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* Herman B Wells Center for Pediatric Research and Departments of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics,
Biochemistry and Molecular Biology, and
Walther Oncology Center and Walther Cancer Institute,
James Whitcomb Riley Hospital for Children, Indiana University Medical School, Indianapolis, IN 46202
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4-fold more
Rac2-GTP was detected than Rac1-GTP. Wild-type and Rac2-deficient
neutrophils have similar levels of total Rac1. FMLP-induced Rac1-GTP in
rac2-/- neutrophils was 3-fold greater
than in wild-type cells, which have similar levels of total Rac1, yet
FMLP-stimulated F-actin, chemotaxis, and superoxide production are
markedly impaired in rac2-/- neutrophils.
Heterozygous rac2+/- neutrophils, which had
intermediate levels of total and FMLP-induced activated Rac2, exhibited
intermediate functional responses to FMLP, suggesting that Rac2 was
rate limiting for these functions. Thus, phenotypic defects in
FMLP-stimulated Rac2-deficient neutrophils appear to reflect distinct
activation and signaling profiles of Rac 1 and Rac2, rather than a
reduction in the total cellular level of Rac. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are three Rac isoforms, Rac1, Rac2, and Rac3 (20, 21, 22, 23). Rac1 is the most studied isoform and is ubiquitously expressed, whereas Rac2 expression is highly restricted to hemopoietic cells (20, 22, 23). These two isoforms share 92% identity overall and differ primarily in the C-terminal 10 residues, where Rac1, but not Rac2, contains a highly basic sequence adjacent to a prenylated cysteine that can insert into cellular membranes. Murine Rac1 and Rac2 differ by only one and two amino acids from human Rac1 and Rac2, respectively (20, 23). The more recently discovered Rac3 shares 72% identity with Rac1 and 83% identity with Rac2 (20, 21, 22, 23); like Rac1, it is expressed in a variety of tissues but its functions are relatively uncharacterized.
rac2-/- mice generated by gene targeting exhibit multiple functional defects in different hemopoietic lineages (24, 25, 26, 27). In vivo, rac2-/- mice had decreased exudate formation and increased mortality in invasive aspergillosis (27). rac2-/- neutrophils displayed impaired F-actin generation, lamellipodia formation, and directed cell movement in response to FMLP and other agonists signaling through G-protein-coupled receptors, decreased NADPH oxidase activity in response to FMLP, phorbol ester, and IgG-opsonized particles and diminished L-selectin-mediated adhesion (24, 27). The requirement for Rac2 in these functions was not absolute and was selective for specific signaling pathways. For example, F-actin formation in neutrophils stimulated with tyrosine kinase-coupled growth factors was normal, as was opsonized zymosan-stimulated NADPH oxidase activity (24, 27). Rac2 is also likely to play an important role in human neutrophil function, given that a patient with recurrent bacterial infections and a neutrophil phenotype similar to that of the rac2-/- mouse was found to have a dominant-negative Rac2 mutation (28, 29). Taken together, these observations demonstrate that Rac is a critical regulator of specific phagocyte signaling pathways and suggest the hypothesis that Rac2 has nonoverlapping functions with other Rac isoforms. Alternatively, the functional defects in Rac2-deficient cells could reflect an overall reduction in the cellular level of Rac, and the impaired neutrophil function resulting from expression of a dominant-negative Rac2 mutation includes effects on other Rac isoforms.
