Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612
NITRIC OXIDE (NO) is a biologically
active gas generated from the precursor arginine by one of three known
NO synthases. NO produced by the endothelial nitric oxide synthase
(eNOS) plays a crucial role in the regulation of a variety of
cardiovascular and pulmonary functions in both normal and pathological
conditions (14). It was noted early on that NO generation
from endothelial cells requires calcium, and the dependence of eNOS
activity on calcium/calmodulin binding was proven after purification
and characterization of the enzyme (23). Increased
intracellular calcium is thus an important regulator of eNOS activity
and explains the ability of agonists such as bradykinin, acetylcholine,
and thrombin to stimulate NO production via interaction with their
respective G protein-coupled receptors. However, it is now becoming
clear that the control of eNOS activity is much more complex, as
reflected in the titles of two recent reviews on the subject (8,
19).
One important regulator of eNOS function is caveolin-1. Caveolin is an
essential component of caveolae, 50-100 nm of flask-shaped microinvaginations in the cell membrane that are the location of an
important pool of eNOS in endothelial cells (9). Binding of eNOS to the scaffolding domain of caveolin-1 inhibits its activity both in vitro and in vivo, and this effect can be mimicked by a
synthetic peptide containing the scaffolding domain sequence (8). It is thought that activation of eNOS by the binding
of calcium/calmodulin releases the caveolin inhibition due either to a
conformational change or to dissociation of eNOS from caveolin (8). The tonic inhibition of eNOS by caveolin-1 in vivo
was recently confirmed in studies on caveolin-1 knockout mice that exhibited blunted responses to vasoconstrictors and enhanced responses to acetylcholine due to increased vascular NO production (5, 25,
26).
Caveolin-1 is not the only protein that can regulate eNOS activity. The
bradykinin receptor inhibits eNOS by binding to it through
intracellular domain 4, and the interaction is disrupted by the binding
of bradykinin to the receptor (17). NOSIP (eNOS interacting protein) is a 34-kDa protein that binds with high affinity
to eNOS, and the binding can be dissociated with a synthetic caveolin-1
scaffolding domain peptide (4). Although NOSIP does not
directly inhibit eNOS activity in vitro, coexpression with eNOS reduces
ionomycin-induced NO production by causing redistribution of eNOS from
the plasma membrane to intracellular compartments (4).
Dynamin-2, a large GTPase thought to be involved in vesicle formation
and trafficking, can associate directly with eNOS and augment its
activity (2). The physiological relevance of these protein-eNOS interactions remains to be defined.
A recently discovered mechanism for activating eNOS is by
phosphorylation of the protein, stimulated by agonists such as vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate, or by
shear stress (8, 19). In general, this activation is slower in onset and more prolonged than that which is stimulated by
agonist-induced increases in intracellular calcium. A critical phosphorylation site in eNOS has been identified as Ser1179
(bovine) or Ser1177 (human) (8). Although
several protein kinases have been reported to phosphorylate eNOS, a
pathway of primary importance involves activation of Akt or protein
kinase B (8). Akt can directly phosphorylate eNOS,
resulting in enzyme that is up to 20 times more active at optimal
calcium/calmodulin concentrations (10) and is activated at
lower levels of calcium/calmodulin (7). It has also been
reported that phosphorylation at Thr497 negatively
regulates eNOS activity (8). For example, eNOS activation
by bradykinin does not result in phosphorylation of Ser1179
but does require dephosphorylation of Thr497
(13). Protein kinase A signaling also activates eNOS due
to dephosphorylation of Thr497 and phosphorylation of
Ser1179, whereas protein kinase C activation has the
opposite effect on phosphorylation at these sites, causing inactivation
of eNOS (22).
Another important factor that regulates eNOS activity is its
localization within the cell (21). It has been known for
some time that eNOS is both myristoylated and palmitoylated and that appropriate acylation is required for the correct targeting of eNOS to
the caveolar microdomains of the plasma membrane and intracellular membranes (8, 20). Elimination of the appropriate
acylation signals inhibits NO release, eNOS activity, and eNOS
phosphorylation in response to agonists, indicating that proper
subcellular localization is necessary for optimal functioning and
receptor-mediated stimulation of eNOS activity (6, 8, 20).
