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)

GnRH, acts via the GnRH receptor (GnRHR), plays a pivotal role in human reproduction by stimulating the synthesis and secretion of gonadotropins from pituitary gonadotropes. Studies have also suggested that it has other extra-pituitary functions. To date, the transcriptional regulation of human GnRHR gene in the brain remains largely unknown. Recently, the human cerebellar medulloblastoma cell line TE-671 is found to express GnRH. We report here for the first time that GnRHR is also expressed in this neuronal cell line. Treatment with GnRHR agonist stimulated the phosphorylation of both ERK1/2 and JNK in the cells. Moreover, transient transfection of various human GnRHR promoter-luciferase constructs into the cells identified an upstream promoter region located between -2197 and -1018. Important cis-acting regulatory elements were found at -1300/-1018 and -2197/- 1900, as deletion of either region caused a dramatic decrease in the promoter activity. An upstream GnRHR promoter element was identified to be important for basal transcription in the human neuronal TE-671 cells, in contrast to the previous finding that a downstream promoter is responsible for the gonadotrope-specific expression. Furthermore, we showed that antide (GnRHR antagonist) significantly stimulated the GnRHR promoter activity and inhibition of protein kinase C (PKC) pathway by staurosporine could also up-regulate the promoter activity in dose- and time-dependent manners. Taken together, these data suggest that activation of the GnRHR by interacting with GnRH may transcriptionally down-regulate itself via the PKC pathway in human neuronal cells.
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PMID:Expression and transcriptional regulation of the GnRH receptor gene in human neuronal cells. 1636 74

Necrotizing enterocolitis (NEC), a severe intestinal inflammation in neonates, occurs following bacterial colonization of the gut. LPS-induced production of inflammatory factors in immature enterocytes may be a factor in NEC. Previously, we described LPS-induced p38 MAPK-dependent expression of cyclooxygenase-2 (COX-2) in rat IEC-6 cells. In this study, we examine COX-2 expression in newborn rat intestinal epithelium and further characterize the mechanisms of COX-2 regulation in enterocytes. Induction of NEC by formula feeding/hypoxia increased phospho-p38 and COX-2 levels in the intestinal mucosa. Celecoxib, a selective COX-2 inhibitor, exacerbated the disease, suggesting a protective role for COX-2. COX-2 was induced in the intestinal epithelium by LPS in vivo and ex vivo. The latter response was attenuated by the p38 inhibitor SB202190, but not by inhibitors of ERK, JNK, or NF-kappaB. In IEC-6 enterocytes, COX-2 was induced by the expression of MAPK kinase 3 EE (MKK3EE), a constitutive activator of p38, but not of activators of ERK or JNK pathways. However, neither MKK3/6 nor MKK4, the known p38 upstream kinases, were activated by LPS. Dominant-negative MKK3 or MKK4 or SB202190 failed to prevent LPS-induced, p38-activating phosphorylation, ruling out important roles of these kinases or p38 autophosphorylation. LPS increased COX-2 and activating phosphorylation of p38 with similar dose-response. Blockade of LPS-induced expression of COX-2-luciferase reporter and destabilization of COX-2 message by SB202190 indicate that p38 regulates COX-2 at transcription and mRNA stability levels. Our data indicate that p38-mediated expression of COX-2 proceeds through a novel upstream pathway and support the role of the neonate's enterocytes as bacterial sensors.
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PMID:Lipopolysaccharide induces cyclooxygenase-2 in intestinal epithelium via a noncanonical p38 MAPK pathway. 1636 53

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.
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PMID:Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation. 1640 1

Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-alpha (TNF-alpha), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFkappaB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFkappaB nuclear entry. The forced expression of high levels of NFkappaB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis.
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PMID:Transcriptional regulation of the osterix (Osx, Sp7) promoter by tumor necrosis factor identifies disparate effects of mitogen-activated protein kinase and NF kappa B pathways. 1641 Feb 54

