1 Department of Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Ave, Denver, CO 80262, USA
2 Department of Craniofacial Biology, University of Colorado Health Sciences Center, 4200 East Ninth Ave, Denver, CO 80262, USA
3 Bonfils Blood Center and the Department of Pediatrics, University of Colorado School of Medicine, Denver, CO 80230, USA
4 Program in Experimental Pathology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA
Authors for correspondence (e-mail: amy_abell{at}med.unc.edu; glj{at}med.unc.edu)
Accepted 4 September 2003
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Summary |
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Key words: Rac2, Bone-marrow-derived macrophage, p38 kinase, Actin, Human mutation
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Introduction |
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Rac signaling is regulated by three classes of proteins: guanine-nucleotide-dissociation inhibitors (GDIs), guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) (Bishop and Hall, 2000; Sprang, 2001
). In the resting (inactive) state, Rac remains bound to GDP owing to tight association with GDIs (Bishop and Hall, 2000
; Sprang, 2001
). Activation by chemoattractants, growth factors, cytokines and selective stress stimuli results in the dissociation of Rac from GDIs and the association of Rac with GEFs, which stimulate GDP release (Bishop and Hall, 2000
; Olofsson, 1999
; Sprang, 2001
). GEF-catalysed dissociation of GDP from Rac allows GTP binding. In the GTP-bound state, Rac is active and exerts its effects on actin polymerization and several signaling pathways, including the p38 kinase and c-Jun N-terminal kinase (JNK) pathways, through its association with various effector proteins (Allen et al., 1997
; Minden et al., 1995
; Roberts et al., 1999
; Zhang et al., 1995
). GAPs terminate Rac signaling by promoting the hydrolysis of bound GTP, returning Rac to the resting GDP-bound state (Bishop and Hall, 2000
; Sprang, 2001
).
There are three highly homologous forms of Rac: Rac1, Rac2 and Rac3 (Courjal et al., 1997; Didsbury et al., 1989
). Rac1 and Rac2 exhibit 92% amino acid identity (Courjal et al., 1997
; Didsbury et al., 1989
). Also, murine and human Rac2 are 99% identical (Shirsat et al., 1990
). Unlike the ubiquitously expressed Rac1, Rac2 expression is restricted to hematopoietic cells (Courjal et al., 1997
; Didsbury et al., 1989
). Northern-blot analysis has demonstrated Rac2 expression in peripheral blood mononuclear cells and differentiated monocytic U937 cells (Didsbury et al., 1989
). Rac2 protein expression has been shown in neutrophils, monocytes and macrophages with different Rac2-specific antibodies (Abo et al., 1994
; Kuncewicz et al., 2001
; Prada-Delgado et al., 2001
).
The roles of Rac1 and Rac2 in cell types that express both remain controversial. Several studies have demonstrated an essential role for Rac2 in regulating superoxide production and chemotaxis in neutrophils (Diebold and Bokoch, 2001; Irani and Goldschmidt-Clermont, 1998
; Knall et al., 1997
). These studies have been further confirmed by the identification of a patient whose cells expressed both wild-type Rac2 and a mutant Rac2 wherein an aspartate at position 57 was substituted with an asparagine (Ambruso et al., 2000
). Aspartate at this position is conserved in all GTP-binding proteins and is predicted to coordinate the catalytic Mg2+ ion through an intervening water molecule (Bourne et al., 1991
). This patient suffered from recurrent bacterial infections, and neutrophils from this patient exhibited defects in chemotaxis, F-actin assembly, polarization, azurophilic granule secretion and superoxide anion production (Ambruso et al., 2000
; Williams et al., 2000
). The ability of the Rac2D57N mutant to decrease superoxide production and to inhibit neutrophil migration suggests that Rac2D57N acts as a dominant inhibitory mutant. Furthermore, neutrophils from Rac2-deficient mice have defects in actin remodeling and mitogen-activated protein kinase (MAPK) activation, indicating a role for Rac2 in these processes (Roberts et al., 1999
). Collectively, these data demonstrate that Rac2 plays a crucial role in both human and murine hematopoietic cell homeostasis (Ambruso et al., 2000
; Roberts et al., 1999
). The expression of Rac2 in multiple hematopoietic cell types suggests the possibility of abnormalities induced by Rac2D57N in different hematopoietic cell lineages, prompting examination of the effect of Rac2D57N expression in these cell types.
In this study, we examine the effects of Rac2D57N expression in primary murine bone-marrow-derived macrophages. Collectively, the data presented here demonstrate that Rac2D57N is a potent dominant inhibitory mutant of Rac signaling that is capable of promoting significant alterations in cell morphology and MAPK signaling, probably through the sequestration of upstream regulators of Rac and a decreased ability to bind Rac effectors.