It has been proposed that downregulation of eNOS activity after agonist
stimulation is a result of redistribution of the enzyme from the
caveolae/plasma membrane to either the cytosol or internal membranes
(20), although some studies have failed to show such a
redistribution. For example, it was recently reported (6)
that activation of eNOS by VEGF or by overexpression of Akt increased
phosphorylated eNOS in both the plasma membrane/caveolar fraction as
well as intracellular membranes (likely Golgi) without a change in
cellular distribution of the enzyme. Furthermore, NO was generated not
only in the vicinity of the plasma membrane but also at high levels
intracellularly, in the perinuclear region. Thus eNOS on both the
plasma membrane/caveolae and intracellular membranes can be activated
by agonists without a net change in subcellular distribution
(6).
Heat shock protein 90 (HSP90) is an essential, highly expressed
cytosolic protein that can be upregulated during stress
(24). Similar to other heat shock proteins, HSP90 plays an
important role as a chaperone in regulating protein folding and
maturation, but it also interacts with signal transduction proteins
such as Src kinases, Raf, or G As can be seen from this brief overview, eNOS activity is regulated by
a complex, coordinated interplay between many different proteins.
However, the details by which these control mechanisms work are not
well understood. In addition, the physical localization of eNOS is
likely an important factor that regulates its ability to interact with
these modulators and mediators. In this context, the paper by Su et
al., one of the current articles in focus (Ref. 27, see p.
L1183 in this issue), reveals another level of regulation for eNOS that
connects the activation process to the integrity of the cytoskeleton.
Besides adding another piece to the eNOS regulation puzzle, it opens up
a potentially fruitful area for investigation. The essence of the study
is the demonstration that the state of microtubule polymerization
affects endothelial cell NO production and eNOS activity and,
furthermore, that this response is mediated through HSP90.
The investigators (27) treated porcine pulmonary artery
endothelial cells for 2-4 h with either taxol (to stabilize
microtubules) or nocodazole (to inhibit microtubule polymerization) and
then measured NO production using the intracellular fluorescent probe 4,5-diaminofluorescein diacetate and assayed eNOS activity by measuring [3H]arginine to [3H]citrulline
conversion. Pretreatment of cells with taxol resulted in an increase in
NO production, whereas treatment with nocodazole reduced NO production
(27). The effect of nocodazole was blocked when
coadministered with taxol, indicating a specific effect on microtubule
formation was responsible for the results. Interestingly, this effect
is mediated by HSP90, as shown by two pieces of evidence. First,
coimmunoprecipitation of eNOS and HSP90 was increased by taxol and
decreased by nocodazole. Second, geldanamycin, an antibiotic that binds
to the nucleotide-binding site of HSP90 and blocks its activity
(24), inhibited the ability of taxol to increase eNOS
activity (27). These treatments had no effect on protein levels of HSP90, eNOS, or tubulin, indicating the results were due to
changes in the association of existing molecules (27). Surprisingly, there was also no effect of taxol or nocodazole on the
coimmunoprecipitation of eNOS with tubulin or HSP90 with tubulin. These
data indicate that tubulin polymerization strengthens the interaction
between eNOS and HSP90 or increases the amount of HSP90 associated with
eNOS without changing the bulk association of these molecules with
tubulin. However, there remains the possibility that microtubule
depolymerization might alter tubulin binding to HSP90/eNOS at a
restricted local level (e.g., in the vicinity of caveolae) that could
affect NO production but would not be detectable by immunoprecipitation
of the proteins from the whole cell. Immunoprecipitation studies of
caveolar preparations from cells treated with microtubule-active agents
would be informative in this regard.
In a recently published paper (30), investigators from the
same group provided evidence for another connection between
eNOS-mediated NO production and the cytoskeleton. They reported that
stabilization of actin polymerization with jasplankinolide increased
arginine transport and NO production in porcine pulmonary artery
endothelial cells, whereas treatment with swinholide A to disrupt actin
microfilaments decreased arginine transport and NO production
(30). These treatments had no effect on expression of eNOS
or the arginine transporter and did not affect eNOS activity in
isolated membrane fractions. Thus the state of actin polymerization
likely affected the efficiency of arginine transport and substrate
supply for eNOS, possibly by changing the proximity of the arginine
transporter to eNOS.