In the current study, to better define the relative role of the
different Rac isoforms in regulating neutrophil functions, we used an
affinity precipitation assay for Rac-GTP to compare Rac1 and Rac2
activation in chemoattractant-stimulated wild-type and Rac2-deficient
murine neutrophils. We found that similar amounts of Rac1 and Rac2 were
present in wild-type murine neutrophils, unlike human neutrophils in
which Rac2 is the predominant isoform (14). However,
although FMLP induced the activation of Rac1 and Rac2 in wild-type
murine neutrophils, 4-fold more activated Rac2 was detected than in
Rac1. Both basal and FMLP-stimulated levels of Rac1-GTP were increased
in rac2-/- neutrophils compared with
wild-type neutrophils, although FMLP-stimulated chemotaxis and
superoxide production are markedly impaired in
rac2-/- neutrophils. Heterozygous
rac2+/- mice had intermediate levels
of FMLP-stimulated F-actin formation, chemotaxis, and NADPH oxidase
activity, with decreased levels of activated Rac2 and similar Rac1
activation compared with wild-type neutrophils. These data suggest that
the hemopoietic-specific Rac2 plays a dominant role in FMLP-activated
responses in murine neutrophils, which appears to reflect in part a
greater activation of Rac2 and in part a preferred role for Rac2 in
stimulating downstream functional responses that cannot be compensated
for by enhanced Rac1 activation in Rac2null
neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A polyclonal Rac2 Ab raised in rabbits was a gift from G. Bokoch
and U. Knaus (The Scripps Research Institute, La Jolla, CA). A mouse
mAb against Rac1 was purchased from Upstate Biotechnology (Lake Placid,
NY). Highly purified recombinant prenylated human Rac1 and Rac2 were
provided by E. Pick (Tel Aviv University, Tel Aviv, Israel) and R.
Erickson and J. Curnutte (Genentech, South San Francisco, CA),
respectively. An expression vector for a 6.4-kDa fragment from
p21-activated kinase 3 containing the Cdc42/Rac binding motif and
tagged with glutathione-S-transferase was from R. Cerione
(Cornell University, Ithaca, NY) (30). BL21 competent
cells were obtained from Novagen (Madison, WI). PBS (pH 7.2),
ddH2O, glycerol, HBSS, and HEPES (125 mM, pH 7.5)
were from Life Technologies (Gaithersburg, MD). Polymorphprep was
purchased from Accurate Chemical and Scientific (Westbury, NY). Other
chemicals were purchased from Sigma (St. Louis, MO) unless otherwise
stated. Other buffers used in this article included: PBS with 0.1% BSA
and 1% glucose (pH 7.2–7.4); PBS with 0.9 mM
CaCl2, 0.5 mM MgCl2, and
7.5 mM glucose (PBSG); Triton IPB lysis buffer (20 mM Tris-Cl (pH 8.0),
150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 20 µg/ml chymostain, 2 mM
PMSF, 10 µM leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl
fluoride); and 5x MLB lysis buffer (125 mM HEPES (pH 7.5), 750 mM
NaCl, 5% Igepal CA-630, 25 mM MgCl2, 25 mM EDTA,
50% glycerol, 100 µg/ml chymostatin, and 10 mM PMSF).
Animals
C57BL/6J mice purchased from The Jackson Laboratory (Bar Harbor, ME) or 129SV mice from an in house colony were used for wild-type controls. rac2-/- mice had previously been generated by targeted disruption of the rac2 gene (27). Strains of rac2-/- and heterozygous rac2+/- mice used in this study either had been backcrossed into C57BL/6J mice for >12 generations or were descendants from a 129SV cross with the original blastocyst injection founder. Mice were housed in microisolator cages under specific pathogen-free conditions and were fed autoclaved food and acidified water ad libitum. Both male and female mice 8–12 wk of age were used in these experiments.
Isolation of neutrophils
Murine neutrophils were purified from the bone marrow (BM)
storage pool using sequential Percoll and Histopaque 1119 gradients as
described (24, 27), except that PBS buffers were used
instead of HBSS in some experiments. Freshly isolated murine neutrophil
preparations (80% neutrophils as assessed by Diff-Quik staining
(Dade, Miami, FL)) were resuspended in PBS (without
Ca2+ and Mg2+) and kept on
ice until further use. Human neutrophils were isolated from heparinized
whole blood using Polymorphprep according to the manufacturer’s
protocol.
Quantification of Rac1 and Rac2 by immunoblot assay
Cell lysates of diisofluorophosphate-treated murine BM neutrophils and human peripheral blood neutrophils were prepared and subjected to 12% SDS-PAGE and immunoblotting as previously described (24, 31). For quantification of Rac1 and Rac2 levels in cells, serial dilutions of recombinant isoprenlyated Rac1 and Rac2 were loaded in adjacent lanes. Blots were probed with either a mouse mAb for Rac1 or a rabbit polyclonal Ab for Rac2 and with either an anti-mouse or anti-rabbit secondary Ab conjugated with HRP and developed using ECL (Amersham Pharmacia Biotech, Piscataway, NJ). Integrated densitometry was used to determine the intensity of signals using the Eagle Eye II Still Video System and associated software (Stratagene, La Jolla, CA) or, for scanned films, NIH Image software (Research Services Branch, National Institute of Mental Health, Bethesda, MD). Multiple exposures were analyzed to ensure that relative signal intensities measured were in the linear range.