Previous studies suggest that adenosine possesses anti-inflammatory properties, however, the mechanisms by which adenosine affects immune function remain unclear, particularly in the intestine. In this study, we hypothesized that adenosine directly affects pro-inflammatory gene expression in intestinal epithelial cells through modulation of NF-kappaB signaling. HT-29 cells were treated with adenosine prior to incubation with various stimuli and pro-inflammatory gene expression and signal transduction analyzed. Adenosine pretreatment resulted in a reduction in IL-8 expression and secretion in response to TNF-alpha, IL-1, LPS, and PMA. This effect was paralleled by inhibition of kappaB-driven luciferase expression and a reduction in recruitment of NF-kappaB to the IL-8 promoter. Pretreatment of HT-29 cells also resulted in reduced ERK, p38, and JNK MAPK phosphorylation, following TNF-alpha treatment. The observed effects in this study occurred independently of known surface adenosine receptors. This study identifies adenosine as a potent negative regulator of pro-inflammatory signaling in intestinal epithelial cells.
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PMID:Adenosine is a negative regulator of NF-kappaB and MAPK signaling in human intestinal epithelial cells. 1641 16

It is well documented that manganese neurotoxicity induces clinical symptoms similar to those of idiopathic Parkinson's disease. Although microglial cytotoxic mediator-induced neurotoxicity is suggested, the mechanism by which manganese up-regulates cytotoxic mediator, such as nitric oxide (NO), remains poorly understood. Therefore, in this study, we investigated the mechanism of manganese on induction of iNOS in microglial cells. iNOS promoter/luciferase assay revealed that manganese (500 (M) regulated the iNOS expression at the transcriptional level. Immunoblot analysis also revealed that phosphorylation levels of ERK, JNK MAPKs and Akt (PKB, PI 3-kinase downstream effector), were increased. Both protein and mRNA levels of iNOS expression were abrogated by specific inhibitors, SP600125 (JNK inhibitor, 20 microM), PD98059 (ERKs inhibitor, 50 microM), or LY294002 (PI 3-kinase inhibitor, 20 microM), but not by SB203580 (20 microM), a p38 specific inhibitor. These data lead to the conclusion that manganese regulates the iNOS expression at the transcriptional level in BV2 microglial cells and the increased iNOS protein expression is mediated via both JNK-ERK MAPK and PI3K/Akt signaling pathways, but not via p38 MAPK pathway. Increased iNOS protein level was also found in RAW264.7 murine macrophage cells.
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PMID:Manganese induces inducible nitric oxide synthase (iNOS) expression via activation of both MAP kinase and PI3K/Akt pathways in BV2 microglial cells. 1641 67

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 muM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.
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PMID:PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK. 1642 39

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde that is present in cigarette smoke. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by various such electrophilic compounds. In this study, the regulatory effects of acrolein upon the expression of HO-1 were investigated in endothelial cells (ECs). We demonstrate that acrolein induces the elevation of HO-1 protein levels, and subsequent enzyme activity, at non-cytotoxic concentrations. An additional alpha,beta-unsaturated aldehyde, cinnamaldehyde, was also found to increase HO-1 expression and have less cytotoxicity than acrolein. Moreover, acrolein-mediated HO-1 induction is abrogated in the presence of actinomycin D and cycloheximide. Nrf2 is a transcription factor involved in the induction of HO-1 through an antioxidant response element (ARE) in the promoter region of the HO-1 gene. We show that acrolein induces Nrf2 translocation and ARE-luciferase reporter activity. Acrolein was also found to induce the production of both superoxide and H2O2 at levels greater than 100 microM. However, with the exception of NAC, no antioxidant generated any effect upon acrolein-dependent HO-1 expression in ECs. Our present findings suggest that reactive oxygen species (ROS) may not be a major modulator for HO-1 induction. Using buthionine sulfoximine to deplete the intracellular GSH levels further enhanced the effects of acrolein. We also found that cellular GSH level was rapidly reduced after both 10 and 100 microM acrolein treatment. However, after 6 h of exposure to ECs, only 10 microM acrolein treatment increases GSH level. In addition, only the JNK inhibitor SP600125 and tyrosine kinase inhibitor genistein had any significant inhibitory impact upon the upregulation of HO-1 by acrolein. Pretreatment with a range of other PI3 kinase inhibitors, including wortmannin and LY294002, showed no effects. Hence, we show in our current experiments that a sublethal concentration of acrolein is in fact a novel HO-1 inducer, and we further identify the principal underlying mechanisms involved in this process.
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PMID:Upregulation of endothelial heme oxygenase-1 expression through the activation of the JNK pathway by sublethal concentrations of acrolein. 1648 Jul 51

