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.

Thyrotropin modifies activation of nuclear factor κB by tumour necrosis factor α in rat thyroid cell line

2001 ◽  
Vol 354 (3) ◽  
pp. 573-579 ◽  
Author(s):  
Toyone KIKUMORI ◽  
Fukushi KAMBE ◽  
Takashi NAGAYA ◽  
Hiroomi FUNAHASHI ◽  
Hisao SEO
Keyword(s):  
Tumour Necrosis Factor ◽  
Tumour Necrosis ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Tumour Necrosis Factor Α ◽  
Tnf Α ◽  
Rat Thyroid ◽  
Factor Α ◽  
Necrosis Factor

We have recently demonstrated that nuclear factor κB (NF-κB) mediates the tumour necrosis factor α (TNF-α)-dependent expression of the gene encoding interleukin 6 (IL-6) in rat thyroid FRTL-5 cells cultured in the presence of thyrotropin (TSH). In the present study we investigated how TSH is involved in the activation of NF-κB by TNF-α in the cells. Electrophoretic mobility-shift assay revealed that, in the absence of TSH, TNF-α activated a single protein–DNA complex containing the p50 subunit but not other NF-κB subunits such as p65. In contrast, two distinct protein–DNA complexes were activated in the presence of TSH: the faster-migrating complex contained only p50 subunit; the slower-migrating complex consisted of p65–p50heterodimer. This TSH effect was mimicked by forskolin and thyroid-stimulating antibodies obtained from patients with Graves's disease, suggesting that an increase in intracellular cAMP is responsible for the induction of different NF-κBs by TNF-α. A transient transfection study with a luciferase reporter gene driven by multimerized NF-κB sites demonstrated that TNF-α increased the luciferase activities only in the presence of TSH, and that this increase was inhibited by the co-transfection of mutant p65, which prevented the function of wild-type p65 in a dominant-negative manner. Accordingly, TNF-α activated the expression of the IL-6 gene in the presence of TSH but not in its absence. Although the expression of the p105 gene, another known target for NF-κB, was increased by TNF-α in the absence of TSH, the presence of TSH further increased the mRNA level. Taken together, these observations indicate that the presence of TSH is crucial for the NF-κB-mediated actions of TNF-α on thyroid follicular cells.

Post-transcriptional effects of extracellular pH on tumour necrosis factor-α production in RAW 246.7 and J774 A.1 cells

2001 ◽  
Vol 100 (3) ◽  
pp. 259-266 ◽  
Author(s):  
Thomas A. HEMING ◽  
Divina M. TUAZON ◽  
Sanat K. DAVÉ ◽  
Ashok K. CHOPRA ◽  
Johnny W. PETERSON ◽  
...  
Keyword(s):  
Tumour Necrosis Factor ◽  
Cell Lines ◽  
Gene Transcription ◽  
Tumour Necrosis ◽  
Extracellular Ph ◽  
Tumour Necrosis Factor Α ◽  
Tnf Α ◽  
J774 Cells ◽  
Factor Α ◽  
Necrosis Factor

The present studies determined the effects of extracellular pH (pHo) on the production of tumour necrosis factor-α (TNF-α) in the macrophage-like cell lines RAW 246.7 and J774 A.1. The cells were activated with lipopolysaccharide (LPS) at pHo 5.5, 6.5 or 7.4. TNF-α gene transcription was monitored by Northern blot analysis. Synthesis of the cytokine was monitored by ELISA measurements of the TNF-α content of cell-conditioned media (extracellularly released TNF-α) and cell lysates (cytosolic TNF-α). The magnitude of the TNF-α response differed markedly between the two cell lines. RAW cells were more responsive to LPS than were J774 cells. However, the effects of pHo on TNF-α production were similar in the two cell lines. TNF-α gene transcription was insensitive to experimental pHo. The pHo had no effect on the abundance of TNF-α mRNA at 2, 4 or 18 h. Nonetheless, synthesis of TNF-α was affected significantly by pHo. The TNF-α contents of cell-conditioned medium and cell lysate at 18 h were reduced progressively at lower pHo values. The data indicate that pHo alters TNF-α production in RAW and J774 cells at a post-transcriptional level. These findings suggest that pHo influences the phenotypic responses of macrophages to activating stimuli and modifies the role that macrophages play in inflammatory and immune actions.

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