPP1/PTP-oc gene system is one of the very few systems in which two important tissue-specific enzymes are derived from the same gene by the use of alternative intronic promoters.

Arsenite induces delayed mutagenesis and transformation in human osteosarcoma cells at extremely low concentrations.

Arsenite is a human multisite carcinogen, but its mechanism of action is not known. We recently found that extremely low concentrations (</=0.1 microM) of arsenite transform human osteosarcoma TE85 (HOS) cells to anchorage-independence. In contrast to other carcinogens which transform these cells within days of exposure, almost 8 weeks of arsenite exposure are required for transformation. We decided to reexamine the question of arsenite mutagenicity using chronic exposure in a spontaneous mutagenesis assay we previously developed. Arsenite was able to cause a delayed increase in mutagenesis at extremely low concentrations (</=0.1 microM) in a dose-dependent manner. The increase in mutant frequency occurred after almost 20 generations of growth in arsenite. Transformation required more than 30 generations of continuous exposure. We also found that arsenite induced gene amplification of the dihydrofolate reductase (DHFR) gene in a dose-dependent manner. Since HOS cells are able to methylate arsenite at a very low rate, it was possible that active metabolites such as monomethylarsonous acid (MMA(III)) contributed to the delayed mutagenesis and transformation in these cells. However, when the assay was repeated with MMA(III), we found no significant increase in mutagenesis or transformation, suggesting that arsenite-induced delayed mutagenesis and transformation are not caused by arsenite's metabolites, but by arsenite itself. Our results suggest that long-term exposure to low concentrations of arsenite may affect signaling pathways that result in a progressive genomic instability.

Bone cell mitogenic action of fluoroaluminate and aluminum fluoride but not that of sodium fluoride involves upregulation of the insulin-like growth factor system

The fluoroaluminate (AlF 4 −) ion and sodium fluoride (NaF) have previously been shown to be bone cell mitogens. This study sought to determine whether the bone cell mitogenic action of AlF 4 − and/or NaF would involve the insulin-like growth factor (IGF) regulatory system. We evaluated the effect of mitogenic doses of AlF 4 − and NaF on the mRNA levels and the protein level (in conditioned media [CM]) of several components of the IGF system (i.e., IGF-2, IGF binding protein [IGFBP]-4, and IGFBP-5) in human TE85 osteosarcoma cells. Aluminum fluoride (AlF 3) was included for comparison. NaF, AlF 3, and AlF 4 −, each at 50–100 μmol/L, increased [ 3H]thymidine incorporation in TE85 cells. Mitogenic concentrations of AlF 3 and AlF 4 −: (1) increased the mRNA (up to twofold after 24 h treatment) and protein (in CM) levels (up to 2.5-fold after 48 h treatment) of IGF-2; (2) increased the mRNA level (twofold) and the protein level in CM (up to threefold) of stimulatory IGFBP-5; and (3) either reduced slightly or had no effect on the mRNA and protein (in CM) levels of the inhibitory IGFBP-4. Conversely, mitogenic concentrations of NaF had no significant effects on the protein (in CM) or mRNA level of IGF-2, IGFBP-4, or IGFBP-5. The addition of an inhibitory concentration of IGFBP-4 completely abolished the bone cell mitogenic activity of AlF 3 and AlF 4 − but not that of NaF. The findings of this study provide strong evidence that the bone cell mitogenic activity of AlF 4 − and AlF 3, but not that of NaF, is mediated by the upregulation of the IGF regulatory system.

Human osteoblasts' proliferative responses to strain and 17beta-estradiol are mediated by the estrogen receptor and the receptor for insulin-like growth factor I.