Measurement of F-actin and chemotaxis
Neutrophil F-actin content and chemotaxis were examined as previously described (27). To measure relative F-actin levels, neutrophils were incubated in the absence or presence of 10 µM FMLP at 37°C for either 10 or 30 s and then fixed with PBS containing 4.6% paraformaldehyde and 0.1% BSA on ice, stained with 160 nM FITC-phalloidin for 30 min, and analyzed by flow cytometry. The results are reported as mean cellular fluorescence. For chemotaxis assays, 27 µl of FMLP (at 10, 1, or 0.1 µM) or DMSO vehicle diluted in HBSS (with Ca2+, Mg2+, and glucose) were placed in each lower chamber of a 48-well microchemotaxis device (NeuroProbes, Cabin John, MD), and 1 x 105 neutrophils in 50 µl of HBSS (with Ca2+, Mg2+, and glucose) were placed in each upper chamber, which were separated by a 3-µm pore size polycarbonate filter. The chamber was incubated at 37°C for 45 min; then the filter was fixed and stained with Diff-Quik. The number of migrated cells per high power view field (x400) was counted for a minimum of three fields per well, and a mean estimate for individual samples was calculated from data of replicate wells.
Measurement of NADPH oxidase activity
Superoxide dismutase-inhibitable FMLP-elicited superoxide
production was measured by an isoluminol chemiluminescence assay
(32) in 96-well plates using an Lmax microplate
luminometer (Molecular Devices, Sunnyvale, CA). A 2 x
106 cells/ml suspension (50 µl) in PBSG was
added to each well with 80 µl of 125 µM isoluminol in PBSG, 40 µl
of 100 U/ml HRP (Roche Applied Science, Indianapolis, IN) in 0.9%
NaCl, and either 5 µl of 3 mg/ml superoxide dismutase or PBSG.
After cells were incubated at 37°C for 10 min, 25 µl of 80 µM
FMLP (final concentration 10 µM) in PBSG or 25 µl of PBSG were
injected into each well by the automatic injector of the luminometer.
Chemiluminescence was detected as relative luminescence units by fast
kinetic mode, and the relative total amount of superoxide produced
during 1 min was determined using SoftMax PRO software (Molecular
Devices). Under these conditions, 
97.5% of chemiluminescence
was inhibited by superoxide dismutase. Superoxide dismutase-inhibitable
superoxide production elicited by 200 ng/ml PMA was measured using a
quantitative kinetic assay based on the reduction of cytochrome
c, as previously described (24, 31, 33).
Rac activation assays
An affinity precipitation or pull-down assay for Rac activation was performed as described (34) with slight modifications. The p21-binding domain (PBD) of p21-activated kinase 3 was expressed as a fusion protein with glutathione-S-transferase in the BL21 strain of Escherichia coli (30). GST-PBD was purified from glutathione-Sepharose beads with 10 mM reduced glutathione and stored at -80°C in 25 mM Tris-HCl (pH 8.0), 0.2 M DTT, 1 mM MgCl2, and 10% glycerol until use. Diisofluorophosphate- treated murine neutrophils (6 x 106/tube) were suspended in 0.5 ml of PBSG and incubated at 37°C for 5 min before the addition of 5 µl of either DMSO or 200 µM FMLP in DMSO (final concentration, 2 µM) or 20 µg/ml PMA in DMSO (final concentration, 200 ng/ml). In some experiments, cells were pretreated at 37°C for 20 min before stimulation with the following inhibitors: 100 µM genistein; 20 µM 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002); 100 nM wortmannin; or 10 µM, 25 µM, or 50 µM 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1). Stimulus- or DMSO-treated cells were incubated at 37°C for the indicated times and then lysed with ice cold 125 µl 5x MLB lysis buffer containing a total of 10 µg GST-PBD. Cell lysates were immediately placed on ice, clarified by centrifugation at 10,000 rpm for 5 min at 4°C, and transferred to a fresh microfuge tube. For precipitation of GST-PBD-bound Rac, 20 µl of glutathione-Sepharose 4B beads (50%) was added to the 0.5 ml of clarified cell lysate and incubated for 1 h at 4°C with agitation before centrifugation for 2 min at 9000 rpm. The bead pellet was then washed three times with 1x MLB lysis buffer before the final resuspension in 35 µl of Laemmli sample buffer and stored, if needed, at -80°C. Before SDS-PAGE, samples were heated to 100°C for 10 min. GST-PBD affinity-precipitated proteins were separated by 12% SDS-PAGE in parallel with aliquots of total MLB cell lysate, then transferred to nitrocellulose membrane, and probed for Rac1 or Rac2, followed by densitometry, as described above.
