calcimycin and Osteosarcoma

Tissue factor (TF), a transmembrane glycoprotein, plays a role in the initiation of blood coagulation at sites of vascular injury. Activated products of coagulation may then enhance inflammatory responses. The present investigation assesses the ability of rat osteosarcoma (UMR-106) cells cultured on titanium alloy (Ti6Al4V) to express differential surface TF activity in response to cyclic mechanical strain. Strains ranged from -2000 micro-strain to +2000 micro-strain, and durations from 5, 10, and 20 min per day over 5 days to 24 h continuous stimulation. ROS cells exhibited significant TF activity as demonstrated by the conversion of Factor X to Factor Xa. Strains of +2000 micro-strain with 5-20-min duration exhibited decreased TF activity with duration from 1.4E-04 nM/cell to 8.7E-05 nM/cell. Additionally, ROS cells stimulated with calcium ionophore (A23187) exhibited at least twice the activity of nonstimulated cells. Strains of +1340 micro-strain with 5-20-min duration exhibited an increasing trend with 4.15E-05 nM/cell to 7.38E-05 nM/cell. Strain direction had no significant effect on TF activity. Thus, both mechanical and chemical stimuli induce differential expression of TF activity by ROS cells cultured on Ti6Al4V, a phenomenon that may potentiate or regulate the inflammatory responses associated with the implantation of orthopedic biomaterials.

Topics: Alloys; Animals; Biocompatible Materials; Calcimycin; Cell Culture Techniques; Cell Line, Tumor; Ionophores; Osteosarcoma; Rats; Stress, Mechanical; Surface Properties; Thromboplastin; Titanium

Tissue factor (TF), a transmembrane glycoprotein expressed by numerous cell types, plays a critical role in the initiation of blood coagulation at sites of vascular injury. Activated products of the coagulation cascade may then enhance the inflammatory responses associated with wound healing. In the present investigation the ability of rat osteosarcoma (ROS) cells to express TF activity was examined following their growth on tissue-culture polystyrene (TCPS) and selected orthopedic biomaterials (titanium and zirconium alloys, and stainless steel). ROS cells exhibited significant TF activity as evidenced by the conversion of Factor X to Factor Xa in the presence of TF, Factor VIIa, and Ca2+. Factor Xa concentrations ranged from 1.0 fM per cell at 10 min to 6.0 fM per cell after 60 min. Additionally, ROS cells stimulated with calcium ionophore (A23187) exhibited approximately twice the activity of non-stimulated cells when grown on TCPS but not on the metallic substrates. ROS cells (stimulated or unstimulated) adherent to the zirconium alloy generated lower amounts of Factor Xa compared to those bound to the other alloys and unstimulated cells grown on TCPS. These results indicate that ROS cells cultured on these synthetic surfaces differentially express procoagulant activity and that cells grown on TCPS, but not the metallic alloys, exhibit increased TF activity in response to stimulation by calcium ionophore. This procoagulant activity may potentiate subsequent inflammatory responses associated with the use of orthopedic biomaterials and thereby influence the tissue compatibility of the implant.

Topics: Alloys; Animals; Biocompatible Materials; Calcimycin; Cell Adhesion; Colorimetry; Factor VII; Factor X; Gene Expression Regulation; Ionophores; Orthotic Devices; Osteosarcoma; Polystyrenes; Rats; Stainless Steel; Thromboplastin; Titanium; Tumor Cells, Cultured; Zirconium

