ets-2 is a target for an akt (Protein kinase B)/jun N-terminal kinase signaling pathway in macrophages of motheaten-viable mutant mice.

The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase-stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase-Akt-JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival.

Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis.

Anti-CD20 antibodies may reduce or eliminate non-Hodgkin's lymphoma B cells in patients, although the mechanism of action is not clear. To explore mechanism(s), we examined the induction of signal transduction events using anti-CD20 monoclonal antibodies (mAb) in the human non-Hodgkin's lymphoma Ramos B cell line. We found that while Rituximab (a human-mouse hybrid mAb) alone induced apoptotic cell death, other murine anti-CD20 mAbs induced apoptosis of Ramos B cells only upon clustering with a secondary antibody. CD20 clustering was accompanied by activation of tyrosine protein kinase activity, PLCgamma2 phosphorylation, influx of Ca(2+), and activation of caspase 3. All signaling events, as well as the subsequent apoptosis, were blocked by PP2, a selective inhibitor of Src-family kinases. Treatment of Ramos with EGTA and BAPTA to block changes in cytoplasmic Ca(2+) likewise prevented CD20-induced apoptosis. Our findings support a model in which CD20 clustering activates members of the Src family of protein tyrosine kinases, leading to phosphorylation of PLCgamma2 and increased cytoplasmic Ca(2+). These early signal transduction events activate caspase 3 to promote apoptotic cell death of NHL B cells.

Internalization of anti-nucleolin antibody into viable HEp-2 cells.

Anti-nucleolin antibodies have been detected in patients with systemic connective tissue diseases (SCTD) including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). In vivo bound autoantibodies to nucleoli of epidermal keratinocytes have been demonstrated in skin from patients with SCTD. In this study, monoclonal antibody to nucleolin (D-3) was used to determine the distribution of nucleolin in different culture cells including HEp-2, HepG2, HRCC, Molt-4 and Wil2 cells. Nucleolin was found to be present on the surface of HEp-2 and HepG2 cells, but not on the surface of HRCC and lymphoblastoid (Molt-4 and Wil2) cells; in contrast, nucleolin was detected in the nucleoli of all permeabilized cells examined. In immunoprecipitation, using extracts from 32P-labeled HEp-2 cells as antigenic source, cell membrane as well as nuclear nucleolins were found to be phosphorylated with a molecular weight of 105 kDa. Viable HEp-2 and HepG2 cells were cocultured with IgG fraction of D-3 in a CO2 incubator for 1 to 24 h, and then permeabilized with acetone followed by immunofluorescence staining with FITC-labeled goat anti-mouse IgG antibodies. Nucleolar staining was observed in cells after 10 h or longer of coculture. These data indicated that D-3 antibody reacted with cell membrane nucleolin and subsequently gain access into cells in a process related to pinocytosis.