Biomedical subjects

T C Spelsberg

Publications and source records attributed to T C Spelsberg.

At least 55 records · Page 3Linked to original sources

PubMed1998

Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma.

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34 degrees C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5 degrees C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5 degrees C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10(-8) M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10(-8) M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5 degrees C with ascorbate and beta1-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.

Adipocytes↗

PubMed1998

Steroid receptors at the nexus of transcriptional regulation.

During the past few years, our understanding of nuclear receptor action has dramatically improved as a result of the identification and functional analysis of co-regulators such as factors involved in chromatin remodeling, transcription intermediary factors (co-repressors and co-activators), and direct interactions with the basal transcriptional machinery. Furthermore, the elucidation of the crystal structures of the empty ligand-binding domains of the nuclear receptor and of complexes formed by the nuclear receptor's ligand-binding domain bound to agonists and antagonists has contributed significantly to our understanding of the early events of nuclear receptor action. However, the picture of hormone- and hormone receptor-mediated mechanisms of gene regulation remain incomplete and extremely complicated when one also considers the "nontraditional" interactions of hormone-activated nuclear receptors, for example, interactions between the activated steroid receptors and components of the chromatin/nuclear matrix; and finally the nongenomic effects that steroid hormones can exhibit with other signaling pathways. In this prospectus on steroid receptors, we discuss the implications of various steroid hormone and nuclear receptor interactions and potential future directions of investigation.

Animals↗

PubMed1997

A DNA-binding element for a steroid receptor-binding factor is flanked by dual nuclear matrix DNA attachment sites in the c-myc gene promoter.

The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-myc gene promoter. This element contains a 5'-GC-rich domain and a 3'-AT-rich domain, the latter of which has a homopurine/homopyrimidine structure. The gel shift assays required the generation of an RBF-maltose fusion protein (RBF-MBP), which specifically binds this element and is supershifted when the anti-RBF polyclonal antibody is added. Computer analysis of the full-length amino acid sequence for RBF predicts a DNA-binding motif involving a beta-sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-myc promoter, which flank an intervening domain containing the RBF element. The co-transfection of this MAR sequence, containing the RBF element and cloned into a luciferase reporter vector, together with an RBF expression vector construct, into steroid treated human MCF-7 cells, results in a decrease of the c-myc promoter activity relative to control transfections containing only the parent vector of the RBF expression construct. These data suggest that a unique chromatin/nuclear matrix structure, composed of the RBF-DNA element complex which is flanked by nuclear matrix attachment sites, serves to bind the PR and repress the c-myc promoter.

Animals↗

PubMed1997

Overexpression of the TGFbeta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells.

Members of the TGFbeta family of peptides exert antiproliferative effects and induce apoptosis in epithelial cell populations. In the exocrine pancreas, these peptides not only regulate normal cell growth, but alterations in these pathways have been associated with neoplastic transformation. Therefore, the identification of molecules that regulate exocrine pancreatic cell proliferation and apoptotic cell death in response to TGFbeta peptides is necessary for a better understanding of normal morphogenesis as well as carcinogenesis of the pancreas. In this study, we have characterized the expression and function in exocrine pancreatic epithelial cells of the TGFbeta-inducible early gene (TIEG), a Krüppel-like zinc finger transcription factor encoding gene previously isolated from mesodermally derived osteoblastic cells. We demonstrate that this gene is expressed in both acinar and ductular epithelial cell populations from the exocrine pancreas. In addition, we show that the expression of TIEG is regulated by TGFbeta1 as an early response gene in pancreatic epithelial cell lines. Moreover, overexpression of TIEG in the TGFbeta-sensitive epithelial cell line PANC1 is sufficient to induce apoptosis. Together, these results support a role for TIEG in linking TGFbeta-mediated signaling cascades to the regulation of pancreatic epithelial cell growth.

Adult↗

PubMed1997

Estrogen regulation of human osteoblastic cell proliferation and differentiation.