Statistical analysis
Student’s t test (either paired or unpaired, as indicated) was performed using Microsoft Excel software (Redmond, WA) and correlation analysis performed using Cricket Graph III (Computer Associates International, New York, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported (24) that murine neutrophils
have 3 to 5 times more Rac1 on a milligram of protein basis compared
with human neutrophils, in which Rac2 has been estimated at 60
ng/106 cells and accounts for at least 90% of
total cellular Rac (14). Murine neutrophils do not express
Rac3 (U. Knaus, personal communication). To directly compare
total cellular levels of the Rac1 and Rac2 isoforms in murine
neutrophils, we used serial dilutions of recombinant isoprenylated Rac1
and Rac2 as standards on immunoblots to estimate the quantity of Rac1
and Rac2 (Fig. 1
A), using Abs
that are relatively specific for Rac1 and Rac2. Although there is a
small amount (5%) of cross-reactivity of the Rac1 Ab with Rac2, and
vice versa (Ref. 27 and unpublished observations), this
should have only a negligible effect on our analyses. Rac1 and Rac2
were detected in approximately equal amounts in murine neutrophils
(Fig. 1B), whereas human neutrophils contained 3-fold
more Rac2 than Rac1, consistent with our previous results
(24) and also in general agreement with published data for
human neutrophil Rac2 (14). Somewhat more Rac1 was
detected in human neutrophils than in the study of Heyworth et al.
(14) which could reflect differences in the approaches
used to estimate the quantity of Rac1 and/or the small degree of Ab
cross-reactivity. As we have previously reported (24, 27),
the amount of Rac1 in rac2-/- (Fig. 1)
and heterozygous rac2+/- (not shown)
neutrophils was similar to that in wild-type cells, and
rac2+/- neutrophils contained
50% of wild-type levels of Rac2 (not shown). The similar levels of
Rac1 in primary neutrophils isolated from all three rac2
genotypes contrast with the increased expression of Rac1 observed in
long term rac2-/- mast cell cultures
(26) or in marrow progenitor-derived
rac2-/- neutrophils generated in vitro
using high concentrations of hemopoietic cytokines
(35).
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To examine whether there was a rac2 gene dosage effect on neutrophil responses induced by the chemoattractant FMLP, we compared F-actin formation, chemotaxis, and NADPH oxidase activity in rac2+/+, rac2+/-, and rac2-/- neutrophils. We previously observed a small but not statistically significant decrease in PMA-elicited O2- production in rac2+/- neutrophils and failed to detect any differences in FMLP-elicited F-actin formation or chemotaxis between wild-type and rac2+/- mice (27). However, our original studies were done in mixed 129SV x C57BL/6J littermates, in which variability introduced by strain to strain differences might have obscured any gene dosage effect. In subsequent studies, we observed that the defect in PMA-elicited O2- production in rac2-/- exudate neutrophils became more severe as mice were backcrossed into C57BL/6J (24). Therefore, we re-examined responses elicited by either FMLP or PMA in freshly isolated BM neutrophils containing differing levels of Rac2 in an otherwise homogeneous genetic background, using C57BL/6J rac2+/+, rac2+/-, and rac2-/- mice.
