EC:1.14.14.3 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dysregulation of apoptosis is one of the likely underlying mechanisms of neointimal thickening, a disorder in which proinflammatory cytokines may influence the function of vascular smooth muscle cells (VSMCs) and contribute to atherogenesis. One of these cytokines, tumor necrosis factor-alpha (TNF-alpha), induces 2 possibly conflicting pathways, 1 leading to the activation of nuclear factor-kappaB (NF-kappaB) and the other leading to caspase-mediated apoptosis. We investigated whether specific inhibition of NF-kappaB affects TNF-alpha-dependent apoptosis in human VSMCs. To inhibit NF-kappaB activation specifically, we constructed a recombinant adenovirus vector expressing a truncated form of the inhibitor protein IkappaBalpha (AdexIkappaBDeltaN) that lacks the phosphorylation sites essential for activation of NF-kappaB. The IkappaBDeltaN was overexpressed by adenoviral infection and was resistant to stimulus-dependent degradation. Electromobility gel shift and luciferase assays demonstrated that overexpression of IkappaBDeltaN inhibited NF-kappaB activation induced by TNF-alpha or interleukin-1beta (IL-1beta). In cells overexpressing IkappaBDeltaN, TNF-alpha dramatically induced apoptosis, whereas IL-1beta had no effect. The induction was suppressed by treatment with a selective inhibitor of the caspase-3 family, Z-DEVD-fmk, and the overexpression of IkappaBDeltaN induced TNF-alpha-mediated caspase-3 and caspase-2 activity. These results indicate that overexpression of IkappaBDeltaN induces TNF-alpha-dependent apoptosis by efficient and specific suppression of NF-kappaB and upregulation of caspase-3 and caspase-2 activity in human VSMCs. Our findings suggest that adenovirus-mediated IkappaBDeltaN gene transfer may be useful in the treatment of disorders associated with inflammatory conditions, such as the response to vascular injury and atherosclerosis.
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PMID:Overexpression of truncated IkappaBalpha induces TNF-alpha-dependent apoptosis in human vascular smooth muscle cells. 1103 Dec 4

The immunosuppressant cyclosporin A inhibits transcription mediated by the nuclear factor of activated T-cells (NFAT), a key regulator of cytokine gene expression in lymphocytes that integrates phospholipase C signaling. NFAT is also expressed in vascular smooth muscle cells, but the genes it regulates there are unknown. Here we show that Galpha(q)-coupled P2Y nucleotide receptor signaling in rat vascular smooth muscle cells increases NFAT-mediated luciferase reporter expression. It also induces interleukin (IL)-6 gene expression but not other cytokine mRNAs including IL-1, IL-2, IL-3, IL-4, IL-10, gamma-interferon, tumor necrosis factor-alpha, or tumor necrosis factor-beta. IL-6 mRNA induction by UTP is more rapid and transient then that caused by IL-1beta stimulation and is partially blocked by cyclosporin A or by expression of a trans-dominant NFAT inhibitor. Expression of recombinant NFATc1 markedly augments IL-6 mRNA induction by these and other agonists, which is partially attributable to NFAT-regulated paracrine mediators. However, trans-dominant NFkappaB inhibitors strongly interfere with IL-6 mRNA induction both by IL-1beta and by UTP, which synergistically evoke IL-6 mRNA expression. These findings suggest that NFAT is among the cofactors involved in NFkappaB-dependent IL-6 gene induction by Ca(2+)-mobilizing receptors in vascular smooth muscle cells.
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PMID:Evidence that Galpha(q)-coupled receptor-induced interleukin-6 mRNA in vascular smooth muscle cells involves the nuclear factor of activated T cells. 1104 41

