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)

A nonradioactive receptor binding assay for ciliary neurotrophic factor (CNTF) is described. The assay is based on the interaction between biotinylated human CNTF, soluble gp130, and soluble myc-tagged CNTF receptor captured on a microtiter plate via an antibody against the myc epitope tag. Bound cytokine is revealed by alkaline phosphatase-conjugated avidin. Purified human and rat CNTF competed with biotinylated CNTF for receptor binding, with IC50 values of 29 and 2 nM, respectively. Since the higher affinity of rat vs human CNTF has been previously shown to be conferred by the arginine residue at position 63 of the rat protein, we also tested a human CNTF mutant carrying a Q63R substitution. Secreted forms of wild-type and mutant CNTF were expressed in Escherichia coli, and the amount of cytokines in periplasmic extracts was determined by quantitative Western blotting analysis. The human CNTF mutant (Q63R, N137S) was found to compete with biotinylated CNTF for binding to soluble CNTF receptor with an eightfold higher apparent affinity than wild-type human CNTF. The present method thus faithfully reproduces the relative activities of CNTF analogs determined in other assay systems. The possibility of assaying cytokines in crude bacterial extracts makes the new technique particularly suitable for rapidly determining the receptor binding potencies of genetically engineered CNTF variants.
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PMID:Nonradioactive receptor binding assay for ciliary neurotrophic factor. 781 Aug 82

Leukemia inhibitory factor (LIF) is structurally related to interleukin-6 (IL-6), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF). Since LIF-deficient mice do not exhibit overt phenotypic effects in cell types known to be targets for LIF action in vitro, we examined the ability of IL-6, OSM, and CNTF to reproduce the effects of LIF in five different bioassays. OSM, CNTF, and LIF are able to promote embryonic stem cell growth and to maintain them in an undifferentiated state as marked by a high alkaline phosphatase activity (ED50 are, respectively, 0.5, 3 and 1 ng/ml). Whereas LIF and OSM maintain close to 100% of ES cells in an undifferentiated state, CNTF, at optimal concentrations, prevents differentiation of only 60% of the ES population. Murine 7TD1 hybridoma cell growth is induced only in the presence of IL-6 (ED50 = 0.1 ng/ml). Both LIF and OSM stimulate DA1a cell proliferation (ED50 are, respectively, 1 and 12 ng/ml). OSM appears, therefore, to act as a weak agonist of LIF-dependent processes on murine cells, however, with a 10-fold lower specific activity than that of LIF, which is in agreement with human OSM cross-reacting with the murine LIF-R. Though IL-6, LIF, and OSM all stimulate haptoglobin and fibrinogen production by human HepG2 hepatoma cells, the dose-response curves of these three factors exhibit very different characteristics. CNTF stimulates acute-phase protein production by HepG2 cells only at high concentrations (greater than 1 microgram/ml). A549 epithelial cells are subjected to growth inhibition only in the presence of OSM (ED50 = 6 ng/ml), even though they expressed LIF-R and gp130 transcripts. These data suggest that OSM and LIF act on human cells through different receptors. Altogether, these results indicate that none of the factors examined in this study are precisely interchangeable in terms of their biological actions.
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PMID:Are LIF and related cytokines functionally equivalent? 805 Apr 91

The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone-induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor alpha protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.
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PMID:Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein. 812 83

The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.
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PMID:Identification and expression cloning of a leptin receptor, OB-R. 1505 99

Osteoblasts and their precursors respond to specific cytokines, growth factors, and hormones. One facet of this response includes the secretion of additional cytokines, some of which are part of the circuitry involved in the regulation of osteoblast and osteoclast function. Therefore, understanding which cytokines are able to activate osteoblastic cells and the consequences of that activation are central to understanding normal and pathologic bone remodeling. Oncostatin M (OSM) is a glycoprotein belonging to a new subfamily of cytokines related by sequence and structural homology and the use of the signal transducing receptor component gp130. Osteoblastic cells secrete and respond to leukemia-inhibiting factor (LIF) both in vitro and in vivo, suggesting that LIF is an autocrine regulatory factor. OSM is closely related to LIF, and therefore we hypothesized that OSM should regulate the function of cells in the osteoblastic lineage. Primary neonatal murine or fetal rat calvarial osteoblastic cultures were treated with OSM or LIF and a series of biochemical and biological parameters were determined. In these cultures, OSM induced proliferation, collagen synthesis, and interleukin-6 secretion, whereas it inhibited alkaline phosphatase activity. Bone resorption was also inhibited by OSM. These data represent the first report of OSM's effects on bone cell function and indicate that, like some other members of the LIF/interleukin-6 subfamily, OSM has potent bone regulatory activity.
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PMID:Oncostatin-M: a new bone active cytokine that activates osteoblasts and inhibits bone resorption. 862 82