Estrogen (E2) has been shown to prevent bone loss among postmenopausal women. The molecular mechanism(s) by which this is accomplished is not clear. The discovery of E2 receptor (ER) in osteoblasts and osteoclasts has implicated these cells as direct targets for E2. Previous studies on the effects of E2 on osteoblastic cells in vitro or in organ culture present conflicting results, possibly due to heterogeneity in cell types, stage of differentiation, ER levels, and/or species differences. The effects of E2 on gene expression during various stages of human osteoblast cell differentiation has not been investigated extensively. In this study we employed a newly developed human fetal osteoblastic cell line (hFOB/ER9) that contains high levels of ER to examine the effects of E2 on osteoblast proliferation and differentiation. The basal levels and E2 effects on the expression of various extracellular matrix proteins were also characterized throughout different stages of differentiation. These stages include a proliferative/relatively undifferentiated stage (day 6), a matrix maturation stage (days 10-14), and a mineralization/calcified nodule stage (day 18). During the stage of rapid cell proliferation, E2 treatment of hFOB/ER9 cells resulted in a dose-dependent decrease in [3H]thymidine incorporation to a maximum of 72% compared to the vehicle control value. Treatment of hFOB/ER9 cells with 10(-9) M E2 for 48 h resulted in an increase in alkaline phosphatase (AP) activity throughout cell differentiation. The magnitude of AP induction varied from approximately 200-500%. In contrast, E2 decreased osteocalcin protein levels to a minimum of 54% compared to the vehicle control value. The steady state messenger RNA levels for AP increased and osteocalcin decreased after E2 treatment, similar to the responses observed at the protein level. At all stages, there was little or no effect of E2 on type I collagen protein levels or osteonectin steady state messenger RNA levels. The E2 responses on hFOB/ER9 cell matrix protein expression and cell proliferation were mediated through the ER, as cultures cotreated with a 100-fold molar excess of a type II anti-E2 (ICI 182,780) abrogated these effects. These results support the hypothesis that E2 does have an effect on osteoblastic differentiation by decreasing hFOB/ER9 cell proliferation and differentially regulating extracellular matrix expression.

Bone and Bones↗

PubMed1997

Relationship between DNA ploidy and functional estrogen receptors in operable prostate cancer.

OBJECTIVE: To evaluate the relationship between the nuclear DNA content and the tissue estrogen receptor (ER) level in patients with operable adenocarcinoma of the prostate. METHOD: Surgical specimens taken from 73 patients with clinically localized prostate cancer were studied. Tumor DNA ploidy pattern as measured by flow cytometry was correlated with the level of functional ER using the nuclear biopsy assay. RESULTS: Forty-five percent of the tumors were DNA diploid, and 55% had an abnormal ploidy pattern (DNA tetraploid or DNA aneuploid). The ER level ranged from 0 to 6,475 fmol/mg DNA (mean 839 fmol/mg DNA). Twenty-two percent had no functional receptors. Marked association was noted between ER and nuclear DNA content. Seventy-five percent of the tumors with no ER had abnormal ploidy patterns. The mean receptor level for DNA diploid prostate cancer was 1,034 fmol/mg DNA versus 661 fmol/mg DNA for DNA nondiploid tumors (p < 0.008). An inverse correlation was found between ER values and histologic grade or pathologic stage. High-grade and high-stage tumors had lower levels of ER compared to low-grade and early-stage carcinomas. CONCLUSION: Our results demonstrate an association between ER values and variables that predict prognosis in prostate cancer. It is possible that this parameter may be helpful in identification of prognostic groups in patients with prostate cancer.

Adenocarcinoma↗

PubMed1996

Estrogen inhibits interleukin-6 production and gene expression in a human osteoblastic cell line with high levels of estrogen receptors.