Triptolide (PG490, 97% pure) is a diterpenoid triepoxide with potent anti-inflammatory and immunosuppressive effects in transformed human bronchial epithelial cells and T cells (Qiu D, Zhao G, Aoki Y, Shi L, Uyei A, Nazarian S, Ng JC-H, and Kao PN. J Biol Chem 274: 13443-13450, 1999). Triptolide, with an IC(50) of approximately 20-50 ng/ml, inhibits normal and transformed human bronchial epithelial cell expression of interleukin (IL)-6 and IL-8 stimulated by phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha, or IL-1 beta. Nuclear runoff and luciferase reporter gene assays demonstrate that triptolide inhibits IL-8 transcription. Triptolide also inhibits the transcriptional activation, but not the DNA binding, of nuclear factor-kappa B. A cDNA array and clustering algorithm analysis reveals that triptolide inhibits expression of the PMA-induced genes tumor necrosis factor-alpha, IL-8, macrophage inflammatory protein-2 alpha, intercellular adhesion molecule-1, integrin beta(6), vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, GATA-3, fra-1, and NF45. Triptolide also inhibits constitutively expressed cell cycle regulators and survival genes cyclins D1, B1, and A1, cdc-25, bcl-x, and c-jun. Thus anti-inflammatory, antiproliferative, and proapoptotic properties of triptolide are associated with inhibition of nuclear factor-kappa B signaling and inhibition of genes known to regulate cell cycle progression and survival.
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PMID:Anti-inflammatory effects of triptolide in human bronchial epithelial cells. 1105 33

Pimobendan, an inhibitor of phosphodiesterase III with calcium sensitizing properties, inhibits the production of cytokines and nitric oxide. In the present study, the effects of pimobendan and other inotropic agents on the activation of transcription factor NF-kappaB were examined. Pimobendan significantly decreased the expression of luciferase protein in A549 cells transfected with the NF-kappaB reporter plasmid, stimulated with interleukin (IL)-1beta, tumor necrosis factor-alpha, or phorbol 12-myristate 13 acetate. However, high concentrations of amrinone, vesnarinone, or NKH 477 decreased promoter activity only slightly. Electrophoretic mobility shift assay also showed inhibition of NF-kappaB activation by pimobendan. Pimobendan possesses the unique property of inhibiting NF-kappaB, which may be independent of phosphodiesterase inhibition.
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PMID:Pimobendan inhibits the activation of transcription factor NF-kappaB: a mechanism which explains its inhibition of cytokine production and inducible nitric oxide synthase. 1106 73

This study addresses a mechanism by which lymphocytes may promote vascular endothelial growth factor (VEGF) expression and angiogenesis in immune inflammation. Resting human umbilical endothelial cells (HUVECs) were found to express low levels of VEGF messenger RNA (mRNA) by reverse transcription polymerase chain reaction and ribonuclease protection assay with little or no change in expression following activation by cytokines, including tumor necrosis factor-alpha, interleukin (IL)-1, interferon gamma, or IL-4. In contrast, treatment of HUVECs and monocytes with soluble CD40 ligand (sCD40L) resulted in a marked dose-dependent induction of VEGF mRNA (approximately 4-fold), which peaked between 1 and 5 hours post-stimulation. Transient transfection of HUVECs was performed with a luciferase reporter construct under the control of the human VEGF promoter. Treatment of transfected HUVECs with sCD40L was found to enhance luciferase activity (approximately 4-fold) compared with controls, similar to the relative fold induction in mRNA expression in parallel cultures. Thus, CD40-dependent VEGF expression was a result of transcriptional control mechanisms. Treatment of HUVECs with sCD40L was also found to function in vitro to promote growth and proliferation in a VEGF-dependent manner, and CD40-dependent HUVEC growth was comparable to that found following treatment with recombinant human VEGF. Furthermore, subcutaneous injection of sCD40L in severe combined immunodeficient and nude mice induced VEGF expression and marked angiogenesis in vivo. Taken together, these findings are consistent with a function for CD40L-CD40 interactions in VEGF-induced angiogenesis and define a mechanistic link between the immune response and angiogenesis. (Blood. 2000;96:3801-3808)
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PMID:Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo. 1109 63

This study was performed to examine the effects of the calcium channel blockers, nifedipine, amlodipine, diltiazem, and verapamil on the activation of the transcription factor NF-kappaB. A549 cells, a human epithelium-like lung carcinoma cell line, were transfected with the NF-kappaB reporter plasmid, which contains the luciferase gene driven by promoters containing a TATA element and 5 copies of the kappaB cis-acting element, and co-transfected with 0.2 microg of pSV2neo vector using LipofectAMINE. Nifedipine significantly decreased the expression of luciferase protein stimulated with IL-1beta (1 ng/mL) compared with controls: 80+/-4% at 3 micromol/L, 47+/-2% at 10 micromol/L and 30+/-2% at 30 micromol/L (each, n=3, p<0.0001). The inhibitory effect of nifedipine on promoter activity was concentration-dependent, with a maximal effect obtained at 30 micromol/L. In contrast, high concentrations (30 micromol/L) of amlodipine, diltiazem or verapamil decreased promoter activity to only 89+/-3%, 90+/-3% or 87+/-2% of control, respectively. A comparable inhibitory effect of nifedipine was observed when cells were stimulated with tumor necrosis factor (TNF)-alpha (50 ng/mL), or phorbol 12-myristate 13-acetate (PMA, 100 ng/mL). Electrophoretic mobility shift assay by lipopolysaccharide stimulation, using the RAW 264.7 macrophage cell line, also showed inhibition of NF-kappaB activation by nifedipine in concentrations of 30 and 50 micromol/L. Nifedipine possesses the unique property of inhibiting NF-kappaB, which may be independent of its calcium channel blocking activity, and may, in part, explain its immunosuppressive effect.
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PMID:Nifedipine inhibits activation of transcription factor NF-kappaB. 1110 67