The protein zero (P0) glycoprotein is an important component of compact peripheral nerve myelin produced by the glial cells of the mammalian peripheral nervous system. P0 mRNA expression is reduced following exposure of Schwann cells to sublytic C5b-9, the terminal activation complex of the complement cascade. Sublytic complement treatment decreased P0 mRNA by 81% within 6 h and required C5b-9 assembly. C5b-9 induced a threefold increase in both JNK1 activity and c-jun mRNA within 20 and 30 min, respectively, compared with cells treated with either human serum depleted of complement component C7 (C7dHS) or medium alone. Sublytic C5b-9 stimulation, in the presence of the transcription inhibitor Actinomycin D, decreased P0 mRNA expression by 52%, indicating that mRNA was selectively destabilized. This effect was prevented by pretreatment with L-JNK inhibitor 1 (L-JNKI1). To study a potential inhibition of P0 gene transcription, we transfected Schwann cells with a P0 promoter-firefly luciferase construct. Sublytic C5b-9 stimulation of the transfected cells decreased luciferase activity by 82% at 6 h, and this effect was prevented by pretreatment with L-JNKI1 inhibitor. Our results indicate that the ability of C5b-9 in vitro to affect P0 gene expression is mediated via JNK1 activation that leads to enhanced mRNA decay and transcriptional repression of P0.
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PMID:JNK1 activation mediates C5b-9-induced P0 mRNA instability and P0 gene expression in Schwann cells. 1651 86

Interferongamma inducible protein-10 (IP10 or CXCL10), a Th-1 affiliated chemokine, is expressed by activated glial cells and may contribute to the trafficking of immune cells in the inflamed central nervous system. This study examines the regulation of the expression of this chemokine in cultured microglial cells focusing on the roles of mitogen-activated protein (MAP) kinase cascades. Exposure of a mouse microglial cell line, BV-2, to lipopolysaccharide (LPS) and IFNgamma led to an induction of IP10 mRNA and protein as determined by RT-PCR and ELISA, respectively. This induction was suppressed by pharmacological inhibitors of p38 MAPK (i.e., SB203580) and c-Jun N-terminal kinase (JNK, SP600125), suggesting the involvement of the two kinases in IP10 expression. LPS also induced the activity of an IP10 promoter reporter (luciferase) construct transfected into BV-2 cells in a MAP kinase- and NFkappaB-dependent manner. The use of deletion constructs revealed that the kinase-targeted sequences were within the region between -533 bp and -332 bp upstream of the transcriptional start site. Co-transfection of IP10 luciferase with the active forms of the upstream kinases in the MAP kinase cascades, i.e., MAPK kinase-3 (MKK3), MKK6 (the immediately upstream activators of p38 kinase) and a MAP3K, i.e., TGFbeta-activated kinase-1 (TAK1), produced a marked stimulation of the promoter activity. The results of this study indicate that the MAP kinase cascades prominently regulate IP10 gene expression in microglial cells.
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PMID:MAP kinase regulation of IP10/CXCL10 chemokine gene expression in microglial cells. 1663 81


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