Reviews
This Review is part of a thematic series on The Role of Small GTPases in Cardiovascular Biology, which includes
the following articles:
Rho GTPases, Statins and NO: The Role of Small GTPases in Endothelial Cytoskeletal Dynamics and the Sheer Stress
Response
Rho Kinases in Cardiovascular Physiology and Pathophysiology
Regulation of NADPH Oxidases: The Role of Rac Proteins
Rho GTPases and Signaling by Endothelial Receptors
The Rac and Rho Hall of Fame: A Decade of Hypertrophic Signaling Hits
Anne Ridley, Guest Editor
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Regulation of NADPH Oxidases
The Role of Rac Proteins
Peter L. Hordijk
Abstract—The role for reactive oxygen species (ROS) in cellular (patho)physiology, in particular in signal transduction,
is increasingly recognized. The family of NADPH oxidases (NOXes) plays an important role in the production of ROS
in response to receptor agonists such as growth factors or inflammatory cytokines that signal through the Rho-like small
GTPases Rac1 or Rac2. The phagocyte oxidase (gp91phox/NOX2) is the best characterized family member, and its mode
of activation is relatively well understood. Recent work has uncovered novel and increasingly complex modes of control
of the NOX2-related proteins. Some of these, including NOX2, have been implicated in various aspects of
(cardio)vascular disease, including vascular smooth muscle and endothelial cell hypertrophy and proliferation,
inflammation, and atherosclerosis. This review focuses on the role of the Rac1 and Rac2 GTPases in the activation of
the various NOX family members. (Circ Res. 2006;98:453-462.)
Key Words: NADPH oxidase 䡲 Rac1 䡲 reactive oxygen species
T
generate (intra)cellular ROS, including xanthine oxidase,
cyclo-oxygenases, NO synthases, mitochondrial oxidases,
and the NADPH oxidases (NOXes). Biochemically, ROS
act by oxidative modification of nucleic acids, sugars,
lipids, and proteins. This results in, for example, DNA
damage but also in regulated signaling through the inactivation of specific enzymes such as protein tyrosine phosphatases,3 which leads to increased tyrosine kinase activity. To preserve the cellular redox balance, control cell
signaling, and to prevent potential damage, ROS are
removed or neutralized by, for example, glutathione and
vitamins as well as by enzymes such as superoxide
dismutases, catalase, and peroxidases.
he cellular responses to external stimuli involve the
activation of multiple signaling pathways through processes such as receptor dimerization, protein phosphorylation
and the generation of intracellular messenger molecules. The
classical “second messengers” cAMP and intracellular Ca2⫹
have turned out to be among the first of an ever-growing list
of intracellular signaling intermediates that includes protons,
proteins, (phospho)lipids, and reactive oxygen species (ROS).
ROS, a collection of oxygen-derived molecules such as
oxygen radicals, hydrogen peroxide, peroxynitrite, and NO,
are rapidly diffusing, short-lived messenger molecules, of
which their role in signal transduction has only recently been
fully recognized.1,2 There are various enzymes that can
Original received August 29, 2005; resubmission received November 16, 2005; accepted January 12, 2006.
From the Department Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam,
The Netherlands.
Correspondence to Peter L. Hordijk, Department Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center,
University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000204727.46710.5e
453
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Figure 1. Schematic summary of the available information on
the control of NOX1 to NOX5 activity. Superoxide/ROS are produced in cells after stimulation by pathogens, receptor agonists,
and shear stress. NOXes1 through 4 but not NOX5 associate
with p22phox. NOX2 (gp91phox) is simulated by phosphorylationactivated p47phox and by p67phox, in conjunction with activated
Rac. The hematopoietic Rac GEFs Vav1 and P-Rex1 can activate NOX2. NOX1 is activated by the NOXO1 and NOXA1 proteins; NOXO1 is phosphorylation independent, but NOXA1 can
associate with activated Rac1. The Rac GEF ␤-PIX binds and
activates NOX1, although a role for NOXA1 remains to be demonstrated. NOX3 is activated by NOXO1 but is insensitive to
additional stimulation by NOXA1 or activated Rac. Regulation of
NOX4 activity is controversial. There are no data on regulatory
proteins for NOX4, as represented by the black box. NOX5 is
activated by Ca2⫹ and does not require the regulatory subunits.
Dashed lines indicate additional or potential ways of cross-talk
or activation.
It has recently been shown that most, if not all, types of cell
generate intracellular ROS and that the well-established
phagocyte oxidase is in fact member of a family of various
NOXes that show widespread, differential tissue expression.
