Characterization of a Mutant Cell Line That Does Not Activate NF-κB in Response to Multiple Stimuli

Abstract

Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor B (NF-B). Associated with its inhibitor, IB, NF-B resides as an inactive form in the cytoplasm. Upon stimulation by various agents, IB is proteolyzed and NF-B translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to IB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-B in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, IB␣ is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in IB␣ nor a mutation in p50 or relA, the two major subunits of NF-B in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of IB␣ on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce IB␣ degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-B activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other IB molecules, IB␤ and the recently identified IB, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-B. In addition, this analysis suggests a common step in the signaling pathways that trigger IB␣, IB␤, and IB degradation.

The Trimerization Domain of Nemo Is Composed of the Interacting C-terminal CC2 and LZ Coiled-coil Subdomains

“…Stable and Transient Transfections-The 1.3E2 cells were transiently transfected essentially as in a previous study (26). To generate stable 1.3E2 clones, 5 g of HA-nemo or HA-LZ-mut-nemo plasmids were added to 5 ϫ 10 6 cells in a 500-l volume and electroporated at 260 V, 1500 microfarads, and R ϱ. G418 (1 g/ml) was added the following day, and cells were cloned by limited dilution after a week of selection.…”

“…The murine pre-B cell mutant 1.3E2 not expressing NEMO does not respond to LPS activation (6,26). These cells were transiently co-transfected with an NF-B-dependent construct (Ig -luciferase) and plasmids bearing wild-type or mutant nemo cDNAs.…”

Inhibition of NF-κB Activation by Peptides Targeting NF-κB Essential Modulator (NEMO) Oligomerization

“…The human retinoblastoma cell line Y79 (American Type Culture Collection; Manassas, VA), was grown in RPMI 1640 medium supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal calf serum. Jurkat T cells were transiently transfected by a DEAE-dextran method with an SRE-Luc reporter plasmid as described (26). 24 h after transfection, cells were incubated without or with NEMO-derived peptides for 2 h and then mock-stimulated or stimulated with 100 ng/ml phorbol 12-myristate 13-acetate and 1 g/ml ionomycin for 5 h. Luciferase expression was carried out as described in Ref.…”

“…FACS Analysis-Stable cell lines (70Z/3-C3) were obtained after electroporation of 70Z/3 cells (26) in the presence of a cx12lacZ-B plasmid containing three tandem copies of an NF-B-binding site in the interleukin-2 promoter (27) upstream from the lacZ reporter gene (a gift from G. R. Crabtree). The human retinoblastoma cell line Y79 (American Type Culture Collection; Manassas, VA), was grown in RPMI 1640 medium supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal calf serum.…”

Characterization of Calcineurin in Human Neutrophils

“…A more suitable ROI messenger would be the less reactive H 2 O 2 . However, arguments against ROI involvement in NF-B activation have been published (30 -32), and despite the fact that phorbol 12-myristate 13-acetate-dependent NF-B stimulation is cancelled by antioxidants, it has been recently shown that phorbol 12-myristate 13-acetate does not increase intracellular ROI (33). Peroxide-mediated stimulation of NF-B appears to be cell line-specific, since N-acetylcysteine, an antioxidant, elicited up-regulation of NF-B binding activity in monocyte-derived macrophages (34).…”