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Human osteoblastlike cells do not respond to interleukin-6.
Interleukin 6 (IL-6) exerts well-established effects on cells of the immune system as well as on various other cell types. It has been implicated in the control of connective tissue cells in such conditions as rheumatoid arthritis and osteoporosis. We have investigated the effects of recombinant human interleukin-6 (rhIL-6) on human osteoblastlike cells derived from explants of trabecular bone. ROS 17/2.8 cells were used as an additional osteoblastlike cell model system. We were unable to identify any effects of rhIL-6 (5-5000 pg/ml) on the proliferation, alkaline phosphatase activity. osteocalcin production, or release of cytokines or prostaglandins by either osteoblastlike cell model system. Since we have shown previously that these cells release IL-6 in culture, we used a sheep anti-human IL-6 antibody to investigate the possibility that (1) action of added exogenous IL-6 could be masking endogenous production, and (2) endogenous IL-6 may regulate the effects of osteotropic agents on the osteoblastlike cells. Presence of the antibody exerted no detectable effects on 1,25-(OH)2D3-stimulated alkaline phosphatase or on proliferation or TNF production enhanced by IL-1. Thus IL-6 does not appear to be involved in the regulation of osteoblast activity.
Transforming growth factor beta increases ecto-nucleoside triphosphate pyrophosphatase activity of human bone-derived cells.
Inorganic pyrophosphate (PPi) may be involved in the regulation of mineralization. The cell surface enzyme, ecto-NTP pyrophosphatase, could be a major source of extracellular PPi in bone, and agents that influence its activity in osteoblasts may modulate bone mineralization. We studied the effects of serum on the ecto-NTP pyrophosphatase activity of cultured human osteoblast-like cells. Enzyme activity was lowered when the concentration of fetal calf serum (FCS) was reduced from 10 to 2.5% (vol/vol) for 48 h, and a further decrease in activity was observed after 96 h. Relative to enzyme activity in cells cultured in serum-free medium for 96 h, adult human platelet-poor plasma (HPPP; 2.5-10% vol/vol) induced a small increase, similar concentrations of adult human serum (HS) induced much larger increases, and charcoal-depleted FCS was ineffective. In an attempt to identify the factor(s) present in serum that influence ecto-NTP pyrophosphatase activity, we examined transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF). PDGFs AA, AB, and BB (0.1-10 ng/ml) were ineffective, but both TGF-beta 1 and TGF-beta 2 increased enzyme activity. The increase was dose dependent between 0.001 and 10 ng/ml, was enhanced in the presence of 2% vol/vol FCS, and was not potentiated by PDGF or by 1,25-(OH)2D3. Furthermore, the increase was independent of cell density and was blocked by inhibitors of protein and RNA synthesis. Ecto-NTP pyrophosphatase of subject-matched human dermal fibroblasts was unaffected by TGF-beta (10 ng/ml), suggesting that modulation of activity by the growth factor may be tissue specific. Alkaline phosphatase (ALP) probably serves to hydrolyze extracellular PPi in bone. In contrast to effects on NTP pyrophosphatase activity is osteoblast-like cells, TGF-beta 1 and TGF-beta 2 (0.001-10 ng/ml) decreased ALP activity dose dependently after 72 h. By inducing opposing changes in ecto-NTP pyrophosphatase and ALP activities, TGF-beta may increase extracellular PPi concentrations in osseous tissues and consequently modulate bone mineral properties in vivo.
Interleukin-6 does not stimulate bone resorption in neonatal mouse calvariae.
Recombinant human interleukin-6 (IL-6) was assessed for its ability to stimulate bone resorption in prelabeled mouse calvariae in vitro. IL-6 had no effect on bone resorption at concentrations ranging from 300 to 10,000 U/ml (3-1000 pg/ml). Neither the presence of indomethacin nor prolonged incubation periods (96 h) affected this result. IL-6 did not affect resorption stimulated by human recombinant IL-1 alpha (rIL-1 alpha) but inhibited resorption stimulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. rIL-1 alpha, PTH, and 1,25-(OH)2D3 induced IL-6 release by calvariae. We conclude from these studies that IL-6 does not stimulate bone resorption in neonatal mouse calvariae. However, it may act as a locally produced inhibitor and therefore a paracrine regulator of bone resorption induced by osteotropic hormones. IL-6 could also function as a long-range stimulator of systemic reactions and acute-phase responses to local inflammatory and neoplastic lesions in bone.
Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum.
