Neutrophils
Phospholipases
Small GTPases
Lipid mediators of inflammation
Phagocytosis and vesicle trafficking

Interaction of agonists with cell-surface receptors either G protein-coupled receptors or receptor tyrosine kinases often activates the hydrolysis of phosphatidylcholine (PtdCho) by phospholipase D (PLD) enzymes. This pathway generates the second lipid messenger phosphatidic acid (PA). PA-derived PLD regulates a wide variety of physiological processes including membrane vesicle trafficking, cytoskeletal dynamics and serves diverse functions in signal transduction mechanisms. In human neutrophils and macrophages PLD activation has been implicated in agonist-induced exocytosis, phagocytosis of opsonized particles and activation of the NAPDH oxidase. These neutrophil functions constitute the first non-specific response of immune defense against invading pathogens as highlighted by the recurrent infections of chronic granulomatous disease (CGD) patients that have no functional NADPH oxidase. A lot remains to be discovered about the mechanisms by which PA-derived PLD regulates specific functional responses or proteins in human neutrophils. The present proposal will test the hypothesis that PA-derived PLD works as a device for recruiting specific protein(s) to membranes. The main objective of the project is to identify new PA-binding proteins from human neutrophils and to define the specific functions of these proteins in cell physiology.

This project is supported by
the Canadian Institutes of Health Research (CIHR).

The induction of phospholipase D (PLD) activity in human granulocytes is a hallmark of cell activation by agonists. This activation lies at the center of many fundamental cellular processes such as secretion and intravesicular protein transport. G protein-coupled receptor-induced PLD activation is mediated by the recruitment of cytosolic Arf and RhoA to the membrane compartment through a tyrosine kinase-regulated pathway. Arf proteins, like other small GTPases, are regulated by GTPase activating proteins and guanine nucleotide exchange factors (GEF) which act as upstream molecular ON-OFF switches. The Arf-GEFs fall in two groups, based on their susceptibility to inhibition by brefeldin-A (BFA). We recently discovered that fMLP causes the membrane recruitment of cytohesin-1, the major BFA insensitive Arf-GEF in neutrophils. This protein has a pleckstrin homology domain (PH) that binds phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol 3,4,5-triphosphate (PIP3) and a N-terminal coiled-coil motif. We have hypothesized that the activation of Arf 1/6 by cytohesin-1 leads to activation of PLD generating phosphatidic acid (PA) in the vicinity of CD18 and up-regulates the avidity of the integrin LFA-1 to its ligand ICAM-1. The specific objectives are to determine the role of the PH and the coiled-coil motifs in cytohesin-1 translocation to the membrane cytoskeleton and to characterize the cytohesin-1 interacting proteins.

This project is supported by The Arthritis Society and the Canadian Institutes of Health Research.

Autotaxin (ATX) is a 125 kDa pro-angiogenic protein that acts as an autocrine/paracrine motility factor for cancer cells by promoting random as well as directed motility. ATX is proteolytically cleaved by unknown protease(s) and secreted by cells in the extracellular milieu. ATX was initially characterized as a nucleotide pyrophosphatase/phosphatase. The mechanism(s) through which ATX promotes angiogenesis and cell motility remained an enigma until the protein was reported to be a lyso-phospholipase D (lyso-PLD), an enzyme that generates lyso-phosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) from lysophospholipids. This finding was a significant breakthrough since LPA controls various cellular responses such as cell proliferation and survival that can explain the pro-angiogenic and metastatic properties of ATX.

It is worth mentioning that ATX mRNA are expressed at significantly higher levels in fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA) as compared to fibroblast-like synoviocytes from healthy donors. Moreover, our preliminary results reveal that there was significantly more ATX in synovial fluids from patients with RA than in synovial fluids from patients with osteoarthritis. These preliminary data suggest that the amounts of ATX might reflect disease severity and that ATX is a putative prognostic marker for RA. Furthermore, there are increasing evidence suggesting that the effect of ATX correlates with its capacity to hydrolyze lysophospholipids. Indeed, a majority of the responses of cancer cells to ATX is attributable to the activation of specific, seven-transmembrane domain G protein-coupled receptors (GPCRs) for lysophospholipids including LPA and S1P. In addition to investigating the cell biology and the synthesis of ATX by fibroblast-like synoviocytes, we seek to understand the contribution of ATX to the growth of the inflamed synovium and to perpetuation/amplification of the inflammatory response in RA.

This project is supported by the Institute of Muskuloskeletal Health and Arthriris (IMHA), Canadian Institute of Health Research.