The generation of ROS through the activation of the phagocyte NOX (gp91phox/NOX2) is an extensively studied and
well-coordinated process in which various means of regulation, such as phosphorylation, GTPase activation, and protein–protein interactions, act in sequence.4 – 6 The contribution
of Rac proteins to the activation of the phagocyte oxidase has
been studied extensively and has recently seen important
progress in the analysis in mouse models (see below). Many
studies have shown Rac-mediated production of ROS in
nonphagocytic cells, and there are a few recent studies that
indeed link Rac signaling to the activation of the “new”
NOXes (Figure 1).
Rac-Mediated Production of ROS
High levels of ROS are generated in neutrophils upon cell
stimulation and phagocytosis, and these ROS aid in the
killing of ingested pathogens. In most other types of cells,
low levels of ROS are implicated in growth, differentiation,
migration, and angiogenesis, as well as in inflammation,
vascular hypertrophy, and atherosclerosis (see below).7–12
The stimuli that induce the production of intracellular ROS
include growth factors, inflammatory stimuli, integrins and
their ligands, and shear stress.9,13–17 Most of these intracellu-
lar ROS are supposedly derived from NOXes, based on
sensitivity of the responses to the flavoprotein inhibitor
diphenylene iodonium, to a NOX2-inhibitory peptide,18 to
downregulation by antisense oligos19 or through the use of
gene-deficient mice.20 –22
The direct link between Rac activity and ROS production
in nonphagocytic cells was shown for the first time in 1996.13
These authors showed that expression of an activated mutant
of Rac1, V12Rac1, resulted in increased production of ROS
in fibroblasts. This response was mimicked by V12Ras, and
it was suggested that Rac acts downstream of Ras in initiating
ROS production induced by EGF or by platelet-derived
growth factor (PDGF), as well as by cytokines such as tumor
necrosis factor-␣ (TNF-␣) or interleukin-1␤ (IL-1␤).13 Racmediated production of ROS was also found in HeLa cells
and implicated in IL-1␤–mediated activation of nuclear factor
␬B (NF-␬B),9 further strengthening the idea that Racmediated production of intracellular ROS is instrumental in
signal transduction, downstream of (cytokine) receptors.
Several studies have subsequently implicated Racmediated production of ROS in a variety of cellular responses, in particular in endothelial cells. In bovine aortic
endothelial cells, shear stress can activate protein tyrosine
phosphorylation and extracellular-signal regulated kinase activation in a Rac- and ROS-dependent manner.17 These
findings were based on expression of N17Rac1 and the use of
antioxidants without demonstration of direct stimulation of
either Rac1 or ROS production, albeit that Rac1 activation
and ROS synthesis by shear in endothelial cells has been
observed by others as well.23,24 In addition, depolarization of
endothelial cells (seen in hypertensive vessels and implicated
in platelet aggregation) results in ROS production in a
phosphatidylinositol 3-kinase (PI3K) and Rac-dependent
manner.25 ROS also negatively regulate endothelial cell– cell
adhesion, which is controlled by junctional molecules such as
vascular endothelial (VE)-cadherin.26 VE-cadherin associates
to ␤- and ␥-catenin, which link the transmembrane molecules
to the actin cytoskeleton. These complexes are subject to
regulation by (ROS-induced) tyrosine phosphorylation. In
line with this, endothelial ROS production is stimulated by
permeability-increasing agonists such as TNF-␣. In addition,
the immunoglobulin-like cell adhesion molecule vascular cell
adhesion molecule-1 (VCAM-1) in endothelial cells initiates
ROS production that mediate Rac1-induced permeability as
well as leukocyte transendothelial migration.16,19,27 Various
mechanisms of action of the VCAM-1–Rac-induced ROS
have been proposed: Deem and Cook-Mills suggested that
endothelial NOX2 releases these ROS extracellularly and
stimulate matrix metalloprotease activity,19 whereas our laboratory provided evidence for a role of the redox-sensitive
kinase Pyk2 in ROS-mediated loss of cell– cell contact.28 In
conclusion, there is ample evidence for Rac-dependent production of ROS by NOXes in cellular signaling, in particular
in vascular cells.
