Over-expression of p190RhoGEF enhances B-cell activation and germinal center formation in T-cell-dependent humoral immune responses.

Previously, we reported induced expression of the p190 Rho guanine nucleotide exchange factor (p190RhoGEF, ARHGEF28) following CD40 stimulation of B cells isolated from mouse spleen. We also reported that p190RhoGEF and a downstream effector molecule RhoA are required for B-cell differentiation, especially for the induction of the plasma cell (PC) differentiation. This study investigates the role of p190RhoGEF in B-cell biology in vivo, using p190RhoGEF transgenic (TG) mice that overexpress a wild-type full gene in B cells. Immunization of these mice with T-cell-dependent antigen showed that populations of germinal center B cells and PCs were significantly increased in TG mice. Furthermore, similar results were shown in recombination activating 1 (Rag1) knockout mice that were reconstituted with B cells isolated from TG mice in combination with T cells isolated from littermate control mice. Analyses of isotype class switching and transcription factors involved in a germinal center reaction and PC differentiation also supported the findings from the cellular responses. These results suggest that p190RhoGEF may play a role in the stage of PC differentiation during T-cell-dependent humoral immune responses.

Isoproterenol Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Embryonic Kidney Cells through Death Receptor 5 up-Regulation

BACKGROUND AND OBJECTIVES: Chronic impairment of beta-adrenergic receptor signaling increases cardiac apoptosis, hypertrophy and fibrosis. The aim of this study was to investigate whether isoproterenol (ISO), an agonist of the adrenergic receptor, can enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human embryonic kidney (HEK) 293 cells. MATERIALS AND METHODS: HEK 293 cells were treated with ISO and/or TRAIL for 24 hours. Cell viability was evaluated by microscopy and an established viability assay, and apoptotic cell death was analyzed by staining with fluorescein isothiocynate-annexin-V/propidium iodide (PI) and caspase activation. To confirm the mechanism of cell death induced by co-treatment with ISO and TRAIL, expression of TRAIL receptor 2 (death receptor 5, DR5) was evaluated by immunoblotting. RESULTS: Although ISO or TRAIL treatment decreased HEK 293 cell viability by 13% and 17%, respectively, co-treatment with ISO and TRAIL resulted in a markedly higher death rate of 35% after 24 hours. Increases were evident in early apoptotic cells (i.e., annexin-V positive/PI negative; 19.4%), late apoptotic cells (i.e., annexin-V positive/PI positive; 6.3%) and dead cells (i.e., annexin-V negative/PI positive; 1.1%) when cells were co-treated with ISO and TRAIL, compared to cells treated with either ISO or TRAIL. In addition, marked increases of cleaved cas-3, cleaved poly (adenosine diphosphate-ribose) polymerase and DR5 were observed in HEK 293 cells co-treated with ISO and TRAIL. CONCLUSION: Treatments combining ISO with TRAIL may be responsible for death of HEK 293 cells through DR5 up-regulation. Activation of adrenergic receptors is responsible for the synergistic cell death observed with TRAIL.

Over-expression of a RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, in mouse dendritic cells negatively regulates cellular responses to bacterial lipopolysaccharide.

We studied the role of a RhoA-specific guanine nucleotide exchange factor (p190RhoGEF) in dendritic cells (DCs), using transgenic (TG) mice that over-express a full gene of p190RhoGEF under the control of an invariant chain promoter. TG mice lacked localization of activated DCs to the T cell zone in the spleen and had reduced serum levels of IL-6 in response to lipopolysaccharide (LPS) injection. DCs from these mice also showed reduced surface expression of CD86, CD40, and CD205, but not MHCII, as well as a reduced capability to uptake antigen. Moreover, chemokine-driven migration and secretion of IL-6, but not of IL-12, were impaired after LPS-stimulation of TG DCs. Collectively, these results suggest that over-expressing p190RhoGEF negatively regulates conventional DC function in response to bacterial LPS infection.

Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation.

Previously, we demonstrated that the p190 Rho guanine nucleotide exchange factor (p190RhoGEF) was induced following CD40 stimulation of B cells. In this study, we examined whether p190RhoGEF and a downstream effector molecule RhoA are required for B cell differentiation. Expression of p190RhoGEF positively correlated with the expression of surface markers and transcriptional regulators that are characteristic of mature B cells with plasma cell (PC) phenotypes. Moreover, either the overexpression of p190RhoGEF or the expression of a constitutively active RhoA drove cellular differentiation toward PC phenotypes. B cell maturation was abrogated in cells that overexpressed p190RhoGEF and a dominant-negative form of RhoA simultaneously. CD40-mediated maturation events were also abrogated in cells that overexpressed either dominant-negative p190RhoGEF or RhoA. Together, these data provide evidence that p190RhoGEF signaling through RhoA in CD40-activated B cells drives the induction of the PC differentiation.

Ligation of CD40 receptor in human B lymphocytes triggers the 5-lipoxygenase pathway to produce reactive oxygen species and activate p38 MAPK.