Fig. 2A shows the mean
basal levels of F-actin and at 10 and 30 s after stimulation with
10 µM FMLP, in a representative experiment using BM neutrophils
prepared on a single day from each of three mice for each genotype. In
resting neutrophils, F-actin levels were decreased in
rac2-/- compared with wild-type
neutrophils, as previously reported (27), and intermediate
levels were detected in heterozygous
rac2+/- neutrophils (Fig. 2A). Furthermore, the FMLP-stimulated increase in F-actin
followed a similar hierarchy, with very little change from basal levels
observed in rac2-/- neutrophils compared
with the progressive increase seen in
rac2+/- and wild-type neutrophils
(Fig. 2A). These data suggest that Rac2 activation may be
rate limiting for this early burst of F-actin generation stimulated by
FMLP. We next examined FMLP-induced chemotaxis in
rac2+/+,
rac2+/-, and
rac2-/- neutrophils, finding a similar
marked dosage effect for the rac2 gene (Fig. 2B).
Finally, we found that superoxide production elicited by FMLP (Fig. 2C) or PMA (Fig. 2D) in
rac2+/- neutrophils was also
intermediate between wild-type levels and the substantial defects in
rac2-/- neutrophils. Linear regression
analysis showed a highly significant correlation of F-actin content,
chemotaxis, and superoxide production with the number of functional
rac2 genes (p < 0.003). We also
observed a similar rac2 gene dosage effect for
FMLP-stimulated chemotaxis (r = 0.9, p
= 0.001) and superoxide production (r = 0.76,
p = 0.017) in the 129SV strain, although there were
slight differences in the absolute magnitude of the responses compared
with the C57BL/6J strain (data not shown). Taken together, these
results suggest that the cellular level of Rac2 can be rate limiting
for F-actin formation, chemotaxis, and NADPH oxidase
activation induced by FMLP in murine neutrophils.
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Using the p21-binding domain of PAK kinase that contains the
Cdc42/Rac interactive binding (CRIB) motif and interacts specifically
with GTP-bound Rac and Cdc42, several groups have established that FMLP
and PMA induce formation of Rac2-GTP in human neutrophils
(36, 37, 38). We used this affinity precipitation or pull-down
assay to examine the relative levels of Rac1-GTP and Rac2-GTP in DMSO-
and FMLP-stimulated wild-type murine neutrophils, in which total
cellular levels of Rac1 and Rac2 are similar (Fig. 1
). As previously
observed in freshly isolated human peripheral blood neutrophils
(37, 38), a small amount of activated Rac2, and to a
lesser extent Rac1, was detected in DMSO-treated murine neutrophils
(Fig. 3), which varied from experiment to
experiment and probably reflects a basal level of activation in resting
neutrophils or induced by the purification procedure. Stimulation with
FMLP induced the formation of both Rac1- and Rac2-GTP within 10 s
of stimulation, with maximal levels detected at 30–60 s of
stimulation, which began to decline by 2 min (Fig. 3A) and
approached basal levels by 5 min (data not shown), similar to what has
been described for Rac2 activation in FMLP-stimulated human neutrophils
(37, 38). At 60 s after FMLP stimulation, 4.5 ±
1.7% of total cellular Rac2 was recovered as Rac2-GTP (Fig. 3B), in the same range that has been reported for Rac2-GTP
levels in stimulated human neutrophils (37, 38). At all
time points after stimulation, the amount of Rac2-GTP detected in
FMLP-activated murine neutrophils was significantly higher than the
amount of Rac1-GTP, which peaked at 1.1 ± 0.4% of total Rac1 at
60 s (Fig. 3B). Because similar amounts of the Rac1 and
Rac2 isoforms are present in wild-type murine neutrophils (Fig. 1), we
therefore infer that the Rac2-GTP levels are 4-fold higher than
Rac1-GTP after FMLP stimulation. Neither Rac1 nor Rac2 was detected in
the detergent-insoluble pellet isolated after the initial
solubilization of DMSO- or FMLP-stimulated neutrophils in the affinity
precipitation assay (data not shown).
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C.
Although FMLP elicited an increase in Rac2-GTP in
rac2+/- neutrophils, the absolute
amount of Rac2-GTP was 2-fold lower than that detected in
FMLP-stimulated wild-type neutrophils (Fig. 3, C and
D). Thus, a compensatory increase in Rac2 activation does
not occur in the face of the 2-fold reduction in cellular Rac2
levels in rac2+/- neutrophils. Rac1
activation in rac2+/- neutrophils
was similar to that in wild-type neutrophils.