The Role of Rac in the Activation of NOXes
NOX2
The NOX of human neutrophils (NOX2) was identified ⬇20
years ago,29,30 and the control of its activation is intensely
Hordijk
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studied.4 – 6,31 This NOX comprises the flavocytochrome b558,
which is a heme-binding heterodimer composed of a large
(gp91phox) and a small (p22phox) subunit. The gp91phox subunit
has 6 transmembrane regions in its N terminus, whereas its
C-terminal portion contains the flavin-adenine dinucleotide
(FAD) and NADPH binding domains that are essential for
activity.32–34 Oxidase activation is controlled by the recruitment of regulatory proteins to the flavocytochrome, including
the p40phox, p47phox and p67phox, of which p47phox and p67phox are
essential for activity (Figure 1).35,36
A human X-linked genetic disorder, chronic granulomatous disease (CGD) has been invaluable in elucidating the
relative contribution of these regulatory proteins and their
specific domains involved in the activation of the phagocyte
oxidase. CGD is characterized by severely impaired phagocyte function attributable to a defect in the production of
superoxide by phagocytes. CGD results from each of ⬎400
different mutations that have been identified in members of
the oxidase complex, including gp91phox, p22phox, p67phox,
p47phox, or Rac2.37– 40 This leads to a lack of proper complex
assembly or to an inactive gp91phox subunit.
Assembly of the neutrophil–NOX complex is initiated on
phosphorylation of p47phox, releasing it from its autoinhibitory
conformation.36,41,42 The p47phox N-terminal SH3 domain then
binds the proline-rich region in p22phox42,43 and the PX domain
of p47phox binds 3⬘phosphoinositides, the products of
PI3K.44,45 Activation of the small GTPase Rac2 is associated
with recruitment of p67phox, which associates with p47phox and
with the cytochrome.46 – 48 The interaction of activated Rac2
with the neutrophil oxidase complex is mediated by the N
terminus of the p67phox subunit.49 Intriguingly, Rac-mediated
translocation of p67phox to the cytochrome does not occur in
p47phox-deficient cells,36 indicating Rac2 activation is not the
sole event that drives p67phox translocation. Finally, direct
binding of Rac2 to the flavocytochrome has been implicated
in the initial steps of the electron transfer reaction: a Rac2
function, dependent on the insert domain (see below).50
NOX1
The NOX1 protein, initially identified by homology-based
polymerase chain reaction as mitogenic oxidase 1,51 is predominantly expressed in colon epithelium as well as in
vascular smooth muscle cells (VSMCs).52,53 NOX1 is efficiently activated by homologues of p47phox and p67phox, the
NOXO1 (NOX-organizer 1) and NOXA1 proteins (NOX-activator 1), respectively (Figure 1).54,55 The NOXO1 protein
does not contain an autoinhibitory region, which, in p47phox, is
involved in the phosphorylation-induced activation and subsequent binding to the flavocytochrome b558. As a result,
NOXO1 is considered “constitutively active,” and significant
activation of NOX1 can already be observed in coexpression
experiments in the absence of PMA.54,55 NOXA1 and p67phox
both have four so-called tetratricopeptide repeats in their N
termini. These repeats mediate the binding to activated
Rac249 and are required for NOX2 activation. In NOXA1, a
R102E mutation that in p67phox impairs Rac2 binding, also
blocks its interaction with activated Rac1 and Rac2.55 GDPbound Rac1 or Rac2 does not bind NOXA1, nor does active
or inactive Cdc42. These data suggest that Rac proteins
Rac-Mediated Regulation of NADPH Oxidases
455
activate NOX1 through NOXA1. However, whereas NOX2
requires additional activated Rac1 in cotransfection studies in
HEK293 cells,56 NOX1, in the presence of the NOXO1 and
NOXA1 proteins, apparently does not.55 In contrast, in gastric
mucosal cells, lipopolysaccharide-induced superoxide production was blocked by the PI3K inhibitor LY 294002,
which, in turn, was bypassed by activated Rac1, suggesting
that Rac1 activates NOX1 in these cells.57
More direct evidence for a role for the PI3K–Rac pathway
in NOX1 activation was provided by Park et al.58 This study
showed that the Rac– guanine nucleotide exchange factor
(GEF) ␤-PIX is required for and even potentiates EGFinduced ROS production in Caco-2 and HEK293T cells. Park
et al showed that ␤-PIX or activated Rac1 binds the
C-terminal domain of NOX1, and that, in particular, the
membrane proximal portion acts as a dominant negative in
that it blocks EGF-induced ROS production. The relevance
for NOX1 was further demonstrated by depletion of NOX1 in
Caco-2 cells, which prevented ROS production by EGF.
