Cell cycling determines integrin-mediated adhesion in osteoblastic ROS 17/2.8 cells exposed to space-related conditions.

Six days of microgravity (Bion10 mission) induced dramatic shape changes in ROS 17/2.8 osteoblasts (7). During the Foton 11 and 12 space flights, we studied the kinetics (0-4 days) of ROS 17/2.8 morphology and adhesion, the relationships between adhesion and cell cycle progression after 4 days in space, and osteoblastic growth and activity after 6 days in space. Quantitative analysis of high-resolution adhesion [focal adhesion area imaged by total interference reflection fluorescent microscopy (TIRFM)] and integrin-dependent adhesion (imaged on confocal microscope by vinculin and phosphotyrosine staining) as well as cell cycle phase classification [Ki-67 staining, S-G2, mitotic cells and G1 (postmitotic cells)] were performed using programs validated in parabolic flight and clinostat. We observed disorganization of the cytoskeleton associated with disassembling of vinculin spots and phosphorylated proteins within focal contacts with no major change in TIRFM adhesion after 2 and 4 days of microgravity. Postmitotic cells, alone, accounted for the differences observed in the whole population. They are characterized by immature peripheral contacts with complete loss of central spots and decreased spreading. Osteocalcin, P1CP and alkaline phosphatase, and proliferation were similar in flight cells and 1 g centrifuge and ground controls after 6 days. In conclusion, microgravity substantially affected osteoblastic integrin-mediated cell adhesion. ROS17/2.8 cells responded differently, whether or not they were cycling by reorganizing adhesion plaque topography or morphology. In ROS 17/2.8, this reorganization did not impair osteoblastic phenotype.     

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8)

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)₂D₃) on the intracellular free calcium-ion concentration ([Ca²⁺¹]_i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using ¹⁹F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorephenoxy)ethane-N, N, N′, N′-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca²⁺]_i at doses of 1 to 10 nM 1,25-(OH)₂D₃. At 10 nM, 1,25-(OH)₂D₃ elevated the [Ca²⁺]_i from a control level of 118±4 nM to a peak value of 237±8 nM within 40 min. 1,25-(OH)₂D₃ also increased the intial rate of Ca²⁺ influx into ROs 17/2.8 cells, measured by ⁴⁵Ca uptake, with a dose-response relationship which paralleled its effects on [Ca²⁺]_i. Treatment of ROS 17/2.8 cells with Pb²⁺ at 1 and 5 μM significantly increased [Ca²⁺]_i but significantly reduced the 1,25-(OH)₂D₃-induced elevation of [Ca²⁺]i. Simultaneous treatment of naive cells with 1,25-(OH)₂D₃ and Pb²⁺ produce little reduction of 1,25-(OH)₂D₃ and Pb²⁺ produce little reduction of 1,25-(OH)₂D₃-induced ⁴⁵Ca uptake while 40 min treatment with Pb²⁺ before addition of 1,25-(OH)₂D₃ significantly reduced the 1,25-(OH)₂D₃-induced increase in ⁴⁵Ca influx. These findings suggest that Pb²⁺ acts by inhibiting 1,25-(OH)₂D₃-activation of Ca²⁺ channels and interferes with 1,25-(OH)₂D₃ regulation of Ca²⁺ metabolism in osteoblastic bone cells.     

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8).

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) on the intracellular free calcium-ion concentration ([Ca2+]i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca2+]i at doses of 1 to 10 nM 1,25-(OH)2D3. At 10 nM, 1,25-(OH)2D3 elevated the [Ca2+]i from a control level of 118 +/- 4 nM to a peak value of 237 +/- 8 nM within 40 min. 1,25-(OH)2D3 also increased the initial rate of Ca2+ influx into ROS 17/2.8 cells, measured by 45Ca uptake, with a dose-response relationship which paralleled its effect on [Ca2+]i. Treatment of ROS 17/2.8 cells with Pb2+ at 1 and 5 microM significantly increased [Ca2+]i but significantly reduced the 1,25-(OH)2D3-induced elevation of [Ca2+]i. Simultaneous treatment of naive cells with 1,25-(OH)2D3 and Pb2+ produce little reduction of 1,25-(OH)2D3-induced 45Ca uptake while 40 min treatment with Pb2+ before addition of 1,25-(OH)2D3 significantly reduced the 1,25-(OH)2D3-induced increase in 45Ca influx. These findings suggest that Pb2+ acts by inhibiting 1,25-(OH)2D3-activation of Ca2+ channels and interferes with 1,25-(OH)2D3 regulation of Ca2+ metabolism in osteoblastic bone cells.     

