scholarly journals Nuclear factor-κB regulates expression of platelet phospholipase C-β2 (PLCB2)

2016 ◽  
Vol 116 (11) ◽  
pp. 931-940 ◽  
Author(s):  
Guangfen Mao ◽  
Jianguo Jin ◽  
Satya P. Kunapuli ◽  
A. Koneti Rao
Keyword(s):  
Phospholipase C ◽  
Healthy Subjects ◽  
Consensus Sequence ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Nuclear Factor ◽  
Nuclear Extracts ◽  
Hel Cells ◽  
Activation Mechanisms

SummaryPhospholipase C (PLC)-β2 (gene PLCB2) is a critical regulator of platelet responses upon activation. Mechanisms regulating of PLC-β2 expression in platelets/MKs are unknown. Our studies in a patient with platelet PLC-β2 deficiency revealed the PLCB2 coding sequence to be normal and decreased platelet PLC-β2 mRNA, suggesting a defect in transcriptional regulation. PLCB2 5’- upstream region of the patient revealed a heterozygous 13 bp deletion (-1645/-1633 bp) encompassing a consensus sequence for nuclear factor-κB (NF-κB). This was subsequently detected in three of 50 healthy subjects. To understand the mechanisms regulating PLC-β2, we studied the effect of this variation in the PLCB2. Gel-shift studies using nuclear extracts from human erythroleukaemia (HEL) cells or recombinant p65 showed NF-κB binding to oligonucleotide with NF-κB site; in luciferase reporter studies its deletion reduced PLCB2 promoter activity. PLCB2 expression was decreased by siRNA knockdown of NF-κB p65 subunit and increased by p65 overexpression. By immunoblotting platelet PLC-β2 in 17 healthy subjects correlated with p65 (r=0.76, p=0.0005). These studies provide the first evidence that NF-kB regulates MK/platelet PLC-β2 expression. This interaction is important because of the major role of PLC-β2 in platelet activation and of NF-κB in processes, including inflammation and atherosclerosis, where both are intimately involved.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Gαq promoter during megakaryocytic differentiation

2008 ◽  
Vol 100 (05) ◽  
pp. 821-828 ◽  
Author(s):  
Gauthami Jalagadugula ◽  
Danny N. Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Luciferase Reporter Gene ◽  
Nuclear Extracts ◽  
Upstream Promoter ◽  
Hel Cells ◽  
The Difference ◽  
Responsive Element ◽  
Increased Activity

SummaryGαq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Gαq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5’ upstream region of the Gαq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5’ upstream of the Gαq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Gαq 5’ upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt-1042/-1037 eliminated the difference. Gel-shift studies on Gαq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence –1042/-1037.Silencing of endogenous EGR-1 inhibited Gαq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence –1042/-1037 bp in Gαq promoter mediates the induction of Gαq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Gαq promoter, and new insights into regulation of Gαq gene by EGR-1.

Regulation of Platelet PLC-β2 Expression by NF-κB: Studies in Human Platelet PLC-β2 Deficiency.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 699-699 ◽  
Author(s):  
Guangfen Mao ◽  
Satya P. Kunapuli ◽  
A. Koneti Rao
Keyword(s):  
Protein Binding ◽  
Consensus Sequence ◽  
Pcr Amplification ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Mrna Levels ◽  
Luciferase Gene ◽  
Wild Type ◽  
Control Subjects ◽  
Hel Cells

