Activators of protein kinase A decrease the levels of free arachidonic acid in osteoblasts via stimulation of phosphatidylcholine and phosphatidylethanolamine synthesis (original) (raw)
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Calcified Tissue International, 1991
Phorbol esters were used to evaluate the putative effect of protein kinase C (PKC) activation on prostaglandin E: (PGEz)-induced increases in calcium uptake and cAMP production in the human osteoblastic osteosarcoma cell line, Saos-2. The cells were pretreated for 15 min with phorbol myristate acetate (PMA) followed by a 5 rain incubation with PGE 2. Calcium uptake was measured with 45Ca and cAMP by radioimmunoassay. A significant increase in calcium uptake was noted in the PGE-treated cells compared with controls and preincubation with the PMA caused a significant decrease in this response. Preincubation with PMA also inhibited the PGE2-induced increase in cAMP under identical conditions. The effect of PMA on the cAMP response was not influenced by the addition of a phosphodiesterase inhibitor. PMA had no effect on the basal levels of either calcium uptake or cAMP production. Likewise, the inactive phorbol esters, phorbol 12,13-didecanoate (PDD) and 4 et-phorbol 12-myristate, 13-acetate (4alpha), had no effect on either basal levels of these parameters or on the PGE2-induced increases. These resuits suggest that PKC is involved in the downregulation of PGE2-induced increases in calcium uptake and cAMP production in the Saos-2 osteoblastic cell line.
Journal of Bone and Mineral Research, 1998
We have previously shown that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH) 2 D 3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH) 2 D 3 elicited a dose-dependent increase in both inositol-1,4,5trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH) 2 D 3-treated GCs, indicating that neither G i nor G s proteins were involved. Phospholipase A 2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A 2), and AACOCF 3 (selective for cytosolic phospholipase A 2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A 2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E 2 , two downstream products of phospholipase A 2 action, also increased PKC activity. These results indicate that 1,25(OH) 2 D 3dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A 2. (
Arachidonic acid influences intracellular calcium handling in human osteoblasts
Prostaglandins, Leukotrienes and Essential Fatty Acids, 2006
The effect of arachidonic acid (AA) on intracellular Ca 2+ concentration ([Ca 2+ ]i) in human osteoblasts MG63 was studied. AA caused a concentration-dependent increase in [Ca 2+ ]i, mainly due to inward Ca 2+ transport from extracellular environment. Moreover, AA in Ca 2+-free medium produced a small, transient increase of [Ca 2+ ]i, indicating that AA may also trigger Ca 2+ release from intracellular stores. Because the [Ca 2+ ]i response to AA was inhibited by the cyclooxygenase (COX) inhibitor indomethacin, we tested the effect of prostaglandins (PGs), products of COX pathway. PGs E1 and E2 caused an increase in [Ca 2+ ]i, which, however, was far lower than that obtained with AA. The [Ca 2+ ]i response to AA was not inhibited by nifedipine, suggesting that AA did not activate a voltage-dependent Ca 2+ channel. Our results indicate that AA could modulate [Ca 2+ ]i in MG63 human osteoblasts, where it may influence Ca 2+ transport across both plasma and endoplasmic membranes. Furthermore, they suggest that osteoblast activity may be modulated by AA.
Prostaglandins & other lipid mediators, 1999
Ca 2ϩ-independent phospholipase A 2 (iPLA 2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA 2 in AA remodeling in different cells was evaluated by studying the Ca 2ϩ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA 2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca 2ϩ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca 2ϩ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca 2ϩ. Both events were fully dependent on extra and intracellular Ca 2ϩ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca 2ϩ at short times (Ͻ30 min). The involvement of iPLA 2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced Յ50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA 2 plays a major role in regulating AA turnover in different cell types.
Endocrinology, 1999
Prior studies have shown that 24,25-dihydroxyvitamin D 3 [24,25-(OH) 2 D 3 ] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A 2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A 2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH) 2 D 3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH) 2 D 3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [ 3 H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH) 2 D 3 inhibited [ 3 H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH) 2 D 3 were
Prostaglandins, 1986
Recent experimental studies indicated that prostaglandin E 2 (PGE 2) is the most abundant prostanoid synthesized by rabbit articular chondrocytes. Exogenous PGE 2 stimulates cyclic AMP (cAMP) synthesis in these cells. Analogues of cAMP and forskolin have now been shown to suppress the biosynthesis of PGE 2 in the presence of serum in a time-dependent manner. The most abundant prostanoid, PGE 2 was most markedly affected. PGF2~ was unaffected. These results indicated that intracellular accumulation of cAMP in chondrocytes and relative resistance of cAMP to phosphodiesterases control prostanoid synthesis in a negative feedback loop.