Parathyroid hormone (PTH) functions in part by regulating osteoblast cytokine expression. We recently demonstrated that PTH induced a rapid and transient increase in interleukin-6 (IL-6) mRNA expression in rat bones in vivo. To determine the molecular basis of this effect, we analyzed the human IL-6 promoter fused (-1,179 to +9) with the chloramphenicol acetyltransferase (CAT) reporter gene in stable transfections into human osteoblast-like osteosarcoma SaOS-2 cells. We compared the effects of PTH on IL-6 expression with adenylate cyclase activator forskolin, PKC activator phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, interleukin-1 alpha (IL-1 alpha), prostaglandin E-2 (PGE-2), RS-66271 (a parathyroid hormone-related peptide analog), and platelet-derived growth factor-BB (PDGF-BB). Analyses of cell clones showed that IL-6 promoter expression was extremely low in the unstimulated state. Exposure to PTH (0.001-100 nM) for 12 h stimulated CAT expression in a dose-dependent manner (200-500% of control). Treatment with IL-1 alpha was more potent than PTH in inducing transcription of the IL-6 promoter (900-1,000%). Activation of the cAMP-PKA pathway by treatment with forskolin induced a comparable level of induction with PTH. Together, the effects of PTH and forskolin were additive. RS-66271, previously shown to have PTH-like effects, induced a comparable level of IL-6 promoter expression. When examined together, PTH+RS-66271 effects were comparable to PTH effects alone. Exposure to PGE-2, PMA, PDGF-BB, or A23187 for 12 h did not significantly alter IL-6 promoter expression. These results demonstrate PTH, forskolin, the PTHrP analog RS-66271, and IL-1 alpha stimulate IL-6 expression by stimulating gene transcription. The response to forskolin suggests that the messenger system mediated by PKA is sufficient to induce IL-6 expression.

Topics: Becaplermin; Calcimycin; Colforsin; Dinoprostone; Drug Interactions; Gene Expression; Humans; Interleukin-1; Interleukin-6; Osteosarcoma; Plasmids; Platelet-Derived Growth Factor; Promoter Regions, Genetic; Proto-Oncogene Proteins c-sis; Recombinant Fusion Proteins; Teriparatide; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

Although an excess intake of fluoride has been reported to cause skeletal fluorosis, very little is known about the mechanism of adverse effects of fluoride on bone. In the present study cytotoxic effects of fluoride were studied using the osteosarcoma cell line, UMR 106. The DNA ladder formation upon agarose electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining revealed that UMR 106 underwent apoptosis following exposure to 5 mM fluoride for 8 h. On the other hand exposure to A23187, a calcium ionophore, caused necrosis while co-exposure to fluoride and A23187 inhibited fluoride-mediated apoptosis in UMR 106. The proliferation of UMR 106 cells cultured for 6 days in the presence of 0.5 mM fluoride was significantly decreased compared to the control culture. The cytotoxic effects of fluoride were modulated by both the cell density and the pH of the culture medium. The fluoride-induced viability loss in UMR 106 was enhanced in culture of high cell-density and inversely correlated with pH of the culture medium. Enhancement of fluoride cytotoxicity at acidic pH was also observed in rat alveolar macrophages and RAW 264, a macrophage cell line. The results suggest that fluoride-mediated apoptosis and culture conditions, including pH of the medium, should be taken into consideration to evaluate toxicity of fluoride in vitro.

Topics: Animals; Apoptosis; Bone Neoplasms; Calcimycin; Cell Count; Cell Line; Cell Survival; Culture Media; DNA Fragmentation; Dose-Response Relationship, Drug; Fluorides; Hydrogen-Ion Concentration; Ionophores; Macrophages, Alveolar; Mice; Necrosis; Osteosarcoma; Rats; Tumor Cells, Cultured

The effects of tumor necrosis factor alpha (TNF-alpha) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [3H]AA. TNF-alpha differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-alpha (10(-8)M) significantly increased the cyclooxygenase pathway, with prostaglandin E2 (PGE2) being a major product. However, at the lowest concentration (10(-10)M) of TNF-alpha, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-alpha was increased to 10(-9) M leukotriene B4 (LTB4), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10(-6) M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-alpha (10(-8)M) and A23187 (10(-6)M) caused an inhibitory effect on each agent-induced PGE2 or 15-HETE production.

Topics: Arachidonic Acid; Calcimycin; Dinoprostone; Dose-Response Relationship, Drug; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Leukotriene B4; Lipoxygenase; Osteoblasts; Osteosarcoma; Prostaglandin-Endoperoxide Synthases; Tritium; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP) from ROS cells. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100,000 g. A substantial amount of lipids was detected in the 100,000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage.

Topics: Alkaline Phosphatase; Animals; Bacterial Proteins; Calcimycin; Calcium; Cell Membrane; Cholesterol; Cold Temperature; Dose-Response Relationship, Drug; Escin; Glycosylphosphatidylinositols; Osteosarcoma; Phospholipases; Rats; Streptolysins; Tumor Cells, Cultured

1. Bone resorptive factors, prostaglandin E2 and parathyroid hormone are shown to suppress alkaline phosphatase activity in a rat osteoblastic cell line. 2. Phorbol myristate acetate, but not dibutyryl cAMP or calcium ionophore can suppress alkaline phosphatase activity. 3. The protein kinase C inhibitors (H89, staurosporine) are able to block the suppression of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone. 4. These data suggest that protein kinase C is involved in the inhibition of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.