Some studies suggest that estrogen acts on bone by decreasing the production of interleukin-6 (IL-6), a cytokine that increases bone resorption, by osteoblasts or bone marrow cells. However, other studies have not confirmed this, possibly because of a low and variable number of estrogen receptors (ER) in the model systems used. Thus, we employed a recently developed human fetal osteoblast cell line with high levels of ER. Treatment (n = 4 experiments) with 0.01 to 10 nM of 17 beta-estradiol had no effect on the constitutive production of IL-6. However, stimulated production, induced by treatment with IL-1 beta plus tumor necrosis factor-alpha (TNF-alpha), was reduced in a dose-dependent manner to 74 +/- 3% (mean +/- SEM) of control (p < 0.01). This response was blocked by cotreatment with the type II antiestrogen ICI 182,780. Treatment with hydrocortisone (1 microM), a known inhibitor of IL-6 production in many cell types, reduced IL-6 production to 17 +/- 1% of control (p < 0.001). As assessed by Northern analysis, treatment (n = 3 experiments) with 0.01-10 nM of 17 beta-estradiol decreased steady-state levels of IL-6 mRNA in a dose-dependent manner. These data support the hypothesis that at least part of the antiresorptive action of estrogen in humans is mediated by decreased production of IL-6 by osteoblastic cells.

Base Sequence↗

PubMed1996

Isolation and characterization of osteoblast precursor cells from human bone marrow.

Osteoblasts are derived from precursor cells present in low frequency in the stromal element of bone marrow. Because of the lack of a practical procedure to isolate osteoblast precursors from early cultures of plastic adherent cells from bone marrow, previous studies of marrow stromal cells have been made in confluent cultures of bone marrow when the osteoblast (OB) precursors are already differentiated. Also these studies utilized cultures containing mixed populations of cells including hematopoietic cells. Thus we have employed a negative immunoselection procedure to remove contaminating hematopoietic cells and to isolate nearly homogeneous populations of early human stromal cells derived from the plastic-adherent mononuclear marrow cells cultured in the presence of serum. By reverse transcriptase polymerase chain reaction (RT-PCR) analysis for mRNA, and by immunocytochemical study for protein, we studied the sequential expression in culture of multiple markers of the osteoblast phenotype--alkaline phosphatase, osteopontin, parathyroid hormone receptor, types I and III procollagen, and osteocalcin--as well as lipoprotein lipase (LPL), a marker of the adipocyte phenotype. At an early stage of culture (7-9 days), human OB precursors formed colonies of variable sizes that expressed low levels of mRNA and protein concentrations of OB markers, and their concentration increased on growth to a confluent monolayer (approximately 14 days). LPL mRNA was expressed at high levels in the colony stage, and its level decreased upon confluency, suggesting a loss of potential for commitment to the adipocyte lineage. Interestingly, treatment with dexamethasone at 10(-8) M increased the expression for some of the osteoblast markers and for the LPL gene and was required for the deposition of mineralized matrix and for the formation of adipocytes containing cytoplasmic lipid droplets in confluent cultures. Cloned single early colonies were able to coexpress the osteoblast and adipocyte markers (as assessed by RT-PCR). Thus these immunoselected marrow stromal cells have the characteristics of authentic human osteoblast precursor cells which also are capable of differentiating into adipocytes.

Alkaline Phosphatase↗

PubMed1996

Osteoclasts and transforming growth factor-beta: estrogen-mediated isoform-specific regulation of production.

Estrogen deficiency induced by menopause leads to an increase in bone resorption that is not compensated for by a comparable increase in bone formation, resulting in excessive bone loss. Clinically, estrogen replacement reverses these processes, but the mechanisms by which this takes place are not completely understood. Both osteoclasts and osteoblasts contain functional estrogen receptors and, therefore, may be directly involved in these responses. Because both osteoclasts and osteoblasts secrete transforming growth factor-beta (TGF beta), and because 17 beta-estradiol (E2) treatment increases TGF beta production by osteoblast-like cells in vitro, we have investigated the possibility that E2 also may increase the production of TGF beta by isolated osteoclasts in vitro. Highly purified avian osteoclasts were treated with either vehicle or E2, and TGF beta protein accumulation in culture was measured by bioassay. Although an E2 dose-dependent increase in TGF beta protein accumulation in osteoclast-conditioned medium was measured at 4 h of treatment, a steroid dose-dependent decrease in the accumulation of active TGF beta was measured after 18 h of estrogen treatment. The steroid specificity of the increased TGF beta accumulation was confirmed by demonstrating that the E2-induced increase in TGF beta protein levels in the medium was inhibited by cotreatment with a specific E2 antagonist. Interestingly, E2 treatment induced a TGF beta isoform change from TGF beta 2 to predominantly TGF beta 3. Thus, the data suggest that a direct action of E2 on osteoclasts to lower resorption activity may be mediated by autocrine/paracrine production and activation of TGF beta, perhaps including modulation of specific isoform production.