The mechanism by which mechanical strain and estrogen stimulate bone cell proliferation was investigated using monolayer cultures of human osteoblastic TE85 cells and female human primary (first-passage) osteoblasts (fHOBs). Both cell types showed small but statistically significant dose-dependent increases in [3H]thymidine incorporation in response to 17beta-estradiol and to a single 10-minute period of uniaxial cyclic strain (1 Hz). In both cell types, the peak response to 17beta-estradiol occurred at 10(-8) - 10(-7) M and the peak response to strain occurred at 3500 microstrain ((mu)epsilon). Both strain-related and 17beta-estradiol-related increases in [3H]thymidine incorporation were abolished by the estrogen receptor (ER) modulator ICI 182,780 (10-8 M). Tamoxifen (10(-9) - 10(-8) M) increased [3H]thymidine incorporation in both cell types but had no effect on their response to strain. In TE85 cells, tamoxifen reduced the increase in [3H]thymidine incorporation associated with 17beta-estradiol to that of tamoxifen alone but had no such effect in fHOBs. In TE85 cells, strain increased medium concentrations of insulin-like growth factor (IGF) II but not IGF-I, whereas 17beta-estradiol increased medium concentrations of IGF-I but not IGF-II. Neutralizing monoclonal antibody (MNAb) to IGF-I (3 microg/ml) blocked the effects of 17beta-estradiol and exogenous truncated IGF-I (tIGF-I; 50 ng/ml) but not those of strain or tIGF-II (50 ng/ml). Neutralizing antibody to IGF-II (3 microg/ml) blocked the effects of strain and tIGF-II but not those of 17beta-estradiol or tIGF-I. MAb aIR-3 (100 ng/ml) to the IGF-I receptor blocked the effects on [3H]thymidine incorporation of strain, tIGF-II, 17beta-estradiol, and tIGF-I. HOBs and TE85 cells, act similarly to rat primary osteoblasts and ROS 17/2.8 cells in their dose-related proliferative responses to strain and 17beta-estradiol, both of which can be blocked by the ER modulator ICI 182,780. In TE85 cells (as in rat primaries and ROS 17/2.8 cells), the response to 17beta-estradiol is mediated by IGF-I, and the response to strain is mediated by IGF-II. Human cells differ from rat cells in that tamoxifen does not block their response to strain and reduces the response to 17beta-estradiol in TE85s but not primaries. In both human cell types (unlike rat cells) the effects of strain and IGF-II as well as estradiol and IGF-I can be blocked at the IGF-I receptor.

Characterization of the progestin receptors in the human TE85 and murine MC3T3-E1 osteoblast-like cell lines.

Progestins are believed to exert positive effects on bone density through receptors located in osteoblasts. In the present studies, the binding characteristics and regulation of the progestin receptors in two osteoblast-like cell lines were compared with those in human breast lines. Human TE85 and murine MC3T3-E1 osteoblast-like cells contain a single, high-affinity progestin binding site whose affinity and concentration are lower than in human breast cells. The osteoblastic progestin binding sites showed the expected steroid specificity and associated with the cell nuclei when occupied by ligand. The progestin receptors in osteoblastic cells also had sedimentation coefficients similar to those receptors in breast cells. The regulation of the progestin receptor in the osteoblast-like cells was explored by treating them with estradiol. In contrast to the large, rapid change seen in the breast cells, the progestin receptor levels in the MC3T3-E1 cells showed only a small, delayed up-regulation with estradiol treatment. The progestin receptor number in the TE85 cells was unaffected by estradiol. Down-regulation of the progestin receptors was explored by treating the cells with the progestin, norethindrone (NET). NET administration produced a rapid drop in progestin binding sites in the breast cells and a smaller, more gradual decline in MC3T3-E1 progestin binding. While the maximal decrease in receptor number occurred within 24 h in the breast cells, the receptor number was still continuing to fall after 72 h in the MC3T3-E1 cells. The data presented here demonstrate that both human and murine osteoblast-like cells contain a functional progestin receptor whose binding characteristics and regulation are similar, but not identical, to those receptors in other progestin target tissues such as the breast.

