Marie Körner

RelB/p50 dimers are differentially regulated by TNF-α and lymphotoxin-β receptor activation: critical roles for p100

Tumor necrosis factor-α (TNF-α) and lymphotoxin-β receptor (LTβR) signaling both play important roles in inflammatory and immune responses through activation of NF-κB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-κB induction in response to TNF-α and LTβR activation. We demonstrate that LTβR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-α induces only RelA/p50 dimers. LTβR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKα but is independent of IKKβ, NEMO/IKKγ, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-α but not LTβ signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-κB pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved.

NF-κB activation by tumor necrosis factor α in the jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca2+-regulated kinases

Abstract NF-κB is a DNA-binding regulatory factor able to control transcription of a number of genes, including human immunodeficiency virus (HIV) genes. In T cells, NF-κB is activated upon cellular treatment by phorbol esters and the cytokine tumor necrosis factor α (TNFα). In the present work, we investigated the molecular events leading to NF-κB activation by TNFα in a human T cell line (Jurkat) and its subclone JCT6, which presents a deficiency in the PKA transduction pathway. We found that in both cell lines, both phorbol ester and TNFα were able to activate NF-κB. Phorbol activation was positively modulated by CA 1+ influx while TNFα activation was not. Furthermore, while PMA activation was inhibited by the PKC inhibitor staurosporin, the TNFα effect was unchanged. TNFα did not activate cAMP production and its signal was not modulated by cAMP activators. Moreover, cAMP activators did not activate NF-κB in Jurkat cells. Thus, TNFα-induced NF-gkB activation was found to be mediated by none of the major signal-mediating kinases such as protein kinase C (PKC), protein kinase A, or Ca 2 -regulated kinases. Furthermore, we found that cytoplasmic acidification facilitated NF-κB activation by both TNFα and PKC, by a mechanism that increases NF-κB/IκB dissociation without afflecting the NF-κB translocation step.

Impairment of Fas-antigen expression in adriamycin-resistant but not TNF-resistant MCF7 tumor cells

Anti‐cancer drugs and cytotoxic cytokines such as members of the TNF/Fas‐ligand family play a predominant role in apoptosis induction in tumor cells, and are critical in cancer therapy. In this study we used the human breast‐carcinoma cell line MCF7, its derivatives MCF7Adr (resistant to adriamycin) and R‐A1 (resistant to TNF), to determine the impact of acquired drug and cytokine resistance on susceptibility to Fas‐induced cytotoxicity and Fas‐antigen expression. While MCF7 and R‐A1 cells were killed by anti‐Fas in the presence of IFN‐γ, MCF7Adr was found to be resistant to Fas‐mediated apoptosis. This resistance was correlated with a loss of surface Fas‐protein expression. Fas‐gene transfer in MCF7Adr resulted in high sensitivity to Fas‐mediated cytotoxicity, indicating that the Fas signalling pathway is virtually intact in this cell line. Over‐expression of the MDRI gene in MCF7 following gene transfer did not affect Fas expression and anti‐Fas sensitivity, suggesting that the P‐gp‐mediated multidrug‐resistance phenotype is not directly involved in the loss of Fas expression, contrary to what has been observed by others in T‐cell lines. Furthermore, the down‐regulation of Fas expression and subsequent resistance to anti‐Fas were observed in drug‐resistant human ovarian‐carcinoma IGR‐OVI/VCR cells and leukemic lymphoblast CEM/VLB cells, suggesting that the alteration of Fas expression following drug‐resistance selection is not restricted to one cell type.

Discrimination between RelA and RelB Transcriptional Regulation by a Dominant Negative Mutant of IκBα

RelA and RelB belong to the nuclear factor-κB (NF-κB-Rel) transcription factor family. Both proteins are structurally and functionally related, but their intracellular and tissue distributions are different. In resting cells, RelB is found mostly in the nucleus, whereas RelA is sequestered in the cytosol by protein inhibitors, among which IκBα is the dominant form in lymphocytes. Upon cellular activation IκBα is proteolyzed, allowing RelA dimers to enter the nucleus and activate target genes. To study the selectivity of gene regulation by RelA and RelB, we generated T cell lines stably expressing a dominant negative mutant of IκBα. We show that selective inhibition of RelA-NF-κB decreased induction ofNFKB1, interleukin-2, and interleukin-2Rα genes but not c-myc. Transcription driven by the IκBα promoter was blocked by the transgenic IκBα; however, wild type IκBα was expressed in the transgenic cell clones but with much slower kinetics than that in control cells. Wild type IκBα expression was concomitant with RelB up-regulation, suggesting that RelB could be involved in transcription of IκBα through binding to an alternative site. These results indicate that RelB and RelA have both distinct and overlapping effects on gene expression.