Although 3':5' cyclic adenosine monophosphate (cAMP) is known to modulate cytokine production in a number of cell types, little information exists regarding cAMP-mediated effects on this synthetic function of human airway smooth-muscle (HASM) cells. We examined the effect of increasing intracellular cAMP concentration ([cAMP](i)) on tumor necrosis factor (TNF)-alpha-induced regulated on activation, normal T cells expressed and secreted (RANTES) and interleukin (IL)-6 secretion from cultured HASM cells. Pretreatment of HASM with prostaglandin (PG) E(2), forskolin, or dibutyryl cAMP inhibited TNF-alpha-induced RANTES secretion but increased TNF-alpha-induced IL-6 secretion. Moreover, stimulation with PGE(2), forskolin, or dibutyryl cAMP alone increased basal IL-6 secretion in a concentration-dependent manner. SB 207499, a specific phosphodiesterase type 4 inhibitor, augmented the inhibitory effects of PGE(2) and forskolin on TNF-alpha-induced RANTES. Collectively, these data demonstrate that increasing [cAMP](i) in HASM effectively increases IL-6 secretion but reduces RANTES secretion promoted by TNF-alpha. Reverse transcriptase/polymerase chain reaction and ribonuclease protection assays suggested that these opposite effects of increased [cAMP](i) on TNF-alpha- induced IL-6 and RANTES secretion may occur at the transcriptional level. Accordingly, we examined the effects of TNF- alpha and cAMP on the regulation of nuclear factor (NF)-kappaB, a transcription factor known to modulate cytokine synthesis in numerous cell types. Stimulation of HASM cells with TNF-alpha increased NF-kappaB DNA-binding activity. However, increased [cAMP](i) in HASM neither activated NF-kappaB nor altered TNF-alpha- induced NF-kappaB DNA-binding activity. These results were confirmed using a NF-kappaB-luciferase reporter assay. Together, our data suggest that TNF-alpha-induced IL-6 and RANTES secretion may be associated with NF-kappaB activation, and that inhibition of TNF-alpha-stimulated RANTES secretion and augmentation of IL-6 secretion by increased [cAMP](i) in HASM cells occurs via an NF-kappaB-independent mechanism.
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PMID:Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smooth-muscle cells. Modulation by cyclic adenosine monophosphate. 1110 33

Inducible nitric-oxide synthase (iNOS) is an important signaling protein involved in the regulation of biological processes (e.g. vasodilation, inflammation) and is subject to transcriptional regulation by cytokines and lipopolysaccharide (LPS). Full activation of the human iNOS (hiNOS) promoter by cytokines (i.e., tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma (IFN-gamma)) required downstream and upstream nuclear factor-kappaB (-115, -8283) and activator protein-1 (AP-1) (-5115, -5301) transcription factor binding sites. Human lung epithelial (A549) cells were transiently transfected with luciferase reporter plasmids containing an 8.3-kilobase human iNOS promoter to examine the molecular signaling events necessary for hiNOS transcriptional activation. The combination of LPS and IFN-gamma, but neither alone, increased hiNOS promoter activity 28-fold, in a reaction requiring two critical AP-1 (JunD-Fra-2) promoter binding sites. Mitogen-activated protein kinases (MAPKs) were assessed as potential activators of AP-1 and the hiNOS promoter. Both pharmacological and molecular inhibitors of the extracellular signal-related kinase (ERK) and p38 pathways reduced cytokine mixture (CM)- and LPS/IFN-gamma-induced promoter activation. By gel retardation analysis, the addition of MAP/ERK kinase-1 and p38 inhibitors significantly diminished AP-1 binding in both CM- and LPS/IFN-gamma-stimulated cells. Thus, p38- and ERK-dependent pathways, through effects on the AP-1 complex, activate the hiNOS promoter in cells stimulated with CM or LPS/IFN-gamma.
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PMID:Mitogen-activated protein kinases mediate activator protein-1-dependent human inducible nitric-oxide synthase promoter activation. 1111 84

Activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), which forms a heterodimer with retinoic X receptor (RXR), inhibit the production of certain inflammatory mediators. To clarify the role of the PPAR gamma:RXR signaling pathway in Kupffer cells, we studied the effect of an RXR agonist and PPARgamma agonist on LPS-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production. An RXR-specific agonist, Ro47-5944, and a PPAR gamma-specific agonist, AD4833 (pioglitazone hydrochloride), each inhibited LPS-induced NO and TNF-alpha production. The combined treatment of Ro47-5944 and AD4833 resulted in enhanced inhibition, and suppressed the mRNA levels of NO and TNF-alpha. PPAR gamma:RXR activation did not affect the level of LPS-induced phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase. PPAR gamma:RXR activation also did not affect nuclear factor kappa B (NF-kappa B) nuclear translocation nor NF-kappa B and activator protein 1 (AP-1) activation in the electrophoretic mobility-shift assay. Finally, PPAR gamma:RXR activation suppressed the LPS-induced promoter activity of the NF-kappa B-luciferase reporter gene in RAW 264.7 cells. These data imply that PPARgamma:RXR activation suppresses LPS-induced NO and TNF-alpha production in Kupffer cells, and that this inhibition occurred at the transcriptional level. Although no consensus PPAR gamma:RXR-responsive element in the promoter regions of the inducible isoform of nitric oxide synthase (iNOS) and TNF-alpha genes was found, PPAR gamma:RXR may interfere with NF-kappa B and AP-1 transcriptional activity. Our data also suggest a potential therapeutic approach for moderating hepatic injury such as endotoxin shock in which Kupffer cell activation has been implicated.
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PMID:Activation of retinoic X receptor and peroxisome proliferator-activated receptor-gamma inhibits nitric oxide and tumor necrosis factor-alpha production in rat Kupffer cells. 1112 25

Recent evidence suggests the possible involvement of inducible nitric oxide synthase (iNOS) in the development and maintenance of hypertension in certain animal models. Inflammatory cytokines activate nuclear factor (NF)-kappaB, which plays a major role in transactivation of the inducible nitric oxide synthase (iNOS) gene. However, it remains unknown whether cytokine-mediated iNOS expression in vascular smooth muscle cells (VSMCs) requires signaling pathway(s) other than NF-kappaB activation. The purpose of this study was to determine whether the p42/p44 MAP kinase pathway is involved in cytokine-induced NF-kappaB activation and/or iNOS expression in cultured rat VSMCs. Nitrite/nitrate (NOx) production stimulated by interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha in VSMCs was markedly suppressed by inhibiting MAP kinase by pretreatment with a p42/p44 MAP kinase kinase (MAPKK)-1 inhibitor (PD98059) or by transfecting the dominant-interfering form of the nonphosphorylated MAPKK-1 expressing construct (MAPKK S222A). Inhibition of p42/p44 MAP kinase also antagonized the upregulation of iNOS mRNA and protein, as demonstrated by the quantitative RT-PCR method and Western blot analysis, respectively. Furthermore, rat iNOS promoter activity using an iNOS-luciferase construct stimulated by cytokines was inhibited by MAPKK-1 inhibition. However, kappaB-dependent transcription analysis revealed that cytokine-stimulated NF-kappaB activity was unaffected by MAP kinase inhibition. Western blot analysis using anti-IkappaB-alpha and anti-phospho-IkappaB-alpha antibodies showed that PD98059 had no effect on transient phosphorylation or degradation of IkappaB-alpha by cytokines. An electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-kappaB site of rat iNOS promoter as a probe showed that MAP kinase inhibition did not block cytokine-stimulated activation of NF-kappaB. These data suggest that the MAP kinase pathway is in part involved in cytokine-induced iNOS expression independent from NF-kappaB activation in rat VSMCs.
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PMID:Cytokine-activated p42/p44 MAP kinase is involved in inducible nitric oxide synthase gene expression independent from NF-kappaB activation in vascular smooth muscle cells. 1113 Dec 79

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