Abstract We have previously described a patient with platelet phospholipase C (PLC)-β2 deficiency characterized by impaired platelet responses to activation with multiple G-protein coupled receptor agonists. The PLC-β2 coding sequence was normal and platelet PLC-β2 mRNA levels were decreased in the patient (Blood, 2002, 99:905). Very little is currently known regarding the transcriptional regulation of PLC-β2. PCR-amplification of patient leukocyte DNA and sequencing of the PLC-β2 5′-upstream region revealed a heterozygous 13-bp deletion (−1645 to −1633 bp from ATG) that encompasses a consensus binding site (GGGAATTCCC) for nuclear factor-κB, NF-κB. This deletion was present in the propositus and her affected son, but not in control subjects. PCR amplification of region −1791 to −1606 bp of genomic DNA revealed one band in 5 control subjects (size ~186 bp) on agarose gel electrophoresis but 2 bands in the patient and her son, consistent with a heterozygous defect. Luciferase reporter gene studies were performed in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA, 30 nM) to induce megakaryocytic transformation. Genomic fragment (−1648/−23 nt) of PLC-β2 5′-upstream sequence and its truncated form without the 13 nt region (−1633/−23 nt) were inserted upstream of luciferase gene in a promoterless expression vector PGL3-basic (Promega) and transiently transfected into HEL cells. Truncation of the wild-type −1648/−23 fragment at 1631 bp resulted in a consistent decrease in promoter activity by ~ 25% (6 experiments, p<0.05). Protein binding assay (EMSA) was performed using PMA-treated HEL cell nuclear extracts and oligonucleotide probes (−1652/−1628 bp) with wild-type and mutated NF-κB consensus sites. Specific protein binding to the wild-type oligonucleotide was abolished when the NF-κB consensus sequence was deleted or mutated. Protein binding to wild-type probe was not competed by the unlabeled mutant oligonucleotide lacking NF-κB consensus sequence. In supershift assay, antibody targeted against the p65 subunit of NF-κB abolished protein binding, indicating a role for NF-κB. In summary, our studies demonstrate in the 5′-upstream region of PLC-β2 gene of the patient a 13-bp deletion that has a consensus site for NF-κB. Luciferase gene promoter assays demonstrate loss of activity when the 13-bp site is truncated. These studies provide evidence that impaired regulation of PLC-β2 gene by NF-κB may be the basis for the PLC-β2 deficiency in our patient. They show for the first time that PLC-β2, the most abundant β-PLC in platelets, is regulated by NF-κB. These findings are highly relevant because of the important role of PLC-β2 in platelet function, and of NF-κB in megakaryocytic differentiation and atherosclerosis.

scholarly journals Oxygen-evoked changes in transcriptional activity of the 5′-flanking region of the human amiloride-sensitive sodium channel (αENaC) gene: role of nuclear factor κB

2002 ◽  
Vol 364 (2) ◽  
pp. 537-545 ◽  
Author(s):  
Deborah L. BAINES ◽  
Mandy JANES ◽  
David J. NEWMAN ◽  
Oliver G. BEST
Keyword(s):  
Sodium Channel ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Consensus Sequence ◽  
A549 Cells ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Α Subunit

Expression of the α-subunit of the amiloride-sensitive sodium channel (αENaC) is regulated by a number of factors in the lung, including oxygen partial pressure (Po2). As transcriptional activation is a mechanism for raising cellular mRNA levels, we investigated the effect of physiological changes in Po2 on the activity of the redox-sensitive transcription factor nuclear factor κB (NF-κB) and transcriptional activity of 5′-flanking regions of the human αENaC gene using luciferase reporter-gene vectors transiently transfected into human adult alveolar carcinoma A549 cells. By Western blotting we confirmed the presence of NF-κB p65 but not p50 in these cells. Transiently increasing Po2 from 23 to 42mmHg for 24h evoked a significant increase in NF-κB DNA-binding activity and transactivation of a NF-κB-driven luciferase construct (pGLNF-κBpro), which was blocked by the NF-κB activation inhibitor sulphasalazine (5mM). Transcriptional activity of αENaC-luciferase constructs containing 5′-flanking sequences (including the NF-κB consensus) were increased by raising Po2 from 23 to 142mm Hg if they contained transcriptional initiation sites (TIS) for exons 1A and 1B (pGL3E2.2) or the 3′ TIS of exon 1B alone (pGL3E0.8). Sulphasalazine had no significant effect on the activity of these constructs, suggesting that the Po2-evoked rise in activity was not a direct consequence of NF-κB activation. Conversely, the relative luciferase activity of a construct that lacked the 3′ TIS, a 3′ intron and splice site but still retained the 5′ TIS and NF-κB consensus sequence was suppressed significantly by raising Po2. This effect was reversed by sulphasalazine, suggesting that activation of NF-κB mediated Po2-evoked suppression of transcription from the exon 1A TIS of αENaC.

