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)

Cytokine-induced expression of the endothelial cell surface adhesion molecule E-selectin is inhibited by glucocorticoids (GCs). To investigate possible mechanisms for steroid inhibition, a reporter gene (ESAP) was constructed, comprising the cytokine responsive region of the E-selectin gene (nt -383 to +81) coupled to alkaline phosphatase (AP). In A549 cells stably transfected with the ESAP gene, AP production was highly responsive to the cytokines interleukin 1beta (IL-1beta) and tumour necrosis factor alpha, with ED50 values of 3 pM and 1000 pM respectively. Furthermore the cytokine-induced AP responses were inhibited by GCs, indicating that both transcriptional activation and GC suppression of the E-selectin gene were mediated via regulatory elements within the same region of the promoter. The relative potencies of GC drugs as inhibitors of IL-1beta (10 pM)-stimulated ESAP-gene activation were fluticasone> beclomethasone>dexamethasone, with IC50 values of 0.13, 1.1 and 2.7 nM respectively. Inhibition by fluticasone was blocked by the GC receptor (GR) antagonist drug mifepristone (Ru486), which is consistent with the suppressive effects of GCs being mediated via the GR. However, because the E-selectin promoter lacks a consensus glucocorticoid responsive element, mechanisms for inhibition independent of GR-DNA binding were investigated. Evidence that GCs also inhibited cytokine activation of a synthetic nuclear factor kappaB (NFkappaB)-driven reporter gene transiently transfected into A549 cells suggested that interference with the activation and/or function of this transcription factor was important for GC inhibition of ESAP. However, in A549-ESAP cells, fluticasone (100 nM) did not affect IL-1beta (10 pM)-induced IkBalpha degradation, NFkappaB-p65 nuclear translocation or the DNA-binding capacity of nuclear NFkappaB complexes, over a period during which cytokine-induced ESAP-gene activation was inhibited. Finally, there was no evidence to suggest that GC enhancement of IkBalpha gene expression contributed to the suppression of the cytokine response. We conclude that interference by GR with the transcriptional activation potential of DNA-bound NFkappaB complexes might contribute to mechanisms underlying the anti-inflammatory effects of GCs.
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PMID:Induction of the E-selectin promoter by interleukin 1 and tumour necrosis factor alpha, and inhibition by glucocorticoids. 937 35

Reduced cell surface levels of major histocompatibility complex class I antigens enable adenovirus type 12 (Ad12)-transformed cells to escape immunosurveillance by cytotoxic T lymphocytes (CTL), contributing to their tumorigenic potential. In contrast, nontumorigenic Ad5-transformed cells harbor significant cell surface levels of class I antigens and are susceptible to CTL lysis. Ad12 E1A mediates down-regulation of class I transcription by increasing COUP-TF repressor binding and decreasing NF-kappaB activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-kappaB in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-kappaB dimers reconstituted from denatured and renatured p50 and p65 subunits from Ad12- and Ad5-transformed cell nuclear extracts demonstrated that p50, and not p65, is responsible for the decreased ability of NF-kappaB to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-kappaB to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-kappaB binding was further demonstrated by showing that an NF-kappaB dimer composed of p65 and alkaline phosphatase-treated p50 from Ad5-transformed cell nuclear extracts could not bind to DNA. These results suggest that phosphorylation of p50 is a key step in the nuclear regulation of NF-kappaB in adenovirus-transformed cells.
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PMID:Reduced phosphorylation of p50 is responsible for diminished NF-kappaB binding to the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells. 1002 3