Differential regulation of syndecan expression by osteosarcoma cell lines in response to cytokines but not osteotropic hormones

Bone cells are regulated by interactions with both growth factors and components of the extracellular matrix (ECM). Syndecans are cell-surface heparan sulfate proteoglycans known to play a role in cell adhesion and migration, and binding of growth factors. This study was performed to investigate the expression of syndecans by osteoblasts. Reverse transcription-linked polymerase chain reaction (RT-PCR) and Northern analysis detected syndecan transcripts in the human osteosarcoma cell lines MG-63, TE-85, SaOS-2, and U2OS; human osteoblast-like cells; rat calvarial osteoblasts; and in human bone. Western blot analysis of proteoglycans from MG-63 and TE-85 cells detected multiple heparan sulfate proteoglycan core proteins consistent with syndecan expression. Regulation of syndecan-1, -2, and -4 expression was investigated in TE-85, MG-63, and SaOS-2 cells, in response to interleukin (IL)-1β, and IL-6, parathyroid hormone [PTH(1-34)], and 1,25(OH) 2-vitamin D 3. Northern analysis demonstrated that in the osteosarcoma cell lines there was no regulation of syndecan transcript levels in response to PTH(1-34) or 1,25(OH) 2-vitamin D 3 for 24 or 48 h. In contrast, when MG-63 and SaOS-2 cells were incubated with IL-1β (0.01–10 ng/mL) and IL-6 (0.1–50 ng/mL) there was a dose-dependent decrease in mRNA levels for syndecan-1 and -2 at 24 and 48 h, but in response to IL-1β upregulation in the levels of syndecan-4 transcripts. In addition, Northern analysis was performed on RNA isolated from neonatal rat calvarial osteoblasts cultured under conditions that promote osteogenesis for 0, 5, 13, 21, and 35 days. Syndecan-1 expression was observed to decrease during the culture period, syndecan-2 transcript levels increased, and there appeared to be no overall change in syndecan-4 levels. Controlled expression of syndecans by cells of the osteoblast lineage may be important in the regulation of osteoblastic proliferation and differentiation.

Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells

Previous studies have shown evidence of constitutive and cytokine-inducible nitric oxide (NO) synthase activity in cultured osteoblast-like cells from various species. Although cytokine-induced NO production has been found to inhibit osteoblast growth, the role of constitutive NO production in regulating osteoblast function is less clear and the isoforms of nitric oxide synthase (NOS) that are expressed by human osteoblasts have not been determined. Here, we investigated NOS expression in cultured human osteoblast-like cells and studied the effects of constitutive and cytokine-induced NO on osteoblast growth and differentiation. Low levels of NO were produced constitutively by osteoblast-like cells as reflected by analysis of medium nitrite concentrations, and evidence of ecNOS mRNA, protein, and bioactivity was found in primary osteoblasts (hOBs), TE85, and MG63 osteosarcoma cells. None of the osteoblast-like cells expressed nNOS, however, and iNOS was produced only by hOB cells after stimulation with the cytokines IL-1β, TNF-α, and IFN-γ. The NOS inhibitor, L-NMMA, did not affect growth or alkaline phosphatase activity in unstimulated osteoblasts. Incubation of hOB cells with cytokines inhibited growth and stimulated alkaline phosphatase activity and these effects were abrogated by L-NMMA. Cytokines also inhibited growth of TE85 cells and MG63 cells, but these effects appeared to be NO independent because they were not influenced by L-NMMA. Our experiments show that human osteoblasts constitutively produce NO through the ecNOS pathway, but demonstrate that this does not appear to exert an appreciable effect on osteoblast growth or differentiation under basal conditions. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting and differentiation-inducing effects on osteoblasts that were partly NO dependent, indicating that NO may act predominantly as a modulator of cytokine-induced effects on osteoblast function.