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Materials and Methods |
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Quantitative RT-PCR
Total RNA was extracted from day 5 murine bone-marrow-derived macrophages or murine fibroblasts using RNA-Bee from TEL-TEST (Friendswood, TX). cDNA generated from 6 µg RNA using Super Script First-Strand Synthesis System from Invitrogen (San Diego, CA) was used for quantitative reverse-transcription polymerase chain reactions (RT-PCRs) using the ABI Prism 7700 sequence detector (Perkin Elmer/Applied Biosystems). The mRac1 and mRac2 cDNAs were generated with primers for cloning the full-length coding sequences. The following primers and probes were used: mRac1 forward 5'-AGCGAGGACTCAAGACAGTGTTT-3'; mRac1 reverse 3'-TTTCTCTTCCTCTTCTTGACAGGAG-5'; the mRac1 probe FAMACGAAGCTATCCGAGCGGTTCTCTGTC-TAMRA; mRac2 forward 5'-CGAGGCCTGAAGACCGTCT-3'; mRac2 reverse 3'-TTCTGCTGTCGTGTGGGCT-5'; and the mRac2 probe FAMCGATGAGGCAATCCGCGCAGTC-TAMRA. The TaqMan probe was purchased from Perkin-Elmer 5' labeled with 6-carboxyfluorescein (FAM) and 3' labeled with 6-carboxytetramethylrhodamine (TAMRA). Reactions contained 8% glycerol, 1x TaqMan Buffer A (500 mM KCL, 100 mM Tris-HCl, 0.1 M EDTA, 600 nM passive reference dye ROX, 300 µM each dATP, dGTP and dCTP, 600 µM dUTP, 5.5 mM MgCl2, 900 nM forward primer, 900 nM reverse primer, 200 nM probe, 1.25 U AmpliTaq Gold DNA Polymerase (Perkin Elmer, Foster City, CA) 12.5 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD), 20 U RNAsin ribonuclease inhibitor (Promega, Madison, WI) and the template cDNA prepared from total cell RNA. Reverse transcription was performed at 48°C for 30 minutes and activation of TaqGold at 95°C for 10 minutes, then 40 cycles of amplification were performed at 95°C for 15 seconds and 60°C for 1 minute. Serial dilutions of purified plasmid cDNA for mRac1 and mRac2 were used to construct a standard curve to compare sample cDNA concentrations. The quantities of mRac1 and mRac2 were normalized to the amount of rRNA in each sample.
Cell culture and transfections
Bone-marrow-derived macrophages were prepared from C57Bl6 mice as previously described (Riches and Underwood, 1991). Macrophages were plated on glass coverslips and grown for four days at 37°C and 10% CO2 in macrophage medium with high-glucose Dulbecco's modified Eagle's medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), 10% L cell-conditioned medium and 1% penicillin-streptomycin-glutamine (PSG). On day 4, macrophages were transfected with 2 µg of each expression construct in Optimem using Lipofectamine Plus. After 3 hours, macrophage medium was added and cells were grown for 24 hours. On day 5, macrophages were treated as described in the figure legends. COS7 cells were cultured in DMEM with high glucose, 10% FBS, and 1% PSG. RAW macrophages were grown in DMEM with high glucose, 10% heat-inactivated FBS and 1% PSG.
Immunofluorescence microscopy
Macrophages plated on coverslips were fixed for 10 minutes in PBS, pH 7.4, containing 3% paraformaldehyde and 3% sucrose. Cells were subsequently washed and permeabilized for 7 minutes in PBS containing 0.2% Triton X-100. Cells were blocked for 30 minutes in DMEM containing 10% donkey serum and were incubated with 0.2 µM rhodamine phalloidin for 30 minutes. As indicated in the figure legends, cells were immunostained with M5 or anti--tubulin antibodies for 1 hour, followed by incubation with FITC-conjugated anti-mouse secondary antibody. Coverslips were mounted in mounting medium (1x PBS, 50% glycerol, 10 mM Tris, pH 7.3). Cells were visualized using a Leica DM/RXA fluorescent microscope and digital images were acquired with a SensiCam digital camera (The Cooke Corp., Tonawanda, NY) driven by software from Intelligent Imaging Innovations (Denver, CO).
Macropinocytosis assays
On day 5, bone-marrow-derived macrophages were washed twice and incubated for 3 hours in DMEM containing 10% heat-inactivated FBS to deprive macrophages of macrophage colony-stimulating factor (MCSF). Macrophages were stimulated for 5 minutes with 100 ng ml-1 recombinant M-CSF followed by a 5-minute incubation with 0.5 mg of lucifer yellow (LY). Coverslips were placed on ice and washed four times with ice-cold PBS. Cells were fixed and processed as described above for immunofluorescence microscopy and incubated for 1 hour with a rabbit polyclonal antibody to LY followed by a CY3 donkey anti-rabbit antibody.
