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)

Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.
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PMID:Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells. 1134 48

When osteoblasts are cultured on surfaces of increasing microroughness, they exhibit decreases in proliferation, increases in differentiation and local factor production, and enhanced response to 1alpha,25(OH)(2)D(3). The cells interact with surfaces through integrins, which signal by the same pathways used by 1alpha,25(OH)(2)D(3), including protein kinase C via phospholipase C and protein kinase A via phospholipase A(2). This provides opportunities for crosstalk that may contribute to the synergistic effects of surface roughness and the vitamin D metabolite. Because these pathways converge at mitogen-activated protein kinase (MAPK), we tested the hypothesis that the extracellular signal-regulated kinase (ERK1/2) subclass of MAPKs mediates the effects of surface roughness and 1alpha,25(OH)(2)D(3). MG63 osteoblast-like osteosarcoma cells were cultured on commercially pure Ti disks with various surface roughnesses: pretreatment (PT; 0.6 microm average roughness [Ra]), coarse grit-blasted and acid-etched (SLA; 4 microm RA), and titanium plasma-sprayed (TPS; 5.2-microm R(a)). At confluence, cells were treated for 24 h with control media or media containing 10(-7) M 1alpha,25(OH)(2)D(3). One-half of the cultures received 1 microm or 10 microm PD98059, a specific inhibitor of the ERK family of MAPKs. PD98059 alone did not affect proliferation, osteocalcin production, or production of transforming growth factor-beta1 or nitric oxide, regardless of the surface roughness. Alkaline phosphatase was reduced by the inhibition of the ERK family kinases on all surfaces to a comparable extent. However, when PD98059 was added to the cultures with 1alpha,25(OH)(2)D(3), the effects of the seco-steroid were blocked, including the synergistic increases seen in MG63 cells cultured on SLA or TPS. These results indicate that ERK1/2 MAPK is required for the maintenance of alkaline phosphatase at control levels and that the effects of 1alpha,25(OH)(2)D(3) are mediated by ERK1/2. However, the effects of surface roughness are not due to the ERK family of MAPKs. This suggests that alternative pathways may be used, including those mediated by other MAPK subclasses.
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PMID:Osteoblast response to titanium surface roughness and 1alpha,25-(OH)(2)D(3) is mediated through the mitogen-activated protein kinase (MAPK) pathway. 1137 60

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Galpha, which can sequester betagamma-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the betagamma-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.
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PMID:Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways. 1150 Mar 4

SOX9 is a transcription factor that is essential for chondrocyte differentiation and cartilage formation. We stably overexpressed SOX9 cDNA in the rat chondrocytic cell line CFK2. Compared with the vector control, a greater proportion of SOX9-transfected cells accumulated in the G0/G1 phase. This was associated with an increase in mRNA and protein expression of p21(cip1), an inhibitor of cyclin-dependent kinase activity. SOX9 enhanced p21(cip1) promoter activity in a luciferase reporter assay. CFK2 cells overexpressing SOX9 became more elongated and adhesive and demonstrated a shift in cytoplasmic F-actin distribution. N-cadherin mRNA levels were elevated in the SOX9-transfected cells, and SOX9 enhanced N-cadherin promoter activity. By electrophoretic mobility shift assay, nuclear extracts of SOX9-transfected CFK2 cells specifically bound an oligonucleotide comprising an N-cadherin promoter region containing a consensus SOX9-binding motif. The transcriptional activity of SOX9 depended upon nuclear localization signals required for SOX9 nuclear entry. Differentiation of transfected CFK2 cells was accelerated as evidenced by more rapid accumulation of alkaline phosphatase activity, increased production of proteoglycans, and increased calcium accumulation, and this was associated with decreased ERK1 expression. These studies demonstrate that SOX9 alters the rate of cell cycle progression of chondrocytes and their differentiation by enhancing or inhibiting the expression of selected genes, including p21(cip1) and ERK1, and that N-cadherin is an additional direct target of this transcriptional regulator.
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PMID:The transcription factor SOX9 regulates cell cycle and differentiation genes in chondrocytic CFK2 cells. 1151 54

It has been shown that thyroid hormone stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated whether p44/p42 mitogen-activated protein (MAP) kinase is involved in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. Triiodothyronine (T(3)) markedly induced the phosphorylation of p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly enhanced the T(3)-induced alkaline phosphatase activity in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by T(3) was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase takes part in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblasts and that p44/p42 MAP kinase plays an inhibitory role in the thyroid hormone-effect.
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PMID:Activation of p44/p42 mitogen-activated protein kinase limits triiodothyronine-stimulated alkaline phosphatase activity in osteoblasts. 1152 18

We have characterized the effects of different short-chain fatty acids (SCFAs) on cell growth and differentiation as well as the phosphorylation state of ERK1 and 2 in the human colon adenocarcinoma cell line HT-29. Of the five SCFAs tested, only butyrate and propionate impaired cellular proliferation. Moreover, butyrate and propionate specifically resulted in a decrease in ERK1 and 2 phosphorylation at 3 and 6 hours post-treatment, suggesting a correlation between the ability of these SCFAs to inhibit cellular proliferation and decrease ERK phosphorylation. Notably, the decrease in ERK phosphorylation was observed prior to the induction of the differentiation markers alkaline phosphatase (AP) and carcinoembryonic antigen (CEA) by butyrate and propionate from days 6 to 18 post-treatment. In the case of butyrate- and propionate-induced differentiation, ERK phosphorylation is a marker and may play a role in the proliferation and/or differentiation states of this cell line.
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PMID:Butyrate and propionate downregulate ERK phosphorylation in HT-29 colon carcinoma cells prior to differentiation. 1153 73