Nuclear factor-κB activation by the photochemotherapeutic agent verteporfin

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 256-262 ◽  
Author(s):  
D. J. Granville ◽  
C. M. Carthy ◽  
H. Jiang ◽  
J. G. Levy ◽  
B. M. McManus ◽  
...  
Keyword(s):  
Stress Responses ◽  
Nuclear Factor Κb ◽  
Light Irradiation ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Luciferase Reporter Construct ◽  
Nuclear Extracts ◽  
The Status ◽  
General Caspase Inhibitor ◽  
Induced Apoptosis

The nuclear factor-kappa B (NF-κB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-κB principally resides within the cytoplasm in association with inhibitory κ (IκB) proteins. The status of IκB and NF-κB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IκB proteins may be caspase substrates during apoptosis. However, the level of IκBβ was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IκB levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IκB levels were transiently depressed after PDT. At these times, p50 and RelA NF-κB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a κB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-, a well-characterized NF-κB activator. Productive NF-κB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IκB is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)

Nuclear factor-κB activation by the photochemotherapeutic agent verteporfin

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 256-262 ◽  
Author(s):  
D. J. Granville ◽  
C. M. Carthy ◽  
H. Jiang ◽  
J. G. Levy ◽  
B. M. McManus ◽  
...  
Keyword(s):  
Stress Responses ◽  
Nuclear Factor Κb ◽  
Light Irradiation ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Luciferase Reporter Construct ◽  
Nuclear Extracts ◽  
The Status ◽  
General Caspase Inhibitor ◽  
Induced Apoptosis

Abstract The nuclear factor-kappa B (NF-κB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-κB principally resides within the cytoplasm in association with inhibitory κ (IκB) proteins. The status of IκB and NF-κB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IκB proteins may be caspase substrates during apoptosis. However, the level of IκBβ was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IκB levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IκB levels were transiently depressed after PDT. At these times, p50 and RelA NF-κB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a κB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-, a well-characterized NF-κB activator. Productive NF-κB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IκB is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)

scholarly journals The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

2012 ◽  
Vol 32 (6) ◽  
pp. 531-537 ◽  
Author(s):  
Albert Braeuning ◽  
Silvia Vetter
Keyword(s):  
Photon Emission ◽  
Gene Promoter ◽  
Firefly Luciferase ◽  
Nuclear Factor Κb ◽  
Signalling Pathway ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Reporter System ◽  
Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

scholarly journals Developmental regulation of specific protein interactions with an enhancerlike binding site far upstream from the avian very-low-density apolipoprotein II gene.

1990 ◽  
Vol 10 (1) ◽  
pp. 154-164 ◽  
Author(s):  
P A Hoodless ◽  
R N Roy ◽  
A K Ryan ◽  
R J Haché ◽  
M Z Vasa ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Developmental Regulation ◽  
Consensus Sequence ◽  
Specific Protein ◽  
Low Density ◽  
Nuclear Factor ◽  
Stage Of Development ◽  
Nuclear Extracts ◽  
Enhancer Binding Protein ◽  
Nuclear Factor 1

