Involvement of Phosphoinositide 3-Kinase γ, Rac, and PAK Signaling in Chemokine-induced Macrophage Migration

Abstract

In macrophages, chemotactic stimuli cause the activation of Rac and PAK, but little is known about the signaling pathways involved and their role in chemotactic gradient sensing. Herein, we report that in macrophages, the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 activates the small GTPase Rac and its downstream target PAK2 within seconds. This response depends on G i activation and largely on the subsequent triggering of phosphoinositide 3-kinase ␥ (PI3K␥) and Rac. Retroviral transduction of tagged Rac1 and -2 indicates that RAN-TES/CCL5-mediated activation of PI3K␥ triggers Rac1 but not Rac2. In agreement, silencing of Rac1 by shRNA blocks PAK2 activity and inhibits RANTES/CCL5-induced macrophage polarization and directional migration. On the other hand, the tyrosine kinase receptor agonist CSF-1 activates PAK2 independently of PI3K␥ and Rac. Our results thus demonstrate a chemokinespecific signaling pathway in which G i and PI3K␥ coordinate to drive Rac1 and PAK2 activation that eventually controls the chemotactic response.Rac1, Rac2, and Rac3 constitute a subfamily of the Rho family of monomeric GTPases and cycle between active GTP-bound (Rac GTP ) and inactive GDP-bound (Rac GDP ) states (1-3). Activation is accomplished by guanine-nucleotide exchange factors (GEFs), 1 which catalyze GDP dissociation (4), and inactivation by GTPase-activating proteins, which increase the intrinsic GTPase activity (5). Rho GTPases integrate signals from cellular receptors and membrane components to regulate the cytoskeleton dynamics required for cell locomotion during chemotaxis, phagocytosis, and many other cellular responses (2, 6).Leukocyte chemotaxis toward sites of inflammation (7) is primarily mediated by chemokine signaling. RANTES CCL5 is a disease-relevant and potent chemoattractant for macrophages. Moreover, it has been shown that RANTES-mediated T-cell activation and chemotaxis requires Rho GTPase activity (8,9). RANTES is a member of the CC-subfamily of chemokines and activates the seven-transmembrane CC-chemokine receptors CCR1, CCR3, CCR4, and CCR5, which are coupled to pertussis toxin sensitive heterotrimeric G i␣ proteins (10). PI3K was identified as a target of these G-protein-coupled chemokine receptors because chemotaxis and polarization of T cells could be inhibited by isoform non-selective class I PI3K inhibitors, such as LY294002 (11). All class I PI3Ks are heterodimers consisting of a 110-kDa (p110) catalytic subunit and an 85-(p85) or 101-kDa (p101) regulatory subunit (12). The two catalytic subunits p110␣ and -␤ are ubiquitously expressed, whereas p110␥ and -␦ expression is largely confined to leukocytes. In addition to their characteristic expression, PI3Ks are differentially regulated by GPCRs and receptor tyrosine kinases (RTKs) (13). RTKs have been shown to activate p110␣, -␤, and -␦ via the p85 regulatory subunit (class I A: PI3K-␣, -␤, and -␦). In contrast, it has been shown that regulation of p110␥ (class I B: PI3K␥) is mediated via the p101 adapter, ...

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration

“…Therefore, based on our previous and present findings, it may be speculated that the Gab1, p115 RhoGEF, Rac1, and RhoA signaling may be diverging at Pak1/2 levels in the modulation of HASMC versus THP-1 cell migration in response to thrombin (17). A role for Pak2 in macrophage migration has also been demonstrated previously (54). Furthermore, the activation of Pak2 by heterotrimeric G proteins and the adaptor protein NCK may indicate that several signaling events influence Pak2 in the mediation of cell migration (55).…”

Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase γ

“…Our data showing reduced Rac1 activity in mutant BMDMs suggest that impaired PIP3 polarization was caused by alteration of the positive feed back loop that involves Rac/PI3K reciprocal activation (37). In mouse macrophages, PI3K␥ deficiency does not affect Rac2 and only halves Rac1 activity (22), thus indicating that PI3K␥ only partially contributes to chemoattractant-mediated Rac activation. Our results are in agreement with a role of PIP3 in controlling the duration rather than the triggering of Rac activity (7) and suggest an involvement of PI3K in mediating GAP activation in response to chemoattractants.…”

“…Downstream of PI3K, the small GTPase Rac is a major player in cell spreading, polarization and leading edge formation (5,22). Analysis by pull-down assay of Rac1 activity induced by C5a showed that in wild-type cells, Rac-GTP rapidly increased, peaked at 1 min and declined within 5 min (Fig.…”

Defective Macrophage Migration in Gαi2- but Not Gαi3-Deficient Mice

“…Although Ga i1 is primarily found in the brain, Ga i2 and Ga i3 are abundantly expressed in the immune system. Many biological functions of leukocytes, including macrophage phagocytosis and migration, involve Ga i -mediated signaling (11,12). Previous studies in mice lacking Ga i2 or Ga i3 suggested key roles of these two G i isoforms in distinct cellular responses (13)(14)(15)(16).…”