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

Enhanced Constitutive PI3Kγ Activity Is Compatible with Life.

To investigate the effects of constitutively active PI3Kγ expression, knockin mice were generated by gene targeting (18) and engineered to express PI3Kγ fused with the CAAX-box of K-Ras (PI3Kγ^CX allele) (19). Mice homozygous for the mutation (PI3Kγ^CX/CX) were viable, fertile and did not show any overt phenotype or alteration of life span. Only the mutant PI3Kγ RNA was expressed but the amount of the mutant protein was reduced: PI3Kγ^CX/CX macrophages showed a 50% decrease in protein amounts (Fig. 1A). This reduction was not due to the targeting strategy, as a similar construct, aimed at expressing a kinase-dead protein (PI3Kγ^KD/KD) (18), did not alter PI3Kγ levels (Fig. 1A). In resting wild-type cells, PI3Kγ was found to be more abundant in the cytoplasm but PI3Kγ_CX was mainly located in the membrane fraction, thus confirming that the addition of the CAAX box caused delocalization of the enzyme (Fig. 1B). Analysis of PIP3 distribution by immunofluorescence revealed plasma membrane staining in resting PI3Kγ^CX/CX bone marrow-derived macrophages (BMDMs) but not in wild-type controls (Fig. 1C). Similarly, mutant BMDM showed significantly increased PIP3 levels in resting conditions, thus indicating that PI3Kγ_CX constitutively enhanced PIP3 production (Fig. 1D). The peak of PIP3 production upon GPCR stimulation was similar in the two genotypes but, with time, PIP3 remained higher in PI3Kγ^CX/CX cells. Consistently, resting PI3Kγ^CX/CX cells showed increased PKB/Akt phosphorylation [Fig. 1E and supporting information (SI) Fig. 6A]. However, PI3Kγ^CX/CX cells were still able to respond to GPCR agonists like CCL5 and C5a by further increasing PKB/Akt phosphorylation, thus suggesting that for PI3Kγ activation, Gβγ-dependent signals are stronger than membrane targeting. Other signaling pathways, like induction of intracellular Ca²⁺ transients (data not shown), or phosphorylation of p38, JNK and Erk1/2 or PTEN or SHIP-1 expression were not affected by the mutation (SI Fig. 6). Nonetheless, PI3Kγ^CX/CX peritoneal macrophages displayed an average 2-fold increase in the production of IL-1β, IL-6, and TNFα cytokines after LPS stimulation (Fig. 2A), thus evidencing an enhancement in inflammatory responses in mutant mice.

PI3Kγ_CX Enhances Leukocyte Proliferation and Survival.

Despite the established role of PIP3–dependent signaling in the regulation of cell proliferation and survival (10), PI3Kγ^CX/CX mice did not show predisposition to develop leukemia or other types of neoplasia. Nevertheless, counts of circulating blood cell populations revealed a generalized leukocytosis (SI Table 1). Consistent with enhanced leukocyte proliferation, PI3Kγ^CX/CX BMDMs incorporated more thymidine than wild-type controls in the presence of GM-CSF and CSF-1 (SI Table 2). On the other hand, analysis of apoptosis by Annexin V detection (SI Table 3) revealed a significant increase in PI3Kγ^CX/CX leukocyte survival (40% and 56% reduction of Annexin V⁺ neutrophils and macrophages, respectively). These data thus indicate that PI3Kγ_CX expression causes bone marrow hyperplasia due to enhanced cell proliferation and survival.

Consistent with higher inflammatory responses and abnormal leukocyte survival, PI3Kγ^CX/CX mice rarely showed spontaneous development of skin lesions with accumulation of inflammatory cells (Fig. 2B). Analysis of wound healing, indeed, revealed that the mutation caused a delayed resolution of the inflammatory reaction and clearance of granulation tissue at 10 days after skin excision (Fig. 2C), because of a 2-fold decrease in apoptotic removal of inflammatory cells (Fig. 2D).

Constitutive PIP3 Production Selectively Blocks Chemotaxis.

