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)

Using the c-fos enhancer as a model to analyze growth hormone (GH)-promoted gene expression, the roles of CCAAT/enhancer-binding proteins (C/EBPs) in GH-regulated transcription were investigated. In 3T3-F442A fibroblasts stably expressing the c-fos promoter mutated at the C/EBP binding site upstream of luciferase, c-fos promoter activity is stimulated by GH 6-7-fold; wild type c-fos promoter shows only a 2-fold induction by GH. This suggests that C/EBP restrains GH-stimulated expression of c-fos. Electrophoretic mobility shift assays with nuclear extracts from 3T3-F442A cells indicate that GH rapidly (2-5 min) increases binding of C/EBPbeta and C/EBPdelta, to the c-fos C/EBP binding site. Both liver activating protein (LAP) and liver inhibitory protein (LIP), forms of C/EBPbeta, are detected in 3T3-F442A cells by immunoblotting. GH increases the binding of LAP/LAP and LAP/LIP dimers. Overexpression of LIP interferes with GH-promoted reporter expression in CHO cells expressing GH receptors, consistent with the possibility that LIP restrains GH-stimulated c-fos expression. Overexpression of LAP elevates basal luciferase activity but does not influence promoter activation by GH, while overexpressed C/EBPdelta elevates basal promoter activity and enhances the stimulation by GH. GH stimulates the expression of mRNA for C/EBPbeta and -delta and increases levels of C/EBPdelta. Although C/EBPbeta is not detectably altered, GH induces a shift to more rapidly migrating forms of LIP and LAP upon immunoblotting. Treatment of extracts from GH-treated cells with alkaline phosphatase causes a shift of the slower migrating form to the rapidly migrating form, consistent with GH promoting dephosphorylation of LIP and LAP. These studies implicate C/EBPbeta and -delta in GH-regulated gene expression. They also indicate that GH stimulates the binding of C/EBPbeta and -delta to the c-fos promoter and promotes the dephosphorylation of LIP and LAP. These events may contribute to the ability of C/EBPbeta and -delta to regulate GH-stimulated expression of c-fos.
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PMID:CCAAT/enhancer-binding protein beta (C/EBPbeta) and C/EBPdelta contribute to growth hormone-regulated transcription of c-fos. 1053 66

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.
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PMID:Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2. 1054 21

The premise that bone cells are able to perceive and respond to mechanical forces is well accepted. This article describes the use of an in vivo hydraulic bone chamber for investigations of mechanical signal transduction. The servohydraulic loading mechanism was activated to apply a controlled compressive load to the woven trabecular bone that formed in one chamber, while the contralateral chamber served as an unloaded control. Specimens were harvested at a series of postload time points, and the cellular response to loading was evaluated by cytochemical, histomorphometric, and Northern blot analysis. A repetitive daily load stimulus elicited osteoblast biosynthetic activity characterized by an initial increase in type I procollagen by day 3 and a subsequent rise in alkaline phosphatase (ALP) activity after the sixth daily load episode. Application of a single load episode induced a biphasic pattern of c-fos and zif-268 gene expression with up-regulation at 30 minutes, down-regulation at 12 h, and up-regulation 24 h after the mechanical stimulus. The results show that a synchronized pattern of bone cell activity and gene expression occurs in response to controlled mechanical stimulation and that candidate load-responsive molecular mediators can be evaluated easily by this model.
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PMID:An in vivo model for investigations of mechanical signal transduction in trabecular bone. 1089 83

Vascular endothelial growth factor (VEGF) is a potent mitogen in endothelial cells, but little is known about its activity in other cell types. To clarify the role of VEGF in human dental pulp cells and pulp tissue, we investigated the effects of VEGF on the chemotaxis, proliferation, and differentiation of human dental pulp cells. VEGF induced a strong chemotactic response in human dental pulp cells in a dose-dependent manner. VEGF also marginally enhanced the proliferation of human dental pulp cells and induced an increase in alkaline phosphatase in human dental pulp cells. However, these effects of VEGF were not observed in reference to human skin fibroblasts. Analyses by the reverse-transcription/polymerase-chain-reaction method and flow cytometry showed that the mRNAs of two VEGF receptors, fins-like tyrosine kinase and kinase insert domain-containing receptor, were expressed in human dental pulp cells, whereas only fms-like tyrosine kinase mRNA was expressed in human skin fibroblasts. VEGF induced the activation of activator protein 1 (AP-1) and c-fos mRNA expression in human dental pulp cells. The AP-1 inhibitor curcumin strongly inhibited VEGF-induced alkaline phosphatase production in human dental pulp cells. In addition, VEGF antisense oligonucleotide suppressed the production of VEGF and alkaline phosphatase in human dental pulp cells. These results suggest that VEGF produced by human dental pulp cells acts directly upon human dental pulp cells in an autocrine manner, and may promote the chemotaxis, proliferation, and/or differentiation of human dental pulp cells via the utilization of kinase insert domain-containing receptor and in part through AP-1 by increasing c-fos.
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PMID:The role of vascular endothelial growth factor in human dental pulp cells: induction of chemotaxis, proliferation, and differentiation and activation of the AP-1-dependent signaling pathway. 1102 81