Quantitative immunofluorescence
Macrophages were equilibrated for 3 hours in 2 ml macrophage medium. Hanks Balanced Salt Solution (HBSS) or 10 ng ml-1 lipopolysaccharide (LPS) in HBSS was added and cells were incubated for 20 minutes. All subsequent steps were performed at room temperature unless otherwise specified. Coverslips were washed once with PBS, pH 7.4, and fixed for 14 minutes in PBS containing 3% paraformaldehyde and 3% sucrose. Coverslips were washed three times with PBS and permeabilized for 7 minutes in 0.2% Triton X-100 in TBS, pH 7.4. Subsequently, coverslips were washed twice with 0.1% Triton X-100 in TBS and once with TBS, and blocked for 1.5 hours in 10% FBS. Cells were washed three times in TBS and incubated overnight at 4°C with anti-phosphorylated-p38 antibody in TBS and 3% bovine serum albumen. Cells were subsequently washed five times with PBS and incubated for 1 hour with 0.1 ng ml-1 DAPI, 1.5 µg ml-1 Cy3-conjugated donkey anti-rabbit antibody and 10% FBS.
To quantify phosphorylated p38 kinase intensity in the nuclei of macrophages, 12-16 0.5 µm sections were taken of each macrophage. All images were taken at the same exposure times. Images were deconvolved using a constrained iterative according to specifications from Intelligent Imaging Innovations. Total nuclear intensity of phosphorylated p38 kinase was determined for the sum of all nuclear sections. To normalize for differences in nuclear size, the total nuclear phosphorylated-p38-kinase intensity per cell was divided by the total nuclear volume per cell to give the average nuclear phosphorylated p38 kinase intensity per cell. Total nuclear volume was calculated from the nuclear DAPI intensity for each nuclei. For each treatment, five independent experiments with duplicate coverslips were evaluated. Statistical significance was determined using a Student's t test.
p38 kinase and JNK assays
p38 kinase and JNK assays were performed as previously described (Gerwins et al., 1997).
Rac activity assays
Day-5 macrophages were incubated in DMEM containing 10% heat-inactivated FBS for 3 hours. Subsequently, cells were stimulated for the indicated time with 100 ng ml-1 recombinant murine M-CSF. Alternatively, cells were equilibrated for 3 hours in fresh macrophage medium, followed by stimulation for the indicated times with 10 ng ml-1 LPS. Rac activity assays were performed as described previously (Arthur and Burridge, 2001).
Guanine-nucleotide binding
GST fusion proteins were prepared as previously described (Ambruso et al., 2000). The binding of [3H]GDP (specific activity of 3465 cpm pmol-1) and [35S]GTP
S (specific activity of 5520 cpm pmol-1) to GST-Rac2 fusions was measured in 50 mM Hepes, pH 8.0, 1 mM DTT, 2 mM EDTA. 2 µg GST-Rac2 wild-type or mutant was incubated at 30°C for the indicated times with 1 µM [35S]GTP
S in the absence or presence of 10 µM unlabeled GTP
S. Binding reactions were terminated on ice in 2 ml 25 mM Tris, pH 8.0, 100 mM NaCl, 30 mM MgCl2, 2 mM DTT, 1 mg ml-1 bovine serum albumen. Samples were filtered through nitrocellulose filters, washed, dried and counted. [3H]GDP binding was measured as described for [35S]GTP
S except that 2 µM [3H]GDP was used in the absence or presence of 20 µM unlabeled GDP.
Dissociation of [3H]GDP
To measure the dissociation of [3H]GDP, wild-type or mutant GSTRac2 was incubated for 20 minutes at 30°C with 2 µM [3H]GDP as described above for the binding reactions. Tubes were placed on ice and unlabeled GDP or GTPS was added to yield a final concentration of 170 µM. Samples were incubated at 30°C for the indicated times. Binding reactions were terminated as described above.
Coimmunoprecipitation of Rac with RhoGDI or Tiam1
COS7 cells were co-transfected with expression constructs encoding FLAG-tagged Rac and expression constructs encoding either RhoGDI or Myc-tagged Tiam1. After 24 hours, cells were lysed in 20 mM Tris, pH 7.4, 100 mM NaCl, 1% Triton X-100, 10% glycerol, 0.1 mM EDTA, 0.1 mM EGTA, 25 mM NaF, 5 mM MgCl2, 1 mM PMSF, 0.01 mg ml-1 leupeptin and aprotinin, 1 mM DTT, 1 mM sodium vanadate. Lysates were immunoprecipitated and probed with the antibodies as described in figure legends.