In leukocytes, PI3Kγ is known to control inflammatory responses primarily by sustaining cell recruitment (20). To test the effect of constitutive activation of PI3Kγ on granulocyte chemotaxis in vivo, PI3Kγ^CX/CX mice were challenged in a model of septic peritonitis. Counts of mutant neutrophils and macrophages at 12 h after infection, unexpectedly revealed a significant reduction (Fig. 3A). Similar results were obtained in a model of urinary tract Escherichia coli infection (Fig. 3B). Boyden chambers analysis of BMDM and neutrophil migration toward chemokines (CCL5, CXCL8) and chemoattractants (C5a) confirmed that PI3Kγ^CX/CX cells display an abnormally reduced chemotactic response (Fig. 3 C–F).

To understand the nature of the chemotactic defect, mutant and wild-type cells were analyzed in the Dunn chamber assay (21). In gradients of GPCR agonists like CCL5 and C5a, as well as tyrosine kinase receptor ligands like colony-stimulating factor-1 (CSF-1), measurement of cells reaching a fixed horizon (radial displacement from the origin) after 18 h revealed that the number of mutant cells was significantly lower than wild-type controls at distances up to 70 μm (Fig. 3G), thus indicating that mutant cells fail to efficiently follow the chemotactic gradient. Measurement of the ratio of cell displacement in the correct direction to the actual length of its migration path indicated that mutant BMDMs turned more often than wild-type controls (Fig. 3H). Although BMDMs of the two genotypes did not show differences in mean velocity (data not shown), PI3Kγ^CX/CX cells showed a less persistent orientation toward the gradient than wild-type controls (Fig. 3I). Interestingly, this defect was not only observed with GPCR agonists but also with the tyrosine kinase receptor agonist CSF-1. On the other hand, mutant cells were still able to follow a net migration in the direction of the GPCR ligand C5a, although with a markedly lower efficiency.

Constitutive and Delocalized PIP3 Production Impairs Leading Edge Formation.

In chemotaxing cells, preferential localization of PIP3 at the leading edge is thought to guide directional movement. We thus analyzed PIP3 distribution in PI3Kγ^CX/CX and PI3Kγ^+/+ BMDMs stimulated with C5a. In contrast to wild-type controls, PI3Kγ^CX/CX cells showed a constitutive presence of PIP3 at the plasma membrane without any evident preferential accumulation (Fig. 4A). At 1 and 5 min, mutant BMDMs showed partial PIP3 polarization at multiple membrane locations but failed to generate a clear leading edge and, at longer time points, PIP3 did not disappear (Fig. 4A). In agreement with defective organization of the leading edge, PI3Kγ^CX/CX BMDMs did not increase the elongation ratio of the cell (Fig. 4B) and failed to properly spread (Fig. 4C).

PI3Kγ_CX Shortens Rac Activity Peak by Activating RacGAPs.

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. 4 D and E). In mutant cells, Rac1 activity was similar to wild-type controls at 30 s but thereafter declined at a faster rate (Fig. 4E). As activation of Rac GEFs, like Vav, by tyrosine phosphorylation was equal in the two genotypes (SI Fig. 7), these findings suggested that the mutation did not alter Rac activating mechanisms but rather enhanced Rac deactivation. To find a potential PI3Kγ-regulated RacGAP, bioinformatic analysis of coexpression data was performed (23). This approach led to the identification of ArhGAP15, a leukocyte-expressed PH-domain containing GTPase-activating protein (GAP) for Rac, triggered by plasma membrane translocation (24). The expression of a myc-tagged ArhGAP15 by lentiviral transduction in wild-type BMDMs (expression analysis not shown) revealed that this protein, although increasing cell rounding and blocking polarization, was in the cytoplasm in resting conditions but localized to plasma membrane after C5a stimulation (Fig. 5A). This process was PI3K-dependent as treatment with the inhibitor LY294002 significantly reduced ArhGAP15 membrane recruitment in stimulated cells. In PI3Kγ^CX/CX BMDMs, ArhGAP15 was constitutively localized at the plasma membrane, did not change location upon C5a stimulation but redistributed to the cytoplasm after treatment with LY294002 (Fig. 5A). Consistently, wild-type BMDMs showed up-regulated global RacGAP activity (40% over basal) 90 s after C5a stimulation (Fig. 5B), but PI3Kγ^CX/CX cells showed enhanced basal RacGAP activity that was not altered by agonist stimulation (Fig. 5C). LY294002 blunted this effect in mutant BMDMs and blocked agonist-induced RacGAP activation in wild-type cells (Fig. 5D).