Finally, we used the PBD-GST precipitation assay to compare the
activation of Rac1 and Rac2 in wild-type and
rac2-/- murine BM neutrophils that were
stimulated with either 2 µM FMLP or 200 ng/ml PMA. Each agonist
induced Rac1 activation in rac2-/-
neutrophils with kinetics that was similar to that of wild-type
neutrophils. Compared with FMLP, the PMA-induced increases in Rac1-GTP
were never as high as those detected after FMLP stimulation in either
wild-type or rac2-/- neutrophils, and
very little Rac activation was detected before 1 min (not shown),
although elevated Rac1-GTP levels persisted for at least 5 min. In
wild-type neutrophils, the kinetics and relative magnitude of Rac2
activation after FMLP or PMA stimulation paralleled that observed for
Rac1 (Fig. 4, A and
B).
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, A and
B). Peak Rac1-GTP levels were 3-fold higher (range, 2- to
5-fold) in FMLP-stimulated rac2-/- cells
than in wild-type cells (Fig. 4C). The basal level of
Rac1-GTP in DMSO-treated polymorphonuclear neutrophils (PMNs) was also
consistently higher in rac2-/- than in
wild-type PMNs (Fig. 4C). FMLP-induced activation of Rac1 and Rac2 in murine neutrophils is sensitive to inhibitors of phosphatidylinositol 3-kinase (PI3K) and Src family tyrosine kinases
The neutrophil FMLP receptor is coupled to heterotrimeric
G
i
GTP-binding proteins and transduces
signals via multiple pathways, including the src-related
kinases, Lyn and Hck, and PI3K (6, 39, 40, 41). In
chemoattractant-stimulated human neutrophils, Rac2 activation has been
reported to be sensitive to either the tyrosine kinase inhibitor
genistein or PI3K inhibitors wortmannin or LY294002 (37, 38). Consistent with these observations, we found that the
increase in activated Rac above basal levels in FMLP-stimulated
wild-type murine neutrophils was substantially inhibited by LY294002 or
the src family tyrosine kinase inhibitor PP1 (Fig. 5A). The activation of Rac1 in
both wild-type and rac2-/- neutrophils
appeared to be more sensitive to either LY294002 or PP1 than did Rac2
activation (p < 0.03), with Rac1-GTP levels
decreasing in inhibitor-treated FMLP-stimulated cells to even below the
basal levels detected in DMSO-only treated cells (Fig. 5A).
In two additional experiments, similar results were obtained for
inhibition of FMLP-induced Rac1 and Rac2 activation using either
wortmannin (100 nM), another PI3K inhibitor, or the tyrosine kinase
inhibitor, genistein (100 µM) (data not shown). Inhibition of Rac1
and Rac2 activation in wild-type neutrophils by PP1 was dose dependent
(Fig. 5B). PP1 (10 µM) inhibited 50% of the
FMLP-induced increase in Rac2-GTP over basal levels, which increased to
90% inhibition of the FMLP response with 50 µM PP1. Rac1
activation in wild-type neutrophils was relatively more sensitive to
PP1 at all doses tested (Fig. 5B), which was statistically significant
at 50 µM PP1 (p < 0.001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). We further
demonstrate that stimulation of wild-type murine neutrophils with
either FMLP or PMA results in activation of both Rac isoforms but found
that substantially more Rac2-GTP was detected than Rac1-GTP (Figs. 3 and 4). This is the first evidence to indicate that these highly
similar GTPases are differentially activated by agonist-induced
signals, which suggests one mechanism that contributes to a
nonoverlapping role for Rac1 and Rac2 in coupling chemoattractant
receptor signals to downstream functions.