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Results |
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Rac2wt and Rac2D57N expression in bone marrow-derived macrophages alters the steady-state actin cytoskeleton
Allen et al. have shown that injecting constitutively active Rac1 into a macrophage cell line causes prominent cell ruffling and lamellipodium formation, whereas injection of a dominant inhibitory mutant Rac1T17N inhibits M-CSF-stimulated lamellipodium formation and causes cell rounding (Allen et al., 1997). Although most studies have used macrophage cell lines, they do not fully represent differentiated macrophages in function and regulation, including growth-factor dependence and the expression of adhesion molecules (Schmidt et al., 2001
). Therefore, we used primary murine bone-marrow-derived macrophages that express endogenous Rac1 and Rac2 to examine phenotypic changes resulting from Rac2D57N expression. Coexpression of Rac2 and EGFP by pIRES2-EGFP expression constructs was verified in 293 cells (data not shown). After transfection, cells were grown for 24 hours in macrophage medium containing M-CSF, macrophages were fixed and F-actin was stained with rhodamine-phalloidin. Transfected macrophages were identified by EGFP expression (Fig. 1A,B). GFP expression levels were similar in both Rac2wt- and Rac2D57N-expressing macrophages (data not shown). Fig. 1A,C,D illustrates the actin staining of three separate macrophages expressing Rac2wt. Transfected macrophages displayed three main types of actin structures: surface ruffling (Fig. 1A), lamellipodia (Fig. 1C) and filopodia/retraction fibers (Fig. 1D). Table 2 shows the proportions of transfected cells expressing these actin structures, quantified in a minimum of 50 transfected cells from two independent experiments. In empty-vector-expressing cells, 64% of the cells displayed prominent filopodia/retraction fibers, whereas fewer than 20% of the cells exhibited surface ruffling or an elongated phenotype (Table 2). Rac2wt-expressing cells exhibited fewer cells with filopodia/retraction fibers and a concomitant 2.5-times enhancement in the proportion of cells with F-actin-rich surface ruffles relative to macrophages transfected with empty vector alone (Table 2). Rac2D57N-expressing macrophages also showed a threefold reduction in the expression of filopodia/retraction fibers but lacked F-actin containing surface ruffles (Table 2). Unexpectedly, there was a twofold increase in the proportion of Rac2D57N-expressing macrophages that exhibited an elongated, spread morphology compared with either vector- or Rac2wt-expressing cells (Table 2). However, these elongated Rac2D57N-expressing cells lacked peripheral and filamentous actin structures including lamellipodia and filopodia (Fig. 1E). Although Rac2D57N-expressing macrophages exhibited perturbation of actin structures relative to Rac2wt- or vector-expressing macrophages, these cells maintained microtubule networks that protruded into the leading edge (Fig. 1F) and were indistinguishable from Rac2wt-or vector-expressing macrophages (Fig. 1G,H).
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FLAG-Rac2D57N localized to both the cytoplasm and the plasma membrane (Fig. 1I,K). FLAG-Rac2D57N also localized with large perinuclear actin aggregates, suggesting that Rac2D57N expression perturbs the macrophage actin cytoskeleton (Fig. 1I-L). The effects of Rac2D57N expression were compared with those of Rac1D57N, Rac1T17N and Rac2T17N (Table 3). All inhibitory mutants tested prevented the accumulation of F-actin in surface ruffles compared with vector (Table 3). Rac1D57N expression resulted in a twofold increase in elongated macrophages, similar to Rac2D57N. By contrast, Rac1T17N and Rac2T17N expression did not induce macrophage elongation and spreading (Table 3).
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Rac2D57N promotes an elongated phenotype with enhanced spreading in RAW macrophages
Bone marrow-derived macrophages are highly heterogeneous in morphology, making it difficult to ascertain whether the expression of Rac2D57N promotes or stabilizes the extension of the macrophage. To examine this question further, we used RAW murine macrophages, which exhibit a homogeneously round morphology (Fig. 2). Expression of Rac1wt or Rac2wt did not promote spreading of RAW cells (Fig. 2). Expression of either Rac2D57N or Rac1D57N promoted the elongation of the RAW macrophage with the appearance of leading and trailing edges (Fig. 2). Similar to expression in bone-marrow-derived macrophages, Rac1T17N was ineffective at promoting macrophage extension (Fig. 2). Taken together, the data from bone-marrow-derived and RAW macrophages demonstrate that Rac2D57N expression in macrophages results in an elongated and spread morphology concurrent with abnormal remodeling of the actin cytoskeleton.