In agreement with a role of ArgGAP15 in chemotaxis, RNAi-mediated knock-down of this GAP in BMDMs (SI Fig. 8) enhanced chemotactic responses in both wild-type and mutant cells (Fig. 5E), thus significantly rescuing the defect of PI3Kγ^CX/CX BMDMs and demonstrating a role of PI3K function in RacGAP modulation during chemotaxis.

Generation of the PI3Kγ^CX allele.

To generate knockin animals, the locus encoding the catalytic subunit of PI3Kγ was replaced as described (18) with a chimeric minigene containing a mutant form of the human cDNA, in which the CAAX motif, derived from K-Ras, was added at its 5′ end (19) and followed by a neomycin resistance gene (Neo^r) cassette sandwiched between loxP sequences. Two of three recombinant clones were used to generate germ line-transmitting chimeras. Heterozygous mice were bred with Balancer Cre mice to delete the Neo^r cassette.

Antibodies.

Anti PI3Kγ and anti PIP3 (no. Z-P345B; Echelon, Salt Lake City, UT) monoclonal antibodies were kindly provided by R. Wetzker (Friedrich Schiller University, Jena, Germany) and P.O.N., respectively. The following antibodies were used: phospho-Ser-473-Akt, total Akt, phospho-Erk1/2, JNK, phospho-JNK, and PTEN (Cell Signaling Technology, Danvers, MA); Erk1, PI3Kβ and cyclinE (Santa Cruz Biotechnology, Santa Cruz, CA); p38 and phospho-p38 (Calbiochem, Darmstadt, Germany); Rac1 (05-389) and SHIP (Upstate Biotechnology; Lake Placid, NY); anti-vinculin and TRITC-phalloidin (Sigma, St. Louis, MO); BiP (BD PharMingen, San Diego, CA); CD18 (BMA, Augst, Switzerland), myc-tag [American Type Culture Collection, Manassas, VA]; Annexin V-FITC (BD PharMingen, San Diego, CA). Bioplex phosphoprotein detection assay (Bio-ad, Hercules, CA) was used with antibodies provided by the manufacturer.

Immunofluorescence.

BMDMs were plated on glass coverslips, and, after 24 h, cells were starved for 12 h. After stimulation, cells were fixed in 4% paraformaldehyde and permeabilized in TBS-0.3% Triton X-100, soaked in TBS-3% BSA, and stained with primary antibody followed by TRITC–conjugated secondary antibody. Both confocal and brightfield images were acquired with Leica TCS SP2 laser-scanning confocal microscope. Images were acquired by means of a ×63/1.32 NA, PL APO objective (Leica, Heidelberg, Germany).

Macrophage Culture and Analysis.

BMDMs were derived as described (21). For PIP3 measurement, an ELISA kit was used (K-2500; Echelon) following the manufacturer's instructions. For cytokine production analysis, peritoneal macrophages were stimulated after 24 h culture with 100 units/ml IFNγ for 4 h (for TNFα measurement), followed by 1 μg/ml LPS for a further 24 h. Supernatants were collected, and cytokines were measured by ELISA by using BD-PharMingen kits, according to the manufacturer's instructions.

Migration Assays.

