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Protein kinase C (PKC) isoforms have been shown to have specific expression profiles and individual isoforms are believed to play distinct roles in the cells in which they are found. The goal here was to determine which specific isoform(s) is involved in proliferation of primary human osteoblasts. In primary human osteoblasts, 10 μM of acute sphingosine‐1‐phosphate (S1P) treatment induced an increase in proliferation that correlated with an increase in PKCα and PKCι expression. To further delineate which isoforms are involved in osteoblastic cell proliferation, the effect of low versus high serum culture conditions on PKC isoform expression was determined. Likewise, the effect of antisense oligodeoxynucleotides (ODNs) to specific PKC isoforms on proliferation and MAPK activation was studied. The effect of S1P on intracellular translocation of activated PKC isoforms was also evaluated. The results indicated that in primary human osteoblasts, PKCα was not expressed under conditions of low proliferative rate while PKC° and PKCι expression was not affected. The specific inhibition of PKCα by antisense ODNs resulted in inhibition of MAPK activity leading to a significant decrease in proliferation. S1P up‐regulated antisense ODN inhibited PKCα expression and MAPK activity and led to an increase in proliferation. Subsequent experiments using platelet‐derived growth factor (PDGF) as an additional mitogen generated similar data. PDGF stimulation resulted in a significant increase in proliferation that correlated with an up‐regulation of inhibited PKCα expression in antisense ODN‐treated cells. Immunofluorescence methods showed that mitogenic stimulation of PKCα resulted in nuclear translocation. Our findings present original data that PKCα is the isoform specifically involved in the proliferation of primary human osteoblasts.

Sphingosine-1-phosphate (S1P) has been shown to participate in the proliferative process in human osteoblasts.1The mitogenic effect of S1P has been postulated to involve two signaling pathways, the Gi linked protein receptor pathway and the PKC pathway. To define the possible role of PKC isoforms in osteoblastic cell proliferation, the effects of S1P on PKC isoform expression was determined. While PKC λwas minimally detected, the isoforms α, δand ιwere all found to be highly expressed by the human osteoblast. In human osteoblastic cells, S1P induced a 25% increase in the expression of PKC αand approximately a 30% increase in the expression of PKC ι. S1P did not have an effect on PKC δ expression. Pretreatment with pertussis toxin (PT) led to an inhibition of the observed S1P effects on the expression of the αand ιisoforms.

Sphingolipids mediate a number of cellular functions in a variety of cell systems. The role they play in osteoblast signaling is yet unknown. This study investigated the effects of epidermal growth factor (EGF) on the levels of ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P) in rat calvariae osteoblastic cells, and whether these metabolites mediated cytosolic calcium ([Ca2+]I) mobilization in these cells. EGF significantly (P⪡0.05) increased the levels of all three sphingolipids, and the phorbol ester PMA partially inhibited these effects. SPH and S1P markedly increased [Ca2+]Ilevels, with thapsigargin (depletes [Ca2+]Ipools) decreasing the response by 60%. Verapamil (calcium channel blocker) only inhibited ceramide's effects on [Ca2+]I. Furthermore, SPH enhanced the EGF' induced increase in [Ca2+]I. This study demonstrates that ceramide, SPH and S1P mediate [Ca2+]Imobilization in rat calvarial osteoblastic cells, and that EGF induces changes in the levels of these metabolites with PKC playing an important role in the mechanisms regulating these events.

Ceramide, ceramide-1-phosphate (C1P) sphingosine (SPH) and sphingosine-1-phosphate (S1P) effects on proliferation and extracellular-signal regulated kinases, ERKs (also known as MAPKs), activation were investigated in human and rat osteoblastic cells. MAPK activation was sphingolipid-specific in cells from both species. In human osteoblastic cells, S1P and C1P markedly stimulated ERK2 phosphorylation with a slight increase in phosphorylation of ERK1. SPH nor ceramide induced phosphorylation of either ERK isoform. In rat osteoblastic cells, SIP, ceramide and SPH stimulated phosphorylation of both isoforms. C1P did not induce phosphorylation of ERK1 but produced a mild increase in phosphorylation of ERK2. In human cells, only S1P significantly (P⪡0.05) increased osteoblastic cell proliferation, while in the rat cells all four sphingolipids significantly (P⪡0.05) induced proliferation. The calcium channel blocker verapamil blocked (P⪡0.05) these effects in both cell types. The MAPK inhibitor, PD98059, inhibited (P⪡0.05) the mitogenic effect of SIP in human cells. In rat cells, PD98059 effects were less substantial but significant for S1P and C1P. This study demonstrates that sphingolipids are mitogens for both human and rat osteoblastic cells with the MAPK pathway and calcium mediating in part these effects in a species specific manner.

