Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5''-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions.

We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S-adenosylmethionine-dependent methylation reactions. We further show that the LPS-induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway.     

Interleukin 1 and cyclic AMP induce kappa immunoglobulin light-chain expression via activation of an NF-kappa B-like DNA-binding protein.

Interleukin 1 (IL-1) induces the synthesis of kappa immunoglobulin light chains and the expression of surface immunoglobulin in the murine pre-B-cell line 70Z/3 (J. G. Giri, P. W. Kincade, and S. B. Mizel, J. Immunol. 132:223-228, 1984). In the present study, we found that these effects of IL-1 are mimicked by cyclic AMP (cAMP) analogs and cAMP-elevating drugs. The induction of kappa immunoglobulin light-chain gene expression by IL-1 was associated with an increase in intracellular cAMP levels. Incubation of 70Z/3 cells with IL-1 or cAMP resulted in the activation of the kappa immunoglobulin enhancer, as detected by the induction of chloramphenicol acetyltransferase (CAT) in cells transfected with a kappa enhancer-CAT expression plasmid. In contrast, CAT plasmids lacking a kappa immunoglobulin enhancer were inactive in the presence of IL-1 or cAMP. Furthermore, IL-1 and cAMP analogs and inducers were found to induce the activation of a NF-kappa B-like DNA-binding protein that exhibited specificity for the kappa immunoglobulin enhancer. These results suggest that cAMP may play an important role as a second messenger for IL-1 in the induction of kappa immunoglobulin light-chain synthesis in pre-B cells via the activation of a DNA-binding protein that is similar or identical to NF-kappa B.     

Induction of nuclear NF-kappa B DNA binding activity after exposure of lymphoid cells to soluble tax1 protein

We demonstrate that purified HTLV-I Tax1 protein can be taken up by 70Z/3 lymphoid cells and localized in both the nuclear and cytoplasmic compartments. Introduction of the Tax1 protein into the growth medium of 70Z/3 cells resulted in the rapid and transient induction of NF-kappa B binding activity in the nuclear fraction. Tax1 activation of NF-kappa B was not sensitive to either staurosporin or prolonged stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, suggesting that Tax1-dependent NF-kappa B activation did not require the protein kinase C pathway. Purified Tax1 did not directly increase NF-kappa B binding activity in 70Z/3 cytoplasmic extracts, suggesting that NF-kappa B induction may require cellular factors. Western blot and competitive radioimmunoassays demonstrated that Tax1 protein was present in the tissue culture media of HTLV-I-transformed cell lines. These results show that extracellular Tax1 may regulate cellular gene expression in noninfected cells.     

Interleukin 1 induces NF-kappa B through its type I but not its type II receptor in lymphocytes.

It is not known whether one or both of the interleukin 1 (IL1) receptors mediates the induction of the DNA-binding protein NF-kappa B. Nuclear extracts of the murine lines EL4.NOB.1 and 70Z/3, which bear the type I (80 kDa) and type II (67 kDa) IL1 receptor, respectively, were analyzed by an electrophoretic mobility shift assay. A 265-base pair sequence of the human serum amyloid A gene or a synthetic oligonucleotide each containing the NF-kappa B site were used as the DNA probes. IL1 induction of NF-kappa B was rapid (optimal at 15-30 min) and transient in both cell types. The IL1 receptor antagonist (IL1ra), which binds strongly to the type I receptor, inhibited the NF-kappa B response in both cell lines. IL1ra did not bind to the type II receptor on 70Z/3 cells as judged by competition for binding with 125I-IL1 alpha. When 125I-IL1ra binding to 70Z/3 cells was measured, a small number (10/cell) of high affinity sites (Kd = 5 x 10(-12) M) were detected. These were likely to have been type I receptor because an antibody to this inhibited the NF-kappa B induction in 70Z/3 cells (as well as EL4). Potential signal transduction mechanisms involving protein kinase C or oxygen radicals were studied. Phorbol 12-myristate 13-acetate induced NF-kappa B with a similar time course to IL1 in 70Z/3 but only after 4 h in EL4.IL1 was unaffected by a protein kinase C inhibitor (staurosporine). H2O2 did not mimic IL1, and IL1 was not inhibited by an antioxidant. The type I receptor mediates the induction of NF-kappa B in response to IL1 via a signaling mechanism that still remains to be identified.     