Peritonitis was induced as described (12). In the model of pyelonephritis, E. coli were injected in urinary bladders of female mice through urethral catheterization. Boyden chamber assays were performed as described (12). A Dunn chamber was used as reported (21). Cell tracks were generated from time-lapse images by using the image-processing program Lucida (Kinetic Imaging, Liverpool, U.K.). Cells that translocated <20 μm from their point of origin were excluded from this analysis.

Lentiviral Vector Production and Cell Infection.

Murine ArhGAP15 cDNA (BC034881) was purchased from MRC Geneservice. A myc tag was added at the 5′ end to obtain the MYC-ArhGAP15 fusion protein. MYC-ArhGAP15 was cloned (BamHI-SalI) in pCCL.sin.PPT.hPGK.GFPWpre (kindly provided by L. Naldini, San Raffaele Institute, Milan, Italy) by replacing the GFP cassette. GAP15-1 and GAP15-2 shRNAmir sequences against ArhGAP15 (V2HS_175724 V2MM_60069; Open Biosystems, Huntsville, AL) as well as the control unrelated human sequence (VLHS 156525; Openbiosystems) were cloned in pCMV-GIN(Zeo) vector. High titer lentiviral vector stock was produced in HEK-293T cells by Effectene cotransfection of the MYC-ArhGAP15 or shRNAmir lentivector with the packaging vectors pMDLg/pRRE, pRSV-Rev, and pMD2.VSVG (40). Supernatants were collected and concentrated by ultracentrifugation, and 5 × 10⁶ BMDMs were infected with the concentrated virus.

Measurement of Rac Activation and RacGAP Activity.

Levels of GTP-bound Rac1 were measured by standard protocols based on GST-PAK CRIB domain fusion protein bound to glutathione-coupled Sepharose 4B beads. For RacGAP activity, GTP loaded Rac was prepared by incubating 1 μg of affinity-purified GST-tagged Rac1 in loading buffer [25 mM Tris·HCl, pH 7.5/50 mM NaCl/0.1 mM DTT/1 mg/ml BSA/5 μCi of [γ-³²P]GTP (1 Ci = 37 GBq)] at room temperature for 10 min. Thereafter, to block further loading, the sample was brought to 25 mM MgCl₂ and placed on ice. An aliquot was filtered through a nitrocellulose membrane (0.22-mm pore size; Millipore, Bedford, MA) under vacuum. The filter was washed twice with 5 ml of wash buffer (50 mM Tris·HCl, pH 7.5/50 mM NaCl/20 mM MgCl₂/1 mM DTT), air-dried, and placed in plastic vials with 5 ml of scintillation mixture (Optima Gold; PerkinElmer, Boston, MA). Bound radioactivity was measured by using a scintillation counter and considered as total GTP-loaded Rac (100%). For GAP activity measurements, BMDMs (10⁷ cells) were lysed in 100 μl of a buffer containing 25 mM Tris·HCl (pH 7.5), 1% Nonidet P-40, 100 mM NaCl, 1% glycerol, 10 mM MgCl₂, 1 mM PMSF, 1 mM Na₃VO₄, and protease inhibitors (Roche, Mannheim, Germany). Lysates were clarified by centrifugation (12,500 × g, 10 min), and 700 μg of protein extract were incubated in 100 μl of RacGAP buffer (25 mM Tris·HCl, pH 7.5/1 mM DTT/1 mg/ml BSA/2 mM GTP/100 mM NaCl/11 mM MgCl₂) for 5 min at room temperature. To start the reaction, 130 μl of [γ-³²P]GTP-loaded recombinant Rac were added, and samples were incubated for 5 min at 24°C under shaking. The reaction was stopped by adding 5 ml of ice-cold wash buffer. Samples were filtered through nitrocellulose membranes, washed, and counted as above, and remaining Rac-GTP was expressed as a percentage of total GTP-loaded Rac.

Statistical Analysis.

Values were presented as mean ± standard error (SE) of the mean. P values were calculated by using the nonparametric two-tailed Mann–Whitney U test, Student t test, and one- and two-way ANOVA, when appropriate. The number of experiments was indicated by n. For further details, see SI Materials and Methods.