Polyamines-putrescine, spermidine, and spermine-are involved in the growth of breast cancer cells. A possible target of polyamine action is at the site of interaction of transcription factors with their response elements. NF-kappaB is a member of the rel family of transcription factors that regulate transcription of genes in the proliferative/anti-apoptotic pathways. We performed electrophoretic mobility shift assays to study the role of polyamines in NF-kappaB binding to NF-kappaB response elements (NREs), the consensus sequence of which is GGGGAATTCCCC. Using cellular extract from MCF-7 breast cancer cells, we found very little binding of NF-kappaB to NRE in the absence of polyamines. Addition of 1 mM spermidine or spermine caused a 4- and 6-fold increase in NF-kappaB-NRE binding, respectively. Putrescine induced a 2-fold increase in the binding at 2 mM concentration. Using antibody supershift assays, we identified the p50 subunit of NF-kappaB to be a major component in NF-kappaB-NRE complex formation in the presence of polyamines. However, the decreased intensity of the band corresponding to NF-kappaB-NRE complex in the presence of anti-p65, c-rel, relB and p52 antibodies suggested the participation of these subunits also. Spermine also stimulated NF-kappaB-NRE binding using cellular extracts from other breast cancer cell lines and a normal breast epithelial cell line. A differential effect of spermine analogues on NF-kappaB-NRE binding was observed, with spermine exerting the maximal effect. CD spectra of NRE containing oligonucleotides was asymmetric and distinct from that of a typical B-DNA CD spectrum. A concentration-dependent increase in T(m) of the duplex NRE was seen in the presence of polyamines. In transient transfection experiments using an NF-kappaB driven secreted alkaline phosphatase (SEAP) reporter, spermine induced NF-kappaB activity by approximately 2-fold as compared to controls. Spermine induced activation of NF-kappaB was also confirmed using an NF-kappaB-EGFP (enhanced green fluorescent protein) vector in transient transfections in which expression of the green fluorescent protein was visualized by fluorescence microscopy. These data show a gene regulatory function of polyamines involving enhanced binding of NF-kappaB to NRE and a possible mechanism for the action of polyamines in breast cancer cell proliferation.
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PMID:Activation of nuclear factor kappaB by polyamines in breast cancer cells. 1055 58

More than 80% of human ovarian cancers express LHRH and its receptor as part of a negative autocrine mechanism of growth control. This study was conducted to investigate whether LHRH affects apoptosis in ovarian cancer. EFO-21 and EFO-27 ovarian cancer cells were treated with LHRH agonist Triptorelin or with cytotoxic agent Doxorubicin in the absence or presence of Triptorelin. Apoptotic cells were quantified by flow cytometry. Expression of nuclear factor kappa B (NFkappaB) was assessed by RT-PCR and immunoblotting. For determination of Triptorelin-induced NFkappaB activation, cells were transfected with a NFkappaB-secreted alkaline phosphatase reporter gene plasmid (pNFkappaB-SEAP) and cultured for 96 h with or without Triptorelin. The causal relation between Triptorelin-induced NFkappaB activation and Triptorelin-induced protection against apoptosis was investigated using SN50, an inhibitor for nuclear translocation of activated NFkappaB. Apoptosis induction by Triptorelin was never observed. Treatment with Doxorubicin (1 nmol/L) for 72 h increased the percentage of apoptotic cells in EFO-21 and EFO-27 ovarian cancer cell lines to 31% or 34%, respectively. In cultures treated simultaneously with Triptorelin (100 nmol/L), the percentage of apoptotic cells was reduced significantly, to 17% or 18%, respectively (P < 0.001). RT-PCR and immunoblotting experiments showed that NFkappaB subunits p50 and p65 were expressed by ovarian cancer cell lines EFO-21 and EFO-27. When EFO-21 or EFO-27 cells were transfected with pNFkappaB-SEAP and subsequently treated with Triptorelin (100 nmol/L), NFkappaB-induced SEAP expression increased 5.3-fold or 4.7-fold, respectively (P < 0.001). Triptorelin-induced reduction of Doxorubicin-induced apoptosis was blocked by SN50-mediated inhibition of NFkappaB translocation into the nucleus. We conclude that LHRH induces activation of NFkappaB and thus reduces Doxorubicin-induced apoptosis in human ovarian cancer cells. This possibility to protect ovarian cancer cells from programmed cell death is an important feature in LHRH signaling in ovarian tumors, apart from the inhibitory interference with the mitogenic pathway.
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PMID:Luteinizing hormone-releasing hormone induces nuclear factor kappaB-activation and inhibits apoptosis in ovarian cancer cells. 1106 44