The effects of Phenytoin (diphenylhydantoin, DPH) on calcium uptake in osteoblastic cells were studied to elucidate the potential mechanism of action of this antiepileptic drug on bone metabolism. Preincubation of the human osteoblastic osteosarcoma) cell line, SaOS‐2, and normal rat osteoblastic cells with DPH decreased basal calcium uptake. This inhibition occurred at DPH doses from 0.1 to 50 μg/ml. Parathyroid hormone (PTH) and prostaglandin E2(PGE2) increased calcium uptake in the SaOS‐2 cell line. Following preincubation with DPH, calcium uptake in cells treated with PTH or PGE2did not exceed control levels. However, significant increases in the PTH‐ or PGE2‐treated + DPH‐pretreated cells compared to DPH pretreatment alone were still observed. These studies indicate that DPH induces decreases in osteoblastic calcium influx and they add further information on the possible mode of action of this drug on bone.

Summary: The binding of prostaglandin E2 (PGE2) to bone cells was studied to provide direct evidence for the existence of specific receptors in bone. Bone cells were isolated by collagenase digestion of fetal and newborn rat calvaria. Isolated cells were incubated with3H-PGE2 and collected on Millipore filters. Specific binding was determined by subtracting the binding that occurred with 10−6 M non-radioactive PGE2 and3H-PGE2 from that with3H-PGE2 alone. With heterogeneous cell preparations and at PGE2 concentrations from 10−9 − 1.7 × 10−8 M at 37°C, specific binding reached steady state within 10 min. Bound3H-PGE2 was displaced by the addition of increasing amounts of unlabeled PGE2. Inhibition of PGE2 binding was observed with PGE1 and the endoperoxide analog, U44069, but not with PGE2α, a lipopolysaccharide, or 13,14-dihydro 15-keto PGE2. Studies with bone cell populations, obtained by sequential digestions, indicated that an osteoclastic population binds 30-fold more PGE2 than osteoblastic cells. Scatchard analyses revealed that the osteoclastic cells have an affinity constant for PGE2 binding similar to that obtained with heterogeneous populations. However, the PGE2 binding capacity in this osteoclastic population was fivefold greater than in the heterogeneous population. The osteoclastic population responded with an increase in cyclic AMP to lower concentrations of PGE2 than the osteoblastic populations. These studies suggest that differences in the binding capacity of PGE2 receptors exist among bone celltypes and that these differences are reflected in the cellular cyclic AMP response.

Summary: The bone resorptive factor and amphipathic antigen (AcA) previously identified by us in preparations fromActinomyces viscosus have been partially purified, characterized chemically, and compared. They elute at the same location on chromatography with Ac 22. The fatty acid composition of AcA and the bone resorptive factor is the same. Some differences in carbohydrate composition are observed. TheActinomyces factor does not affect calcium influx or cyclic AMP in isolated bone cells. Therefore it is concluded that AcA stimulates resorption either by gaining entrance into bone cells or by way of a yet undetermined second messenger.

An approach based on a Kirchhoff approximation model [D. Jackson etal., J. Acoust. Soc. Am. 79, 1410–1422 (1986)] conveniently relates to a power law the ocean bottom roughness spectrum based on two parameters α and β, i.e., W(k)=βk−γ, where γ=2(α+1). Initial studies [R. Dziak etal., Eos 70, 71 (1989, 1990)] revealed that α and β, solved by multiregression analysis using sea beam data from the Juan de Fuca Ridge, compared favorably with the spectral models derived from similar tectonic provinces by Fox etal. [Rev. Geophys. 23 (1985)]. In comparison to a similar method applied to a heavily sedimented area by de Moustier etal. [J. Acoust. Soc. Am. 89, 1962–1963 (1991)], a lack of volume reverberation in hard‐rock areas aids in deriving solutions for the model since less bottom parameters are involved. On the other hand, the fluctuation of the echo levels between the pings from the rough and hard bottom made it difficult to fit to a realistic bottom roughness model. The statistics of the peak echo amplitudes from the sea beam system and a method adopted to reduce their standard deviation are described.