Growth hormone (GH) regulates transcription factors associated with c-fos, including C/EBPbeta. Two forms of C/EBPbeta, liver-activating protein (LAP) and liver inhibitory protein (LIP), are dephosphorylated in GH-treated 3T3-F442A fibroblasts. GH-induced dephosphorylation of LAP and LIP is reduced when cells are preincubated with phosphatidylinositol 3'-kinase (PI3K) inhibitors. GH activates Akt and inhibits glycogen synthase kinase-3 (GSK-3). Lithium, a GSK-3 inhibitor, increases GH-dependent dephosphorylation of LAP and LIP. Both are in vitro substrates of GSK-3, suggesting that GSK-3 inactivation contributes to GH-promoted dephosphorylation of C/EBPbeta. Alkaline phosphatase increases binding of LAP homodimers and decreases binding of LIP homodimers to c-fos, suggesting that dephosphorylation of C/EBPbeta modifies their ability to bind DNA. Both alkaline phosphatase- and GH-mediated dephosphorylation comparably increase binding of endogenous LAP in 3T3-F442A cells. In cells overexpressing LAP and GSK-3, LAP binding decreases, suggesting that GSK-3-mediated phosphorylation interferes with LAP binding. Expression of constitutively active GSK-3 reduced GH-stimulated c-fos promoter activity. These studies indicate that PI3K/Akt/GSK-3 mediates signaling between GH receptor and the nucleus, promoting dephosphorylation of C/EBPbeta. Dephosphorylation increases binding of LAP complexes to the c-fos promoter and may contribute to the participation of C/EBPbeta in GH-stimulated c-fos expression.
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PMID:Growth hormone regulates phosphorylation and function of CCAAT/enhancer-binding protein beta by modulating Akt and glycogen synthase kinase-3. 1127 38

Several studies in vitro and a few in vivo have suggested that mature osteoblasts heterogeneously express osteoblast markers. In one recent study of the osteoblasts associated with bone nodules formed in vitro in rat calvaria cell populations, extensive diversity was documented in the overall gene repertoires expressed. To address whether comparable heterogeneity is evident in vivo, we investigated the expression of nine osteoblast lineage markers by both in situ hybridization and immunohistochemistry. At 21 days of fetal rat development, the calvaria is a rapidly growing bone with distinct maturational zones that are readily observed in coronal sections; that is, an osteogenic front emerging at sagittal and coronal sutures is adjacent to areas of growing trabeculae of bone, followed by more mature areas of remodeling bone. Based on expression patterns, markers can be divided into two categories. One category comprises markers that are globally expressed by all osteoblasts irrespective of their position in the calvaria. Of those tested, only two, alkaline phosphatase and the pth/pthrp receptor, fit into this category. All other markers analyzed, including transcription factors (c-fos and msx-2), matrix molecules (bone sialoprotein, osteopontin, and osteocalcin), and a hormone (pthrp), were differentially expressed only in subpopulations of osteoblasts, based on cell maturational status, environment (ectocranial vs. endocranial surfaces), and microenvironment (adjacent osteoblasts). Preosteoblasts and osteocytes in different regions of the calvaria also expressed different subsets of the lineage markers. Mechanisms responsible for generating differential gene expression profiles appear to be both transcriptional and posttranscriptional. These results indicate that postproliferative, morphologically indistinguishable osteoblasts are not a homogeneous class of cells, but instead are molecularly diverse. The present results also raise the possibility that lineage progression and/or maintenance of the differentiated state may be adaptable in the calvaria.
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PMID:Individual osteoblasts in the developing calvaria express different gene repertoires. 1133 15

The periodontal ligament is subjected to mechanical loading during occlusion and mastication. Although internuclear transcription factors are associated with the regulatory pathway that converts these extracellular mechanical stimuli into a cellular response, there are no reports on these in human periodontal ligament fibroblasts. In this study, the amounts of c-fos mRNA in human periodontal ligament fibroblasts were investigated shortly after subjecting them to a cyclic tension force in vitro. The mRNA of alkaline phosphatase and the matrix proteins type I collagen, type III collagen, matrix Gla-protein, osteonectin, osteopontin, and osteocalcin were also examined. A significant, rapid, transient increase in c-fos mRNA was detected, which peaked 30 min after the application of mechanical force. However, there was no significant change in the mRNA for alkaline phosphatase or the matrix proteins. These results provide evidence that periodontal ligament fibroblasts and c-fos may play a critical part in the response of periodontal tissue to mechanical stimulation.
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PMID:The induction of c-fos mRNA expression by mechanical stress in human periodontal ligament cells. 1210 63