Diseases requiring frequent and lifelong injections of recombinant proteins would be more efficaciously treated by intramuscular delivery of genes encoding secretable proteins. However, the success of this approach largely depends on our capability to temporally regulate transcription of delivered genes. Therefore, we sought to generate a humanized transcription factor to regulate transgene expression in muscle. A novel 4-hydroxytamoxifen (4-OHT)-dependent transcriptional regulator (called HEA-3) was constructed by fusing in-frame the DNA binding domain of the human hepatocyte nuclear factor-1alpha (HNF1alpha), which is not expressed in muscle cells, a G(521)R mutant of the ligand binding domain of human estrogen receptor-alpha (ERalpha), and the activation domain derived from human nuclear factor-kappaB p65 subunit (NF-kappaB p65). We demonstrate that an artificial promoter containing multimeric HNF1alpha binding sites is silent in muscles and in cell lines that lack endogenous HNF1alpha. HEA-3 stimulated transcription from this target promoter in a stringent 4-OHT-dependent manner. The dynamic range of transgene regulation was high, because of the low basal activity and high inducibility of the system. Ex vivo, HEA-3 increased expression of the transfected reporter gene by more than 1000-fold in a ligand-dependent manner. In vivo, HEA-3 stimulated by more than 100-fold, the expression of secreted alkaline phosphatase after delivery as plasmid DNA into mouse muscles. Moreover, long-term modulation of the expression of intramuscularly delivered mouse erythropoietin was achieved in immunocompetent mice.
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PMID:Long-term and tight control of gene expression in mouse skeletal muscle by a new hybrid human transcription factor. 1240 64

In this report, we describe that NF-kappaB is spontaneously activated in isolated, normal glomeruli. Ex vivo incubation of isolated rat glomeruli triggered expression of a NF-kappaB-dependent gene, monocyte chemoattractant protein-1 (MCP-1), in parallel with downregulation of IkappaBalpha and IkappaBbeta proteins and activation of the p65 NF-kappaB subunit. The induction of MCP-1 was also observed in mesangial cells coincubated with isolated glomeruli or exposed to media conditioned by isolated glomeruli (GCM), which was abrogated by inhibition of NF-kappaB. The activation of NF-kappaB by glomerulus-derived factors was confirmed using reporter mesangial cells that produce secreted alkaline phosphatase (SEAP) under the control of the kappaB enhancer element. When the reporter cells were adoptively transferred into normal glomeruli, expression of SEAP mRNA and activity of SEAP were also upregulated in the explanted glomeruli. The molecular weight of factors responsible for activation of NF-kappaB was >50 kDa, and TNF-alpha was identified as one of glomerulus-derived activators. To examine upstream events involved, we focused on MAP kinases that are spontaneously activated in explanted glomeruli. Selective suppression of ERK or p38 MAP kinase significantly attenuated activation of NF-kappaB in mesangial cells triggered by coculture with isolated glomeruli. Interestingly, the suppressive effects by MAP kinase inhibitors were not observed in mesangial cells treated with GCM. These data suggested that NF-kappaB was spontaneously activated in explanted glomeruli via autocrine/paracrine factors including TNF-alpha and that the production of NF-kappaB activators by glomeruli was, at least in part, through MAP kinase pathways.
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PMID:Spontaneous activation of the NF-kappaB signaling pathway in isolated normal glomeruli. 1670 44