Animals↗

PubMed1996

Cytokine production in the bone marrow microenvironment: failure to demonstrate estrogen regulation in early postmenopausal women.

Recent studies in rodents and in human in vitro systems suggest that the action of estrogen on bone is modulated by various bone-resorbing cytokines produced by osteoblasts, bone marrow cells, or both, but results among studies have been conflicting. Thus, we studied 20 untreated women (controls) and 20 women who had received estrogen replacement therapy since the time of menopause; both groups were 5 +/- 1 yr (mean +/- SE) postmenopausal. From bone marrow aspirates, we obtained marrow plasma to evaluate the production of cytokines by marrow and trabecular bone cells in vivo and marrow mononuclear cells to assess in vitro the constitutive and lipopolysaccharide-stimulated production of cytokines and also to test the in vitro effects of 17 beta-estradiol (10(-8) mol/L). Interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-1 receptor antagonist, and IL-6-soluble receptor were measured by specific enzyme-linked immunosorbent assay. No statistically significant difference between the two groups in the level of any of these cytokines was detected in the bone marrow plasma or the conditioned medium in vitro. Furthermore, in vitro treatment with 17 beta-estradiol did not affect basal or lipopolysaccharide-stimulated cytokine production. Thus, our data suggest that for normal post-menopausal women, mediation of the effects of estrogen by a single cytokine is unlikely. Either multiple cytokines are involved and small changes in these are difficult to detect in clinical investigative studies, or other mechanisms are operative.

Antigens, CD↗

PubMed1996

A nuclear matrix acceptor site for the progesterone receptor in the avian c-myc gene promoter.

It has been the goal of this project to determine the location, composition, and biological function of the nuclear acceptor sites (i.e., the nuclear binding sites) for the avian oviduct progesterone (Pg) receptor (PR). Many laboratories have demonstrated a native-(in vivo) like cell-free binding of steroid receptor complexes to specific acceptor sites in nuclei/chromatin in a variety of target tissue systems. These sites appear to involve protein-DNA complexes and some of these have been shown to reside in the nuclear matrix, including the chromatin acceptor sites for the avian oviduct PR. We have purified a nuclear matrix "acceptor protein" for the avian PR. termed receptor binding factor-1 (RBF-1), based on its ability to generate specific, high-affinity PR binding on avian genomic DNA. This 10 kD nuclear matrix protein was found to be unique with minimal homology to a couple of other proteins. Using immunohistochemical techniques and antibodies against the purified RBF-1, the RBF-1 was localized to the nuclei of many avian and rat tissues. Co-localizations of RBF-1 and PR in select cell types in the avian oviduct and rat reproductive organs were also reported. A tissue specificity was found with regard to RBF-1 concentrations. The full length cDNA to RBF-1 has been isolated and used to identify a 0.7 kb mRNA whose levels in various avian tissues reflect the protein levels. Genomic sequences of RBF-1 have been isolated and characterized. Preliminary studies indicate that the over-expression of the RBF in human MCF-7 cells results in an inhibition of the c-myc gene promoter activity which is further inhibited by steroid hormone treatments of the cells. Past studies in our laboratory demonstrated that the c-myc mRNA steady state levels are rapidly (approximately 15 min) reduced by Pg and glucocorticoids in the avian oviduct. Further, partially purified fractions of RBF-1 were shown to generate specific PR binding sites only on the genomic DNAs of certain animal species and on the c-myc gene, but not ovalbumin gene. Using Southwestern blot analyses, the purified RBF-1 was shown to bind specifically to sequences in the 5' end of c-myc, c-jun proto-oncogenes but not to genomic sequences of the ovalbumin gene. A specific DNA binding element in the promoter region of the c-myc proto-oncogene has been identified as AT-rich domain of homopurine/pyrimidine stretches flanked by GC-rich sequences. Southern blot analyses using 200 bp matrix DNA fragments protected by the nuclear matrix structure indicate that the matrix is attached on either side of the RBF-1 binding element. A model is described for a nuclear matrix acceptor site attached to the c-myc promoter which may mediate the Pg-induced down-regulation of the c-myc gene expression.