Stimulation by low concentrations of fluoride of the proliferation and alkaline phosphatase activity of human dental pulp cells in vitro

Fluoride has been used for decades, either systemically or topically, to prevent dental caries. The purpose of this study was to clarify the effects of low concentrations of fluoride on proliferation, differentiation and extracellular-matrix synthesis in normal human dental pulp cells (DP-1 and DP-2) in vitro. The effects were compared with those on a human osteoblastic osteosarcoma cell line, TE-85. Fluoride at micromolar concentrations significantly and dose-dependently stimulated [ 3H]thymidine incorporation into DNA in DP-1, DP-2 and TE-85 cells, with optimal effects around 50 μM, by 127±7%, 124±0.6% and 152±13.4%, respectively. To assess the potential influence of fluoride on cell differentiation, the effects of mitogenic concentrations on alkaline phosphatase activity were measured. Fluoride significantly increased the enzyme’s activity in DP-1 and TE-85 by 177±12% and 144±12.3%. To evaluate the effect on extracellular-matrix synthesis, the synthesis of type I collagen was indirectly determined by an assay of procollagen type I c-peptide production. Fluoride significantly increased that production by 150±8.7% in TE-85, but not in either DP-1 or DP-2. These observations suggest that fluoride, if used at low concentrations, could be a useful therapeutic agent where increased regeneration of dentine is desired, such as after pulp amputation, by stimulating the proliferation and differentiation of the dental pulp cells.

Molecular Cloning and Characterization of LR3, a Novel LDL Receptor Family Protein with Mitogenic Activity

We report molecular cloning and initial functional characterization of a novel member of the low density lipoprotein receptor (LDLR) gene family. The cDNA was isolated from a human osteoblast cDNA library and encoded a 1,615 amino acids protein designated as LR3. It has, in the extracellular region, a cluster of three LDLR ligand binding repeats at a juxtamembrane position and four EGF precursor homology domains separated by YWTD spacer repeats. The entire ectodomain shares the same modular organization with the middle portion of the extracellular regions of two LDLR family members, LDLR-related protein (LRP), and gp330/megalin. LR3 mRNA was expressed in most of the adult and fetal tissues examined. The highest expression level was seen in aorta. In human osteosarcoma cells examined, LR3 mRNA was highly enriched in TE85 cells, moderately expressed in MG63 cells and primary human osteoblasts, and undetectable in SaOS-2 cells. NIH 3T3 cells transfected with either full length LR3 or its ectodomain showed significantly increased proliferation, whereas transfection of intracellular domain had no proliferative effect. We predict that LR3 is a multi-functional protein with potential mitogenic activity.

Formoterol and Isoproterenol Induce c- fos Gene Expression in Osteoblast-Like Cells by Activating β 2-Adrenergic Receptors

Formoterol, a β 2-adrenergic agonist has been shown in ovariectomized rat models to have anabolic effects on bone. However, those studies did not determine whether the effect of formoterol was by a direct action on bone cells themselves or indirectly via anabolic action on muscle. To address the question of whether formoterol could directly affect osteoblast function we investigated the expression patterns of β-adrenergic receptors (βARs) in human osteoblast-like cells and functional coupling to gene expression. Northern blot analysis showed that βAR subtypes are expressed at different levels in the osteoblast-like cell lines TE-85, SaOS-2, MG-63, and OHS-4. β 1AR expression was found in SaOS-2, OHS-4, and TE-85, but not MG-63 cells. β 2ARs are expressed at higher levels in MG-63 cells than in TE-85 and SaOS-2 cells, but were not detected in OHS-4 cells. PCR analysis paralleled the northern blot analysis except that β 3AR expression was found in one of three human primary osteoblast cDNAs tested. β 3AR expression was not found in any of the osteoblast-like cell lines. The nonspecific βAR agonist, isoproterenol, and the β 2AR-specific agonist, formoterol, induced c- fos gene expression in cultured SaOS-2 cells in an immediate early fashion. This effect was inhibited by the β 2AR-specific antagonist, ICI 118551, but not by the β 1AR-specific antagonist, CGP 20712, indicating that induction of c- fos gene expression is specifically mediated by β 2ARs. c- fos gene expression was induced by both isoproterenol and formoterol via increases in cAMP, which in turn activated the cAMP/PKA pathway; the PKA inhibitor, H89, inhibited c- fos gene expression. Thus, βARs are expressed in osteoblast-like cells and are coupled to c- fos gene expression via the β 2AR, increases in cAMP levels and activation of a PKA-dependent pathway.