Although NOXO1 and NOXA1 proteins further potentiated
EGF-induced ROS in a ␤-PIX dependent fashion, it was not
clarified whether for NOX1 activation, Rac1 interaction with
NOXA1 is required or whether direct Rac1 binding to NOX1
is sufficient.58
NOX3
For NOX3, the situation is quite different. NOX3 is expressed
in the vestibular and cochlear epithelia of the inner ear and is
involved in the morphogenesis of otoconia and thus in control
of the sense of balance,59,60 and it is not clear which, if any,
regulatory subunits are coexpressed with NOX3. Studies on
its regulation have so far relied on cotransfection experiments, mainly in HEK293, COS, or Chinese hamster ovary
(CHO) cells. For CHO cells, which lack p22phox, coexpression
of p22phox is required to induce a basal level of NOX3
activity.61 Different from NOX1, NOX3 is already activated
after coexpression with the NOXO1 protein, whereas coexpression of just NOXA1 does not activate NOX3 (Figure 2).56
In contrast, p67phox by itself does activate NOX3, and this is
further potentiated by p47phox. Ueno et al showed that p67phox
R102E, which does not bind Rac proteins, also stimulated
NOX3.61 Although the role of p67phox or NOXA1 in NOX3
activation remains unclarified, activation of NOX3 apparently is Rac independent. In line with this notion, expression
of N17Rac1 had little effect on NOX3 activity in CHO
cells.61
NOX4
NOX4 was originally identified in the kidney and was named
Renox. Expression of NOX4 by itself already results in ROS
production in NIH3T3 cells, suggesting that NOX4 might be
constitutively active.62 Later studies have shown that NOX4,
similar to NOX1, NOX2, and NOX3, at least requires p22phox
for its activity.63,64 Stable expression of NOX4 in HEK293
tk;2cells, which have endogenous p22phox, results in significant ROS production, up to 100-fold over basal levels. NOX4
was not further activated by p47phox/p67phox or the NOXO1/
NOXA1 proteins. Using the Cos–PHOX system,65 it was
shown that whereas NOX2 in that system is inactivated after
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a Rac1 knockdown, activation of NOX4 is undisturbed,
supporting the notion that, like NOX3, NOX4 is Rac independent.64 Also, expression of Rac1N17, Rac1V12, or of a
region of p21-activated kinase (PAK) that binds activated
Rac1, did not affect NOX4 activity in the COS cell system.
Thus, the lack of effect of coexpressed Rac1, p67phox, or
NOXA1 indeed suggests that NOX4 is Rac1 independent.
In contrast to studies that claim NOX4 to be constitutively
active, some recent reports suggest that NOX4 is, in fact,
regulated. In mesangial cells, which express high levels of
endogenous NOX4, antisense oligos to NOX4 reduced intracellular ROS production by arachidonic acid (AA),66 which is
produced upon angiotensin II (Ang II) stimulation and leads
to Rac1 activation. Similarly, Ang II/AA-induced and
L61Rac1-induced activation of Akt was blocked by reduction
of NOX4 expression. Although this study did not show direct
activation of NOX4 by Rac1, it suggests that in mesangial
cells, NOX4 activation is part of a Rac1-dependent signaling
pathway. Along similar lines, Mahadev et al showed in
3T3L1 adipocytes, which also express high levels of NOX4,
that insulin induces a 10-fold increase in intracellular ROS
production, which was further increased by expression of
NOX4.67 Importantly, NOX4 mutants that were mutated in
their FAD- or NADPH-binding regions blocked insulininduced ROS production for 80%, suggesting that these
mutants act as dominant negatives, something one would not
expect for an unregulated protein. Competition for p22phox
does not explain this effect because expression of NOX4,
which is p22phox dependent, increased ROS production, suggesting that p22phox was not limiting.
Clearly, the mechanism of NOX4 regulation is not solved
at this point. It is important to note that in all their experiments, Mahadev et al never found elevated NOX4-mediated
ROS production in the absence of insulin, arguing against a
constitutively active NOX4.67 Also, the studies by Gorin et
al66 and Mahadev et al67 were based on cells that express
endogenous NOX4, whereas the work by Geiszt et al62 and
Martyn et al64 was based on overexpression of NOX4 in 3T3
or HEK293 cells. Whether these differences in experimental
setup may relate to the findings on Rac-mediated regulation
of NOX4 activity remains to be seen. In addition, several
groups have noted clear cell type–specific differences in the
mechanisms of NOX activation. Future studies on endogenously expressed NOXes, in combination with knockdown
and genetic approaches,20,60 likely will provide the most
informative results.
NOX5
For NOX5, little information is available. NOX5 is expressed
in lymphoid cells and the testis68 as well as in prostate cancer
cells.69 NOX5 contains four EF-hand domains in its N
terminus, is FAD and NADPH dependent but p22 phox independent,70 and is activated by calcium (Figure 1).71 The N
terminus of NOX5 binds its C terminus, and the binding of
calcium disrupts this intramolecular interaction, resulting in
activation of the oxidase. There are currently no data on the
role of the various regulatory proteins or Rac in the activation
of NOX5.