Topics: Alkaline Phosphatase; Alkaloids; Animals; Bucladesine; Calcimycin; Cyclic AMP; Cycloheximide; Dinoprostone; Isoquinolines; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Protein Kinase C; Rats; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In the present study, we investigated the role of parathyroid hormone-related peptide (PTHrP)-responsive dual signal transduction systems in the regulation of sodium-dependent phosphate (Pi) transport by PTHrP in UMR-106 cells. Exposure of the cells to 10(-7) M human (h) PTHrP-(1-34) induced a significant increase in Pi uptake within 15 min of incubation. The peptide stimulated Pi uptake dose-dependently at the range of 10(-11)-10(-7) M. Activation of protein kinase C (PKC) by 12-O-Tetradecanoyl phorbol-13-acetate (TPA) also increased Pi uptake in time- and dose-dependent manners similar to PTHrP. In contrast, neither activation of adenylate cyclase by 10(-5) M forskolin nor calcium ionophore treatment with 10(-7) M A23187 affect Pi uptake. These agents failed to influence on Pi uptake even in combined treatment with TPA. The PTHrP-induced increase in Pi uptake was strongly inhibited by pretreating cells with PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) (50 microM), and by down-regulating PKC with a prolonged TPA pretreatment. These results indicate that the messenger system mediated by PKC, rather than adenylate cyclase or cytosolic calcium, plays a crucial role in the regulation of sodium-dependent Pi transport by PTHrP in the osteoblast-like cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenylyl Cyclases; Animals; Biological Transport; Bone Neoplasms; Calcimycin; Calcium; Cell Line; Colforsin; Cytosol; Humans; Isoquinolines; Kinetics; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Phosphates; Piperazines; Protein Kinase C; Proteins; Rats; Sodium; Tetradecanoylphorbol Acetate

There has been recent evidence that calcium/protein kinase C (Ca/PKC) messenger system as well as adenylate cyclase are involved in the signal transduction stimulated by PTH. We therefore examined the role of these dual-signal transduction systems and the interaction of these systems in the regulation of DNA synthesis by PTH in the osteoblastic osteosarcoma cells, UMR-106. As recently reported, 10(-4) M Sp-cAMPS, a direct activator of cAMP-dependent protein kinase (PKA), and 10(-4) M dibutyryl-cAMP, as well as hPTH-(1-34), caused the significant inhibition of [3H]thymidine incorporation (TdR). Both A23187 and ionomycin (10(-8)-10(-6) M) inhibited TdR in a dose-dependent manner, with a minimal effective dose at 10(-7) M. Although 10(-6) M phorbol 12-myristate 13-acetate (PMA) caused slight but significant stimulation of TdR by itself, it augmented not only dibutyryl-cAMP- but also Sp-cAMPS-induced inhibition of TdR. On the other hand, 4 alpha-phorbol 12,13-didecanoate, incapable of activating PKC, failed to augment these cAMP analogs-induced effects. Pretreatment with 50 microM H-7, an inhibitor of PKC, not only abolished the PMA-induced augmentation of effect by cAMP analogs but also significantly blocked the PTH-induced inhibitory effect on TdR. Pretreatment with 10(-6) M PMA, which downregulates PKC, significantly inhibited the PTH-induced suppression of TdR. Combined treatment with cAMP analog (dibutyryl-cAMP or Sp-cAMPS) and calcium ionophore (A23187 or ionomycin) caused additive effects on TdR, and PMA used in combination with both cAMP analog and calcium ionophore induced the further inhibition of TdR.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bucladesine; Calcimycin; Cyclic AMP; DNA; Dose-Response Relationship, Drug; Ionomycin; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The present study was performed to examine whether parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) would stimulate osteoclast-like cell formation via soluble factor(s) released from osteoblasts and, if so, to characterize the involvement of PTH/PTHrP-responsive dual signal transduction systems [cAMP-dependent protein kinase (PKA) and calcium/protein kinase C(PKC)]. Osteoblasts-conditioned medium (CM) was obtained from rat osteoblastic osteosarcoma cells (UMR-106 cells), which had been cultured in serum free medium for 24 hrs after treatment with various kinds of reagents. The CM of osteoblasts treated with either 10(-7) M human(h) PTH-(1-34) or 10(-7)M hPTHrP-(1-34) equally stimulated osteoclast-like cell formation from hemopoietic blast cells derived from mouse spleen cells, although the CM treated with 10(-8) M 1,25dihydroxyvitamin D3 failed to affect it. The CM treated with both 10(-4) M dibutyryl-cAMP and a direct PKA activator, 10(-4)M Sp-cAMPS significantly increased osteoclast-like cell formation. The CM treated with a PKC activator, 10(-7)M phorbol 12-myristate 13-acetate (PMA) and calcium ionophores (10(-7)M A23187 and 10(-7)M ionomycin) also significantly enhanced osteoclast-like cell formation. The present study first indicated that osteoblast-mediated stimulation of osteoclast-like cell formation by PTH and PTHrP, and the participation of PTH/PTHrP-responsive dual signal transduction systems of osteoblasts in the stimulation of osteoclast-like cell formation by PTH and PTHrP.