The mitogenic effect of parathyroid hormone is associated with E2F-dependent activation of cyclin-dependent kinase 1 (cdc2) in osteoblast precursors.

Injections of parathyroid hormone (PTH) have been reported to stimulate skeletal accretion through increased bone formation in several species, and osteoblast proliferation is a critical component of bone formation. However, the biological mechanisms of PTH-stimulated bone cell proliferation are largely unknown. In this study, we demonstrated that PTH stimulates proliferation of the osteoblast precursor cell line, TE-85, in association with increasing cdc2 protein levels and its kinase activity. cdc2 antisense oligonucleotides blocked PTH-induced DNA synthesis and cell cycle progression. Analysis of the time course of PTH-stimulated cdc2 message levels demonstrated that cdc2 mRNA levels were increased 1.5- to 4-fold between 3-18 h following release from cell synchronization. Transfections of TE-85 cells with a series of cdc2 promoter-luciferase deletion constructs revealed PTH stimulation of the cdc2 promoter. Promoter constructs containing a mutation in the E2F binding site were not stimulated by PTH. Gel mobility shift assays demonstrated increased free E2F levels in TE-85 nuclear extracts in response to PTH. Furthermore, the ratios of hyperphosphorylated to hypophosphorylated forms of Rb protein were increased by PTH treatment. These results demonstrate that PTH-stimulated cdc2 expression was associated with TE-85 cell proliferation and that the mechanism of stimulating cdc2 gene expression involved increasing the levels of free E2F.

Quantitative study of calcium uptake by tumorigenic bone (TE-85) and neuroblastoma × glioma (NG108-15) cells exposed to extremely-low-frequency (ELF) electric fields

To verify the effect of cell culture state on frequency dependent increase in proliferation as well as Ca 2+ flux across the plasma membrane, tumorigenic bone (TE-85) and neuroblastoma × glioma (NG108-15) cells cultured in the presence of fetal bovine serum (FBS) were exposed to capacitively coupled electric (CCEF) fields in the extremely low frequency (ELF) range of 10 to 18 Hz. [ 3H]Thymidine incorporation and 45Ca 2+ uptake were used as endpoints. TE-85 cells cultured in the presence of 10% FBS did not exhibit a frequency dependent increase in proliferation in contrast to previous studies under growth arrested culture conditions, in which the cells were deprived of FBS. However, both TE-85 and NG108-15 cells had an increase in 45Ca 2+ uptake in response to a 16 Hz 18.3 mV/cm CCEF. Fura-2 digital imaging microscopy was used to verify addition of 0.5 mM La 3+ and 0.5 mM ionomycin as negative and positive controls, respectively. Imaging microscopy data was combined with 45Ca 2+ incorporation results to quantify free intracellular calcium ([Ca 2+] i) increase in response to CCEF exposure. TE-85 [Ca 2+] i increased from 140 to 189–210 nM where as NG108-15 [Ca 2+] i increased from 67 to 189–210 nM. These results suggested that serum deprivation may be a requirement for a frequency dependent increase in proliferation in TE-85 cells but is not necessary for the electric field induced increase in 45Ca 2+ uptake in both TE-85 adn NG108 cells. The present study also represents the first demonstration of increased 45Ca 2+ uptake by neuroblastoma and/or glioma cells in response to an electric field exposure.

