Toxicology 143 (2000) 249 – 261 Beryllium-stimulation does not activate transcription factors in a mouse hybrid macrophage cell line Hironobu Hamada a, *, Richard T. Sawyer a,b , Lori A. Kittle a , Lee S. Newman a,b,c a Diision of Enironmental and Occupational Health Sciences, Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Dener, CO 80206, USA b Diision of Pulmonary and Critical Care Medicine, Department of Medicine, Uniersity of Colorado Health Sciences Center, Dener, CO USA c Department of Preentie Medicine and Biometrics, Uniersity of Colorado Health Sciences Center, Dener, CO, USA Received 14 August 1999; accepted 22 October 1999 Abstract We tested the hypothesis that beryllium (Be) could stimulate H36.12j cell (12j) TNF- production by transcription factor-mediated pathways similar to those induced by either LPS- or IFN- stimulation. Unstimulated 12j cells produce constitutive levels of TNF- (175 18 pg/ml, mean SEM) detected by ELISA of culture supernatants after 24 h. Beryllium-stimulated (100 M BeSO 4 ) 12j cell TNF- (724 47 pg/ml) was observed after 24 h while LPS-stimulated (1 g/ml) TNF- (515 151 pg/ml) after 6 h. Recombinant-Mu-IFN- (10 U) stimulated 12j cell TNF- at lower levels (284 31 pg/ml) while rMu-IFN- +Be-stimulated 12j cells produced 1195 225 pg/ml TNF-. Constitutive levels of transcription factors were observed in unstimulated 12j cell nuclei. In LPS-stimulated 12j cells IB was degraded in the cytoplasm and increased levels of NF-B 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-B, 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-. However, NF-B 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- +Be increased the levels of NF-B 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- 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- synthesis via a transcription factor-independent pathway. Beryllium may induce novel pathways of macrophage cytokine gene regulation. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Beryllium; Chronic beryllium disease; Transcription factors; Macrophages; Hybrid macrophages; NF-B/IB; AP-1; CREB; Bzip proteins; Lipopolysaccharide; Interferon-; Tumor necrosis factor-; TNF- promoter www.elsevier.com/locate/toxicol * Corresponding author. Tel.: +1-303-398-1167; fax: +1-303-398-1851. E-mail address: sawyerr@njc.org (H. Hamada) 0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII:S0300-483X(99)00183-3