Topics: Animals; Bucladesine; Calcimycin; Cells, Cultured; Culture Media, Conditioned; Culture Media, Serum-Free; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Ionomycin; Mice; Mice, Inbred Strains; Osteoblasts; Osteoclasts; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Protein Kinase C; Protein Kinases; Proteins; Rats; Second Messenger Systems; Signal Transduction; Teriparatide; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The present study was performed to characterize the direct involvement of cAMP-dependent protein kinase (PKA) in the regulation of collagen synthesis by parathyroid hormone (PTH) and PTH-related peptide (PTHrP) in osteoblastic osteosarcoma cells, UMR-106. Sp-cAMPS (10(-4)M), a direct activator of PKA, as well as dibutyryl cAMP (dbcAMP, 10(-4)M) significantly inhibited collagen synthesis. Human (h) PTH-(1-34) (10(-7)M) and hPTHrP (10(-7) M) inhibited collagen synthesis to the same degree. Although Rp-cAMPS, which acted directly as an antagonist in the activation of PKA, did not affect collagen synthesis by itself, it significantly antagonized dbcAMP- and Sp-cAMPS-induced inhibition of collagen synthesis. Moreover, Rp-cAMPS antagonized PTH- and PTHrP-induced inhibition of collagen synthesis to the same degree. The present study first indicated that the activation of PKA was directly linked to the regulation of collagen synthesis by PTH in osteoblast and that PTHrP had the same effect on collagen synthesis presumably through the same mechanism as PTH.

Topics: Animals; Bucladesine; Calcimycin; Cell Line; Collagen; Cyclic AMP; Ionomycin; Kinetics; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proline; Protein Kinases; Proteins; Rats; Tetradecanoylphorbol Acetate; Thionucleotides; Tritium

The present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH-related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR-106 which possessed PTH-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amiloride-inhibitable Na+/H+ exchange system. Both human (h)PTH-(1-34) and hPTHrP-(1-34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose-dependent manner with a minimal effective dose of 10(-10) M. Dibutyryl cAMP (10(-4) M and Sp-cAMPS (10(-4) M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10(-6) M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP- and Sp-cAMPS-induced decrease in pHi but did not affect calcium ionophore-induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore-induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA-inhibited Na+/H+ exchange and increased cytosolic calcium-induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/H+ exchange system.

Topics: Amiloride; Bone Neoplasms; Bucladesine; Calcimycin; Carrier Proteins; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Ionomycin; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Protein Kinase C; Proteins; Second Messenger Systems; Signal Transduction; Sodium-Hydrogen Exchangers; Teriparatide; Thymidine; Tritium; Tumor Cells, Cultured