PCR phenotyping of cytokines, growth factors and their receptors and bone matrix proteins in human osteoblast-like cell lines

The expression of a total of 58 cytokines, growth factors, and their corresponding receptors and bone matrix proteins was assessed using reverse transcription-linked polymerase chain reaction (RT-PCR) analysis to determine the similarity in the expression profile between clonal osteosarcoma-derived human osteoblast-like cell lines and primary human osteoblast-like cell cultures derived from human trabecular bone explants. The spectrum of cytokines, growth factors, and bone-related proteins expressed by three human osteosarcoma-derived cell lines, TE-85, MG-63, SaOS-2, and primary human osteoblast-like cells was found to be highly comparable and for the first time the expression of EGF, ECGF, FGFβ, oncostatin M, TNFβ, and SCF by human osteoblast-like cells was detected. Also the expression of several receptor types including IL-4R, IL-7R, IFNα/βR, and SCFR was detected that has not been previously described for human osteoblast-like cells. For the factors examined, no qualitative variations in the expression profile were observed in the six primary human osteoblast-like cell cultures used in this study. Of the 58 factors examined, only 13 showed some degree of nonuniformity of expression between all of the three cell lines and primary cell cultures. These differences were seen especially in the expression of cytokine receptor mRNA and to a lesser extent with some cytokines. Differences in receptor expression would suggest that the possible spectrum of response to exogenously added factors, or even autocrine/paracrine networks would be determined by the repertoire of receptors expressed by each cell type. Whether the differences are related to the status of cell maturation within the osteoblast development lineage or to their abberant regulation of expression cannot be concluded at this stage. However, this PCR-phenotyping approach rapidly provides a resource of information, which can be subsequently used for further in depth studies to facilitate the analysis of the molecular mechanisms, whereby the target gene of interest is modulated in a model cell line. In addition, this study indicates that at least based on the transcript expression profile of the factors analyzed, human osteosarcoma-derived osteoblast-like cells are useful as models for their nontransformed counterparts.

A molecular mechanism of integrin regulation from bone cells stimulated by orthodontic forces.

The purpose of this paper is to discuss a molecular mechanism in the signal transduction pathways of the regulation of integrin genes taking place in bones cells as a result of orthodontic or mechanical stimulation. Human osteosarcoma (HOS) TE-85 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and grown to confluency in Flexercell type I dishes and orthodontic forces were applied to the cells via an intermittent strain of 3 cycles/minute using the Flexercell Strain Unite System for periods of 15 and 30 minutes, 2 and 24 hours and 3 days. Antibodies against beta 1 and alpha v integrins were immunolocalized in strained and unstrained cultures. Total RNA was extracted and cDNA probes were used to measure at various mRNA expression of beta 1 (1.2 kb) and alpha v (1.1 kb) integrins. A cDNA probe for cyclophylin (750 b) was used for controls of gene expression. Results showed that mechanical stimulation caused a reorganization of integrin distribution in comparison with non-stimulated controls. mRNA for beta 1 expression showed a marked increase at 30 minutes and 3 days, while mRNA levels for alpha v did not change with strain. The selective expression of integrins mRNA is indicative of a specific gene regulation by mechanical stimulation in the cells studied.

Dexamethasone enhances the osteogenic effects of fluoride in human TE85 osteosarcoma cells in vitro.