Expression of the avian very-low-density apolipoprotein II (apoVLDLII) gene is completely dependent on estrogen and restricted to the liver. We have identified binding sites for nonhistone nuclear proteins located between -1.96 and -2.61 kilobases. One of these sites, located at -2.6 kilobases (designated site 1), was found to span an MspI site that becomes demethylated between days 7 and 9 of embryogenesis, the stage of development at which competence to express the apoVLDLII gene begins to be acquired. Levels of the factor(s) involved were high at day 7 of embryogenesis, decreased two- to threefold by days 9 to 11, and continued to decline more slowly until hatching. Furthermore, the mobility of the complex formed underwent a well-defined shift between days 11 to 13 embryogenesis. Methylation interference studies showed that modification of the outer guanosines of the MspI site resulted in marked inhibition of the formation of the protein-DNA complex. Competition studies, fractionation of nuclear extracts, and tissue distribution indicated that the factor was not the avian homolog of hepatocyte nuclear factor 1, nuclear factor 1, or CCAAT/enhancer-binding protein (C/EBP). However, site 1 could complete for binding to an oligonucleotide, previously shown to be recognized by C/EBP, in a nonreciprocal fashion. These studies demonstrate that the sequence recognized by the protein includes a C/EBP consensus sequence but that elements in addition to the core enhancer motif are essential for binding.

scholarly journals Transcriptional regulation of alpha(1b) adrenergic receptors (alpha(1b)AR) by nuclear factor 1 (NF1): a decline in the concentration of NF1 correlates with the downregulation of alpha(1b)AR gene expression in regenerating liver.

1996 ◽  
Vol 16 (11) ◽  
pp. 5997-6008 ◽  
Author(s):  
B Gao ◽  
L Jiang ◽  
G Kunos
Keyword(s):  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Upstream Region ◽  
Remnant Liver ◽  
Nuclear Factor ◽  
Nucleotide Substitutions ◽  
P2 Promoter ◽  
Hep3b Cells ◽  
Nuclear Factor 1

The 5' upstream region from --490 to --540 (footprint II) within the dominant P2 promoter of the rat alpha(1b) adrenergic receptor (alpha(1b)AR) gene is recognized by a sequence-specific DNA-binding protein (B. Gao, M. S. Spector, and G. Kunos, J. Biol. Chem. 270:5614-5619, 1995). This protein, detectable in Southwestern (DNA-protein) blots of crude nuclear extracts as 32- and 34-kDa bands, has been purified 6,000-fold from rat livers by DEAE-Sepharose, heparin-Sepharose, and DNA affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV cross-linking of the purified protein indicated the same molecular mass as that in crude extracts. Methylation interference analysis revealed strong contact with a TTGGCT hexamer and weak contact with a TGGCGT hexamer in the 3' and 5' portions of footprint II, respectively. Nucleotide substitutions within these hexamers significantly reduced protein binding to footprint II and the promoter activity of P2 in Hep3B cells. The purified protein also bound to the nuclear factor 1 (NF1)/CTF consensus sequence, albeit with lower affinity. Gel mobility supershift and Western blotting (immunoblotting) analyses using an antibody against the NF1/CTF protein identified the purified 32- and 34-kDa polypeptides as NF1 or a related protein. Cotransfection into Hep3B cells or primary rat hepatocytes of cDNAs of the NF1-like proteins NF1/L, NF1/X, and NF1/Redl resulted in a three- to fivefold increase in transcription directed by wild-type P2 but not by the mutated P2. Partial hepatectomy markedly decreased the levels of NF1 in the remnant liver and its binding to P2, which paralleled declines in the rate of transcription of the alpha(1b)AR gene and in the steady-state levels of its mRNA. These observations indicate that NF1 activates transcription of the rat alpha(1b)AR gene via interacting with its P2 promoter and that a decline in the expression of NF1 is one of the mechanisms responsible for the reduced expression of the alpha(1b)AR gene during liver regeneration.

scholarly journals MicroRNA-218 Negatively Regulates Osteoclastogenic Differentiation by Repressing the Nuclear Factor-κB Signaling Pathway and Targeting Tumor Necrosis Factor Receptor 1

2018 ◽  
Vol 48 (1) ◽  
pp. 339-347 ◽  
Author(s):  
Weiwei Wang ◽  
Lei Yang ◽  
Dan Zhang ◽  
Chao Gao ◽  
Jie Wu ◽  
...  
Keyword(s):  
Bioinformatics Analysis ◽  
Tumor Necrosis Factor Receptor ◽  
Nuclear Factor Κb ◽  
Raw 264.7 Cells ◽  
Luciferase Reporter ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Reporter Assay ◽  
Trap Staining ◽  
Necrosis Factor