Localization of NOX Proteins
In neutrophils, NOX2 resides in intracellular vesicles in
resting cells. NOX2 can be recruited to the plasma membrane
after cell stimulation and is found in the membrane of
phagosomes after phagocytosis. The topology of NOX2 with
six transmembrane domains predicts that the superoxide is
released either into the extracellular milieu or into the
phagosome. Based on sequence homology, this organization
is likely similar for the other NOXes.72 In VSMCs and
endothelial cells, NOX proteins have been detected at the
plasma membrane and in the nucleus, the endoplasmic
reticulum (ER), caveolae, and focal adhesions.73,74 NOX
localization presently is a controversial issue because
whereas Ambasta et al63 could not detect NOX1 in the plasma
membrane of transfected HEK293 cells, Cheng and Lambeth75 found GFP-tagged as well as untagged versions of
NOX1 and its regulator NOXO1 in the plasma membrane.
NOX2 has been claimed in endothelial cells to reside in the
plasma membrane, leading to extracellular release of ROS
after cell stimulation.19 Others have detected NOX2 and its
regulatory proteins as a preassembled complex in an intracellular, perinuclear compartment in resting endothelial
cells.76 Thus, it may well be that, comparable to the situation
in neutrophils, NOX proteins in nonhematopoietic cells reside
in ER-associated vesicles that can fuse with the plasma
membrane upon stimulation. This would also correlate with
the membrane recruitment of activated Rac proteins.
Structural Requirements for Rac-Mediated
Production of ROS
Several isoforms of Rac proteins exist: Rac1, Rac1b, Rac2,
and Rac3. For the studies on oxidase activation, most information has been obtained on the Rac1 and Rac2 proteins.
Rac1b is a Rac1 splice variant that is constitutively active77
and that has recently been linked to mitochondria-derived
production of ROS in a model for epithelial–mesenchymal
transition.78 Rac2 is expressed in hematopoietic cells only and
is the most relevant Rac GTPase for activation of NOX2 in
human neutrophils. In contrast, Rac1 is ubiquitously expressed and is likely the main Rac GTPase for NOX activation in nonhematopoietic cells. Expression studies have
shown that Rac1 can also activate NOX2 in heterologous
systems. However, Rac1 and Rac2 are not completely redundant. Various studies have shown that the intracellular targeting of Rac1 and Rac2 proteins, mediated by their hypervariable C termini, is not identical and that this, in part,
determines differential effects on cell functions such as
superoxide production and chemotaxis (discussed in more
detail below).
The structure–function analysis of Rac2 in NOX2 activation has initially been studied with the semirecombinant
cell-free system.79,80 In this assay, recombinant Rac proteins,
or mutants thereof, are added, together with recombinant
p47phox or p67phox, to isolated neutrophil membranes that
contain the NOX2 and p22phox subunits. An anionic amphiphile is also added, which induces a conformational change in
p47phox, to initiate activation of the oxidase. Superoxide
production can subsequently be detected by a variety of
methods. Later studies have used a reconstitution model in
Hordijk
Rac-Mediated Regulation of NADPH Oxidases
457
Figure 2. Overview of regions and residues in Rac that were found to be involved in NOX2 activation. Rac is represented by the black
bar, and the switch I and switch II insert region and the C-terminal domain are indicated in white. Individual residues and domains that
are required for activation of the NADPH oxidase are indicated in the top of the figure. The unique D57 mutation (#) was identified in a
CGD patient. The bottom part of the figure indicates the amino acid differences between the Rac2 and Rac1 proteins. The hypervariable C termini are shown for comparison, with the divergent residues indicated by an asterisk. The curved line indicates the lipid
anchor attached to the C-terminal cysteine residue.
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COS cells65,81,82 or used elegant mouse models.83,84 Although
Rac proteins are relatively small, 21-kDa proteins, a large
series of residues and regions throughout the GTPase, have
been implicated in the activation of NOX2 (Figure 2). Rac
proteins, like other small GTPases, contain an effector region
in the N terminus (approximately residues 20 to 40), which
mediates interactions with effector proteins including
p67phox.85– 87 This part of the protein overlaps with the socalled switch I region (Figure 2; amino acids 32 to 40) that
undergoes a strong conformational change on Rac activation.