We studied the of effects raloxifene alone or in combination with human PTH (hPTH) 1-34 in mineralizing cultures of SaOS-2 cells. Raloxifene (10(-8)-10(-6) M) increased bone nodule formation in cultures of SaOS-2 cells when added intermittently from day 8 to day 17. A single 24-h treatment with 10(-8) M hPTH (1-34) at day 8 reduced the nodule area by 75.6% at day 17, and raloxifene added concomitantly with hPTH (1-34) reduced this inhibitory effect in a dose-dependent manner. Raloxifene also reduced the hPTH (1-34)-induced inhibition of alkaline phosphatase (ALP) activity. The 10-fold stimulation of c-fos mRNA expression by hPTH (1-34) was not influenced by raloxifene co-treatment. The protein kinase A (PKA) inhibitor 6-22 amide (1.7 nM) and the protein kinase C (PKC) inhibitor-bisindolylmaleimide 1 (10 nM) did not influence the separate effects of PTH and raloxifene on mineralized bone nodule formation. This is the first report on the interaction of PTH and raloxifene in an osteoblast culture system.
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PMID:Effect of raloxifene and its interaction with human PTH on bone formation. 1527 72

Marrow stroma cells (MSC) play a major role in osteogenesis. The potential of the MSC to differentiate to bone-forming cells relies upon molecular regulation. This study analyzed MBA-15 cells for the expression of genes and proteins that are key regulators of osteoblast differentiation. These cells express Cbfa1 and c-fos transcription factors (TF) of osteoprogenitor proliferating cells. RT-PCR and immunohistochemistry were used to demonstrate the message and protein expression of extracellular matrix proteins that are a prerequisite for matrix formation and mineralization, including alkaline phosphatase (ALP), osteocalcin, osteopontin, biglycan, and bone sialoprotein (BSP). The activity of ALP was correlated at various cell densities with co-expression of osteocalcin or osteopontin. Adhering cells must attach to the appropriate matrix to enable survival and differentiation. Using attachment assays, we demonstrated that MBA-15 cells adhered to collagenous matrix and the effect on survival measured by changes in intracellular calcium (Ca) levels. The cells' adhesion to matrix is mediated via cell surface molecules. We quantified the expression of cells surface molecules that are important players in mediating cell-matrix interaction. Flow cytometry analysis (FACS) was used to determine the expression of CD-31 (36%), and lower levels were identified for CD-62E and CD11b. In summary, the present study demonstrates the expression of molecular markers that are distinctive for the osteoblastic phenotype in MBA-15 marrow stroma cells and have crucial role in cell-matrix interaction, in establishing the cellular osteogenic phenotype and their survival.
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PMID:Characterization of adhesion and differentiation markers of osteogenic marrow stromal cells. 1538 28

Ca(2+)/calmodulin-dependent protein kinase IIalpha (alpha-CaMKII) was once thought to be exclusively expressed in neuronal tissue, but it is becoming increasingly evident that CaMKII is also expressed in various extraneural cells. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. The purpose of this study was to examine the expression of CaMKII in osteoblast-like cells (MC4) and to elucidate its role in osteoblast differentiation. We demonstrated that CaMKII, specifically the alpha isoform, is expressed in osteoblasts both in vitro and in vivo. Inhibition of CaMKII by the calmodulin antagonist trifluoperazine or the CaMKII antagonist KN93 reduces alkaline phosphatase activity and mineralization, as well as causes 85 and 56% decreases in alkaline phosphatase and osteocalcin gene expression, respectively. CaM and CaMKII antagonists, using the newborn mouse calvaria in vivo model, cause a 50% decrease in osteoblast number (N.Ob-BS) and a 32% decrease in mineralization (BV/TV). Pharmacologic and genetic inhibition of alpha-CaMKII by using trifluoperazine, KN93, and alpha-CaMKII small interfering RNA decreases the phosphorylation of ERK and of cAMP-response element-binding protein, leading to a significant decrease in the transactivation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII decreases the expression of c-fos, AP-1 transactivation, and AP-1 DNA binding activity. Our findings demonstrated that alpha-CaMKII is expressed in osteoblasts and is involved in c-fos expression via regulation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII results in a decrease in c-fos expression and AP-1 activation, leading to inhibition of osteoblast differentiation.
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PMID:Calmodulin and calmodulin-dependent kinase IIalpha regulate osteoblast differentiation by controlling c-fos expression. 1559 Jun 32


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