Changes in sphingolipid levels induced by epidermal growth factor in osteoblastic cells. Effects of these metabolites on cytosolic calcium levels.

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+]i levels, with thapsigargin (depletes [Ca2+]i pools) 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+]i mobilization 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.

Molecular characterization of the alpha1 subunit of the L type voltage calcium channel expressed in rat calvarial osteoblasts.

Voltage-activated calcium channels (VACCs) regulate extracellular calcium influx in many cells. VACCs are composed of five subunits. The alpha1 subunit is considered the most important in regulating channel function. Three isoforms of this subunit have been described: skeletal, cardiac, and neuroendocrine. It was the purpose of the present study to determine the molecular identity of the alpha1 subunit of the VACCs in rat calvarial osteoblasts and to study the nature of the regulation of these channels as a function of cellular growth. We also attempted to identify which isoform of the alpha1 subunit of the VACCs mediates the effects of epidermal growth factor (EGF) on osteoblastic cell proliferation. Reverse transcription-polymerase chain reaction was used to detect the isoforms of the VACCs that are expressed in osteoblastic cells. These analyses showed that the proliferative state of the cell and the time in culture influence RNA expression. The only alpha1 subunit detected in osteoblasts corresponds to the cardiac isoform. In additional experiments, the effects of EGF on cytosolic calcium and osteoblast proliferation were determined. For these experiments, the synthesis of the different isoforms of the VACCs was selectively blocked by antisense oligonucleotides prior to EGF stimulation. These studies showed that the cardiac isoform mediates the effects of EGF on cytosolic calcium and cellular proliferation in rat calvarial osteoblasts.

Sphingolipids stimulate cell growth via MAP kinase activation in osteoblastic cells.

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.

Activation of phospholipase D signaling pathway by epidermal growth factor in osteoblastic cells.

The receptor-mediated activation of phospholipase D (PLD) is a major signaling pathway in several cell systems. This study determined the effects of epidermal growth factor (EGF) on PLD activity in normal rat osteoblastic cells. Primary cultures were obtained from fetal rat calvaria by sequential collagenase digestion and seeded in BGJb media supplemented with 10% fetal calf serum. PLD activity was assayed by the transphosphatidylation reaction in [H3]myristic acid (5 microCi/ml)-labeled cells treated with EGF in the presence of 5% ethanol and measuring the production of phosphatidylethanol (PEtOH). Lipids were extracted and separated by thin-layer chromatography, detected by iodine staining, and the areas of interest were scraped off and transferred to vials for scintillation counting. EGF significantly increased PEtOH production in a dose-dependent manner and at short (10-60 s) and long (up to 30 minutes) incubation periods (p < 0.05). Phosphatidic acid levels were also significantly increased (p < 0.05) compared with unstimulated controls, but the levels were approximately 60% less than those of PEtOH. 4b-phorbol 12-myristate, 13-acetate (PMA) also produced a significant increase in PEtOH levels when compared with unstimulated control cultures, but when PMA was added together with EGF, the production of PEtOH was reduced about 30%. Pretreatment of cells with the protein kinase C (PKC) inhibitor H-7 caused a significant increase in PEtOH levels, compared with cells stimulated with EGF alone. Preincubation of cells with pertussis toxin produced a partial decrease in PEtOH levels. This study demonstrates that EGF activates the PLD signaling cascade in normal rat osteoblastic cells and that the pathway appears to involve, at least in part, a PKC- and Gi protein-dependent mechanism.

Phosphatidic acid effects on cytosolic calcium and proliferation in osteoblastic cells.

Our previous studies show that epidermal growth factor (EGF) stimulates phospholipase D (PLD)-induced phosphatidic acid (PA) formation in rat calvarial osteoblastic cells. This study investigated the effects of PA on cytosolic calcium ([Ca2+]i) and proliferation, and the possible involvement of the PLD pathway in EGF effects on [Ca2+]i and proliferation in rat calvarial osteoblastic cells. PA markedly increased [Ca2+]i. This response was unaffected by thapsigargin, which depletes [Ca2+]i pools, blocked by verapamil, a calcium channel blocker, and enhanced by propanolol, an inhibitor of PA-phosphohydrolase. PA also reduced the EGF dependent-[Ca2+]i increase by 60%, while a PLD inhibitor blocked these effects. Furthermore, PA significantly increased cell proliferation (P < 0.05) which was inhibited by verapamil and enhanced by H-7 (PKC inhibitor). The PLD inhibitor significantly (P < 0.05) reduced the EGF-induced increase in proliferation. In summary, PA stimulates rat calvarial osteoblastic cell proliferation and mobilization of [Ca2+]i using extracellular pools, and EGF's mitogenic effect on these cells requires activation of PLD.

Osteoporosis and its relationship to oral bone loss.

Osteoporosis, an age-related condition, is classified into primary and secondary types. Primary osteoporosis encompasses the postmenopausal and senile types; secondary osteoporosis occurs "secondary" to endocrine and renal diseases. Subjects affected by osteoporosis have an overall reduced bone mass and become highly susceptible to bone fractures. Dual energy x-ray absorptiometry is the method most often used to determine the risk for osteoporotic fractures. In the past decade, a number of studies have suggested a possible correlation between systemic osteoporosis and alveolar bone loss in periodontal disease pathogenesis. It appears that a clear correlation between periodontal health and the general mineral status of the skeleton is still lacking. This review addresses the pathogenesis of osteoporosis and emphasizes the multifactorial nature of bone loss. The current concepts in alveolar bone loss resulting from osteoporosis and its implications as a risk factor in periodontal disease development are also presented.

Studies of the angular reproducibility of positioning patients adjacent to an x-ray tube: 1. Stent-rod based and extra-oral systems.

The usefulness of subtraction radiography for detecting small changes in crestal bone is dependent upon achieving reproducible geometry between x-ray beam and patient structures when serial radiographs are taken. This study evaluates 2 methods currently employed to maintain geometric correspondence: 1) a stent-based system which rigidly fixes a custom-made stent to the x-ray tube by the use of a rod and 2) an extra-oral system which positions a patient in the x-ray unit by means of ear rods. The projection of a light beam from a fixed subject reference was used to measure the change of the orientation of the reference at 2 different measurement times. The rod-stent system was able to maintain a discrepancy of less than 2 degrees 75% of the time over a time period of 6 months. For the extra-oral system this ranged from 72% to 92% during a 1-month period.

Evaluation of a simple modified radiographic alignment system for routine use.

Radiographic frames used for longitudinal studies may be in part unreadable for measuring crestal bone change. Sites may not be present on the film, or the measurement reliability may be compromised because of dissimilar geometry. Several techniques used to address this problem are expensive, time-consuming, and required great skill. For the present study a commercially-available alignment system was simply modified by addition of a reference pin in the bite block, facilitating the repositioning of the film holder for a second exposure. This study determined the ability of the modified instrument to: 1) improve the geometrical correspondence between serial radiographs; and 2) reduce the frequency of missed sites in the film. Two pairs of x-rays were taken for each of 40 subjects, 1 pair with the standard alignment instrument of an assigned site and 1 pair with the modified instrument of the contralateral site. Measurements of alveolar bone height were performed using the "side by side" technique. The modified instrument yielded significantly smaller measurement differences and a significantly better geometrical correspondence than the conventional system (P < 0.05). Also, the modified instrument yielded significantly greater (P < 0.01) readable sites (86%) as compared to the conventional instrument (62%). The simply-modified instrument facilitates the correct interpretation of serial radiographs.