Previously, we reported that CD40-induced production of reactive oxygen species (ROS) by NADPH oxidase requires the TNF receptor-associated factor (TRAF) 3, as well as the activities of phosphatidylinositol 3-kinase (PI3K) and Rac1. Here we investigated the possible mechanisms of the production of ROS after CD40 ligation in B cells. We describe an alternative ROS production pathway that is triggered by CD40 ligation, involves 5-lipoxygenase (5-LO), and results in activation of p38 MAPK. Our studies in Raji human B lymphomas revealed that CD40-induced ROS production by 5-LO also requires the activities of PI3K and Rac1. In contrast to the NADPH oxidase pathway, however, TRAF molecules are not required for the CD40-induced ROS production by 5-LO. The association of CD40 with 5-LO is dependent on CD40 ligation in Raji B cells, and co-immunoprecipitation experiments using epitope- tagged proteins transiently expressed in human embryonic kidney 293T cells revealed the role of the regulatory subunit of PI3K, p85, in this association. Collectively, these data suggest a separate pathway for the CD40-induced ROS production in B cells and demonstrate that this pathway requires 5-LO via direct association of p85 with both CD40 and 5-LO.

Characterization of phenotypically distinct B-cell subsets and receptor-stimulated mitogen-activated protein kinase activation in human cord blood B cells.

Human cord blood (CB) is a valuable source of hematopoietic stem cells, but clinical reports have indicated slow recovery of B-cell development and function after CB transplantation. To investigate the basis of these B-cell defects in reconstitution, we characterized B cells purified from CB. We compared B-cell receptor activation and B-cell subsets in CB, bone marrow (BM), and peripheral blood (PB). We found that in CB B cells activation of extracellular signal-regulated kinase (ERK) and p38 following ligation of CD40 but not of the B-cell antigen receptor (BCR) was inefficient. The patterns of expression of CD5, CD34, and CD40 in the B-cell population of CB were similar to those in PB rather than in BM. The B cells in CB contained an increased proportion of B cells expressing a high level of CD24 and a low proportion of B cells expressing CD27, pointing to the presence of circulating CD24high immature transitional and CD27(-) naive B cells. CD40-mediated activation of ERK and p38 was also minimal in these B cells of CB. These findings may account for the functional defects of B cells in transplanted CB.

Association of the Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) with the p85 subunit of phosphoinositide 3-kinase.

To investigate additional functions of the T cell adaptor, Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD (SLP-76), we performed a yeast two-hybrid assay using the N-terminal region of SLP-76 fused with the kinase domain of Syk. By screening a human leukemia cDNA library, we identified the p85 subunit of phosphoinositide 3-kinase (PI3K) as one of the interacting molecules. Unlike the SH2 domain of Vav or Nck, tyrosine phosphorylation of SLP-76 at position 113 or 128 was sufficient for it to associate with the N-terminal SH2 of p85. Collectively, these data suggest that SLP-76 may play a role in PI3K signaling pathways.

Role of TNF receptor-associated factor 3 in the CD40 signaling by production of reactive oxygen species through association with p40phox, a cytosolic subunit of nicotinamide adenine dinucleotide phosphate oxidase.

To extend our previous report, which showed the production of the reactive oxygen species (ROS) after the CD40 ligation in the B cells, we further examined the possible mechanisms for ROS production and the involvement of CD40-induced ROS in p38 activation. Our research shows that the stimulation of WEHI 231 B lymphomas with anti-CD40 induced ROS production and p38 activation. An antioxidant N-acetyl-L-cysteine or an inhibitor for NADPH oxidase blocked both of these, but the inhibitors for 5-lipoxygenase did not. We also show that the treatment of cells with inhibitors for the phosphatidylinositol 3-kinase (PI3-K) interfered with the CD40-induced ROS production and p38 activation. In addition, when overexpressed with a dominant negative form of either Rac1 (N17Rac1) or the TNFR-associated factor (TRAF) 3, the WEHI 231 B cells did not show a full response to the CD40 stimulation to produce ROS. Molecular association studies further revealed that the TRAF3 association with p40(phox), a cytosolic subunit of NADPH oxidase and p85 (a subunit of PI3-K), may possibly be responsible for the production of ROS by CD40 stimulation in WEHI 231 B cells. Collectively, these data suggest that the CD40-induced ROS production by NADPH oxidase in WEHI 231 requires the role of TRAF3, as well as activities of PI3-K and Rac1.

Cutting edge: induced expression of a RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, following CD40 stimulation and WEHI 231 B cell activation.

Stimulation of the B cell surface receptor CD40 induces transcriptional activation and protein expression. To determine which proteins are required for the CD40-mediated B cell activation, we performed a two-dimensional gel electrophoresis of the WEHI 231 B cell lysates. We report in this study the identification of one protein in which the expression was remarkably induced following CD40 stimulation. It was the p190 Rho guanine nucleotide exchange factor (GEF), p190RhoGEF, a recently identified GEF that is specific for RhoA. Overexpression of either p190RhoGEF or RhoA (Q63L), a constitutively active form of RhoA, mimics the effects of CD40 stimulation, such as changes in cellular structure and NF-kappaB activation. These p190RhoGEF overexpression effects are abrogated when coexpressed with a dominant negative form of RhoA (T19N). We also provide evidence for the CD40-mediated cellular changes that are abrogated in cells that are overexpressed with the dominant negative form of either p190RhoGEF (Y1003A) or RhoA (T19N).