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Rac2D57N inhibits M-CSF-stimulated surface ruffling
M-CSF has been shown to stimulate the formation of filopodial structures, lamellipodia and active cell ruffling (Allen et al., 1997; Racoosin and Swanson, 1989
; Racoosin and Swanson, 1992
). In bone-marrow-derived macrophages, 3 minutes of MCSF stimulation activated Rac (Fig. 3A), as measured by GST-PAKcrib binding, and promoted surface ruffling (Fig. 3B). 5 minutes of M-CSF stimulation of macrophages transfected with empty vector increased by 3.5 times the proportion of cells with surface ruffling that decreased by 30 minutes of stimulation (Fig. 3C). In the basal state, the proportion of Rac2wt-expressing cells with surface ruffles was 2.3-times greater than empty vector expressing cells (Fig. 2C). M-CSF stimulation further increased the number of Rac2wt-expressing macrophages with surface ruffles (Fig. 3B,C). Rac2D57N macrophages lacked F-actin-containing surface ruffles, even after M-CSF stimulation (Fig. 3C). Similar to Rac2D57N, membrane ruffles containing F-actin were absent from M-CSF-stimulated Rac1T17N-expressing cells (data not shown). MCSF withdrawal did not alter the proportion of cells with filopodia/retraction fibers relative to steady-state macrophages (Table 2, Fig. 3D). M-CSF stimulation of empty vector expressing cells resulted in a twofold decrease in the proportion of cells expressing filopodia/retraction fibers at 5 minutes that returned back to basal levels after 30 minutes (Fig. 3D). Expression of either Rac2wt or Rac2D57N in macrophages reduced the proportion of cells exhibiting filopodia/retraction fibers (Fig. 3D). M-CSF deprivation followed by the addition of M-CSF did not result in a statistically significant increase in the number of transfected cells with an elongated morphology, suggesting that M-CSF does not stimulate macrophage polarization (Fig. 3E). Approximately 50% of the Rac2D57N-expressing macrophages exhibited an elongated morphology (Fig. 3E). This represents a greater than twofold increase in elongated cells in Rac2D57N-expressing cells relative to vector- or Rac2wt-expressing macrophages (Fig. 3E). Together, the loss of M-CSF-stimulated membrane ruffling in Rac2D57N-expressing cells and the increase in elongated cells is consistent with inhibition of actin remodeling by Rac2D57N shown in Fig. 1.
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Rac2D57N expression in bone-marrow-derived macrophages inhibits M-CSF-stimulated macropinocytosis
M-CSF stimulates solute pinocytosis that results in the formation and accumulation of fluid-filled endocytic vesicles called macropinosomes (Murray et al., 2000; Racoosin and Swanson, 1989
). LY is used as a probe to monitor solute endocytosis (Racoosin and Swanson, 1992
). After M-CSF deprivation, basal uptake of LY by macrophages is seen within smaller pinosomes (Fig. 4A). M-CSF stimulates the uptake of LY in large macropinosomes (Fig. 4A). Expression of either the empty vector or Rac2wt did not perturb M-CSF-stimulated macropinocytosis of LY (Fig. 4B). Although Rac2D57N-expressing macrophages could form small LY-containing pinosomes, macropinocytosis of LY was inhibited (Fig. 4B). Macropinosomes were quantified and stimulation of macropinocytosis was defined as macrophages with five or more macropinosomes. M-CSF stimulation of empty-vector-expressing cells resulted in an eightfold stimulation of macropinocytosis (Fig. 4C). Rac2wt-expressing cells exhibited a fourfold elevation in basal macropinocytosis, whereas MCSF-stimulated macropinocytosis was not significantly different from that in empty-vector-expressing cells (P>0.154) (Fig. 4C). Basal macropinocytosis by Rac2D57N-expressing macrophages was not significantly different from basal macropinosome formation in vector-expressing cells (P>0.344) (Fig. 4C). By contrast, Rac2D57N-expressing macrophages exhibited an eightfold decrease in M-CSF-stimulated macropinocytosis (P<0.005) (Fig. 4C). This dramatic loss of macropinocytosis is similar to the block of macropinocytosis by Rac1T17N in NIH-3T3 cells (Dharmawardhane et al., 2000
). Macropinosomes are known to form at sites of active membrane ruffling (Racoosin and Swanson, 1989
; Racoosin and Swanson, 1992
; Racoosin and Swanson, 1993
). Thus, the increased basal macropinocytosis in Rac2wt-expressing cells suggests that the ruffles in these cells are productive actin structures, whereas the lack of macropinocytosis in Rac2D57N-expressing cells is consistent with the loss of F-actin from surface ruffles (Figs 3, 4). Therefore, disruption of Rac signaling by Rac2D57N causes a defect in the formation of actin structures required for macropinocytosis.