Bisphosphonates are a class of synthetic pyrophosphate analogues. Some are known to be potent inhibitors of osteoclast-mediated bone resorption in vivo, but their mechanisms of action are unclear. The order of potency of bisphosphonates as inhibitors of bone resorption closely matches the order of potency as inhibitors of growth of amoebae of the slime mould Dictyostelium discoideum, indicating that bisphosphonates may have a mechanism of action that is similar in both osteoclasts and Dictyostelium. Methylenebisphosphonate and several halogenated derivatives, which have low potency as antiresorptive agents and as growth inhibitors of Dictyostelium, are metabolized intracellularly by Dictyostelium amoebae into methylene-containing adenine nucleotides. We have used a combination of n.m.r. and f.p.l.c. analysis to determine whether incorporation into nucleotides is a feature of other bisphosphonates, especially those that are potent antiresorptive agents. Only bisphosphonates with short side chains or of low potency are incorporated into adenine nucleotides, whereas those with long side chains or of high potency are not metabolized. Bisphosphonate metabolism in cell-free extracts of Dictyostelium was accompanied by inhibition of aminoacylation of tRNA by several aminoacyl-tRNA synthetases. These enzymes were barely affected by the bisphosphonates that were not metabolized. The results indicate that some bisphosphonates are not metabolically inert analogues of pyrophosphate and appear to be metabolized by aminoacyl-tRNA synthetases. The cellular effects of some bisphosphonates may be the result of their incorporation into adenine nucleotides or inhibition of aminoacyl-tRNA synthetases, although the potent bisphosphonates appear to act by a different mechanism.
Cloning of a novel membrane-linked metalloproteinase from human myeloma cells.
We have isolated a novel cDNA from human myeloma cells encoding a member of the reprolysin family of metalloproteinases. Derived amino acid sequence predicts a protein of approx. 76 kDa. The open reading frame predicts the presence of a leader peptide, a pro-peptide with a 'cysteine switch', a metalloproteinase domain, a disintegrin-like domain, a cysteine-rich domain, an epidermal growth factor-like domain and a putative transmembrane sequence. Expression of the mRNA for this metalloproteinase has been demonstrated in human myeloma cells.
Effect of aspirin on urinary excretion of 6-ketoprostaglandin F1 alpha.
1. We studied the effect of oral administration of acetylsalicylic acid (1200 mg/day for 3 days) on the urinary excretion of 6-ketoprostaglandin F1 alpha in normal human subjects as an index of prostacyclin production in vivo. 2. The concentrations and excretion rate in urine fell to 45% of pretreatment levels in 3 days, but returned to pretreatment values after 7 days. 3. These results suggest that production of prostacyclin in vivo is only partially inhibited by high doses of aspirin and that there are sites of production of prostacyclin which are protected from inhibition by aspirin and which contribute to urinary 6-ketoprostaglandin F1 alpha. The measurement of 6-ketoprostaglandin F1 alpha in urine may therefore be of only limited value as an index of the metabolism of vascular tissue in vivo.
Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage.
Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.
The anti-resorptive drug clodronate is metabolised to a non-hydrolysable ATP analogue by mammalian cells in vitro
Bisphosphonates (BPs) are stable analogues of pyrophosphate that contain P-C-P bonds. Clodronate (dichloromethylenebisphosphonate) is widely used in the treatment of bone diseases characterised by excessive bone résorption such as Paget's disease, tumour-induced osteolysis and hypercalcaemia of malignancy. The mechanisms of action of BPs have not been identified but may involve a toxic effect on bone-resorbing osteociast cells due to induction of apoptosis. It is generally considered that BPs are not metabolised. We have found that mammalian cells in vitro (human MG63 osteosarcoma cells and mouse J774 macrophage-like cells) can metabolise clodronate to a non-hydrolysable ATP analogue, β,γ-dichloromethylene ATP. This metabolite could be detected in deproteinised cell extracts prepared from J774 and MG63 cells that had been incubated for 48 hours with 250μM or 750μM clodronate respectively. These concentrations significantly inhibited cell proliferation. An additional peak of absorbance at 254nm that was not present in control extracts could be detected after anion-exchange f.p.l.c. analysis of extracts from clodronate-treated cells. This peak, corresponding to the metabolite of clodronate, co-eluted with synthetic β,β-dichloromethylene ATP. This is the first report that clodronate can be metabolised by mammalian cells. Similarly, 1007mu;M methylenebisphosphonate (MBP) was metabolised into β,γ-methylene ATP and also a methylene analogue of Ap4A, diadenosine 5′5‴-P1,P4-(p2/p 3-methylene tetraphosphate). We have obtained similar results with amoebae of the eukaryotic slime mould Dictyostelium discoidemn, the growth of which is inhibited by BPs. In Didyostelium, the metabolites of clodronate and MBP appear to be produced by a back-reaction catalysed by certain aminoacyl-tRNA synthetases. The toxic and anti-proliferative effects of clodronate on osteoclasts and other cells may therefore be due to accumulation of a non-hydrolysable ATP analogue or to inhibition of aminoacyl-tRNA synthetase enzymes.