Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PGE2 is one of the key molecules in the osteoblast. It is the major prostanoid in the bone, and its production is under the control of both systemic and local factors. PGE2 has been reported to have multiple actions in the osteoblast, such as growth promotion and cell differentiation. To better understand the action of PGE2 in the osteoblast, we determined the PGE receptor subtypes in MC3T3-E1, an osteoblastic cell line derived from the normal mouse calvaria. Northern blot analysis revealed that EP1 and EP4 subtypes are expressed in MC3T3-E1. In contrast, EP3 subtype was not detected by either Northern blot analysis or RT-PCR. The contribution of each subtype was evaluated by studying the effects of subtype-specific analogs on osteoblastic function at confluency and 5 days after confluency. An EP1 agonist, 17-phenyl-omega-trinor PGE2, increased DNA synthesis and decreased alkaline phosphatase activity. 11-Deoxy-PGE1, and EP2 and EP4 agonist, decreased DNA synthesis and increased alkaline phosphatase activity at both stages. Butaprost, an EP2-selective agonist, showed effects similar to those of 11-deoxy-PGE1 only at confluency. Another and more differentiated osteoblastic marker, osteocalcin production, was detectable and was stimulated by 11-deoxy-PGE1 only 5 days after confluency. The exposure of these cells to EP1 agonist changed the cell shape to a more fibroblastic appearance. These results indicate that EP1, EP4, and probably EP2 are present in MC3T3-E1 cells; EP1 promotes cell growth, and EP2 and EP4 mediate differentiation of the osteoblast. Furthermore, the decreased response to EP2-specific agonist 5 days after confluency suggests that the expression of PGE receptor subtype is dependent on the stage of osteoblastic differentiation. This is the first report to determine PGE receptor subtypes in the bone.
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PMID:Prostaglandin E receptor subtypes in mouse osteoblastic cell line. 861 4

Prostaglandin E2 (PGE2) is an anabolic agent in vivo that stimulates bone formation by recruiting osteoblasts from bone marrow precursors. To understand which of the known PGE2 receptors (EP1-4) is involved in this process, we tested the effect of PGE2 and various EP agonists and/or antagonists on osteoblastic differentiation in cultures of bone marrow cells by counting bone nodules and measuring alkaline phosphatase activity. PGE2 increased both parameters, peaking at 100 nM, an effect that was mimicked by forskolin and was abolished by 2',3'-dideoxyadenosine (an adenylate cyclase inhibitor) and was thus cAMP dependent, pointing to the involvement of EP2 or EP4. Consistently, 17-phenyl-omega-trinor PGE2 (EP1 agonist) and sulprostone (EP3/EP1 agonist) lacked any anabolic activity. Furthermore, butaprost (EP2 agonist) was inactive, 11-deoxy-PGE1 (EP4/EP2 agonist) was as effective as PGE2, and the PGE2 effect was abolished dose dependently by the selective EP4 antagonist AH-23848B, suggesting the involvement of EP4. We also found that PGE2 increased nodule formation and AP activity when added for the initial attachment period of 24 h only. Thus this study shows that PGE2 stimulates osteoblastic differentiation in bone marrow cultures, probably by activating the EP4 receptor, and that this effect may involve recruitment of noncommitted (nonadherent) osteogenic precursors, in agreement with its suggested mode of operation in vivo.
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PMID:The anabolic effect of PGE2 in rat bone marrow cultures is mediated via the EP4 receptor subtype. 995 Jul 99