Background/Aims: Postmenopausal osteoporosis is a common disease associated with estrogen deficiency leading to bone loss and bone tissue changes. The resultant bone fragility and increased risk of fracture has serious adverse effects on health and quality of life of the elderly, making it an important health issue. MicroRNA-218 (miR-218) is closely related to the development of osteoporosis. In this study, we investigated the regulatory mechanisms of miR-218 in osteoclastogenesis. Methods: We investigated miR-218 levels on differentiation of RAW 264.7 cells into osteoclasts compared with normal cells. Next, RAW 264.7 cells were transfected with miR-218 mimics or inhibitors to study the role of miR-218 in osteoclastogenic differentiation. Tartrate-resistant acid phosphatase (TRAP) staining was performed to determine osteoclastogenic differentiation. Bioinformatics analysis and luciferase reporter assay were used to identify and validate miR-218 target genes. Results: miR-218 was downregulated following RAW 264.7 cell differentiation into osteoclasts. miR-218 overexpression attenuated osteoclast differentiation, whereas low miR-218 expression promoted it as demonstrated by increased expression of osteoclast-specific genes and TRAP staining. Bioinformatics analysis and the luciferase reporter assay showed that tumor necrosis factor receptor 1 (TNFR1), a cell membrane receptor of TNF (TNF is an activator of nuclear factor-κB [NF-κB]), is a direct target of miR-218. Conclusions: Our findings indicate that miR-218 regulates osteoclastogenic differentiation negatively by repressing NF-κB signaling by targeting TNFR1, suggesting that targeting miR-218 may be a therapeutic approach in postmenopausal osteoporosis.

scholarly journals JFC1 is transcriptionally activated by nuclear factor-κB and up-regulated by tumour necrosis factor α in prostate carcinoma cells

2002 ◽  
Vol 367 (3) ◽  
pp. 791-799 ◽  
Author(s):  
Sergio D. CATZ ◽  
Bernard M. BABIOR ◽  
Jennifer L. JOHNSON
Keyword(s):  
Tumour Necrosis Factor ◽  
Cell Lines ◽  
Prostate Carcinoma ◽  
Tumour Necrosis ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Tumour Necrosis Factor Α ◽  
Factor Α ◽  
Necrosis Factor

The human promoter region of JFC1, a phosphatidylinositol 3,4,5-trisphosphate binding ATPase, was isolated by amplification of a 549bp region upstream of the jfc1 gene by the use of a double-PCR system. By primer extension analysis we mapped the transcription initiation site at nucleotide −321 relative to the translation start site. Putative regulatory elements were identified in the jfc1 TATA-less promoter, including three consensus sites for nuclear factor-κB (NF-κB). We analysed the three putative NF-κB binding sites by gel retardation and supershift assays. Each of the putative NF-κB sites interacted specifically with recombinant NF-κB p50, and the complexes co-migrated with those formed by the NF-κB consensus sequence and p50. An antibody to p50 generated a supershifted complex for these NF-κB sites. These sites formed specific complexes with nuclear proteins from tumour necrosis factor α (TNFα)-treated WEHI 231 cells, which were supershifted with antibodies against p50 and p65. The jfc1 promoter was transcriptionally active in various cell lines, as determined by luciferase reporter assays following transfection with a jfc1 promoter luciferase vector. Co-transfection with NF-κB expression vectors or stimulation with TNFα resulted in significant transactivation of the jfc1 promoter construct, although transactivation of a mutated jfc1 promoter was negligible. The expression of a dominant negative IκB (inhibitor κB) decreased basal jfc1 promoter activity. The cell lines PC-3, LNCaP and DU-145, but not Epstein—Barr virus-transformed lymphocytes, showed a dramatic increase in the expression of JFC1 after treatment with TNFα, suggesting that transcriptional activation of JFC1 by the TNFα/NF-κB pathway is significant in prostate carcinoma cell lines.

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