Analysis of FMLP-stimulated Rac2-deficient neutrophils indicates that
more efficient activation of downstream pathways by Rac2 accounts for a
second mechanism leading to a preferred role for Rac2 in regulating
chemoattractant-induced responses. The severity of defects in
FMLP-elicited F-actin generation, chemotaxis, and superoxide production
was strongly correlated with the level of Rac2 expression and number of
functional rac2 genes (Fig. 2), despite the presence of
Rac1. In rac2+/- neutrophils, total
activated Rac2 levels after FMLP stimulation were 2-fold less than
in wild-type neutrophils (Fig. 3), consistent with reduced total levels
of Rac2 protein in the rac2 heterozygotes and suggesting
that Rac2 activation is rate limiting for chemoattractant-induced
responses. There was not always a 1:1 relationship between the
magnitude of each response and Rac2 levels, but this likely reflects
the complexity of the signaling cascades leading to these functional
responses. Because <5% of Rac2 is activated in stimulated
rac2+/+ neutrophils, one might also
anticipate that the 50% reduction in Rac2 protein level in
rac2+/- cells would still be adequate to
generate a full response. However, subcellular compartmentalization or
the efficient activity of Rac GTPase-activating proteins may contribute
to the observed gene dosage effect. In
rac2-/- neutrophils, higher levels of
activated Rac1 were detected after FMLP stimulation relative to
wild-type or rac2+/- neutrophils (Figs. 3 and 4). Comparing the relative amounts of Rac1-GTP and Rac2-GTP after
FMLP stimulation, we estimate that the total cellular level of
activated Rac in wild-type neutrophils still exceeds that in
rac2-/- neutrophils by up to 2-fold,
despite the increased amount of Rac1-GTP in
rac2-/- neutrophils. Hence, it is
possible that an overall decrease in total cellular levels of activated
Rac in rac2-/- neutrophils contributes to
the observed impairment in FMLP-elicited chemotaxis and superoxide
production. However, the functional defects in
rac2-/- neutrophils are substantially
more severe compared with rac2+/- cells
(Fig. 2), indicating that activation of even a limited amount of Rac2
is more effective at stimulating downstream functions than
Rac1-GTP. By inference, this suggests that Rac2-GTP plays a preferred
role compared with Rac1-GTP in signaling to downstream effectors that
regulate actin polymerization, chemotaxis, and superoxide
production.
The mechanism(s) which underlies the apparent differential
activation of Rac1 and Rac2 in FMLP-stimulated neutrophils is unknown
but likely reflects different affinities for proteins that regulate
overall level of Rac-GTP (GEFs or GTPase-activating proteins), or
subtle differences in membrane binding or subcellular location that
influence accessibility to these regulatory proteins. Similar
mechanisms may account for a preferential role for Rac2-GTP in
activating downstream responses. Of interest in this latter regard are
the findings of Heyworth et al. (42), who compared
the ability of purified recombinant prenylated preparations of Rac1 or
Rac2 to stimulate NADPH oxidase activity under cell-free conditions in
the presence of GTP-S and neutrophil membranes. Both isoforms were
equivalent when using recombinant p47phox and
p67phox. However, Rac2 was substantially more
active in the presence of otherwise limiting amounts of neutrophil
cytosol (42), suggesting that the cytosol contains a
factor(s) that either suppresses Rac1 activity or promotes Rac2
function.
Although Rac1 and Rac2 isoforms have 92% sequence identity, there are
a number of candidate regions that could permit discrimination between
the two isoforms. These regions lie outside of the N-terminal switch I
and II regions that change conformation on guanine nucleotide exchange,
which are identical in Rac1 and Rac2 except for a Gly vs Ser residue at
position 49, located between the 2 and 3 strands linking switch I
and II. These domains, particularly switch I, constitute a major
binding interface with target effector proteins and with Rac GEFs
(5, 43, 44). The greatest divergence between Rac1 and Rac2
is in the polybasic region located just upstream of the C-terminal CAAX
box, where Rac1 has six adjacent basic amino acids compared with Rac2,
in which three of these residues are uncharged. The four mammalian Ras
proteins also differ primarily in this region, termed the
"hypervariable" domain, and isoform specificities of Ras and Rac
may at least in part be related to differences in localization to
subcellular membrane compartments or microdomains known to be conferred
by this domain (45, 46). In the case of Ras, the
hypervariable domain has been linked to differential activation by the
Ras exchange factors Ras GRF1 and Ras GRP2 (47, 48), efficiency of activating PI3K or the Raf
serine-threonine kinase (49, 50, 51), and specific biologic
responses (52, 53, 54, 55, 56). The Rac polybasic region may also
modulate interactions with downstream protein targets independent from
effects on membrane localization, as has been shown for Pak1
(57). Regions outside of the hypervariable domain in Ras
also strongly influence the oncogenic potency of H-Ras
(54) and in an analogous manner may contribute to
isoform-specific Rac functions. There are three amino acid differences
between Rac1 and Rac2 clustered in residues 145–151, a domain shown to
be important for transducing signals through PAK (58).