Resting zone chondrocyte differentiation is modulated by the vitamin D metabolite, 24,25-(OH)(2)D(3), via activation of protein kinase C (PKC). In previous studies, inhibition of prostaglandin production with indomethacin caused an increase in PKC activity, suggesting that changes in prostaglandin levels may mediate the 24, 25-(OH)(2)D(3)-dependent response and act as autocrine or paracrine regulators of chondrocyte metabolism. Supporting this hypothesis is the fact that resting zone cells respond directly to prostaglandin E(2) (PGE(2)). The aim of the present study was to identify which PGE(2) receptor subtypes (EP) mediate the effects of PGE(2) on resting zone cells. Using primers specific for EP1-EP4, reverse transcription-polymerase chain reaction (RT-PCR) amplified EP1 and EP2 cDNA in a RT-dependent manner. A variant form of the EP1 cDNA, EPlv, was also amplified in an RT-dependent manner. In parallel experiments, we used EP subtype-specific agonists to examine the role of EP receptors in 24,25-(OH)(2)D(3)-mediated cell proliferation and differentiation. 17-phenyl-trinor-PGE(2) (PTPGE(2)), an EP1 agonist, increased [(3)H]-thymidine incorporation in a dose-dependent manner and reversed the 24, 25-(OH)(2)D(2)-induced inhibition of [(3)H]-thymidine incorporation. SC-19220, an EP1 antagonist, caused a further dose-dependent decrease in 24,25-(OH)(2)D(3)-induced inhibition of [(3)H]-thymidine incorporation. PTPGE(2) also caused a biphasic increase in [(35)S]-sulfate incorporation and increased alkaline phosphatase enzyme activity at high concentrations (10(-8) M). 24, 25-(OH)(2)D(3)-induced alkaline phosphatase activity was synergistically stimulated in a dose-dependent manner by PTPGE(2). In contrast, 24,25-(OH)(2)D(3)-induced PKC activity was inhibited in a dose-dependent manner by PTPGE(2) and SC-19220, the EP1 antagonist, elevated PKC activity at high concentrations (10(-8) M). The EP2 agonist, misoprostol, only affected [(35)S]-sulfate incorporation, but in a dose-dependent manner. The EP3 and EP4 agonists had no effect on cell response. These results suggest that the EP1 receptor subtype mediates some of the PGE(2)-induced cellular responses in resting zone cells that lead to both increased proliferation and differentiation. Because 24,25-(OH)(2)D(3) inhibits PGE(2) synthesis in these cells, EP1-mediated induction of proliferation is blocked, encouraging cellular maturation and activation of PKC activity.
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PMID:Characterization of prostaglandin E(2) receptors and their role in 24,25-(OH)(2)D(3)-mediated effects on resting zone chondrocytes. 1062 83

Previous studies have shown that transforming growth factor-beta1 (TGF-beta1) stimulates protein kinase C (PKC) via a mechanism that is independent of phospholipase C or tyrosine kinase, but involves a pertussis toxin-sensitive G-protein. Maximal activation occurs at 12 h and requires new gene expression. To understand the signaling pathways involved, resting zone chondrocytes were incubated with TGF-beta1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7. [(35)S]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-beta1 on matrix production. However, there was little, if any, effect on TGF-beta1-dependent increases in [(3)H]thymidine incorporation, and TGF-beta1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC. TGF-beta1 caused a dose-dependent increase in prostaglandin E(2) (PGE(2)) production which was further increased by PKC inhibition. The increase was regulated by TGF-beta1-dependent effects on phospholipase A(2) (PLA(2)). Activation of PLA(2) inhibited TGF-beta1 effects on PKC, and inhibition of PLA(2) activated TGF-beta1-dependent PKC. Exogenous arachidonic acid also inhibited TGF-beta1-dependent increases in PKC. The effects of TGF-beta1 on PKC involve genomic mechanisms, but not regulation of existing membrane-associated enzyme, since no direct effect of the growth factor on plasma membrane or matrix vesicle PKC was observed. These results support the hypothesis that TGF-beta1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE(2) and protein kinase A (PKA). Inhibition of PKA also decreases TGF-beta1-dependent proliferation. We have previously shown that PGE(2) stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways.
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PMID:Transforming growth factor-beta1 regulation of resting zone chondrocytes is mediated by two separate but interacting pathways. 1077 Oct 99

Growth plate chondrocyte function is modulated by the vitamin D metabolite 1alpha,25-(OH)(2)D(3) via activation of protein kinase C (PKC). In previous studies with cells derived from prehypertrophic and upper hypertrophic zones of rat costochondral cartilage (growth zone cells), inhibition of prostaglandin production with indomethacin caused a decrease in the stimulation of PKC activity, suggesting that changes in prostaglandin levels mediate the 1alpha,25-(OH)(2)D(3)-dependent response in these cells. Growth zone cells also respond to PGE(2) directly, indicating that prostaglandins act as autocrine or paracrine regulators of chondrocyte metabolism in the growth plate. The aim of the present study was to identify which PGE(2) receptor subtypes (EP) mediate the effects of PGE(2) on growth zone cells. Using primers specific for EP1-EP4, reverse transcription-polymerase chain reaction (RT-PCR) amplified EP1 and EP2 cDNA in a RT-dependent manner. In parallel experiments, we used EP subtype-specific agonists to examine the role of EP receptors in 1alpha,25-(OH)(2)D(3)-mediated cell proliferation and differentiation. 17-Phenyl-trinor-PGE(2) (PTPGE(2)), an EP1 agonist, decreased [3H]-thymidine incorporation in a dose-dependent manner and augmented the 1alpha,25-(OH)(2)D(2)-induced inhibition of [3H]-thymidine incorporation. PTPGE(2) also caused significant increases in proteoglycan production, as measured by [35S]-sulfate incorporation, and alkaline phosphatase specific activity. 1alpha,25-(OH)(2)D(3)-induced alkaline phosphatase activity was only slightly stimulated by PTPGE(2). In contrast, 1alpha,25-(OH)(2)D(3)-induced PKC activity was synergistically increased by PTPGE(2), whereas EP1 antagonists SC-19220 and AH6809 inhibited PKC activity in a dose-dependent manner. The EP2, EP3 and EP4 agonists had no effect on the various cell-induced responses measured. EP1 receptor-induced responses were blocked by the phospholipase C inhibitor U73122, and reduced by PKA inhibitors. EP1 receptor-induced PKC activity was insensitive to pertussis toxin or choleratoxin but blocked by the G-protein inhibitor GDPbetaS, suggesting the involvement of G(q). These results suggest that the EP1 receptor subtype mediates various PGE(2)-induced cellular responses in growth zone chondrocytes leading to decreased proliferation and enhanced differentiation, as well as the effect of 1alpha,25-(OH)(2)D(3) on cellular maturation.
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PMID:Characterization of PGE(2) receptors (EP) and their role as mediators of 1alpha,25-(OH)(2)D(3) effects on growth zone chondrocytes. 1159 7

