Fc epsilonRI-mediated induction of TNF-alpha gene expression in the RBL-2H3 mast cell line: regulation by a novel NF-kappaB-like nuclear binding complex.
Using rat basophilic leukemia (RBL-2H3) cells as a model, we investigated how aggregation of the high affinity receptor for IgE (Fc epsilonRI) regulates TNF-alpha gene expression. Antigenic stimulation of RBL-2H3 cells led to an increase in newly synthesized TNF-alpha mRNA that was dependent on continuous receptor aggregation and did not require de novo protein synthesis. Kinetic analysis showed that maximal levels were achieved at 60 min and waned by 180 min of stimulation. Concomitant with the transcriptional activation of the TNF-alpha gene, the rapid appearance and disappearance of a previously uncharacterized nuclear NF-kappaB DNA binding activity, comprised of two distinct protein complexes, were observed. These protein complexes bound to NF-kappaB sites within the TNF-alpha gene and contained novel proteins (three species of Mr between 90,000-110,000) distinct from the classical proteins in NF-kappaB complexes. The induced NF-kappaB binding activity required continuous receptor stimulation and induced NF-kappaB-dependent reporter gene expression. Consistent with a role for the novel NF-kappaB nuclear binding activity in TNF-alpha gene expression, deletion of several 5' kappaB elements in the TNF-alpha promoter abolished all measurable Fc epsilonRI-dependent induction of a reporter construct. Pharmacologic agents that inhibited the NF-kappaB binding activity also inhibited TNF-alpha mRNA expression. Our results demonstrate that a novel NF-kappaB-like nuclear binding activity plays an important role in regulation of the rapid and transient transcriptional activation of the TNF-alpha gene via Fc epsilonRI.
Pelletier
C
C
Unité Immuno-Allergie, Institut Pasteur, Paris, France.
Varin-Blank
N
N
Rivera
J
J
Iannascoli
B
B
Marchand
F
F
David
B
B
Weyer
A
A
Blank
U
U
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D015973:Gene Expression Regulation, Leukemic / Q000502:physiology*; D017930:Genes, Reporter; D007073:Immunoglobulin E / Q000502:physiology*; D015471:Leukemia, Basophilic, Acute / Q000473:pathology; D046911:Macromolecular Substances; D008407:Mast Cells / Q000378:metabolism*; D008970:Molecular Weight; D016328:NF-kappa B; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011940:Receptor Aggregation; D017455:Receptors, IgE / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D016328:NF-kappa B; D009363:Neoplasm Proteins; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D007073:Immunoglobulin E; D015500:Chloramphenicol O-Acetyltransferase