We studied the effect of parathyroid hormone (PTH) and activation of the cAMP signal pathway on vitamin D receptor (VDR) mRNA levels in the phenotypically osteoblast cell line UMR 106. PTH caused a time- and dose-dependent increase of the VDR mRNA content with a maximum after 2 h. After 24 h the VDR mRNA level in PTH-treated cells returned to control level. In contrast, the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced increase in VDR mRNA did not decline after 24 h. Inhibition of transcription with actinomycin D (10 micrograms/ml) completely abolished the PTH-induced increase of VDR mRNA and inhibition of translation with cycloheximide (1 microgram/ml) resulted in superinduction of VDR mRNA. The role of cAMP in the induction of VDR mRNA was studied with several agents acting via the cAMP pathway. Incubation for 2 and 4 h with forskolin, Bt2cAMP, PTHrP or prostaglandin E2 caused an increase in the level of VDR mRNA comparable to that caused by PTH. The calcium ionophore A23187 did not affect VDR mRNA level. The present study demonstrates that PTH and activation of the cAMP signal pathway cause up-regulation of VDR via induction of VDR gene expression. The effect of cAMP on the VDR gene is suggestive for a cAMP responsive element in the VDR gene.

Topics: Animals; Blotting, Northern; Bucladesine; Calcimycin; Calcitriol; Cell Line; Colforsin; Cyclic AMP; Dinoprostone; Gene Expression Regulation, Neoplastic; Kinetics; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Protein Biosynthesis; Proteins; Rats; Receptors, Calcitriol; Receptors, Steroid; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Teriparatide; Transcription, Genetic

In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation).

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Biological Transport; Calcimycin; Carrier Proteins; Cell Compartmentation; Enzyme Activation; Gene Expression; Humans; Indoles; Leukotriene Antagonists; Leukotrienes; Membrane Proteins; Membranes; Models, Biological; Osteosarcoma; Transfection; Tumor Cells, Cultured

Effects of increase in intracellular calcium on PTH-induced homologous desensitization were investigated using calcium ionophores. Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with calcium ionophores (A23187 or ionomycin) for 6h resulted in approximately 50% decrease of PTH-stimulated cAMP production. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was significantly decreased in 10(-6) M calcium ionophore-pretreated (for 6h) cells without affecting the dissociation constant (Kd) for PTH. Minimal effective treatment period was 2h and similar inhibitory effect was observed in 12h-treated cells. These data suggest that increase in intracellular calcium might also act on PTH receptor in the similar manner as protein kinase C activation to induce desensitization.

Topics: Animals; Calcimycin; Calcium; Cell Line; Cyclic AMP; Ionomycin; Kinetics; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Rats; Receptors, Cell Surface; Receptors, Parathyroid Hormone

Platelet-derived growth factor (PDGF) is generally considered to stimulate phosphoinositide turnover resulting in activation of protein kinase C and increased cytoplasmic [Ca2+]. We have examined the role of these secondary effects in regulation of c-myc mRNA accumulation in the MG-63 human osteogenic sarcoma line. Treatment of quiescent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to down-regulate protein kinase C inhibited TPA-stimulated c-myc expression but did not affect the PDGF-modulated process. When cytoplasmic [Ca2+] was increased by addition of a Ca2+ ionophore (A23187 or ionomycin), no stimulation of c-myc RNA was seen; furthermore, these agents did not enhance the PDGF-modulated c-myc expression. Addition of EGTA to cultures treated with both PDGF and a Ca2+ ionophore did not inhibit c-myc induction but rather caused a superinduction of c-myc RNA accumulation. Superinduction occurred only if the [EGTA] was greater than [Ca2+] in the medium. This superinduction was distinct from the increased induction caused by inhibition of protein synthesis. Because PDGF-induced c-myc expression is independent of protein kinase C and increased cytoplasmic [Ca2+], the evidence suggests that PDGF modulates c-myc RNA accumulation in MG-63 cells via a novel pathway, seemingly uncoupled from the classic action of increased phosphoinositide metabolism.

Topics: Calcimycin; Cycloheximide; Egtazic Acid; Ethers; Gene Expression Regulation; Humans; Ionomycin; Oncogenes; Osteosarcoma; Platelet Activating Factor; Protein Kinase C; RNA; Tumor Cells, Cultured