For stimulation of the oxidase, Ala 27, Phe28, Gly30, Thr35,
Val36, Asp38, and Tyr40 in Rac have been implicated after
mutational analysis by various groups.81,87–91 Interestingly,
there is a marked difference in the V12Rac2 or the L61Rac2
mutants, in that the D38 mutation blocks activity in the V12
background but has little effect in the L61 version.88 This may
relate to the fact that the L61Rac is more efficient in
activation of the oxidase81 (these authors used the Rac1
isoform) and that L61Rac2 has a much higher affinity for the
p67phox subunit compared with the V12Rac2 mutant,91 suggesting that the switch II region (amino acids 60 to 76) is
required for activation of the oxidase. In line with this,
Freeman et al86 showed that a Thr75Asn mutation also
impaired oxidase activation. However, more recently,
Carstanjen et al92 showed that Rac2 mutants at positions 36,
37, and 39 were all capable of in vitro binding to p67phox as
well as to the Rac effector PAK1 but were unable to rescue
the defect in oxidase activation in Rac2⫺/⫺ cells. These data
suggest that p67phox binding by Rac2 is not sufficient for
oxidase activation but that additional interactions are also
required (see below). A crucial requirement for Asp57 in
Rac2 was identified recently based on a mutation found in a
CGD patient.38,40 This Asp57Asn mutation (Figure 2) resulted in severely impaired fMLP- or IL-8 –induced neutrophil responsiveness, including adhesion, chemotaxis, and
superoxide production, whereas PMA responses were normal. Interestingly, the Asp57 mutant Rac2 did not bind GTP
and was found to act in a dominant-negative fashion for both
Rac1 and Rac2 because of its tight GEF binding.38,40
The insert region (residues 124 to 135) is unique for the
Rho-like GTPases and is also required for oxidase activation.86 This region is not required for Rac1 binding to p67phox,
implicating another protein, binding to this domain, in the
activation of the oxidase.85 This was proposed to be the oxidase
itself, perhaps analogous to Rac1 binding to the C-terminal
region of NOX1.58 This notion is further supported by studies
showing that Rac2 binds directly to the cytochrome, through
its insert region, before interacting with p67phox. The Rac2–
cytochrome complex was proposed to have increased affinity
for p67phox.50 Extensive mutational analysis of Rac and
peptide-walking experiments have implicated amino acids
103 to 107, 163 to 169, and 183 to 188 in Rac1-mediated
oxidase activation (Figure 2).93,94 Together with the switch I
and II regions and the insert region, these domains are likely
exposed on the outside of the GTPase.95 In particular,
mutation of His103 was found to result in a dramatically
reduced activation of the oxidase.93
Finally, there have been extensive studies on the
C-terminal domain of Rac proteins in the activation of the
oxidase. This domain is hypervariable and is the most
divergent region among the usually highly homologous Rholike GTPases (Figure 2). For example, 6 of the 15 amino acid
differences between Rac1 and Rac2 are in the C-terminal
region (Figure 2). The C terminus of Rho-like GTPases
mediates membrane association, in part through the lipid
anchor that is post-translationally added to the C-terminal
cysteine residue of the so-called CAAX box.96 However, for
oxidase activation by Rac1 in the cell-free assay, the lipid
anchor was found to be dispensable.97 The polybasic region in
the Rac1 C-terminus has also been implicated in membrane
association because of its net positive charge. However, these
C termini additionally mediate specific protein–protein interactions, relevant for proper intracellular targeting of Rho-like
GTPases.98,99
The isolated Rac1 C terminus was found to efficiently
interfere with oxidase activation in the cell-free assay.97
Deletion of the entire C terminus of Rac1 or Rac2 or mutation
of Lys183 or Lys186 blocks their capacity to activate the
oxidase.97 Using a variety of peptides, derived from Rac1,
RhoA, or RhoC, Joseph et al showed that the inhibitory effect
of the Rac1 C-terminal peptide was most likely because of its
basic nature.100 Although these data would suggest a nonspecific role for the C termini of Rac1 or Rac2, more recent
studies show that an additional function of the C-terminal
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Figure 3. Role of Rac proteins in cellular
physiology and cardiovascular disease.
Schematic drawing summarizing the
physiological effects of Rac activity,
resulting in adhesion and migration,
effects on growth and differentiation, as
well as bacterial killing. The relevance for
Rac-mediated production of ROS in various (cardio)vascular disorders is indicated in the gray box. See text for
details.