Endocrinology, 2001
The simple glycerophospholipid lysophosphatidic acid (LPA) acts both as an intermediary in phospholipid metabolism and as an intercellular signaling molecule in its own right. In various cell types, LPA signals through its membrane-bound, G protein-coupled receptors to influence cellular processes such as proliferation, survival, and cytoskeletal function. Its actions in bone cells have not been studied. Here we show that the LPA receptor, LP A1 /edg-2/vzg-1, is expressed in primary rat osteoblasts and the UMR 106-01 osteoblastic cell line. LPA potently induces DNA synthesis and an increase in cell number in cultures of osteoblastic cells. LPA rapidly (within 10 min) stimulates phosphorylation of p42/44 mitogen-activated protein (MAP) ki-nases in osteoblastic cells, an effect that is sensitive to inhibition of G i proteins, inhibition of influx of extracellular calcium, and inhibition of protein kinase C. LPA-induced DNA synthesis is partially inhibited by either pertussis toxin or calphostin C, but is insensitive to specific inhibitors of MEK, the kinase upstream of p42/44 MAP kinases, or of phosphatidylinositol-3 kinases. These data demonstrate that LPA is an osteoblast mitogen whose signaling effects in osteoblastic cells include activation of p42/44 MAP kinases. However, the LPA mitogenic signal in osteoblastic cells, while requiring G i proteins and protein kinase C, is independent of the activity of p42/44 MAP kinases.
Journal of Nutritional Biochemistry, 2007
During bone remodelling bone is resorbed by osteoclasts and replaced again by osteoblasts through the process of bone formation. Clinical trials and in vivo animal studies suggest that specific polyunsaturated fatty acids (PUFAs) might benefit bone health. As the number of functional osteoblasts is important for bone formation the effects of specific PUFAs on in vitro osteoblastic cell proliferation were investigated. Morphological studies were conducted to determine whether exposure of the cells to these agents caused structural damage to the cells thereby yielding invalid results. Results from this study showed that arachidonic acid (AA) and docosahexaenoic acid (DHA) both inhibit cell growth significantly at high concentrations. The anti-mitotic effect of AA is possibly independent of PGE 2 production, as PGE 2 per se had little effect on proliferation. Further study is required to determine whether reduced proliferation due to fatty acids could be due to increased differentiation of osteoblasts to the mature mineralising osteoblastic phenotype.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
Previous studies have shown that phospholipase A 2 activity in rat costochondral chondrocyte cultures is differentially regulated by 1,25-(OH)2D 3 and 24,25-(OH)2D 3. 1,25-(OH)2D 3 stimulates enzyme activity in growth zone chondrocytes but has no effect on the resting zone chondrocyte enzyme activity. 24,25-(OH)2D 3 inhibits the resting zone enzyme but has no effect on the growth zone chondrocyte phospholipase A 2-This study examined whether the metabolites affect arachidonic acid turnover in their target cell populations. Incorporation and release of [ 14C]arachidonate was measured at various times following addition of hormone to the cultures. Acylation and reacylation were measured independently by incubating half of the [14C]arachidonate-labeled cultures with p-chloromercuribenzoate. The results demonstrated that the distribution of [14C]arachidonate in membrane phospholipids differed between growth zone and resting zone chondrocytes and between the plasma membranes and matrix vesicles isolated from the growth zone chondrocyte cultures. Plasma membrane phospholipids were more susceptible to the release of [14Clarachidonic acid by exogenous phospholipases than were matrix vesicle phospholipids. The effect of 1,25-(OH)2D 3 on growth zone chondrocytes was observed within 5 min. Incorporation was greatest after 60 min; release was greatest after 30 min. 24,25-(OH)2D 3 stimulated consistently elevated incorporation throughout the incubation period, peaking at 15 min. Peak release was at 60 min. The results confirm that resting zone chondrocytes and growth zone chondrocytes retain a differential phenotype in culture and demonstrate that matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25-(OH)2D 3 and 24,25-(OH)2D 3 appear to stimulate arachidonic acid turnover in their target cells by different mechanisms. Changes in fatty acid acylation and reacylation may be one mode of vitamin D-3 action in cartilage.