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P A Hill and A Tumber

Programmed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathway induced in a variety of cell types. We determined whether ceramide treatment contributes to reduced cell viability and increased PCD in primary osteoblasts and the signalling pathways that are involved. Cell viability was determined by the 3-(4,5-dimethyl-thiozol-2-yl)-2,5-diphenyl tetrazolium bromide assay. We found that C2-ceramide (≤10−7 M) promoted osteoblast viability, whilst concentrations ≥2×10−6 M significantly reduced osteoblast viability in a dose- and time-dependent manner. The effect of ceramide on cell viability was specific since C2-dihydroceramide had no effect. Increasing intracellular ceramide levels with either sphingomyelinase (SMase) or an inhibitor of ceramide metabolism also increased osteoblast apoptosis. Ceramide-induced PCD in osteoblasts was determined by nuclear appearance and DNA fragmentation. PCD was induced by both C2-ceramide and SMase. The ability of ceramide (5×10−8 M) to promote osteoblast survival was prevented by a general protein kinase C (PKC) inhibitor and by a PKC ζ inhibitor, whilst osteoblast survival was enhanced in the presence of a protein phosphatase 1 (PP1) inhibitor. Phosphatidylinositol-3 kinase (PI3K) inhibitors had no effect on osteoblast survival. The ability of ceramide (5×10−5 M) to induce apoptosis was prevented by the inhibitors of PP1 and PKC δ, whilst the general PKC and PI3K inhibitors had no effect on it. Our findings suggest that ceramide signals osteoblast survival and apoptosis through different intracellular pathways, and that alteration in the intracellular levels of ceramide may play an important role in bone remodelling.

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A Tumber, MC Meikle, and PA Hill

We have studied the survival requirements of osteoblasts to test the hypothesis that osteoblasts undergo programmed cell death (PCD) or apoptosis unless they are continuously signalled by other cells not to do so. Osteoblasts survived for 6 days in culture at high cell density in the absence of other cell types, serum or exogenous proteins, but they died with the morphological features of apoptosis in these conditions at low cell density. Osteoblast survival was enhanced during the first 2 days of culture by the addition of the sulphydryl compound, cysteine to the culture medium which was converted intracellularly to the antioxidant glutathione. Catalase, an enzyme decomposing hydrogen peroxide, also protected the cells, whereas superoxide dismutase had no effect. Therefore, osteoblasts in culture are sensitive to toxic compounds derived from molecular oxygen, i.e. hydroxyl radicals or hydrogen peroxide spontaneously generated in CMRL medium containing ascorbate and ferrous ions. Conditioned medium from high density cultures prevented osteoblast apoptosis in low density cultures, as long as antioxidants were also present. The enhancing effect of conditioned medium on osteoblast survival was prevented by neutralizing antibodies to insulin-like growth factor-I (IGF-I) and IGF-II but not by antibodies to either platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). These results suggest that in addition to regulating cell growth and differentiation, IGF-I and IGF-II also function as survival factors for osteoblasts. Our data also indicate that antioxidants are required for osteoblast survival and that they enhance growth factor mediated osteoblast survival.

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A Tumber, S Papaioannou, J Breckon, MC Meikle, JJ Reynolds, and PA Hill

The aims of this study were to identify the role and sites of action of serine proteinases (SPs) in bone resorption, a process which involves a cascade of events, the central step of which is the removal of bone matrix by osteoclasts (OCs). This resorbing activity, however, is also determined by recruitment of new OCs to future resorption sites and removal of the osteoid layer by osteoblasts (OBs), which enables OCs to gain access to the underlying mineralized bone. The resorption systems we have studied consisted of (i) neonatal calvarial explants, (ii) isolated OCs cultured on ivory slices, (iii) mouse OBs cultured on either radiolabelled type I collagen films or bone-like matrix, (iv) bone marrow cultures to assess OC formation and (v) 17-day-old fetal mouse metatarsal bone rudiments to assess OC migration and fusion. Two separate SP inhibitors, aprotinin and alpha(2)-antiplasmin dose-dependently inhibited (45)Ca release from neonatal calvarial explants: aprotinin (10(-6) M) was the most effective SP inhibitor, producing a maximum inhibitory effect of 55.9%.Neither of the SP inhibitors influenced either OC formation or OC resorptive activity. In contrast, each SP inhibitor dose-dependently inhibited OB-mediated degradation of both type I collagen fibrils and non-mineralized bone matrix. In 17-day-old metatarsal explants aprotinin produced a 55% reduction in the migration of OCs from the periosteum to the mineralized matrix after 3 days in culture but after 6 days in culture aprotinin was without effect on OC migration. Primary mouse osteoblasts expressed mRNA for urokinase type plasminogen activator (uPA), tIssue type plasminogen activator (tPA), the type I receptor for uPA, plasminogen activator inhibitor types I and II and the broad spectrum serine proteinase inhibitor, protease nexin I. In situ hybridization demonstrated expression of tPA and uPA in osteoclasts disaggregated from 6-day-old mouse long bones. We propose that the regulation of these various enzyme systems within bone tIssue determines the sites where bone resorption will be initiated.