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domain is to ensure proper post-translation prenylation of the
Rac2 protein.101
Rac1 and Rac2 proteins are 92% homologous and obviously show great overlap in their biological effects. However,
mice that were made deficient in Rac2 show defects in
neutrophil function that could not be rescued by the remaining fraction of Rac1.20 These defects include chemoattractantstimulated actin polymerization, chemotaxis, and NOX activity.20,102 These defects were also not rescued by fMLPinduced Rac1 activation in these cells,103 and it was
concluded that the specific defects that were observed in
neutrophils from Rac2⫺/⫺ mice were the result of isoformspecific signaling, rather than of the reduction in overall Rac
protein levels.103 This is also in line with previous findings
that Rac2 binds ⬇6-fold better to p67phox compared with Rac1
and is more efficient in activating the oxidase.85,91,104 In
human neutrophils, Rac2 is the major isoform expressed and
thus the key GTPase for oxidase activation by chemoattractants and the Fc␥ receptor.103,105 But in human monocytes,
Rac1 is the predominant isoform, and in these cells, Rac1 was
shown to be the most important GTPase for oxidase activation.106 Clearly, expression levels, together with specificity of
signaling, control the relative importance of the Rac isoforms
in NOX2 activation.
Recent studies in mouse models have provided further
insight in the control of differential signaling by the Rac1 and
Rac2 GTPases. In Rac2⫺/⫺ progenitor cell-derived neutrophils, reintroduction of Rac2 but not of Rac1 restores the
fMLP-induced production of superoxide.84,105,107 Similarly,
the defect in fMLP-induced chemotaxis was restored by Rac2
but not by Rac1, in line with the notion that Rac1 in
neutrophils,84,102,107 like in macrophages,108 is not essential
for migration. In contrast, deletion of Rac1 in murine neutrophils impairs actin assembly and chemotaxis but leaves
PMA- or fMLP-stimulated oxidase activity normal.109 Using
chimeric Rac proteins, Filippi et al showed that the Rac2 C
terminus in the background of Rac1 is sufficient for restoration of superoxide production but not of fMLP-induced
chemotaxis.84 Detailed mutational analysis revealed that a
negatively charged residue at position 150 (aspartic acid in
Rac2) is required to for cell migration. Rac1 has a glycine at
this position, and mutating this to an aspartic acid, in
conjunction with a Rac2 C terminus, allowed the Rac1
chimera to fully restore migration as well as actin polymerization in Rac2⫺/⫺ neutrophils. In addition, the C terminus and
residue 150 were shown to be critical determinants for the
intracellular localization of Rac2.84
Finally, the complex activation of the neutrophil NOX is
complicated even more by additional modes of regulation.
Diebold et al recently found that Cdc42, which, by itself, is
not able to activate the oxidase, acts as a negative regulator in
the cell-free assays as well as in the Cos–Phox system
because it can bind, just like Rac1 and Rac2, to the flavocytochrome b558 via its insert region.110 In line with this, a
Cdc42-binding domain of Wiskott-Aldrich syndrome protein
increased fMLP-induced superoxide in human neutrophils,
indicating that Cdc42 may act as a “tonic regulator” to keep
oxidase activation at a low level, for instance during migration of neutrophils through tissues.
The activation of Rac proteins is mediated by GEFs.111 For
neutrophils, the recently identified Rac GEF P-Rex1 has been
proposed to be required for C5a-mediated production of
ROS.112 Price et al81 have shown in the COS–phox system
that expression of the Rac GEFs Vav1, Vav2, or Tiam1
resulted in different efficiencies of oxidase activation by Rac.
Strikingly, whereas Tiam1 and Vav2 were most efficient in
activating Rac GTP loading, Vav1 was most efficient
in inducing oxidase activity. This suggests that these GEFs, in
addition to activating Rac proteins, serve additional roles,
either by controlling localized Rac activation at different sites
in the cell, or perhaps even by interacting with Rac effectors
directly, as was proposed for the RacGEF ␤-PIX, binding to
NOX1.58
Rac and ROS in Cardiovascular Disease
There is a vast body of literature that links vascular ROS
production to cardiovascular disease.53,113 ROS production
can be detected in VSMCs as well as in endothelial cells, and
ROS may also be derived from infiltrating leukocytes. Important stimuli for ROS production in the vasculature are
cytokines (eg, TNF-␣), growth factors (eg, PDGF), and
G-protein– coupled receptor agonists (eg, Ang II). Vascular
ROS production as well as Rac1 activation have been
associated with hypertrophy and smooth muscle cell prolif-
Hordijk
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eration, endothelial dysfunction as well as endothelial cell
migration and proliferation, hypertension inflammation, and
atherosclerosis (Figure 3).7,53,113,114
Vascular hypertrophy has been ascribed to the effects of
various receptor agonists, including the extensively studied
Ang II. Ang II induces ROS production in VSMCs in a