Mechanisms underlying spontaneous calcium spiking in aequorin-loaded ROS 17/2.8 cells

In earlier studies, McLeod and coworkers reported the detection of spontaneous calcium spiking in ROS 17/2.8 cells which they suggested was derived from individual cells progressing through mitosis or the cell cycle. They also indicated that the degree of spiking could be modulated by exposure of the cells to time-varying extremely low frequency electric fields. Given the implications of such observations for our understanding of the effects of electromagnetic fields on biological systems, it appeared important for mechanistic reasons to understand the basis of this spiking. In this study, we were able to confirm that spontaneous calcium spiking activity could be detected in ROS 17/2.8 cells and that this appeared to emanate from individual cells. We found this spiking to be completely dependent on extracellular calcium ions and to be independent of the inositol 1,4,5-trisphosphate-sensitive intracellular calcium store. This spiking is not reduced by treatments which slow down or block the passage of cells through the cell cycle. Further, we found that spiking was only detectable in the most highly aequorin-loaded subpopulation of cells whose growth rate is reduced and whose morphological appearance is abnormal. In conjunction with what is known about calcium spiking in other, nonexcitable mammalian cells in culture, the data presented strongly argue that the spontaneous calcium spiking observed in ROS 17/2.8 cells is unrelated to normal events of the cell cycle and most likely result from the damaging effects of excessive loading with aequorin.     

Effects of GDF7/BMP12 on proliferation and alkaline phosphatase expression in rat osteoblastic osteosarcoma ROS 17/2.8 cells

Growth and differentiation factor 7(GDF7), also later called as bone morphogenetic protein (BMP)12, is a new member of the BMP superfamily, which induces formation of tendon-like tissue formation in the ectopic implantation experiments. We examined the effect of BMP12 on proliferation and expression of phenotype-related genes in rat osteoblastic osteosarcoma ROS17/2.8 cells. BMP12 treatment enhanced proliferation of ROS17/2.8 cells within 3 days and this effect was observed at least up to day 6 of the treatment. The cell number was increased by about 50% on day 3 and about two-fold by day 6. These effects were observed at the dose range between 40 and 1,000 ng/ml. Treatment with BMP12 also enhanced alkaline phosphatase activity by about 50% in ROS17/2.8 cells within 24 h of the treatment. The effect peaked at 48 h and was still observed at 72 h. The enhancing effect of BMP12 on alkaline phosphatase was observed similarly at the doses ranging from 40 to 1,000 ng/ml. These data indicate that BMP12 has positive effects on proliferation and phenotypic expression of ROS 17/2.8 cells.     

Effect of lead on parathyroid hormone-induced responses in rat osteoblastic osteosarcoma cells (ROS 17/2.8) using 19F-NMR

Using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, we have recently demonstrated that Pb2+ treatment elevates the intracellular free calcium ion concentration ([Ca2+]i) of rat osteoblastic osteosarcoma cells (ROS 17/2.8) (Proc. Natl. Acad. Sci. USA (1989) 86, 5133-5135). In this study, we have examined the effects of Pb2+ on the basal and parathyroid hormone (PTH)-stimulated levels of [Ca2+]i and cAMP in cultured ROS 17/2.8 cells. PTH treatment (400 ng/ml) stimulated a 150% elevation in [Ca2+]i from a control level of 105 +/- 25 nM to a concentration of 260 +/- 24 nM. Treatment of ROS 17/2.8 cells with Pb2+ (5 microM) alone produced a 50% elevation in the [Ca2+]i to 155 +/- 23 nM. Pb2+ treatment diminished subsequent elevation in [Ca2+]i in response to PTH administration thereby limiting the peak increase in [Ca2+]i to only 25% or 193 +/- 22 nM. In contrast to the dampening effect of Pb2+ on the peak rise in [Ca2+]i produced by PTH, Pb2+ (1 to 25 microM) had no effect on PTH-induced increments in intracellular cAMP levels. Hence, Pb2+ dissociated the PTH stimulation of adenylate cyclase from PTH effects on [Ca2+]i and shifted the regulation of [Ca2+]i beyond the control of PTH modulation. These observations further extend the hypothesis that an early toxic effect of Pb2+ at the cellular level is perturbation of [Ca2+]i homeostasis.     

