B lymphocytes from mice null for the Rho-family guanine-nucleotide exchange factor, Vav, are defective in their ability to proliferate in response to BCR cross-linking, but are able to proliferate normally in response to LPS. In addition, they have a depletion of CD5(+) (B1) lymphocytes and defective IgG class switching. This phenotype is reminiscent of that observed in mice null for the cell cycle regulatory protein, cyclin D2. We demonstrate here that the inability of vav(-/-) B cells to proliferate in response to BCR ligation is due to an inability to induce cyclin D2. In addition, we show that the proliferative defect of these cells occurs after the cells have entered early G1 phase. Analyses of potential down-stream signaling intermediates revealed differential activation of the stress-activated MAP kinases in the absence of Vav, normal activation of the ERK, MAPK, and phosphatidylinositol 3-kinase pathways, and defective intracellular calcium mobilization. We further demonstrate that intracellular calcium homeostasis is required for cyclin D2 induction, implicating a possible link with the defective calcium response of vav(-/-) B cells and their inability to induce cyclin D2.
Abstract We show in this study that B cell activation following high avidity ligation of IgM or coligation of membrane Ig with CD19 elicits similar levels of Ca2+ flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca2+ flux independently of PI3K following anti-IgM cross-linking. By contrast, Vav function is essential for PI3K activation following membrane Ig (mIg)/CD19 coligation. Inhibition of PI3K revealed anti-IgM-stimulated Ca2+ flux has a PI3K-independent component, while Ca2+ flux following mIg/CD19 coligation is totally PI3K dependent. The p85α and p110δ subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca2+ flux, although the defects are not as severe as observed after pharmacological inhibition. This may reflect the recruitment of additional PI3K subunits, as we found that p110α becomes associated with CD19 upon B cell activation. These data show that the nature of the Ag encountered by B cells determines the contribution of Vav proteins to PI3K activation. Our results indicate that the strong signals delivered by multivalent cross-linking agents activate B cells in a qualitatively different manner from those triggered by coreceptor recruitment.
Purinergic receptors, which bind adenosine 5'-triphosphate (ATP), are expressed on human cutaneous keratinocytes and in squamous cell carcinomas. Studies on normal human epidermis and primary keratinocyte cultures have suggested that P2X(5) receptors are likely to be involved in keratinocyte differentiation and P2X(7) receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of keratinocytes. P2X(7) receptor agonists can significantly reduce primary keratinocyte cell numbers in culture. Human papillomaviruses are increasingly recognised as important human carcinogens in the development of non-melanoma skin cancers. In our study, immunohistochemical analysis for P2X(5) and P2X(7) receptors was performed on paraffin sections of normal human skin, warts, raft cultures of normal human keratinocytes and raft cultures of CIN 612 cells, a model of keratinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of P2X(5) receptors. A similar pattern was seen in the CIN 612 raft cultures. Both P2X(5) and P2X(7) receptors were found in the nuclei of koilocytes, abnormal keratinocytes characteristic of human papillomavirus infection. P2X(5) and P2X(7) receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death.
BCR (B-cell antigen receptor)-induced Ca2+ signalling is initiated by activation of tyrosine kinases, which in concert with adaptor proteins and lipid kinases regulate PLC (phospholipase C) γ2 activation. Vav and PI3K (phosphoinositide 3-kinase) are required for optimal Ca2+ responses, although it has not been established, in primary B-cells, if both proteins are components of the same pathway. In vitro evidence suggests that binding of the PI3K lipid product PIP3 to Vav pleckstrin homology domain contributes to Vav activation. However, pharmacological inhibition of PI3K by wortmannin or deletion of the p110δ catalytic subunit has no effect on Vav activation in response to BCR engagement, suggesting that this mechanism does not operate in vivo. We also show that PI3K recruitment to phosphorylated-tyrosine-containing complexes is Vav-independent. Taken together with our previous observation that protein kinase B phosphorylation is normal in Vav-deficient B-cells, we suggest that PI3K activation is Vav-independent in response to strong signals delivered by multivalent cross-linking.
Objective: Polydeoxyribonucleotide contains a mixture of nucleotides and interacts with adenosine receptors, stimulating vascular endothelial growth factor expression and wound healing. The purpose of this study was to investigate the effect of polydeoxyribonucleotide on experimental burn wounds. Design: Randomized experiment. Setting: Research laboratory at a university hospital. Subjects: Thermal injury in mice. Interventions: Mice were immersed in 80°C water for 10 secs to achieve a deep-dermal second-degree burn. Animals were randomized to receive either polydeoxyribonucleotide (8 mg/kg/day intraperitoneally for 14 days) or its vehicle alone (0.9% NaCl solution at 100 μL/day intraperitoneally). On days 7 and 14 the animals were killed. Blood was collected for tumor necrosis factor-α measurement; burn areas were used for histologic and immunohistochemical examination, for the evaluation of vascular endothelial growth factor and nitric oxide synthases by Western blot, and for the determination of wound nitric oxide products. Measurements and Main Results: Polydeoxyribonucleotide increased burn wound re-epithelialization and reduced the time to final wound closure. Polydeoxyribonucleotide improved healing of burn wound through increased epithelial proliferation and maturation of the extracellular matrix as confirmed by fibronectin and laminin immunostaining. Polydeoxyribonucleotide also improved neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of platelet-endothelial cell adhesion molecule-1. Furthermore, polydeoxyribonucleotide blunted serum tumor necrosis factor-α and enhanced inducible nitric oxide synthase and vascular endothelial growth factor expression and the wound content of nitric oxide products. Conclusions: Our study suggests that polydeoxyribonucleotide may be an effective therapeutic approach to improve clinical outcomes after thermal injury.
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