We have previously established that stromal/osteoblastic cells collectively express receptors for all members of the cytokine subfamily that share the gp130 signal transducer and that different receptor repertoires may be expressed at different stages of differentiation of this lineage. We have now used human (MG-63) and murine (MC3T3-E1) osteoblastic cell lines as well as primary murine calvaria cells to test the hypothesis that these receptors mediate effects of the cytokines on the biology of osteoblasts. We report that as in other cell types, all of the osteoblastic cell models responded to interleukin-6 (IL-6)-type cytokines with activation of both the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and the mitogen-activated protein kinase (MAPK) pathways. In addition, IL-6-type cytokines stimulated alkaline phosphatase activity and osteocalcin expression and inhibited (MG-63), stimulated (MC3T3-E1), or had no effect (calvaria cells) on the rate of cell proliferation. The ability of a given cell type to respond to a particular member of this family of cytokines was strictly dependent on the presence of the corresponding ligand-binding subunit (alpha) of the cytokine receptor, and the magnitude of all the effects was closely correlated with the concentration of this subunit. The relative contribution of the JAK/STAT and MAPK pathways to the biological effects of the cytokines was evaluated using kinase inhibitors. Cytokine-mediated modulation of cell proliferation as well as stimulation of alkaline phosphatase activity were abrogated by tyrosine kinase inhibitors as well as a threonine/serine kinase inhibitor, but were only minimally affected by a specific inhibitor of MAPK phosphorylation. These results demonstrate that IL-6-type cytokines, besides their osteoclastogenic properties, promote differentiation of committed osteoblastic cells toward a more mature phenotype and that this action is mediated primarily via the activation of the JAK/STAT pathway.
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PMID:Activation of the Janus kinase/STAT (signal transducer and activator of transcription) signal transduction pathway by interleukin-6-type cytokines promotes osteoblast differentiation. 927 51

Interleukin (IL)-6, IL-11, leukemia inhibitory factor, and oncostatin M similarly induce osteoclast formation in cocultures of osteoblastic cells and bone marrow cells. These cytokines share a common signal transducer, gp130, which forms a receptor complex with the specific receptor for each cytokine. To investigate the role of gp130 in osteoclast development, we examined bone tissues in gp130-deficient and wild-type newborn mice of the ICR background. Soft x-ray radiographs and microfocus x-ray computed tomographs revealed that bone marrow cavities were present in tibiae and radii of both wild-type and gp130-deficient mice. Microfocus x-ray computed tomography and histological examination demonstrated a decrease in the amount of trabeculae at the metaphysial region in tibiae and radii of the gp130-deficient mice compared with the wild-type mice. The number ofosteoclasts in gp130-deficient mice was about double that in the wild-type mice. There were no apparent differences in the distributions of alkaline phosphatase-positive osteoblasts and the osteoid surface on the trabecular bone at the metaphysial region between the wild-type and gp130-deficient mice. The volume of mineralized trabecular bones was also decreased at mandibulae, accompanied by the increased number of osteoclasts in gp130-deficient mice compared with the wild-type and heterozygous mice. These results suggest that the formation of osteoclasts is not solely dependent on gp130 signaling, at least during fetal development. The osteoclastic bone resorption in gp130-deficient mice may be caused by the functional redundancy of bone-resorbing hormones and cytokines other than those of the IL-6 family.
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PMID:Osteoclasts are present in gp130-deficient mice. 934 27