Animals↗

PubMed1996

Potential mechanism of estrogen-mediated decrease in bone formation: estrogen increases production of inhibitory insulin-like growth factor-binding protein-4.

Using a recently developed human osteoblastic cell line (hFOB/ER9) with high levels (approximately 4,000 per nucleus) of estrogen receptors and the characteristic phenotype of the mature osteoblast, we tested the hypothesis that estrogen decreases bone formation by inhibiting the action of the insulin-like growth factor (IGF) paracrine/autocrine system. IGF-II, the predominant IGF produced by osteoblastic cells, was measurable in hFOB/ER9-conditioned medium (approximately 10 ng/mL) and its level did not change significantly after treatment with 17 beta-estradiol (E2) or anti-estrogens. Treatment with E2 at 0.1-100 nM decreased [3H]thymidine uptake to 53% of control (p < 0.001) in a dose-dependent fashion. The predominant IGF-binding proteins (IGFBPs) produced by hFOB/ER9 and by normal trabecular osteoblasts are IGFBP-3 and IGFBP-4, of which IGFBP-4 is consistently inhibitory of IGF action. Treatment with E2 at 0.01-10 nM for 48 h increased IGFBP-4 mRNA to 346% +/- 90% (mean +/- SE) of control (p < 0.05) and IGFBP-4 protein to 278% +/- 75% of control (p < 0.01) in a dose-dependent fashion but did not alter IGFBP-3 mRNA or protein. E2 treatment also attenuated IGF-dependent, IGFBP-4 specific proteolysis to approximately 50% of control. ICI 182,780, a pure anti-estrogen, completely blocked E2-mediated decreases in cell proliferation and increases in levels of IGFBP-4 mRNA and protein. Treatment of the hFOB/ER9 cells with recombinant human IGFBP-4 (200 ng/mL) decreased cell proliferation to 55% of control (p < 0.01). Thus, E2 acts on osteoblastic cells to increase availability of inhibitory IGFBP-4, by both increasing its production and decreasing its degradation, which may oppose the mitogenic effect of the IGFs on osteoblastic cells. This action may mediate, at least in part, the decreases in bone formation that are observed after estrogen treatment in vivo.

Antibodies↗

PubMed1995

Identification of a novel TGF-beta-regulated gene encoding a putative zinc finger protein in human osteoblasts.

The TGF-beta family of growth factors has been extensively studied and found to play major roles in bone physiology and disease. A novel, TGF-beta-inducible early gene (TIEG) in normal human fetal osteoblasts (hFOB) has been identified using differential-display PCR. Using this differentially expressed cDNA fragment of TIEG to screen a hOB cDNA library, a near full-length cDNA for this gene was isolated. Northern analyses indicated that the steady-state levels of the 3.5 kb TIEG mRNA increased within 30 min of TGF-beta treatment of human osteoblasts and reached a maximum of 10-fold above control levels at 120 min post-treatment. This regulation was independent of new protein synthesis. Computer sequence analyses indicates that TIEG mRNA encodes for a 480 amino-acid protein. The TIEG protein contains three zinc finger motifs, several proline-rich src homology-3 (SH3) binding domains at the C-terminal end, and is homologous in this region to the zinc finger-containing transcription factor family of genes. A growth factor/cytokine-specific induction of TIEG has been shown. TIEG expression in hFOB cells was highly induced by TGF-beta and bone morphogenetic protein-2 (BMP-2), with a moderate induction by epidermal growth factor (EGF), but no induction by other growth factors/cytokines was observed. In addition to osteoblastic cells, high levels of TIEG expression were detected in skeletal muscle tissue, while low or no detectable levels were found in brain, lung, liver or kidney. Because TIEG is an early induced putative transcription factor gene, and shows a growth factor induction and tissue specificity, its protein product might play an important role as a signalling molecule in osteoblastic cells.

Base Sequence↗

PubMed1995

Development and characterization of a conditionally immortalized human fetal osteoblastic cell line.