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Rac2D57N expression alters filopodia/retraction fibers in serum-starved bone-marrow-derived macrophages
Combined serum and M-CSF starvation reduced Rac activity by three times relative to cells in macrophage medium (Fig. 5A). Rac activity increased with the addition of macrophage medium containing serum and L cell-conditioned medium to starved cells. This increase was markedly slower than that observed with M-CSF deprivation alone (Fig. 3A, Fig. 5A). Actin structures under combined serum/M-CSF starvation conditions were also examined. Serum- and M-CSF-starved vector-, Rac2wt- and Rac2D57N-expressing macrophages exhibited a decrease in filopodia/retraction fibers relative to steady-state macrophages (Fig. 5B,C, Table 2). Addition of macrophage medium resulted in a marked increase in filopodia/retraction fibers in both vector- and Rac2wt-expressing cells (Fig. 5B,C). However, Rac2D57N-expressing cells did not respond to the re-addition of macrophage medium (Fig. 5B,C). Combined serum/M-CSF starvation resulted in a fivefold reduction in the number of Rac2D57N-expressing cells with filopodia/retraction fibers relative to steady-state Rac2D57N-expressing macrophages (Fig. 5C, Table 2). These data suggest that Rac2D57N-expressing cells have filopodia/retraction fibers that are lost with combined serum and M-CSF starvation. However, they are unable to regenerate the structures upon re-stimulation with macrophage medium. These data are consistent with the disruption of regulated actin polymerization by the Rac2D57N mutant.
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Rac2D57N inhibits p38 kinase activity in bone-marrow-derived macrophages
Phagocytic cells like macrophages serve a crucial role in the first line of defense against bacterial infections. Stimulation of bone-marrow-derived macrophages with LPS results in an increase in Rac activity (Fig. 6A) and p38-kinase activity (Fig. 6C). Total p38-kinase expression was unaffected by LPS stimulation (data not shown). Similar increases in the phosphorylation of p38 kinase in response to LPS were observed by immunostaining macrophages plated on glass coverslips (Fig. 6B,D,E). LPS stimulation resulted in a three- to fivefold increase in average nuclear phosphorylated p38 kinase immunofluorescence relative to unstimulated cells (Fig. 6B,E). Expression of Rac2wt in macrophages did not alter basal levels of phosphorylated p38 kinase relative to empty-vector-expressing cells (Fig. 6E). Rac2D57N-expressing macrophages exhibited a 50% reduction in basal nuclear phosphorylated p38 kinase immunofluorescence (P<0.005) (Fig. 6E). Stimulation of Rac2wt- or vector-transfected macrophages with LPS resulted in a threefold increase in the average nuclear phosphorylated p38 kinase immunofluorescence relative to unstimulated cells (Fig. 6E). Expression of RacD57N resulted in a statistically significant (P<0.01) reduction of LPS-stimulated phosphorylated p38 kinase relative to vector- and Rac2wt-expressing controls (Fig. 6E). In primary macrophages, LPS stimulates Rac and p38 kinase activities, and Rac2D57N inhibits basal and LPS-stimulated activation of p38 kinase.
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Biochemical demonstration of the dominant inhibitory nature of Rac2D57N in COS7 cells
Rac proteins are important upstream mediators of JNK and p38 kinase pathways in several cell types (Coso et al., 1995; Minden et al., 1995
). In COS7 cells, constitutively active Rac activates JNK and the dominant inhibitory mutant Rac1T17N inhibits epidermal growth factor (EGF)-stimulated JNK activity (Coso et al., 1995
; Minden et al., 1995
). As shown in Fig. 7A, expression of Rac2wt resulted in a slight elevation of basal JNK activity compared with the expression of HAJNK2 alone. Coexpression of HA-JNK2 with Rac2D57N resulted in a strong inhibition of basal JNK activity, even though Rac2D57N was expressed at lower levels than Rac2wt (Fig. 7A). EGF stimulated a threefold increase in JNK activity in HA-JNK2-expressing cells in the absence or presence of Rac2wt (Fig. 7A). By contrast, Rac2D57N expression inhibited EGF-stimulated JNK activity (Fig. 7A), similar to Rac1T17N (Coso et al., 1995
; Minden et al., 1995
). However, ultraviolet (UV)-stimulated JNK activity was unaffected, consistent with previous studies showing that Rac1T17N does not inhibit UV-stimulated JNK activity (Eom et al., 2001
). HA-JNK2 protein expression was not altered by coexpression with Rac2 (Fig. 7A). These data show that Rac2D57N selectively inhibits growth-factor-stimulated Rac1-dependent JNK signaling and indicates that Rac2D57N can inhibit Rac1 signaling pathways.