Transforming growth factor beta 1 (TGF-beta1) affects growth plate chondrocytes through Smad-mediated mechanisms and has been shown to increase protein kinase C (PKC). This study determined if PKC mediates the physiological response of rat costochondral growth zone (GC) chondrocytes to TGF-beta1; if the physiological response occurs via type II or type III TGF-beta receptors, and, if so, which receptor mediates the increase in PKC; and the signal transduction pathways involved. Treatment of confluent GC cells with TGF-beta1 stimulated [(3)H]thymidine and [(35)S]sulfate incorporation as well as alkaline phosphatase (ALPase) and PKC specific activities. Inhibition of PKC with chelerythrine, staurosporine, or H-7 caused a dose-dependent decrease in these parameters, indicating that PKC signaling was involved. TGF-beta1-dependent PKC and the physiological response of GC cells to TGF-beta1 was reversed by anti-type II TGF-beta receptor antibody and soluble type II TGF-beta receptor, showing that TGF-beta1 mediates these effects through the type II receptor. The increase in [3H]thymidine incorporation and ALPase specific activity were also regulated by protein kinase A (PKA) signaling, since the effects of TGF-beta1 were partially blocked by the PKA inhibitor H-8. The mechanism of TGF-beta1 activation of PKC is through phospholipase A(2) (PLA(2)) and not through phospholipase C (PLC). Arachidonic acid increased PKC in control cultures and was additive with TGF-beta1. Prostanoids are required, as indomethacin blocked the effect of TGF-beta1, and Cox-1, but not Cox-2, is involved. TGF-beta1 stimulates prostaglandin E(2) (PGE(2)) production and exogenous PGE(2) stimulates PKC, but not as much as TGF-beta1, suggesting that PGE(2) is not sufficient for all of the prostaglandin effect. In contrast, TGF-beta1 was not regulated by diacylglycerol; neither dioctanoylglycerol (DOG) nor inhibition of diacylglycerol kinase with R59022 had an effect. G-proteins mediate TGF-beta1 signaling at different levels in the cascade. TGF-beta1-dependent increases in PGE(2) levels and PKC were augmented by the G protein activator GTP gamma S, whereas inhibition of G-protein activity via GDP beta S, pertussis toxin, or cholera toxin blocked stimulation of PKC by TGF-beta1, indicating that both G(i) and G(s) are involved. Inhibition of PKA with H-8 partially blocked TGF-beta1-dependent PKC, suggesting that PKA inhibition on the physiological response was via PKA regulation of PKC signaling. This indicates that multiple interacting signaling pathways are involved: TGF-beta1 stimulates PLA(2) and prostaglandin release via the action of Cox-1 on arachidonic acid. PGE(2) activates the EP2 receptor, leading to G-protein-dependent activation of PKA. PKA signaling results in increased PKC activity and PKC signaling regulates proliferation, differentiation, and matrix synthesis.
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PMID:Transforming growth factor-beta1 regulation of growth zone chondrocytes is mediated by multiple interacting pathways. 1206 64