Studies on the role of interleukin-6 (IL-6) in bone metabolism have been accumulating. However, its effects on osteoblasts are still unclear because the results are conflicting depending on the study models employed. We reasoned that these conflicting data are due to variable expression levels of membrane-bound IL-6 receptors (IL-6Rs). In the present study, we found that IL-6 in combination with soluble IL-6R (sIL-6R) consistently caused a marked elevation of alkaline phosphatase and a decrease in proliferation in the human osteoblastic cell line MG-63, which expressed no detectable membrane-bound IL-6R and failed to respond to IL-6. These effects of IL-6/sIL-6R were blocked by neutralizing antibodies to the IL-6 signal transducer gp130, suggesting an involvement of IL-6 signaling in the elicitation of the effects of IL-6/sIL-6R. Upon stimulation with IL-6/sIL-6R, the gp130, cytoplasmic Janus kinases JAK1 and JAK2 were tyrosine phosphorylated. Moreover, signal transducers and activators of transcription STAT1 and STAT3 were also tyrosine phosphorylated, translocated to the nucleus, and bound to the putative STAT-binding DNA elements. In addition, mitogen-activated protein (MAP) kinase was also activated in response to IL-6/sIL-6R These data demonstrate that sIL-6R may enhance the responsiveness of MG-63 cells to IL-6. Thus, IL-6 in collaboration with sIL-6R may modulate differentiation and proliferation of osteoblastic cells, presumably by activating two distinct signaling pathways of JAK-STAT and MAP kinase.
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PMID:Combination of interleukin-6 and soluble interleukin-6 receptors induces differentiation and activation of JAK-STAT and MAP kinase pathways in MG-63 human osteoblastic cells. 961 Jul 41

The cyclin-dependent kinase inhibitor p21(WAF1,CIP1,SDI1) plays a critical role in cell differentiation, and it has been shown to confer resistance to apoptosis. Based on this, and on evidence that activation of the gp130/signal transducer and activator of transcription (STAT) signal transduction pathway by interleukin (IL)-6 type cytokines promotes differentiation and prevents apoptosis in osteoblastic cells, we have investigated the possibility that p21 is a downstream effector of this signaling pathway in osteoblasts. We report that either oncostatin M (OSM) or IL-6 plus soluble IL-6 receptor increased the levels of p21 mRNA and protein in the osteoblast-like human osteosarcoma cell line MG63 and stimulated the activity of a 2.4-kilobase pair segment of the human p21 gene promoter. Further, nuclear extracts from cytokine-stimulated MG63 cells formed protein-DNA complexes with a 19-base pair nucleotide fragment of the p21 promoter containing a single STAT response element. The identity of the binding proteins as Stat3 and Stat1 was demonstrated with specific antibodies. In addition, and in support of a mediating role of STATs in the activation of the p21 promoter, overexpression of Stat3 potentiated the cytokine effect on the p21 promoter; whereas a dominant negative Stat3, or a mutation of the STAT response element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibited OSM-induced stimulation of alkaline phosphatase expression and antagonized the protective effect of OSM on anti-Fas-induced apoptosis. These results demonstrate that p21 is a downstream effector of gp130/Stat3 activation and a critical mediator of the pro-differentiating and anti-apoptotic effects of IL-6 type cytokines on human osteoblastic cells.
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PMID:Transcriptional activation of the p21(WAF1,CIP1,SDI1) gene by interleukin-6 type cytokines. A prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells. 969 69

We previously demonstrated that embryonic stem (ES) cell self-renewal required sustained signaling by leukemia inhibitory factor (LIF) in a concentration-dependent manner, allowing us to hypothesize that thresholds in ligand-receptor signaling modulate stem cell differentiation control. To test this hypothesis, we have experimentally and computationally compared the abilities of two gp130-signaling cytokines (LIF and Hyper-interleukin-6 [HIL-6]) to sustain ES cell self-renewal. Quantitative measurements of ES cell phenotypic markers (stage-specific embryonic antigen-1 and E-cadherin), functional assays (alkaline phosphatase activity and embryoid body formation efficiency), and transcription factor (Oct-4) expression over a range of LIF and HIL-6 concentrations demonstrated a superior ability of LIF to maintain ES cell pluripotentiality at higher concentrations (> or =500 pM). Additionally, we observed distinct qualitative differences in the ES cell self-renewal dose response profiles between the two cytokines. A computational model permitted calculation of the number of signaling complexes as a function of receptor expression, ligand concentration, and ligand/receptor-binding properties, generating predictions for the degree of self-renewal as a function of cytokine concentration by comparison of these calculated complex numbers to experimentally determined threshold cytokine concentrations. Model predictions, consistent with experimental data, indicated that differences in the potencies of these two cytokines were based primarily on differences in receptor-binding stoichiometries and properties. These results support a ligand/receptor signaling threshold model of ES cell fate modulation through appropriate types and levels of cytokine stimulation. Insights from these results may be more generally applicable to tissue-specific stem cells and could aid in the development of stem cell-based technologies.
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PMID:Ligand/receptor signaling threshold (LIST) model accounts for gp130-mediated embryonic stem cell self-renewal responses to LIF and HIL-6. 1189 69


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