Nadine Tarantino

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO–IKK supramolecular structures

The mechanism of NEMO recruitment into supramolecular complexes and its dependence on ubiquitination differs in response to the proinflammatory cytokines TNF and IL-1.

Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia

Boisson et al. report a human homozygous mutation of HOIP, the gene encoding the catalytic component of the linear ubiquitination chain assembly complex, LUBAC. The missense alleles impair the expression of HOIP, destabilizing the LUBAC complex and resulting in immune cell dysfunction leading to multiorgan inflammation, combined immunodeficiency, subclinical amylopectinosis, and systemic lymphangiectactasia.

Relationship between IκBα constitutive expression, TNFα synthesis, and apoptosis in EBV-infected lymphoblastoid cells

In order to understand the role of NF-κB in EBV transformation we have established stably transfected IκBα into lymphoblastoid cells. Two clones were obtained in which the loss of NF-κB binding activity correlated with the constitutive expression of the transgenic IκBα. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFα levels were strongly reduced in transfected clones. Furthermore, 30% of IκBα transfected cells were apoptotic after 8 h of TNFα treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFα treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFα gene could be one of the targets of NF-κB in EBV infected cells and that NF-κB protects EBV-infected cells from apoptosis induced by TNFα, which may favour the proliferative effect of this cytokine.

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.

The 4 Notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors

The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a γ‐secretase inhibitor, at 50 μM for 48 h, reduced cell growth by 50%. S‐phase entry was impaired, but no apoptosis was induced. A systematic paralog‐specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down‐regulation of p27Kip1 and p57Kip2 cyclin‐dependent kinase inhibitors. Conversely, Notch3‐expressing cells proliferate less and express 3‐fold higher levels of p57Kip2. The Notch3 cells present a hepatocyte‐like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic‐lineage commitment of liver progenitors.—Ortica, S., Tarantino, N., Aulner, N., Israël, A., Gupta‐Rossi, N. The 4 Notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors. FASEB J. 28, 603–614 (2014). www.fasebj.org

Stathmin phosphorylation patterns discriminate between distinct transduction pathways of human T lymphocyte activation through CD2 triggering

CD2 triggering of human T lymphocyte activation has been associated with the activation of different interacting protein kinases, including protein kinase C (PKC). However the precise roles of its phosphorylated substrates are still unknown. We show here that PKC‐dependent and ‐independent pathways arc responsible for the CD2‐induced phosphorylation of stathmin, a ubiquitous soluble phosphoprotein, most likely acting as a general intracellular relay integrating various second messenger pathways. The phosphorylated variants of stathmin provide a fingerprint reflecting the second messenger pathway(s) stimulated. The respective roles of both PKC and stathmin in the regulation of T lymphocyte proliferation are discussed.

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.