The effect of ginsenosides on proliferation of chicken primordial germ cells (PGCs) was evaluated and involvement of nuclear factor (NF)-kappaB in the signaling pathway was investigated. PGCs were isolated from the genital ridge of 3.5-4 day embryos and cultured in Medium 199 supplemented with 5% FCS and 10 ng/ml LIF. PGCs subcultured on chicken embryonic fibroblast feeder were challenged with ginsenosides alone or in combination with PKC inhibitor H(7) or activator phorbol 12-myristate 13-acetate (PMA) for 24h. Moreover, the translocation of NF-kappaB and degradation level of IkappaBalpha were investigated by Western blot analysis. Results show that PGCs were identified by periodic acid-Schiff, alkaline phosphatase histochemistry as well as c-kit, SSEA-1 and Oct-4 immunocytochemistry. Treatment with ginsenosides at 1-100 microg/ml significantly increased the number and area of PGC colonies in a dose-dependent manner. However, this proliferating effect was obviously attenuated by combined treatment of H(7) (10(-7)-10(-5)M). Similarly, PKC staining of PGC colonies was more intensive after ginsenosides treatment compared with the control group. In addition, treatment with ginsenosides at 1-10 microg/ml stimulated the translocation of NF-kappaB (p65). However, the NF-kappaB translocation and the degradation of IkappaBalpha were significantly blocked by combined treatment with 10(-6)M H(7). These results indicated that ginsenosides promote proliferation of chicken PGCs through activation of PKC-involved NF-kappaB signaling pathway.
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PMID:Ginsenosides promote proliferation of chicken primordial germ cells via PKC-involved activation of NF-kappaB. 1758 92

Estrogenic effects involve interactions between estrogen receptors (ERs), response elements, and nuclear proteins. It is hypothesized that interaction between ER and NF-kappa B may affect the regulation of responsive genes. Electrophoretic mobility shift assay (EMSA) was performed to assess if the interaction of ERs and NF- kappaB affect their respective DNA-binding activities, and alkaline phosphatase assay was done to evaluate estrogenic activity. EMSA revealed that ERs inhibit DNA-binding of p50 and p65, whereas p50 did not impair ER alpha binding. Stimulation with estradiol inhibited DNA binding of NF-kappaB in ERalpha-transfected endometrial stromal cells (ESCs). Moreover, activation of NF-kappaB significantly decreased estrogen responsiveness of Ishikawa cells and ERalpha-transfected ESC. Our results suggest that ERs downregulate NF-kappaB-dependent gene activation by directly preventing DNA binding. However, NF-kappaB-mediated inhibition of ER-dependent gene activation may be carried out indirectly rather than through a direct inhibition of ER-DNA binding. These findings offer new insight into the specific role of ERalpha and could eventually help in developing therapeutics for endometriosis.
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PMID:DNA-binding ability of NF-kappaB is affected differently by ERalpha and ERbeta and its activation results in inhibition of estrogen responsiveness. 1857 58

Natural compounds with bone-forming (or anabolic) activity have been recently focused on in bone research. The present study investigated the effect of undecylenic acid (UA) on osteoblast differentiation in mouse osteoblastic MC3T3-E1 subclone 4 cells and primary mouse calvarial cells. Low concentrations of UA (up to 5 microM) exhibited no cytotoxicity and significantly increased the expression and activity of alkaline phosphatase (early differentiation marker of osteoblast) and calcium deposition with the induction of expression of the osteocalcin gene in both cells. Interestingly, at low concentration of UA, the induction of NF-kappaB p65 translocation into nucleus and the up-regulation of AP-1 and NFATc1 transcript levels were also observed, suggesting that the stimulatory effect of UA on osteoblast differentiation could be mediated through the activation of transcription factors. Additionally, although the patterns of UA-induced activation of MAP kinases (JNK and p38) were not completely consistent with the increase of both ALP activity and calcium deposition by UA, MAP kinases might be partially involved in the biological function of UA during the early and late stages of osteoblast differentiation.
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PMID:Stimulatory effect of undecylenic acid on mouse osteoblast differentiation. 1977 59

Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor alpha (TNFalpha) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFalpha inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFalpha had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-kappaB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-kappaB by the overexpression of the dominant negative IkappaBalpha. Although TNFalpha failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFalpha suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-kappaB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFalpha inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-kappaB.
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PMID:Tumor necrosis factor alpha represses bone morphogenetic protein (BMP) signaling by interfering with the DNA binding of Smads through the activation of NF-kappaB. 1985 28


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