The Molecular Basis of Phospholipase D2-Induced Chemotaxis: Elucidation of Differential Pathways in Macrophages and Fibroblasts

Abstract

We report the molecular mechanisms that underlie chemotaxis of macrophages and cell migration of fibroblasts, cells that are essential during the body's innate immune response and during wound repair, respectively. Silencing of phospholipase D1 (PLD1) and PLD2 reduced cell migration (both chemokinesis and chemotaxis) by ϳ60% and >80%, respectively; this migration was restored by cell transfection with PLD2 constructs refractory to small interfering RNA (siRNA). Cells overexpressing active phospholipase D1 (PLD1) but, mostly, active PLD2 exhibited cell migration capabilities that were elevated over those elicited by chemoattractants alone. The mechanism for this enhancement is complex. It involves two pathways: one that is dependent on the activity of the lipase (and signals through its product, phosphatidic acid , known to be phosphorylated by epidermal growth factor receptor (EGFR) kinase. Thus, both fibroblasts and macrophages use activity-dependent and activity-independent signaling mechanisms. However, highly mobile cells like macrophages use all signaling machinery available to them to accomplish their required function in rapid immune response, which sets them apart from fibroblasts, cells normally nonmobile that are only briefly involved in wound healing.

PLD1 rather than PLD2 regulates phorbol-ester-, adhesion-dependent and Fcγ-receptor-stimulated ROS production in neutrophils

“…The results we present are surprising in the context of an accumulation of data over previous years which has implicated PLD in many neutrophil (or neutrophil-like) responses, such as polarisation of the actin cytoskeleton (Zouwail et al, 2005;Nishikimi et al, 2009;Su et al, 2009), migration (Zouwail et al, 2005;Lehman et al, 2006;Powner et al, 2007;Su et al, 2009;Knapek et al, 2010), adhesion and spreading (Iyer et al, 2006;Powner et al, 2007;Chae et al, 2008;Su et al, 2009) and production of ROS (Cadwallader et al, 2004;Levy et al, 2005). However, much of this published work was carried out using siRNAs (Iyer et al, 2006;Lehman et al, 2006;Chae et al, 2008;Knapek et al, 2010) or primary alcohols as inhibitors of PLDmediated PA production (Cadwallader et al, 2004;Zouwail et al, 2005;Iyer et al, 2006;Powner et al, 2007;Chae et al, 2008;Knapek et al, 2010).…”

“…However, much of this published work was carried out using siRNAs (Iyer et al, 2006;Lehman et al, 2006;Chae et al, 2008;Knapek et al, 2010) or primary alcohols as inhibitors of PLDmediated PA production (Cadwallader et al, 2004;Zouwail et al, 2005;Iyer et al, 2006;Powner et al, 2007;Chae et al, 2008;Knapek et al, 2010).…”

A Novel Phospholipase D2-Grb2-WASp Heterotrimer Regulates Leukocyte Phagocytosis in a Two-Step Mechanism

“…It is known that PLD2 plays an important role during phagocytosis, yet the mechanism for how this is accomplished is far from being understood (13). Our laboratory has shown previously that PLD2 is a key protein during chemotaxis mediated by ancillary proteins such as Grb2 (15,16). We began this study by hypothesizing that Grb2 could also mediate the effect of PLD2 on phagocytosis.…”

Rab13-dependent Trafficking of RhoA Is Required for Directional Migration and Angiogenesis

“…Consequently, the Grb2-dependent targeting of RhoA to the leading edge of migrating cells may be a general mechanism shared by several growth factors and present in multiple cell types. Depletion of Grb2 has indeed been shown to decrease the EGF-induced migration of fibroblasts and macrophages (53).…”