Constitutive activation of NF-kB in human thymocytes

NF-kB is a eukaryotic transcription regulatory factor. In T cells and T cell lines, NF-kB is bound to a cytoplasmic proteic inhibitor, the IkB. Treatment of T cells with mitogens (phorbol esters) or cytokines (TNF alpha) induces NF-kB nuclear translocation and the subsequent expression of NF-kB dependent T cell genes. Here we examined the activation of NF-kB in human T cell thymic progenitors. We report differences in (Ca2+)i requirement for NF-kB activation in thymocytes as compared to mature T cells. Furthermore, our results indicated that thymocytes have a constitutively active form of NF-kB, suggesting that they are activated in vivo.

Activation of Nuclear Factor KB in Human Neuroblastoma Cell Lines

Abstract: The nuclear factor KB (NF‐kB) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, NF‐KB translocates from cytosol to nucleus as a result of transduction by tumor necrosis factor α (TNFα), phorbol ester, and other polyclonal signals. Using neuro‐blastoma cell lines as models, we have shown that in neural cells NF‐KB was present in the cytosol and translocated into nuclei as a result of TNFa treatment. The TNFα‐activated NF‐KB was transcriptionally functional. NF‐KB activation by TNFα was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12‐myristate 13‐acetate (PMA), which induce phenotypical differentiation of the SH‐SY5Y neuroblastoma cell line, activated NF‐KB, but only in that particular cell line. In a NGF‐responsive rat pheochromocytoma cell line, PC12, PMA activated NF‐KB, whereas NGF did not. In other neuroblastoma cell lines, such as SK‐N‐Be(2), the lack of PMA induction of differentiation was correlated with the lack of NF‐kB activation. We found, moreover, that in SK‐N‐Be(2) cells protein kinase C (PKC) enzymatic activity was much lower compared with that in a control cell line and that the low PKC enzymatic activity was due to low PKC protein expression. NF‐KB was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, NF‐KB activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with TNFα proved that NF‐KB activation was not sufficient for induction of neuroblastoma differentiation.

Identification and characterization of p100HB, a new mutant form of p100/NF-kappa B2

P100, which is encoded by NF-kappa B2, inhibits Rel dimers. It can also be processed into p52, one of the DNA binding sub-units of NF-kappa B/Rel factors. Several p100 C-terminal truncations that result from gene rearrangements are associated with lymphomagenesis. Here, we characterized a new p100 mutant that we termed p100HB. It originates from a point-mutation that generates a premature stop-codon, and thus the protein lacks the last 125 amino acids. We have detected p100HB in several human tumor cell lines. The truncated protein is mainly unprocessed, and although it still binds Rel dimers, it has reduced inhibitory potency compared to p100 and translocates into the nucleus. Thus, p100HB may be associated with deregulated NF-kappa B/Rel functions.

Differential expression of PKCα and PKCβ isozymes in CD4+, CD8+ and CD4+/CD8+ double positive human T cells

Using specific monoclonal and polyclonal antibodies, we have analyzed protein kinase C α and β isozyme expression in human T cells from peripheral blood (PB) and from thymus. While the PKCβ isozyme was present in all T cell sub‐types isolated from both PB and thymus, the α isozyme was found only in single positive CD4+ thymocytes, in PB‐CD4+ lymphocytes and in PB‐CD8+ T cells from several donors. It was absent from both, CD8+and double positive CD4+/CD8+ thymocytes. These results show that PKCα and ‐β are differentially regulated during intra‐thymic development and suggest that PKCα plays a specific role in helper T cell function.

Activation of HIV-enhancer binding activity by mild detergents in human T cells

Binding activity to the HIV enhancer, a sequence highly homologous to the NF-KB enhancer, is inducible by PMA in human Jurkat T cells. We here show that, similar to what is observed for NF-KB in B cells, mild detergents, such as Deoxycholate, reveal a cryptic binding activity in protein extracts from non activated cells. The complex revealed by detergent treatment correspond to the PMA inducible, highly sequence specific species. This result suggest the existence of an inhibitor in non activated cells, which is released by the detergent, as has been demonstrated in B cells. Mild detergent treatment of PMA activated extracts resulted in superinduction of the specific complex. These results suggest that regulation of HIV enhancer binding protein is similar to regulation of NF-KB in B cells.