We report the establishment of a human fetal osteoblast cell line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaline phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB cells at the permissive temperature (33.5 degrees C) resulted in rapid cell division, whereas little or no cell division occurred at the restrictive temperature (39.5 degrees C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB cells cultured at 39.5 degrees C compared with 33.5 degrees C. Treatment of hFOB cells with 1-34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the clonal cell line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.

Alkaline Phosphatase↗

PubMed1995

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)

Aged↗

PubMed1995

TGF-beta regulation of nuclear proto-oncogenes and TGF-beta gene expression in normal human osteoblast-like cells.

Transforming growth factor-beta (TGF-beta) is present in high levels in bone and plays an important role in osteoblast growth and differentiation. In order to dissect the molecular mechanisms of action of TGF-beta on osteoblasts, the effects of TGF-beta on the steady state mRNA levels of c-fos, c-jun, and jun-B proto-oncogenes on normal human osteoblast-like cells (hOB) and a transformed human osteoblast cell line (MG-63) were measured. Treatment of hOBs with 2 ng/ml of TGF-beta 1 resulted in a rapid increase in c-fos mRNA levels as early as 15 min post-treatment. A maximum (10-fold) increase was observed at 30 min after TGF-beta treatment followed by a decrease to control values. Similar responses were measured whether the cells were rapidly proliferating or quiescent. TGF-beta 1 induced jun-B mRNA levels more gradually with steady increase initially observed at 30 min and a maximum induction measured at 2 h post-TGF-beta treatment. In contrast, TGF-beta treatment caused a time dependent decrease in the c-jun mRNA levels, an opposite pattern to that of jun-B mRNA. Treatment of hOBs with TGF-beta 1 in the presence of actinomycin-D abolished TGF-beta 1 induction of c-fos mRNA, suggesting that TGF-beta action is mediated via transcription. In the presence of cycloheximide, TGF-beta causes super-induction of c-fos mRNA at 30 min, indicating that the c-fos expression by TGF-beta is independent of new protein synthesis. Further, transfection of 3 kb upstream region of jun-B promoter linked to a CAT reporter gene into ROS 17/2.8 cells was sufficient to be regulated by TGF-beta 1. Interestingly, TGF-beta treatment also increased the mRNA levels of TGF-beta 1 itself at 4 h post TGF-beta treatment, with a maximum increase observed at 14 h of treatment. TGF-beta 1 treatment for 30 min were sufficient to cause a delayed increase in TGF-beta protein secretion within 24 h. These data support that TGF-beta has major effects on hOB cell proto-oncogene expression and that the nuclear proto-oncogenes respond as rapid, early genes in a cascade model of hormone action.

Animals↗

PubMed1995

Zeolite A inhibits osteoclast-mediated bone resorption in vitro.

The effects of Zeolite A on bone resorbing activity of highly purified avian osteoclasts were analyzed. The present study demonstrates that when 100 micrograms/ml of acid-treated Zeolite A is added to the media the number of pits per osteoclast is reduced 3-fold at 24 h after treatment. Secreted cathepsin B enzyme activity was also reduced 3-fold. A similar reduction in pit number per osteoclast was measured following 48 h of treatment with Zeolite A but there appeared to be less reduction of cathepsin B enzyme activity. The effects on pit number and cathepsin B protein activity were Zeolite dose dependent. The structure of the compound seemed to be responsible for the effects measured since compounds used to represent constituents of Zeolite A (silicon dioxide and aluminum chloride) failed to inhibit bone resorption or reduce the level of secreted cathepsin B enzyme activity. Thus the molecular architecture of Zeolite A or a derivative thereof appears to be important. In conclusion, the data indicate that Zeolite A can inhibit bone resorption. Together with previous data on osteoblasts, this might suggest a potential positive activity of intact Zeolite A or a partial substructure of Zeolite A on bone turnover.

Aluminum Chloride↗

PubMed1995

Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene.

The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (+/- 1,341) 17beta-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (+/- 2) fmol/mg protein. This subclone was examined further for estradiol (E2) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased approximately 25-fold in hFOB/ER9 cells following 10(-9)M E2 treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10(-9)M E2 resulted in a 2.5-4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function.

Base Sequence↗