The role of guanine nucleotide exchange factors in the regulation of immune cell signaling

Abstract

The ARF GEF cytohesin-1 was originally identified as β2-integrin binding protein, and was shown to play an important role in the control of integrin-dependent immune cell migration and adhesion via activating the small GTPase RhoA. To analyze whether the GEF activity of cyohesin-1 is required for regulating adhesive events of dendritic cells, a novel protocol was established, which enables over-expression analysis of cell adhesion and migration events in human mo-DC. It is shown that over-expression of wild type cytohesin-1, but not of the GEF-deficient E157K mutant, markedly enhances adhesion of mo-DC to ICAM-1. These results prove for the first time that the GEF function of cytohesin-1 is required for β2-integrin-mediated adhesion of dendritic cells. <br /> Additionally, there is now strong evidence that cytohesin proteins play major roles in the induction of cellular gene expression, e.g. by regulating insulin signal transduction or MAP kinase activation during tumor cell proliferation. Fully in line with these results, cytohesin-1 is shown here to regulate important intracellular events in the course of T-cell activation. Employment of RNAi reveals that cytohesin-1 expression positively regulates TCR-induced transcriptional activation of the IL-2 gene via activation of the AP-1 transcription factor complex. Cytohesin-1 mediates positive regulation of AP-1 by selectively activating the p38/JNK- but not the ERK1/ERK2-branch of the MAP kinase signaling cascade. The ability of cytohesin-1 to activate p38/JNK signaling depends on the GEF function of the protein, since over-expression of the dominant negative GEF mutant E157K, or treatment with cytohesin-specific chemical GEF inhibitors completely blocks TCR-induced activation of p38. <br /> Similar to what has been described here for cytohesin-1, the Vav-1 GEF plays a dual role in the regulation of T-cell signal transduction and cytoskeletal reorganization via activation of Rho GTPases. Therefore, a possible functional interaction between cytohesin-1 and Vav-1 was investigated. However, co-precipitation experiments did not reveal a direct association of Vav-1 with cytohesin-1. Furthermore, its expression is not required for TCR-induced phosphorylation, and thus for activation of Vav-1. These results indicate that cytohesin- 1 might either be located in a specific signaling branch downstream of Vav-1, or acts in a pathway which alternatively regulates activation of p38/JNK and AP-1.<br /> Vav-1 is known to be essential for proper development of the adaptive immune system. However, less information is available about a potential role of the protein in the regulation of innate immune responses. The so-called dendritic cells bridge innate and adaptive immune defense mechanism through a process known as antigen-presentation. Binding to pathogen-borne structures induces the terminal differentiation (maturation) of this cell type, which is characterized by robust cell surface expression of antigen-presenting MHC peptide complexes and of co-stimulatory molecules, which supports T-cell activation. This study reveals a novel, so far unexpected role of Vav-1 in innate immunity. Vav-1 expression is shown to be essential for TNFalpha-induced terminal differentiation of human mo-DC, since it is involved in the up-regulation of important inflammatory cell surface proteins the expression of which is controlled by NF-KB activity. It has furthermore been established that Vav-1 expression modulates TNFalpha signaling in a cell-type specific manner, since the upregulation of surface markers during TNFalpha-induced maturation of murine Vav-1 knock-out BM-DC is not impaired. Consistent with this hypothesis, Vav-1 is not required for TNFalpha-mediated signaling to NF-KB in T-cells. In conclusion, the presented data suggest that Vav-1 does not directly interact with classical components of the TNFR signaling machinery, but mediates its effects by binding and activating β2-integrins, the co-stimulatory activity of which might augment TNFalpha-induced activation of NF-KB in a cell-type specific manner.