Rac1-dependent fashion.115 Recent studies showed that this
Ang II–induced ROS production also requires the membrane
adapter caveolin, which is involved in Rac1 activation,116,117
and the lipid kinase PI3K-␥.115,118 Ischemia-reperfusion injury also is accompanied by production of high levels of
ROS. Here, Rac1 has clearly been implicated through expression of dominant-negative N17Rac1. Kim et al showed that in
VSMCs, cell death induced by hypoxia/reoxygenation is
prevented by N17Rac1.119 Similarly, Harada et al, showed
that N17Rac1 expression in the liver protects from ischemiareperfusion–induced production of ROS and activation of
NF-␬B.120
Several NOX isoforms have been implicated recently in
Rac1-dependent ROS production. VSMCs express NOX1,
NOX2, and NOX4, but NOX1 appears to be the most relevant
oxidase.52 NOX1 and NOX4 are expressed at higher levels
compared with NOX2, and superoxide-producing agonists
such as PDGF or Ang II downregulated NOX4 expression
and upregulated NOX1.52 In line with the requirement for
Rac1 and ROS production in endothelial cell migration and
proliferation (Figure 3),10,121–123 neovascularization in an
ischemic hindlimb model, which is associated with increased
expression of NOX2 and increased production of ROS, was
found to be impaired after treatment with antioxidants or in
NOX2-deficient animals.22,124 Use of a peptide (gp91ds-tat)
that interferes with the p47phox–NOX2 interaction has provided further in vivo evidence for a role of NOX2 in Ang
II–induced hypertension and smooth muscle cell hypertrophy.18,125 Similarly, in NOX2-deficient mice, induction of
renovascular hypertension resulted in less endothelial dysfunction and reduced hypertension compared with the responses in wild-type animals.21
The Rac1–ROS signaling pathway clearly is a crucial
mediator of cardiovascular disease (Figure 3). Based on
current literature, the most relevant NOX isoforms in vascular
cells appear to be NOX1 and NOX2, which can both be
regulated through Rac1. NOX1 is, in endothelial cells as well
as in smooth muscle cells, expressed at higher levels compared with NOX2.52,74 However, NOX2 appears to be important as well, based on studies with the NOX-inhibitory
peptide, the NOX2-deficient animals, and the observations of
its upregulation after, for example, ischemia and in restenosis.22,126 The role for NOX4, although highly expressed in, for
example, endothelial cells and in VSMCs, is presently unknown. Increased as well as decreased expression of NOX4
has been described in various models for vascular disease as
well as in human atherosclerosis52,126 –129; NOX4-specific
inhibitors or NOX4-deficient mice will likely provide more
detailed insight into its role in cardiovascular disease.
Conclusions
The mechanism of activation of the neutrophil NOX2 has
been the subject of intensive research for ⬇30 years. Still,
Rac-Mediated Regulation of NADPH Oxidases
459
new aspects are being uncovered, and the details of its
activation remain topic of discussion. Elegant mouse models
now allow the testing of specific mutants in a physiologically
relevant background. These approaches have provided important insights in the activation processes for NOX2. In the
meantime, related NOX enzymes and novel regulators have
been discovered, of which the activation or assembly may
prove equally complex as for the neutrophil NOX complex.
Based on currently available evidence, NOX1 is most likely
activated through Rac1 in combination with NOXA1 and
NOXO1. NOX3 appears p67phox/NOXA1, and thus Rac1
independent, and also NOX5 seems to be independent of
Rac1. The regulation of NOX4 remains to be investigated in
detail, also because the suggestion that NOX4 is a constitutively active oxidase is hard to reconcile with its relatively
high expression levels, for example, in endothelial cells.
Although many aspects of NOX regulation are still unknown,
the available data do suggest that the different NOXes are
regulated through distinct mechanisms or regulators. This
suggests that in cells, where several NOXes are coexpressed,
as in VSMCs and in endothelial cells, these may be activated
individually, depending on the stimulus, their localization, or
the condition of the cells. There is increasing evidence for an
important role of Rac-mediated activation of NOXes in
cardiovascular disease, which makes these signaling complexes an important therapeutic target. The challenge lies in
the use of appropriate model systems that allow generation of
consistent data. The use of appropriate mouse models or
knockdown approaches targeting endogenous NOXes or their
regulators will likely provide important progress in our
understanding of the activation of this multimolecular protein
complex.
Acknowledgments
D. Roos is gratefully acknowledged for critical reading. The author
is a fellow of the Landsteiner Foundation for Blood
Transfusion Research.
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Regulation of NADPH Oxidases: The Role of Rac Proteins
Peter L. Hordijk
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Circ Res. 2006;98:453-462
doi: 10.1161/01.RES.0000204727.46710.5e
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