The in vitro osteogenic effects of fluoride have not always been consistently observed in human bone cells. The present study sought to test if dexamethasone (Dex) could potentiate the action of fluoride to increase the detectability of the stimulatory effects of fluoride on [3H]thymidine incorporation, alkaline phosphatase (ALP) specific activity, collagen synthesis, and osteocalcin secretion in human TE85 osteosarcoma cells. Neither Dex at 10(-10)-10(-6) M or fluoride at a mitogenic dose (100 microM) had any consistent stimulatory effects on thymidine incorporation. When the cells were treated with both agents simultaneously, significant and highly reproducible stimulations were observed. The mitogenic effects of the two agents were confirmed with cell number counting. Analysis of variance (ANOVA) revealed a significant interaction (P < 0.001) between fluoride and Dex on cell proliferation. The enhancing effect of Dex on [3H]thymidine incorporation was not due to a shift of the optimal dose response of fluoride. Though fluoride alone or Dex alone also had no consistent effect on ALP specific activity, the co-treatment with fluoride and Dex for 24 hours produced significant (P < 0.001, ANOVA) stimulation in ALP specific activity. Fluoride alone had no consistent effect on collagen synthesis and on 1, 25(OH)2D3-dependent osteocalcin secretion, whereas Dex treatment consistently inhibited these two osteoblastic parameters in a dose-dependent manner. However, both the collagen synthesis and osteocalcin secretion rates were significantly higher (P < 0.001 ANOVA for each) when the cells were co-treated with Dex and fluoride (100 microM) than when they were treated with Dex alone. Thus, these data indicate that the response in collagen synthesis and osteocalcin secretion to fluoride stimulation was more readily observed in the presence of Dex than in its absence. ANOVA analysis revealed that the interaction between fluoride and Dex on collagen synthesis, but not the 1,25(OH)2D3-dependent osteocalcin secretion, was significant (P < 0.02). In summary, we have demonstrated for the first time that in TE85 cells (1) Dex potentiated the effects of fluoride on cell proliferation, ALP specific activity, and collagen synthesis; (2) while Dex at 10(-7)-10(-6) M alone inhibited the collagen synthesis and at 10(-9)-10(-6) M reduced osteocalcin secretion, Dex at 10(-8)-10(-6) M significantly stimulated the proliferation of TE85 cells; and (3) Dex interacted with fluoride to increase the percentage of experiments showing an osteogenic action of fluoride. In conclusion, the in vitro osteogenic actions of fluoride in human TE85 cells are more consistently observed in the presence than in the absence of Dex.

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: effect of tumor necrosis factor-alpha.

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor-alpha (TNF-alpha) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF-alpha inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF-alpha at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-alpha, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF-alpha inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.

Osteosarcoma hybrids can preferentially target alkaline phosphatase activity to matrix vesicles: Evidence for independent membrane biogenesis

Alkaline phosphatase is the marker enzyme for matrix vesicles, extracellular organelles that play a major role in primary bone formation and calcification. Recently, we developed osteosarcoma x fibrosarcoma hybrids in which alkaline phosphatase expression was greatly reduced, a phenomenon known as extinction. In the present study, we used to cell hybrids, LTA-1 and LTA-5, constructed from a human osteoblast-like osteosarcoma. TE85, and a mouse fibrosarcoma, La-t-, to examine the differential distribution of alkaline phosphatase between matrix vesicles and the plasma membrane, postulated to be the parent membrane from which matrix vesicles are derived. While alkaline phosphatase in plasma membranes was extinguished, enzyme activity in matrix vesicles from LTA-1 hybrid cells was 34.2% of that present in matrix vesicles from the TE85 parent cells and 200 times that found in La-t- matrix vesicles. Matrix vesicles from LTA-5 had alkaline phosphatase levels similar to La-t-. When other membrane enzymes (phospholipase A2, 5'-nucleotidase, and Na+/K+ ATPase) were examined, hybrid matrix vesicle and plasma membrane levels were similar to those of TE85 and significantly higher than in La-t- membrane fractions. Northern analysis detected mRNA for alkaline phosphatase in TE85 cells, but not in the hybrids or La-t- cells. In contrast, reverse transcription-polymerase chain reaction (RT-PCR) revealed alkaline phosphatase mRNA in the hybrid cells, but at very low levels. Taken together, the data indicate that regulation of plasma membrane and matrix vesicle alkaline phosphatase is independent and suggest that matrix vesicle biogenesis is independent and distinct from that of plasma membrane biogenesis. Analysis of 1B- and 1L-type alkaline phosphatase mRNA by RT-PCR showed that alternate promoter usage of the alkaline phosphatase gene was not responsible for the differential localization of this enzyme in matrix vesicle. Thus, it is likely that matrix vesicle and plasma membrane alkaline phosphatase are regulated differently at a post-transcriptional level.