The effect of IL-1alpha on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in osteoblastic ROS 17/2.8 cells

Interleukin-1 (IL-1) plays key roles in altering bone matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) , and plasminogen activator inhibitor type-1 (PAI-1). In this study, we examined the effect of IL-1alpha on the expression of the MMPs, TIMPs, tPA, uPA, and PAI-1 genes in osteoblasts derived from the rat osteosarcoma cell line ROS 17/2.8. The cells were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with 0 or 100 U/ml of IL-1alpha for up to 14 days. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 expression were estimated by determining the mRNA levels using real-time RT-PCR and by determining protein levels using ELISA. In IL-1alpha cultures, the expression levels of MMP-1, -2, -3, -13, and -14 exceeded that of the control through day 14 of culture, and the expression of MMPs increased markedly from the proliferative to the later stages of culture. The TIMP-1, -2, and -3 expression levels increased from the initial to the proliferative stages of culture. The expression of tPA increased greatly during the proliferative stage of culture, and uPA expression increased throughout the culture period, increasing markedly from the proliferative to the later stages of culture. In contrast, PAI-1 expression decreased in the presence of IL-1alpha through day 14. These results suggest that IL-1alpha stimulate bone matrix turnover by increasing MMPs, tPA, and uPA production and decreasing PAI-1 production by osteoblasts, and incline the turnover to the resolution.     

Colorimetric Evaluation of Cultured Osteoblast-like Cells (ROS 17/2.8)

We have developed a colorimetric method for evaluating the number of osteoblastic cells in culture without destroying the cells. This assay is based on the staining of basophilic cellular compounds with methylene blue. The dye bound by the cells is released at low pH and measured in a spectrophotometer at 662 nm. Linear correlations exist between the absorbance measured by the methylene blue assay and the number of cells seeded, the total cellular protein content, and thymidine labeling. This colorimetric method has the advantage of preserving cell integrity. After destaining, scanning electron microscopy can be performed on well preserved cell morphology.     

Melatonin inhibits fatty acid-induced triglyceride accumulation in ROS17/2.8 cells: implications for osteoblast differentiation and osteoporosis

Melatonin is produced not only by the pineal gland but by cells of the bone marrow. Moreover, melatonin is known to promote osteogenic differentiation in several cell line models and in multipotential bone marrow mesenchymal stem cells. Fatty acids have been independently shown to direct such cells to acquire the phenotype and molecular characteristics of adipocytes. To examine the effect of melatonin on intracellular triglyceride accumulation, an indicator of adipogenic differentiation in the rat osteoblast-like ROS17/2.8 cell line, cells were incubated with added oleic acid (100 muM), fixed and stained with Oil Red O. Cellular lipid accumulation was quantitated by an Oil Red O method highly specific for triglycerides and expressed as a triglyceride accumulation index (TGAI, triglyceride per cell). Melatonin in nanomolar concentrations inhibited oleic acid-induced triglyceride accumulation. To identify the mechanism by which melatonin reduces triglyceride accumulation, cells were incubated with the two melatonin receptor antagonists, luzindole and S20928, or forskolin, a stimulator of adenylyl cyclase and cAMP production. These compounds prevented the inhibitory effect of melatonin on triglyceride accumulation, indicating that melatonin acts through known melatonin receptor-mediated mechanisms. In view of the previously demonstrated positive effects of melatonin in promoting osteoblastic differentiation in ROS17/2.8 cells and their reciprocal adipocytic differentiation induced by fatty acids, our observations may serve to relate the known age-related decreases of melatonin production, the shift in the bone marrow toward an adipocytic line of cell development, and the development of osteoporosis during aging.     

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8

 Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell’s osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems. Accepted: 25 August 1998     

Regulation of the CYP27B1 5'-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates

We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues. © 1996 Wiley-Liss, Inc.     

Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain.

The human 1,25-dihydroxyvitamin D3 receptor (hVDR) has been recently shown to be phosphorylated in vitro by casein kinase-II. Most of the residues phosphorylated by this enzyme were shown to reside between Asn160 and Asp232, a region near the N-terminal boundary of the hormone-binding domain. We report here that the hVDR is also phosphorylated in vivo after transfection into ROS 17/2.8 cells. In addition to testing full-length hVDR, we analyzed several internally deleted hVDR mutants. The expression and phosphorylation of full-length and mutated hVDRs were monitored in transfected cells by metabolic labeling with either [35S]methionine or [32P]orthophosphate, followed by immunopurification using monoclonal anti-VDR antibody linked to agarose beads. Transfected hVDR is distinguishable from the endogenous rat VDR when the immunoprecipitated proteins are resolved on sodium dodecyl sulfate-polyacrylamide gels. Significant phosphorylation of transfected full-length hVDR was observed in ROS 17/2.8 cells, and it was less dependent on the presence of 1,25-dihydroxyvitamin D3 than that of the endogenous rat receptor. Most importantly, the region of in vivo phosphorylation, as defined by internal deletion mutants, resides between Met197 and Val234. Therefore, we have localized the major site of phosphorylation of hVDR to residues in the N-terminal region of the hormone-binding domain. The boundaries of this region fall within the amino acid segment defined for phosphorylation of hVDR by casein kinase-II in vitro, suggesting that VDR is an in vivo substrate for casein kinase-II or a related protein kinase.     

Osteotoxicity of cadmium and lead in HOS TE 85 and ROS 17/2.8 cells: relation to metallothionein induction and mitochondrial binding

Epidemiological, experimental and clinical data indicate that cadmium and lead are osteotoxins in man and other species. The relative sensitivities of a clonal human osteosarcoma cell line (HOS TE 85) and a clonal rat osteosarcoma cell line (ROS 17.28) to the cytotoxic effects of cadmium and lead were tested in serum-free media without added growth factors. The rat osteosarcoma cells were more sensitive to cadmium with cytotoxicity and inhibition of proliferation at 0.25 versus 0.75 and 1.0 mol l^– cadmium, respectively, for human osteosarcoma cell lines. The lower sensitivity to cadmium of human osteosarcoma cells is attributed, at least partly, to induction of metallothionein synthesis by cadmium and zinc in this cell line; in the rat osteosarcoma cell line, they do not induce metallothionein synthesis. Human osteosarcoma cells were more sensitive than rat osteosarcoma cells to lead with inhibition (IC₅₀) of proliferation at 4 mol l^– lead and cytotoxicity at 20 versus 6 and over 20 moll^– lead, respectively, for these variables in rat osteosarcoma cells. Both cells lines attained the highest lead concentration in the 15 000 × g (mitochondrial) fraction. The lead in the mitochondrial, microsomal, nuclear and cytosolic fractions of the human cell line did not decrease during 24 h post-washout. Binding of lead was much less stable in the less sensitive rat cells, with 50–100% loss of mitochondrial, microsomal and nuclear lead during 24 h post-washout.     

Characterization of an element within the rat parathyroid hormone/parathyroid hormone-related peptide receptor gene promoter that enhances expression in osteoblastic osteosarcoma 17/2.8 cells.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) mediate their actions via a common G-protein-coupled receptor. High levels of PTH/PTHrP receptor expression have been detected in many tissues including bone and kidney. This study has demonstrated specific PTH/PTHrP receptor expression from the U3 promoter in the osteoblastic osteosarcoma ROS 17/2.8 cell line, which expresses the endogenous PTH/PTHrP receptor, compared to rat 2 fibroblasts which do not express the endogenous PTH/PTHrP receptor gene. Transient transfection studies revealed cell-specific expression of a construct containing 4391 bp of DNA upstream of exon U3 of the PTH/PTHrP receptor gene fused to a luciferase reporter gene. Deletion mapping of the 5' region of U3 revealed that a construct containing 206 bp upstream of U3 confers cell-specific expression. These data suggest that cell-specific expression in ROS 17/2.8 involves cell-specific elements within the PTH/PTHrP receptor promoter.     

Regulation of the CYP27B1 5′-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.