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In addition to signaling to JNK, Rac plays an integral role in regulation of p38 kinase (Zhang et al., 1995). Sorbitol has been shown to stimulate both Rac and p38 kinase activity, and to promote cell shape change (Di Ciano et al., 2002
; Lewis et al., 2002
). Coexpression of p38 kinase and Rac2wt resulted in slight increases in the levels of phosphorylated p38 kinase compared with cells expressing only FLAG-p38 kinase (Fig. 7B). Rac2D57N expression reduced phosphorylated-p38-kinase levels under basal and sorbitol-stimulated conditions, whereas UV-stimulated phosphorylation of p38 kinase was not altered relative to cells transfected with FLAG-p38 kinase alone (Fig. 7B). Total Flag-p38 kinase and Rac2 protein expression was not altered (Fig. 7B). Similar results were obtained with in vitro p38 kinase assays (data not shown). These findings show that Rac2D57N expression produces a partial inhibition of basal and sorbitol-stimulated p38 kinase signaling, whereas UV-irradiation-induced p38 kinase activity is unchanged. These data are consistent with sorbitol signaling through Rac to induce cell shape changes (Di Ciano et al., 2002
; Lewis et al., 2002
).
Rac2D57N has altered guanine-nucleotide binding and enhanced association with regulators of Rac signaling
D57 in Rac2 is conserved in all GTP-binding proteins and is predicted to bind the catalytic Mg2+ through an intervening water molecule (Bourne et al., 1991). This Mg2+ binds the oxygen atoms of both ß and
phosphates of GTP (Bourne et al., 1991
; Wittinghofer and Pai, 1991
). As a consequence, mutation of D57 is likely to perturb guanine-nucleotide binding. Guanine-nucleotide association and dissociation with wild-type and mutant Rac2 expressed as GST fusion proteins was measured. As shown in Fig. 8A, the association of [3H]GDP with Rac2D57N was complete by 10 minutes, similar to Rac2wt. However, unlike Rac2wt, which rapidly associated with [35S]GTP
S, Rac2D57N bound [35S]GTP
S weakly (Fig. 8B). Additionally, the dissociation of [3H]GDP from Rac2D57N in response to the addition of a 17-fold molar excess of unlabeled GDP or GTP
S was much slower than Rac2wt (Fig. 8C,D). [3H]GDP completely dissociated from Rac2wt by 10 minutes following the addition of either unlabeled GDP or GTP
S (Fig. 8C,D). By contrast, after 10 minutes, only half of the [3H]GDP bound to Rac2D57N had dissociated in response to the addition of unlabeled GDP, and no [3H]GDP had dissociated following the addition of unlabeled GTP
S (Fig. 8C,D). These data clearly show the alteration in nucleotide binding to Rac2D57N relative to the wild-type protein.
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As shown in Fig. 8, Rac2D57N retains the ability to bind GDP, but the ability to bind GTP and the dissociation of GDP are reduced. Altered nucleotide binding might perturb the interaction of Rac2 with upstream regulators such a GDIs and GEFs. To address this question, Rac2wt or Rac2D57N was coexpressed with RhoGDI followed by immunoprecipitation with an antibody against RhoGDI
. The ability of Rac to coimmunoprecipitate with the GDI was examined. Both Rac2wt and Rac2D57N coimmunoprecipitated with RhoGDI
, but the Rac2D57N association appeared to be more stable (Fig. 8E). Similar experiments were performed with the GEF Tiam1. Unlike Rac2wt, Rac2D57N coimmunoprecipitated with Tiam1, suggesting a more stable interaction with this GEF (Fig. 8F). Similar to Rac2D57N, Rac1D57N and Rac1T17N coimmunoprecipitated with Tiam1 and RhoGDI
(data not shown). Together, these data show that Rac2D57N binds tightly in vivo to both the inhibitor and stimulator of nucleotide exchange. Importantly, both RhoGDI
and Tiam1 are expressed in macrophages (data not shown). The ability of Rac2D57N to bind tightly to both RhoGDI
and Tiam1 might be responsible, at least in part, for its ability to disrupt Rac function in the macrophage.
The GTP-bound form of Rac can regulate Rac effectors (Bishop and Hall, 2000). The reduced GDP dissociation and decreased GTP binding would be predicted to affect the signaling of RacD57N to its effectors. Consistent with this prediction, Rac1wt and Rac2wt bound tightly to GST-PAKcrib, whereas both Rac2D57N and Rac1T17N bound weakly to GST-PAKcrib (Fig. 8G,H), indicating that these dominant inhibitory Racs are unable efficiently to regulate downstream effectors like PAK.
