Regular articleRequirement for PI 3-kinase γ in macrophage migration to MCP-1 and CSF-1☆
Introduction
Cell migration is driven by coordinated responses of the actin cytoskeleton, microtubules, and cell-substratum adhesion [1], [2]. Phosphoinositide 3-kinases (PI3Ks) have been extensively implicated in driving the initial steps of cell polarization and migration [3], [4], [5]. PI3Ks phosphorylate phosphoinositides at the 3′-OH position to generate primarily phosphatidylinositol(3,4,5)trisphosphate (PIP3) and PI(3,4)P2 [4]. Using pleckstrin homology (PH) domains that specifically bind to PIP3 and PI(3,4)P2, it has been shown that these phosphoinositides are concentrated at the leading edge of migrating Dictyostelium cells and neutrophils [6], [7], [8], [9]. Inhibition of PI3Ks using small molecule inhibitors prevents or reduces the migration of many cell types, including macrophages [3], [4], [10]. In addition, mutation or loss of PTEN, which removes the 3′-phosphate and thereby reduces the level of PIP3 and PI(3,4)P2, leads to enhanced cell protrusion in Dictyostelium cells [11], [12], and increased migration of fibroblasts [13]. PIP3 and PI(3,4)P2 are proposed to act as signal transducers by binding to PH domains in a number of signalling proteins, including the serine/threonine kinase Akt and several exchange factors for the small GTPase Rac [4], [14]. Although the molecular mechanism whereby PIP3 and PI(3,4)P2 contribute to cell migration has not been elucidated, it has been proposed that they act at least in part by activating and/or localizing Rac activators (guanine nucleotide exchange factors), and that Rac and/or related GTPases would then further activate/localize PI3Ks to create a positive feedback loop [15]. Indeed, small molecule inhibitors of PI3Ks prevent Rac activation by a number of chemoattractants [3], [8].
There are three classes of PI3Ks, of which class 1 PI3Ks have been most widely studied for their roles in signal transduction [4]. There are four catalytic subunits of class 1 PI3Ks, α, β, γ, and δ. The p110α, β, and δ isoforms are known as class 1A PI3Ks and interact with p85 adaptor subunits, which in turn bind via their SH2 domains to tyrosine kinase receptors and cytoplasmic tyrosine kinases, including the colony stimulating factor-1 (CSF-1) receptor (CSF-1R, also known as c-fms) expressed on macrophages [4], [16]. We have previously shown that in a mouse macrophage cell line responding to the chemoattractant CSF-1, p110β and p110δ but not p110α are required for migration [10]. In contrast to class 1A PI3Ks, the only known class 1B PI3K, PI3Kγ, does not bind to the p85 adaptors but instead interacts with a different regulatory subunit, p101, and is activated by the βγ subunits of heterotrimeric G proteins following stimulation of G protein-coupled receptors (GPCRs) [4], [17]. The PI3Kγ gene is primarily expressed in haematopoeitic cells, and viable mice lacking PI3Kγ have been derived by homologous recombination [18], [19], [20]. Neutrophils and macrophages from these mice develop normally, but show reduced migration toward chemoattractants in Boyden chambers as well as reduced recruitment in in vivo models of inflammation [18], [19], [20].
To examine in more detail how loss of PI3Kγ affects macrophage responses to chemoattractants, we have analysed migration in the Dunn chemotaxis chamber, where cells are directly visualized as they migrate toward a source of chemoattractant [21], [22]. Using mouse bone marrow-derived macrophages (BMMs), we show that PI3Kγ is not required for the early actin reorganization induced by the chemokine monocyte chemotactic protein-1 (MCP-1/CCL2), which acts through the GPCR CCR2 [23], [24]. Similarly, PI3Kγ does not contribute significantly to CSF-1-induced ruffling, mediated by the tyrosine kinase receptor CSF-1R/c-fms. However, subsequent polarization and chemotaxis of PI3Kγ-null macrophages to MCP-1 is significantly impaired, in part because cells turn more and migrate less persistently in one direction. Lack of PI3Kγ also has a small effect on CSF-1-induced migration but not chemotaxis, suggesting that GPCRs contribute to the efficiency of the motile response to the CSF-1R. Consistent with this, inhibition of Gi signalling by B. pertussis toxin inhibits CSF-1-induced migration.
Section snippets
Derivation of macrophages
BMMs were derived as previously described [25]. In brief, bone marrow macrophages were isolated from the femurs of sibling 6- to 8-week-old wild-type (WT) and PI3Kγ-null (γ−/−) 129/Sv mice. Cells were plated on bacteriological plastic plates in macrophage growth medium consisting of RPMI-1640 (Gibco-Invitrogen Ltd., Paisley, UK), 1 mM sodium pyruvate (Invitrogen), 1× nonessential amino acids (Invitrogen), 0.2 mM 2-mercaptoethanol (Sigma-Aldrich Co. Ltd., Gillingham, UK), 10% heat-inactivated
Overall morphology and CSF-1-stimulated ruffling is unaltered in PI3Kγ−/− macrophages
BMMs require a source of the cytokine CSF-1 to survive, proliferate, and differentiate [29], [30]. CSF-1 acts through a tyrosine kinase receptor, CSF-1R (c-fms). We have previously described the derivation and growth of BMM using L-cell-conditioned medium as a source of CSF-1 [25]. BMMs from WT and PI3Kγ−/− mice bound similar levels of the mouse macrophage-specific F4/80 antibody, indicating that lack of PI3Kγ did not grossly affect their differentiation (Fig. 1). In addition, they had similar
Discussion
PI3Ks have been implicated in chemokinesis and/or chemotaxis in a variety of cell types. Here, we show that macrophages lacking PI3Kγ show reduced migration speed and lack of chemotaxis when stimulated by MCP-1, a GPCR agonist, and in addition that their migration is somewhat affected when stimulated by CSF-1, a tyrosine kinase receptor agonist. Interestingly, the cells respond normally to acute stimulation with MCP-1 by membrane ruffling, but cannot polarize efficiently unless CSF-1 is
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Acknowledgements
We are grateful to Claire Wells and Yann Leverrier for discussions and protocols. This work was supported by the Medical Research Council UK, the Wellcome Trust, The Swiss National Science Foundation, EC grants QLRT-2000-02171/BBW00.0564-1 and QLG1-CT-1999-01036, and the Ludwig Institute for Cancer Research. C.H. was supported by an Anatomical Society UK studentship.
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Supplementary data associated with this article can be found at doi:10.1016/S0014-4827(03)00318-5
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These authors contributed equally to this work.