Pretreatment with low doses of norethindrone potentiates the osteogenic effects of fluoride on human osteosarcoma cells

We recently reported that picomolar doses of norethindrone (NET), a synthetic analog of 19-nortestosterone, significantly stimulated human TE85 osteosarcoma cell proliferation, differentiation, and activity in vitro. In the present study, we investigated the possibility that NET interacts with another osteogenic agent, i.e., fluoride, to stimulate human TE85 osteosarcoma cell proliferation, differentiation, and activities. Bone cell proliferation was measured by the stimulation in [3H]thymidine incorporation. Differentiation was monitored by the increase in alkaline phosphatase-specific activity. Osteoblastic activity was assessed by the stimulations in collagen synthesis and in osteocalcin secretion (in the presence of 1 nM 1,25-dihydroxyvitamin D3). When the human TE85 cells were incubated with mitogenic doses of NET and fluoride concurrently, the stimulatory effects of the two agents on these parameters exhibited no significant interaction. The enhancing effect of NET on the osteogenic effect of fluoride was not due to a shift of the fluoride dose response curve. Pretreatment with NET for 24 h followed by a treatment with a mitogenic dose (i.e., 100 microM) of fluoride for an additional 24 h significantly and synergistically potentiated the effects of fluoride on the [3H]thymidine incorporation, alkaline phosphatase-specific activity, collagen synthesis, and osteocalcin secretion, compared with those with the subsequent vehicle (0.05% ethanol) treatments. In contrast, pretreatment with fluoride for 24 h before the addition of NET for 24 h did not produce significant synergistic stimulations in the test parameters. Pretreatment of TE85 cells with the same doses of dihydrotestosterone or progesterone prior to treatment with fluoride under the same conditions did not induce synergistic potentiation of fluoride in [3H]thymidine incorporation, suggesting that the synergistic interaction with fluoride is probably not a common property of anabolic sex steroids. In summary, we found that: (1) the osteogenic effects of fluoride and NET were additive when cells were treated with both agents concurrently; (2) a 24-h pretreatment with picomolar doses of NET potentiated the osteogenic actions of fluoride in human TE85 osteosarcoma cells; and (3) pretreatment with NET produced a subsequent fluoride response that was synergistic. In conclusion, these findings led us to speculate that the osteogenic actions of NET and fluoride act through different mechanisms, and that NET at low doses has a permissive effect on the osteogenic effects of fluoride, and as such NET may be used in concert with fluoride to increase osteoblast proliferation, differentiation, and activity.

IGF-II receptor number is increased in TE-85 osteosarcoma cells by combined magnetic fields.

Human osteosarcoma-derived osteoblast-like cells, TE-85, were used to assess the effect of a low frequency alternating magnetic field in combination with a controlled static magnetic field (combined magnetic fields, CMF) on insulin-like growth factor receptor regulation. In our culture system, application of a 15.3 Hz CMF induces a calculated maximum electrical potential in the culture media of 10(-5) V/m. Initial characterization of TE-85 cells demonstrated that (a) TE-85 cells contain both type I insulin-like growth factor (IGF-I) and IGF-II receptors and (b) dose dependence for IGF-stimulated cell proliferation were comparable to the affinities of the IGF's binding to membrane binding sites (i.e., receptors had dissociation constants in the low nanomolar concentration range). The studies with CMF exposure revealed that CMF treatment for 30 minutes increased the number of IGF-II receptors in a frequency-dependent manner without affecting the number of IGF-I receptors. The CMF-dependent increase in IGF-II receptor number was associated with a significant increase in the IGF-II dissociation constant. These results indicate that a membrane receptor levels can be altered by short-term exposure to low-energy, low-frequency electromagnetic fields and suggest a potential biochemical mechanism for electromagnetic effects on bone formation and remodeling.

Normal human osteoblast-like cells consistently express genes for insulin-like growth factors I and II but transformed human osteoblast cell lines do not.

Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method.(ABSTRACT TRUNCATED AT 250 WORDS)