Control of IgM synthesis in the murine pre-B cell line, 70Z/3'

The murine 70Z/3 tumor resembles a pre-B cell in synthesizing only intracellular mu-chains and no detectable light chain. However, one kappa gene is already rearranged, and after overnight incubation with lipopolysaccharide (LPS), most of the cells are induced to synthesize light chain. The induced cells display IgM on their surface, but do not secrete IgM. Thus, 70Z/3 cells resemble cells poised at the pre-B cell/B lymphocyte border. We have examined synthesis and post-translational modification of mu-chains in uninduced and induced 70Z/3 cells. Isolation of mu-chains and peptide maps demonstrated that both populations synthesize intracellular forms that correspond to membrane-specific mum and secretion-specific mus chains. These intracellular forms have completed only the first of the two glycosylation steps characteristic of eukaryotic cells. After induction by LPS, L chain synthesis commences, mum and mus synthesis are both increased twofold to threefold (due to an increased rate of synthesis rather than decreased degradation), and both complex with L chain to form mu2L2 tetramers. Furthermore, the glycosylation of a subset of the mum chains is completed, and these are placed on the membrane. However, unglycosylated mu2L2 tetramers can be placed on the membrane, so glycosylation is not a requirement. These data suggest that L chain may not be sufficient for externalization of mum and mus chains. These data support the idea that the controls of membrane placement and secretion of mu chains are post-translational and that different mechanisms operate for mum and mus chains.     

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt''s lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.     

Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor

In cells that do not express kappa immunoglobulin light chain genes, the kappa enhancer-binding protein NF-kappa B is not evident in either cytoplasmic or nuclear fractions. By denaturation, size fractionation, and renaturation, however, NF-kappa B activity can be revealed in cytosolic fractions, showing that the DNA-binding protein is present but inhibited in its binding activity. By using a variety of protocols involving the dissociating agents sodium desoxycholate and formamide, as much cytosolic NF-kappa B can be found in the fraction from unstimulated 70Z/3 pre-B cells as is found in the nuclear extract from phorbol ester-activated cells. We conclude that both 70Z/3 and HeLa cells contain apparently cytosolic NF-kappa B in a form with no evident DNA-binding activity, and phorbol esters both release the inhibition of binding and cause a translocation to the nucleus.     

The plasmacytoma J558L lacks constitutively active NF-kappa B and is deficient in early response gene activation.

In mature B cells the nuclear factor NF-kappa B which binds within the kappa enhancer is constitutively present in the nucleus. However, the lambda light chain producing myeloma J558L has been found to lack constitutively functional NF-kappa B. Deoxycholate released functional NF-kappa B from cytoplasmic extracts and functional NF-kappa B was present in J558L following cycloheximide but not phorbol ester treatment. J558L was also unable to respond to phorbol ester stimulation with synthesis of mRNA from the early response gene TIS11. J558L differs from S107, another myeloma which was found to be deficient in the synthesis of NF-kappa B but not in the activation of TIS11. Somatic cell hybrids were used to further define the defect in J558L; hybrids were made with the myelomas S107 and S194 and the pre-B cell line 70Z/3. In general, complementation of the defect in J558L was observed; however there was not a direct correlation between the levels of TIS11 mRNA and NF-kappa B expression in the somatic cell hybrids, suggesting that the pathways of activation of these genes, while possibly sharing common elements, are not identical. The defect in J558L was surprising given that it has frequently been used for the expression of transfected light chain genes.