The Importance of G Protein Signaling in Immune Cell Function: A Novel Role for Active G?i in Cell Adhesion and Migration and the Therapeutic Potential of Targeting G?? in Inflammation PDF Download

Author: Jesi Lee Anne To
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Languages : en
Pages : 128

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Book Description
Chemotaxis is the directional movement of cells towards a gradient of chemoattractant such as chemokines. Chemokines are small peptides secreted by various cell types and play a role in immune surveillance, inflammation, and development. These molecules bind to their cognate receptors, which belong to the Class A, rhodopsin-like family of G protein-coupled receptors (GPCRs). Downstream effects orchestrated by chemokines include directional migration, reactive oxygen species (ROS) production, proliferation, and survival. Chemokine receptors primarily couple to the Gi/o family of G? proteins. Their activation triggers dissociation of the heterotrimeric G protein, G? and G?? dimer. The role of G?? in chemokine signaling is well established as it regulates multiple downstream effectors critical to cell migration such as phosphoinositide 3-kinase gamma (PI3K?), phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger (P-Rex), p21 activated kinase/PAK-interacting exchange factor alpha (PAK/PIX?), and Ras-associating and diluted domain-containing protein (Radil). However, the role of G?i in chemokine signaling is less understood and is thought to only regulate G?? availability and signaling through their association and disassociation cycle. Here we show that both G?i-GTP and G?? signaling are pivotal to cell migration. We also provide evidence for a novel role of G?i-GTP in regulating cell adhesion and migration. Using a small molecule inhibitor of G?? to inhibit migration, we show its anti-inflammatory therapeutic potential against lupus nephritis. Recently, our lab uncovered a novel role for G?i-GTP signaling in neutrophil adhesion through a cAMP-independent pathway. To discover the mechanism for G?i-GTP regulation of adhesion, we used a constitutively active Rap1a(G12V) and its downstream effector Radil to drive adhesion of neutrophil-like HL-60 and HT-1080 fibrosarcoma cells. A G??-Rap1a-Radil complex is known to drive cell adhesion and migration of these cell types. We found that expression of constitutively active G?i1(Q204L) reversed both Rap1a(G12V) and Radil mediated adhesion, suggesting that G?i-GTP regulates adhesion downstream of Rap1a and Radil. Furthermore, Rap1a and Radil reduced migration of HT1080 cells, which is reversed by G?i1(Q204L) expression. These data identify a novel role for G?i-GTP in the regulation of cell adhesion and migration. Multiple chemokines play a role in propagating inflammation and are associated with lupus nephritis pathogenesis by orchestrating immune cell migration toward the kidneys. The chemokine/receptor system has vast and promiscuous properties as one chemokine can bind to multiple receptors and one receptor can bind to more than one ligand. Therefore, valid targeting of individual chemokine receptors associated with a disease may be challenging and display poor efficacy. We used a well-studied G?? inhibitor, gallein, to study its therapeutic potential in alleviating lupus nephritis. Gallein inhibited PI3K?-mediated PIP3 production in neutrophil-like HL-60 cells and inhibited chemotaxis of primary neutrophils and T-cells in response to chemokines. Gallein treatment significantly reduced renal inflammation and proteinuria in NZB/NZW lupus mice. This study illustrates the therapeutic potential of G?? inhibitors against chronic inflammation. Our laboratory has identified a novel small molecule inhibitor of G?? called ellagic acid. Ellagic acid is a naturally occurring polyphenolic compound and that has anti-inflammatory properties. Our evidence showed ellagic acid directly binding to G?? and inhibited downstream G?? signaling. It significantly blocked N-formyl-Met-Leu-Phe (fMLP) chemotactic peptide-induced intracellular calcium release and weakly inhibited G??-mediated phospholipase C ?2 (PLC?2) activation in vitro compared to gallein and peptide SIGKAFKILGYPDYD (SIGK). Ellagic acid also inhibited fMLP-induced PI3K?mediated membrane phosphatidylinositol 3,4,5-triphosphate (PIP3) production, superoxide production, and migration of neutrophil-like HL60 cells. In this study, we identified ellagic acid as a small molecule inhibitor of G?? signaling in neutrophil-like cells and was sufficient to inhibit chemotaxis. This thesis work focuses on the overall importance of G protein signaling in GPCRmediated chemokine signaling and immune cell function. Both G?i and G?? are important mediators of GPCR-mediated chemokine signaling and immune cell function. Furthermore, our studies provide insights into the novel role of G?i in adhesion and migration of both immune cells and cancer cells. Chemokines are not only important during inflammation, but also contribute to the metastatic potential of cancer cells. We illustrate the importance of G?i and G?? in immune cell function such as adhesion and cell migration and we show alleviation of inflammatory lupus nephritis by inhibiting G?? signaling.