Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) beta-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 mug/ml and as an insulin-sensitizer at doses as low as 1 mug/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR beta-subunit, phosphorylation of Akt and glycogen synthase kinase-3beta), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes.

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 p40phox, 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.

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.

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-κB 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).

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.