We have investigated the effect of 1-(5-oxohexyl)-3,7-dimethylxanthine or pentoxifylline (PeTx), a nonselective phosphodiesterase inhibitor, on osteoblastic differentiation in vitro by using two mesenchymal cell lines, C3H10T1/2 and C2C12, which are able to acquire the osteoblastic phenotype in the presence of bone morphogenetic protein-2 (BMP-2). PeTx induced the osteoblastic markers, osteocalcin and Osf2/Cbfa1, in C3H10T1/2 and C2C12 cells and enhanced BMP-2-induced expression of osteocalcin, Osf2/Cbfa1, and alkaline phosphatase. This activity was partially attributed to the fact that PeTx is able to enhance BMP-2-induced Smad1 transcriptional activity. Although PeTx clearly stimulates PKA in these cells, neither pretreatment of cells with the PKA inhibitor H89 nor transfection with the specific PKA inhibitor PKI prevented the induction or enhancement of osteoblast markers by PeTx, demonstrating that these effects were independent of PKA activation. On the other hand, PeTx induced the activation of ERK1/2 and p38 kinase pathways independently of the activation of PKA. Selective inhibitors of these MAPK cascades prevented the induction of osteoblastic markers in cells treated with PeTx, suggesting that the activation of these two pathways plays a role in the effect of PeTx on osteoblastic differentiation.
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PMID:1-(5-oxohexyl)-3,7-Dimethylxanthine, a phosphodiesterase inhibitor, activates MAPK cascades and promotes osteoblast differentiation by a mechanism independent of PKA activation (pentoxifylline promotes osteoblast differentiation). 1160 32

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-induced versus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.
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PMID:The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. 1171 95

Bone morphogenetic proteins (BMPs) transdifferentiate C2C12 cells from the myogenic to the osteogenic lineage. In this work we examine the role of the phosphatidylinositol 3-kinase/p70 S6 kinase (PI3K/p70 S6K) and p38 mitogen-activated protein kinase (p38 MAPK) cascades in the osteogenic effects of BMP-2. BMP-2 stimulated both cascades transiently (maximal at 1 h and decreasing thereafter). In contrast, BMP-2 had no effect on p42/p44 MAPK (Erks) stimulation. We also analyzed the effects of selective inhibitors of these pathways on the expression of osteogenic markers. Inhibitors of p38 MAPK (SB203580) or the PI3K/p70 S6K pathway (Ly294002 and rapamycin) not only fail to block the osteoblast phenotype induced by BMP-2, measured as induction of Cbfa1 expression and transcriptional activity, but also potentiate the effect of BMP-2 on late osteoblast markers, such as alkaline phosphatase activity and osteocalcin expression. These data suggest that, in contrast to their positive effect on myogenic differentiation, PI3K/p70 S6K and p38 MAPK cascades have a negative role in osteoblast differentiation.
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PMID:Inhibition of PI3K/p70 S6K and p38 MAPK cascades increases osteoblastic differentiation induced by BMP-2. 1175 39

Several transmembrane transporters of organic compounds are regulated by phosphorylation/dephosphorylation mechanisms. The aim of this study was to investigate the possible regulation of the human extraneuronal monoamine transporter, hEMT, by these mechanisms. The experiments were performed using HEK293 cells stably transfected with pcDNA3hEMT (293hEMT). The characteristics of hEMT-mediated uptake of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) were studied by incubating the cells at 37 degrees C for 1 min with 200 nM [3H]MPP+. Uptake of [3H]MPP+ by 293hEMT cells was not affected or only slightly reduced by modulators of protein kinase A, protein kinase C, or protein kinase G. It was not affected by an inhibitor of protein tyrosine kinase and was reduced by mitogen-activated protein kinase inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was independent of extracellular Ca2+ and strongly reduced by Ca2+/calmodulin pathway inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced in the presence of non-selective phosphodiesterase inhibitors (IBMX, caffeine, theophylline). The effect of IBMX was independent of extracellular Ca2+ its IC50 was found to be 82.0 microM (66.2-101.6 microM; n=4), and its inhibitory effect resulted from a significant decrease in the maximal velocity of [3H]MPP+ uptake, with no change in the Michaelis-Menten constant. [3H]MPP+ uptake was reduced by 8-methoxy-methyl-IBMX, a selective inhibitor of the Ca2+/calmodulin-dependent phosphodiesterase (PDE1), but not by zaprinast, a selective inhibitor of PDE5. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced by protein tyrosine phosphatase inhibitors, by an alkaline phosphatase inhibitor and, by contrast. showed an increase in the presence of exogenous alkaline phosphatase. In conclusion, these results suggest that hEMT is regulated by phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state.
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PMID:Regulation of human extraneuronal monoamine transporter (hEMT) expressed in HEK293 cells by intracellular second messenger systems. 1177 2


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