Phospholipase A2 (PLA2) is pivotal in the rapid membrane-mediated actions of 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. Microarray analysis indicated that PLA2 activating protein (PLAA) mRNA is upregulated 6-fold before rat growth plate cells exhibit 1alpha,25(OH)2D3-dependent protein kinase C (PKC) increases, suggesting that it plays an important role in 1alpha,25(OH)2D3's mechanism of action. PLAA mRNA was confirmed in 1alpha,25(OH)2D3-responsive growth zone (prehypertrophic and upper hypertrophic cell zones) chondrocytes by RT-PCR and Northern blot in vitro and by in situ hybridization in vivo. PLAA protein was shown by Western blot and immunohistochemistry. PLAAs role in 1alpha,25(OH)2D3 signaling was evaluated in growth zone cell cultures using PLAA peptide. Arachidonic acid release was increased as was PLA2-specific activity in plasma membranes and matrix vesicles. PKCalpha, but not PKCbeta, PKCepsilon, or PKCzeta, was increased. PLAAs effect was comparable to that of 1alpha,25(OH)2D3 and was additive with 1alpha,25(OH)2D3. PLA2 inhibitors quinacrine and AACOCF3, and cyclooxygenase inhibitor indomethacin blocked the effect of PLAA peptide on PKC, indicating arachidonic acid and its metabolites were involved. This was confirmed using exogenous arachidonic acid. Prostaglandin acted via EP1 based on inhibition by SC19220 and not via EP2 since AH6809 had no effect. Like 1alpha,25(OH)2D3, PLAA peptide also increased activity of phospholipase C-specific activity via beta-1 and beta-3 isoforms, but not delta-1 or gamma-1; the effect of PLAA was via lysophospholipid but not via arachidonic acid. PLAA peptide decreased [3H]-thymidine incorporation to 50% of the decrease caused by 1alpha,25(OH)2D3. In contrast, PLAA peptide increased alkaline phosphatase-specific activity and proteoglycan production in a manner similar to 1alpha,25(OH)2D3. This indicates that PLAA is a specific activator of PLA2 in growth plate chondrocytes, and suggests that it mediates the membrane effect of 1alpha,25(OH)2D3, thereby modulating physiological response.
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PMID:Phospholipase A2 activating protein (PLAA) is required for 1alpha,25(OH)2D3 signaling in growth plate chondrocytes. 1536 40

We compared the direct effects of selective EP4 and EP2 receptor agonists (EP4A and EP2A) with prostaglandin E(2) (PGE(2)) on the differentiation of cultured murine calvarial osteoblastic cells. EP4A increased alkaline phosphatase activity and osteocalcin mRNA levels in these cultures similar to PGE(2). This effect was seen with both "direct plating" immediately after isolating the cells, or "indirect plating" in which the cells were grown to confluence and replated. EP2A had a smaller effect, significant only in "indirect plating" experiments. All three agents decreased the DNA and protein content in indirect plating experiments, but not in direct plating experiments. We conclude that the anabolic effect of PGE(2) in calvarial osteoblastic cell cultures is largely mediated by activation of the EP4 receptor, while activation of the EP2 receptor is less effective.
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PMID:Effects of selective prostaglandins E2 receptor agonists on cultured calvarial murine osteoblastic cells. 1708 26

Studies using prostaglandin E receptor (EP) agonists indicate that prostaglandin (PG) E(2) can have anabolic effects through both EP4 and EP2 receptors. We previously found that the anabolic response to a selective EP4 receptor agonist (EP4A, Ono Pharmaceutical) was substantially greater than to a selective EP2 receptor agonist (EP2A) in cultured murine calvarial osteoblastic cells. To further define the role of the EP2 receptor in PG-mediated effects on bone cells, we examined the effects of EP2A and PGE(2) on both calvarial primary osteoblasts (POB) and marrow stromal cells (MSC) cultured from mice with deletion of one (Het) or both (KO) alleles of the EP2 receptor compared to their wild-type (WT) littermates. Deletion of EP2 receptor was confirmed by quantitative real-time PCR, Western blot and immunohistochemistry. The 1 month-old mice used to provide cells in these studies did not show any significant differences in their femurs by static histomorphometry. EP2A was found to enhance osteoblastic differentiation as measured by alkaline phosphatase mRNA expression and activity as well as osteocalcin mRNA expression and mineralization in the WT cell cultures from both marrow and calvariae. The effects were somewhat diminished in cultures from Het mice and abrogated in cultures from KO mice. PGE(2) effects were greater than those of EP2A, particularly in POB cultures and were only moderately diminished in Het and KO cell cultures. We conclude that activation of the EP2 receptor is able to enhance differentiation of osteoblasts, that EP2A is a true selective agonist for this receptor and that PGE(2) has an additional anabolic effect likely mediated by the EP4 receptor.
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PMID:Effect of deletion of the prostaglandin EP2 receptor on the anabolic response to prostaglandin E2 and a selective EP2 receptor agonist. 1840 86

While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the bone morphogenetic protein (BMP) signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2-treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the protein kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-beta to activate the TGF-beta-responsive reporter, 4XSBE. Finally, PGE2 down-regulated BMP-mediated phosphorylation of Smads 1, 5, and 8 in RCJ3.1C5.18 cells and in primary murine sternal chondrocytes. Altogether, the findings show that PGE2 regulates chondrocyte maturation in part by targeting BMP/Smad signaling and suggest an important role for PGE2 in endochondral bone formation.
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PMID:Prostaglandin E2 inhibits BMP signaling and delays chondrocyte maturation. 1902 95


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