This study compares the metabolism of [14C]-arachidonic acid between PGE2 synthesizing (ROS 17/2.8) and nonsynthesizing (ROS 25/1) osteosarcoma cell lines. In both cell lines: (a) 90% of [14C]-arachidonic acid was taken up at 24 h. (b) More than 90% of the label was associated with phospholipids. (c) [14C]-arachidonic acid was rapidly taken up by phosphatidylcholine which reached the highest specific activity around 5 h while the labeling of other phospholipids was still increasing at 24 h. (d) Twenty-four hours after addition of [14C]-arachidonic acid only 4% of the label was associated with triacylglycerols in ROS 25/1 and 0.3% in ROS 17/2.8 cells. The calcium ionophore A23187 enhanced the release of [14C]-arachidonic acid from phospholipids in the PGE synthesizing osteoblastic cells (ROS 17/2.8 and 2/3) but had no effect in nonosteoblastic cells (ROS 24/1 and 25/1). ROS 17/2.8 and 2/3 cells converted the released arachidonic acid as well as exogeneously added arachidonic acid into PGE2. PGE2 synthesis depended on arachidonic acid concentration. Among bone resorbing agents, parathyroid hormone and 1,25(OH)2D3 had no effect on PGE synthesis, whereas thrombin and rabbit serum stimulated PGE2 production. The effect of rabbit serum was abolished by heat inactivation. The findings of this study indicate that the difference in PGE production between the osteoblastic and nonosteoblastic osteosarcoma cells are due mainly to differences in arachidonic acid conversion to PGE2.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Clone Cells; Dinoprostone; Indomethacin; Lipid Metabolism; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Phospholipids; Rabbits; Radioimmunoassay; Rats; Thrombin; Tumor Cells, Cultured

It has been suggested that intracellular Ca2+, in addition to cAMP, plays an important role in PTH-stimulated bone resorption. There is now strong evidence indicating that the osteoblast is the main target cell for PTH action, regulating indirectly, via cell-cell communication, osteoclastic bone resorption. In order to investigate the possible role of free cytosolic calcium in stimulated bone resorption, we studied the effects of the intact hormone (bPTH 1-84) and some of its fragments (bPTH (1-34), bPTH(3-34,) (Nle-8, Nle-18,Tyr-34) bPTH (3-34) amide) on their capacity to modify the cytosolic Ca2+ concentration in rat osteoblast-like cells. The experiments were performed using Quin-2, a fluorescent indicator of free calcium. We found an excellent correlation between the ability of PTH and PTH fragments to transiently increase cytosolic Ca2+ concentration in rat osteoblast-like cells and their ability to stimulate bone resorption in embryonic rat calvaria in vitro. On the other hand, no direct correlation was found for the cAMP and bone-resorbing responses. On the ground of these data we propose a two-receptor model for PTH action in osteoblasts, in which one receptor is coupled to the production of cAMP, whereas the other is involved in the increase of cytosolic Ca2+. Activation of both receptors by PTH (1-84) or PTH (1-34) leads to the full physiological response in osteoblasts, most probably the release of one or more factors which stimulate the activity of existing osteoclasts and others which stimulate the recruitment of additional osteoclasts.

Topics: Animals; Bone Resorption; Calcimycin; Calcium; Cell Line; Cyclic AMP; Intracellular Fluid; Models, Biological; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Rats; Receptors, Cell Surface; Receptors, Parathyroid Hormone

Intracellular microelectrode measurements were made on a well-characterized osteoblast-like clonal cell line isolated from a rat osteosarcoma. In serum-free medium, stable membrane potentials of -42 +/- 9 mV (SD, n = 190) were recorded. Ion substitution experiments suggested that this membrane potential is primarily a Na+/K+ diffusion potential. Input resistance was correlated strongly with colony size, ranging from 49 +/- 18 M omega (SD, n = 14) for colonies of 1-3 cells, to 4 +/- 4 M omega (SD, n = 164) for colonies of 100 or more cells. These results are consistent with the existence of low resistance intercellular junctions. Application of the carboxylic calcium ionophore A23187 by pressure microejection onto the cell surface resulted in a transient hyperpolarization and concomitant decrease in input resistance. Both these effects are consistent with an increased K+ conductance. Ion substitution experiments demonstrated that the degree of hyperpolarization was dependent on the external concentration of both K+ and Ca2+. Quinine, a blocker of Ca2+-activated K+ channels, inhibited the ionophore-induced hyperpolarization in a dose-dependent manner. It was concluded that these cells exhibit a Ca2+-activated K+ conductance.

Topics: Animals; Calcimycin; Calcium; Cell Line; Electric Conductivity; Electrophysiology; Membrane Potentials; Osteoblasts; Osteosarcoma; Potassium; Rats