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Discussion |
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Rac2D57N has dramatic effects on the macrophage actin cytoskeleton. Rac2D57N was found in the cell cytoplasm and periphery, and colocalized with large, perinuclear actin aggregates. Rac2D57N expression inhibited reorganization of the actin cytoskeleton under several conditions, resulting in the loss of F-actin in ruffles, macropinocytosis and actin polymerization. The inhibition of surface ruffles and macropinocytosis is similar to that shown for RacT17N (Dharmawardhane et al., 2000). The effect of Rac2D57N is specific for the actin cytoskeleton because the microtubule cytoskeleton is unaffected. This finding is consistent with data showing that Rac1T17N was unable to block centrosome reorientation, an event that requires microtubules (Etienne-Manneville and Hall, 2001
). Somewhat surprisingly, expression of Rac2D57N and Rac1D57N results in an elongated, spread morphology, whereas Racwt or RacT17N does not cause the same effect. Rac2wt and Rac2D57N are expressed at similar levels, indicating that the differences in actin structures are a function of the D57N mutation in Rac2. Cumulatively, the inhibitory effects of Rac2D57N on the actin cytoskeleton altered macrophage function as demonstrated by the loss of macropinocytosis.
Rac2D57N inhibits MAPK activity. Inhibition of LPS-stimulated p38 kinase activity in macrophages was shown by immunofluorescence. Reductions in JNK and p38 kinase activity were shown biochemically in COS7 cells. Activation of p38 is crucial in the innate pro-inflammatory response (Dong et al., 2002). Activation of p38 is required for the production of pro-inflammatory cytokines, upregulation of adhesion molecules and expression of secreted proteases (Dong et al., 2002
; Schmidt et al., 2001
). p38 is a principal mediator of the inflammatory response to LPS in humans. We show for the first time in mouse primary macrophages that LPS activates Rac. Furthermore, Rac2D57N expression inhibits LPS activation of p38. Dominant inhibitory Rac expression in neurons (Allen et al., 2002
) and COS cells (Zhang et al., 1995
) also inhibited p38 activation, demonstrating the importance of Rac proteins in the regulation of p38 activation in response to multiple stimuli in different cell types. The reduction in responsiveness to LPS is consistent with the patient's impaired response to Gram-negative bacteria.
Inhibitory mutants of small G proteins are predicted to act by competing with the wild-type protein for GEF binding, thus preventing activation of the wild-type protein. For example, because dominant inhibitory RasN17 has a higher affinity for the GEF than for nucleotides, GTP is less likely to displace the GEF, resulting in the enhanced association of RasN17 with the GEF. Similarly, RacD57N bound more tightly to the GEF than does Racwt. The enhanced binding of RacD57N to Tiam1, a GEF expressed in macrophages, would be predicted to perturb nucleotide exchange of wild-type Rac and to disrupt Racdependent processes including actin remodeling and MAPK activation. Rac2D57N can probably disrupt both Rac1- and Rac2-dependent signaling processes. Interestingly, RacD57N also associated tightly with RhoGDI. This finding is in direct contrast with results showing that RhoAT17N binds the GEF smgGDS, but not RhoGDI (Strassheim et al., 2000
). Release of GTPases from GDIs is an important step in allowing small G proteins to be activated (Olofsson, 1999
). The enhanced association of RacD57N with RhoGDI could displace RhoGDI
-binding proteins such as Rac, Rho and Cdc42, potentially promoting the activation of these proteins (Feig, 1999
; Olofsson, 1999
). However, the enhanced binding of RacD57N to Tiam1, a Rac-specific GEF, would be expected specifically to inhibit endogenous Rac activation. Overall, our findings suggest that the complex behavior of RacD57N is related to its enhanced binding to several upstream regulators of Rac and a diminished ability to bind downstream effectors.
In conclusion, our studies have defined the dominant inhibitory functions of the D57N mutation in Rac2. Inhibition of p38 kinase activation and remodeling of the actin cytoskeleton are specific consequences of the inhibitory properties of Rac2D57N. We propose that the properties of the Rac2D57N mutant protein are sufficient to cause the inhibited responses in the infant patient harboring the Rac2D57N mutation. During the initial study, we had access to limited blood samples that precluded the assessment of the effects of this mutant on other Rac2-expressing cell lineages including macrophages. The patient has successfully undergone a bone-marrow transplant. Thus, cells expressing the mutant are unavailable for further study of Rac2D57N expression in patient macrophages. Our current studies both significantly enhance our understanding of the patient's immunodeficiency and define Rac2D57N as a potent dominant inhibitory mutant. In future studies, it will be interesting to see whether somatic mutations in Rho GTPases contribute to the molecular basis of the defects of human syndromes in which specific cell types lose the coordinate regulation of signaling pathways and remodeling of the actin cytoskeleton. Certainly, the study of mutations in effectors for Rho GTPases, such as the WASP protein mutated in Wiskott-Aldrich syndrome (Thrasher and Burns, 1999), indicate that disruption of the Rho GTPase regulatory network results in human syndromes of significant health consequences for the affected individual.
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