Beryllium-stimulation does not activate transcription factors in a mouse hybrid macrophage cell line.
We tested the hypothesis that beryllium (Be) could stimulate H36.12j cell (12j) TNF-alpha production by transcription factor-mediated pathways similar to those induced by either LPS- or IFN-gamma stimulation. Unstimulated 12j cells produce constitutive levels of TNF-alpha (175+/-18 pg/ml, mean +/- SEM) detected by ELISA of culture supernatants after 24 h. Beryllium-stimulated (100 microM BeSO4) 12j cell TNF-alpha (724+/-47 pg/ml) was observed after 24 h while LPS-stimulated (1 microg/ml) TNF-alpha (515+/-151 pg/ml) after 6 h. Recombinant-Mu-IFN-gamma (10 U) stimulated 12j cell TNF-alpha at lower levels (284+/-31 pg/ml) while rMu-IFN-gamma + Be-stimulated 12j cells produced 1195+/-225 pg/ml TNF-alpha. Constitutive levels of transcription factors were observed in unstimulated 12j cell nuclei. In LPS-stimulated 12j cells IkappaBalpha was degraded in the cytoplasm and increased levels of NF-kappaB were found in nuclei after 30 min. After 3 h there were increased levels of AP-1 and CREB, with increased amounts of Fos family, Jun B and Jun D transcription factors. In contrast, Be-stimulation failed to increase the levels of any transcription factor tested, NF-kappaB, AP-1, AP-2, CREB, C/EBP, Sp-1, Egr-1, Ets, NF-Y or Oct-1, in 12j cells. A pattern of increased transcription factors, similar to that observed for LPS-stimulation, was found in 12j cell nuclei after stimulation with rMu-IFN-gamma. However, NF-kappaB was increased at 3 h while AP-1 (Jun B and Jun D) and CREB were increased at 15 h. Co-stimulation of 12j cells with rMu-IFN-gamma + Be increased the levels of NF-KB in 12j cell nuclei at 3 h, and the levels of AP-1 and CREB at 15 h, however, only Jun B was increased. Our data show 12j cell TNF-alpha production was associated with increased levels of transcription factors present in nuclei with disparate kinetics and patterns of expression depending on the trigger. We reject our initial hypothesis and conclude that Be-stimulation signals 12j cell TNF-alpha synthesis via a transcription factor-independent pathway. Beryllium may induce novel pathways of macrophage cytokine gene regulation.
Hamada
H
H
Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206, USA. sawyerr@njc.org
Sawyer
R T
RT
Kittle
L A
LA
Newman
L S
LS
D000818:Animals; D001608:Beryllium / Q000494:pharmacology*; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004586:Electrophoresis; D051790:G-Box Binding Factors; D006822:Hybrid Cells; D007202:Indicators and Reagents; D016898:Interferon-alpha / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects*; D051379:Mice; D016328:NF-kappa B / Q000096:biosynthesis; D011401:Promoter Regions, Genetic / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D013268:Stimulation, Chemical; D014157:Transcription Factors / Q000096:biosynthesis / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D007202:Indicators and Reagents; D016898:Interferon-alpha; D008070:Lipopolysaccharides; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D001608:Beryllium