Rac1 and Rac2 also have a single amino acid difference in the insert
domain (residues 116–136) which has been implicated in regulation of
electron transport in the NADPH oxidase and in mitogenesis (15, 59, 60, 61).
As noted above, Rac1 activation is not defective in rac2-/- neutrophils; in fact, both basal and agonist-stimulated Rac1-GTP levels were elevated in rac2-/- neutrophils compared with wild-type neutrophils. Yang et al. (25) observed similar increases in basal levels of Rac-GTP in early hemopoietic stem/progenitor cells isolated from rac2-/- animals, in addition to elevated Cdc42 in both unstimulated cells and cells activated with the stromal cell-derived factor-1 chemokine, which exhibited increased filopodia and F-actin formation. However, Rac2-deficient neutrophils stimulated with FMLP or other chemoattractants have decreased F-actin formation, and we have not detected elevated levels of Cdc42 in either resting or stimulated PMNs in pull-down experiments (not shown). These differences illustrate some of the variable effects of Rac2 deficiency observed among different hemopoietic lineages (24, 25, 26, 27).
Additional studies using pharmacologic agents suggest that Src kinases and, in either a sequential or parallel pathway, PI3Ks contribute to the regulation of FMLP-induced activation of Rac1 and Rac2 in murine neutrophils. Rac1 activation appeared to be more sensitive to these inhibitors, consistent with the notion that the two isoforms may be differentially activated. Our results confirm and extend previous studies in human neutrophils, where FMLP-stimulated Rac2 activation was reported to be inhibited by PI3K and tyrosine kinase inhibitors (37, 38), and FMLP-induced Rac2 translocation was prevented by tyrosine kinase inhibitors (62).
The mechanism(s) by which Rac-GDP is exchanged for GTP in response to
FMLP is likely to involve the action of one or more GEFs that become
activated downstream of the FMLP receptor. GEFs contain pleckstrin
homology domains believed to be regulated by PI3K-generated
phosphatidylinositol 1,4,5-trisphosphate (63, 64), and the
Vav family of GEFs is additionally regulated by tyrosine
phosphorylation (2, 65). Hence, inhibition of
FMLP-stimulated GEF activation by PI3K and tyrosine kinase inhibitors
might account for the observed effect of these compounds on
FMLP-stimulated Rac activation. One newly discovered exchange factor
that is likely to play an important role in Rac activation
downstream of G protein-coupled chemoattractant receptors in
neutrophils is P-Rex1 (for PIP3-dependent Rac exchanger), which is
directly activated by G subunits and by PI3K, in both an
independent and synergistic manner (66). Stimulation of
guanine nucleotide exchange on Rac by G-activated P-Rex1 at early
time points after chemoattractant stimulation may explain why
PI3K-null mice and PI3K inhibitor-treated human neutrophils have
normal Rac activation at 5–10 s after FMLP (36, 67), in
contrast to the sensitivity to PI3K inhibitors at later times (60 s)
that we and others have observed for murine and human neutrophils
(37, 38).
These studies provide new insights into the basis of the phenotypic defects in Rac2-deficient murine neutrophils and provide the first direct evidence that Rac1 and Rac2 have distinct activation and signaling profiles in agonist-activated cells, despite their high degree of sequence homology. Two different mechanisms were identified that may account for the lack of functional redundancy between these two Rac isoforms. Although murine neutrophils have similar amounts of Rac1 and Rac2, Rac2 appears to be preferentially activated through the FMLP receptor. The relative level of activated Rac2, in turn, was rate limiting for coupling FMLP-induced signals to F-actin formation, chemotaxis, and superoxide production, which could not be compensated for by enhanced Rac1 activation in rac2-/- neutrophils.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mary C. Dinauer, Wells Center for Pediatric Research, 1044 West Walnut Street, R4, Room 402A, Indianapolis, IN 46202-5225. E-mail address: mdinauer{at}iupui.edu
3 Abbreviations used in this paper: GEFs, guanine-nucleotide exchange factors; BM, bone marrow; PBD, p21-binding domain; CRIB motif, Cdc42/Rac interactive binding motif; PMNs, polymorphonuclear neutrophils; PI3K, phosphatidylinositol 3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine.
Received for publication June 13, 2002. Accepted for publication August 19, 2002.
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