Biol. Chem., Vol. 385, pp. 415–417, May 2004 • Copyright  by Walter de Gruyter • Berlin • New York 2004/311120 Short Communication Activation of NF-kB/Rel transcription factors in human primary peripheral blood mononuclear cells by interleukin 7 Figure 1 Activation of NF-kB/Rel by IL-7. (A) Induction of NF-kB/Rel nuclear complexes by IL-7. T lym- phocytes were isolated from normal donors’ heparinized blood by a two-step centrifugation through a Ficoll Hypaque column (ICN Flow, Opera, Italy) (400 g for 30 min) followed by a 50– 53.5% Percoll (Pharmacia, Uppsala, Sweden) density gradient (500 g for 30 min). Cells recovered from the pellet were )95% CD2 q and -1% CD33 q as determined by immunofluorescence. T lymphocytes (10 6 /ml) were incubated for 3 h with control medi- um or 70 ng/ml human recombinant IL-7 (Sanofi-Synthelabo, Labe´ ge, France; specific activity: 3.2=10 7 U/mg). Cell nuclear extracts were prepared and incubated with a w 32 Px-labeled kB oligonucleotide (59-CAA CGG CAG GGG AAT CTC CCT CTC CTT-39). The nuclear extract of IL-7-stimulated cells was incu- bated with the labeled kB oligo in the absence or presence of a 50-fold molar excess of unlabeled AP-1 or kB oligo. DNA/protein complexes were analyzed by electrophoretic mobility shift assay (EMSA; Romano et al., 1999). Arrow 1 indicates a non-specific band, while arrows 2 and 3 indicate specific NF-kB/Rel com- plexes. Similar results were obtained in experiments with T lym- phocytes from four different healthy donors. (B) Time course of NF-kB/Rel activation by IL-7. T lymphocytes (10 6 /ml) were incu- bated with control medium or IL-7 (70 ng/ml) for 3, 16, 24 or 48 h. NF-kB/Rel nuclear complexes were then analyzed by EMSA. Annalisa Lamberti 1 , Antonello Petrella 2 , Maria Pascale 2 , Maria Fiammetta Romano 1 , Rita Bisogni 1 , Natalio Vita 3 , Salvatore Venuta 4 and Maria Caterina Turco 2, * 1 Department of Biochemistry and Biomedical Technologies (DBBM), University ‘Federico II’, via Pansini 5, I-80131 Naples, Italy 2 Department of Pharmacological Sciences (DIFARMA), University of Salerno, Via ponte don Melillo, I-84084 Fisciano (SA), Italy 3 Sanofi Laboratories, F-34184 Montpellier, France 4 Department of Experimental and Clinical Medicine, University of Catanzaro, via Campanella, I-88100 Catanzaro, Italy * Corresponding author e-mail: mcturco@unisa.it Abstract Pathways that regulate the activation of NF-kB/Rel tran- scription factors are known to include signaling through a number of cytokine receptors. Interleukin 7 (IL-7), pro- duced by bone marrow and other stromal cells, is a key factor for differentiation and survival in the lymphoid and other compartments. We found that human recombinant IL-7 induced NF-kB/Rel activation, analyzed by electro- phoretic mobility shift assay (EMSA), in human peripheral blood T lymphocytes from healthy donors. Induced com- plexes included p65 and p50 NF-kB/Rel subunits. These results demonstrate for the first time that IL-7 can partic- ipate in signaling leading to NF-kB/Rel activation. Keywords: interleukin 7; NF-kB/Rel. The activity of NF-kB/Rel transcription factors regulates a number of cellular functions, including cellular prolif- eration, differentiation and survival (Karin and Lin, 2003; Kwon et al., 2003). In human leukocytes, NF-kB/Rel-reg- ulated genes control life/death balance and functional activation in normal and neoplastic progenitor and dif- ferentiated cells (Karin and Lin, 2003; Turco et al., 2004). Among factors that activate NF-kB/Rel in leukocytes, cytokines, including interleukin (IL)-1, -2, GM-CSF, tumor necrosis factors and others mediate paracrine signals originating from the cell environment (Li and Verma, 2002). IL-7, produced by stromal cells from bone marrow, thy- mus and other tissues, is a survival factor of key rele- vance for the differentiation and survival of lymphocytes (Alpdogan et al., 2003; Jaleco et al., 2003; Khaled and Durum, 2003; Napolitano et al., 2003). Here we analyzed Brought to you by | Karolinska Institute Authenticated Download Date | 6/2/15 4:35 PM