Together, the above studies highlight the importance of the
cytoskeleton in regulating the signal transduction process leading to
the generation of NO by endothelial cells. These results are consistent
with an earlier study that showed the cytoskeleton to be important for
mechanotransduction of shear stress-induced NO production in rabbit
aortic rings (15). Cytoskeletal interactions and integrity
are important regulators of a variety of signaling processes and are
now thought to play an important role in vascular changes in
hypertension (16). Determining how these effects are
mediated will require further study, but the physical organization of
the various components into a complex that is efficiently coupled is a
likely possibility.
One way in which the cytoskeleton might interact with and regulate the
eNOS system is through caveolin or caveolar integrity. The possible
interactions of caveolin or caveolae with microtubules have not been
clearly defined, but there are a few studies that hint at a connection.
For example, caveolin-1 expression is highly upregulated in
taxol-resistant A549 human lung carcinoma cells, and taxol induces
upregulation of caveolin-1 expression in the same cell line
(29). In addition, one step in the process of caveolin
cycling between the plasma membrane and Golgi, transfer from the
endoplasmic reticulum/Golgi intermediate compartment (or
ERGIC) to the Golgi, requires microtubules (3).
Although caveolin is obviously an important regulator of eNOS activity (5, 8, 26), these data do not provide a ready explanation for the results reported by Su et al. (27) in which taxol
increased and nocodazole decreased eNOS activity. This is because
increased caveolin-1 expression by taxol should inhibit eNOS activity,
and inhibition of caveolin trafficking by nocodazole would be expected to cause accumulation of caveolin in the ERGIC (3) and
relative depletion in the plasma membrane, which should increase eNOS
activity, opposite of the actual effects of these reagents on
endothelial NO production (27).
The results of Su et al. (27) show that microtubule
integrity affects the ability of HSP90 to bind and activate eNOS. The role of the cytoskeleton in regulating the function of HSP90 is not
well understood. In general, chaperones such as HSP90 can affect the
formation and function of the cytoskeleton under normal conditions and
protect it under stress via their chaperone actions (18).
Although HSP90 is primarily a cytosolic protein, there is evidence that
it can associate with microtubules and possibly cytokeratin
intermediate filaments as well (18). The function of
HSP90-cytoskeletal interactions has not been rigorously investigated, but there is indirect evidence that it may be important. For example, HSP90 plays a critical role in binding and regulating the activity of
steroid hormone receptors, and it may also facilitate trafficking of
the receptors to the nucleus by interacting with the microtubular system (24).
The results of Su et al. (27) raise many interesting
questions that will likely be addressed in future investigations. For example, is the activation of eNOS by agonist-induced increases in
intracellular calcium also affected by microtubule polymerization? This
question was not directly addressed by Su et al. (27). An
earlier study indicates that this may not be the case because administration of nocodazole to isolated arterial ring preparations did
not affect acetylcholine-induced NO production (15). Thus the main role of the cytoskeleton might be to enhance the
phosphorylation-induced activation of eNOS by Akt or other protein
kinases in response to stimuli such as VEGF, sphingosine 1-phosphate,
or shear stress. This is consistent with the known ability of HSP90 to
recruit and enhance Akt-mediated phosphorylation of eNOS
(1). It would thus be interesting to investigate the
phosphorylation state of eNOS in response to treatment with
microtubule-disrupting or -stabilizing agents. Another question raised
by the study of Su et al. (27) is whether microtubule
depolymerization leads to displacement of eNOS from its appropriate
subcellular location by disrupting caveolae or other subcellular
structures. Microtubule disruption might, therefore, have an effect
analogous to that which is seen with acylation-deficient eNOS mutants
that results in mislocalized eNOS with reduced activity. A detailed
investigation of the subcellular distribution of eNOS after microtubule
disruption would be informative in this regard. Another interesting
question arises from the finding that the state of microtubule
polymerization does not affect the ability of tubulin to associate with
HSP90 or eNOS. How, then, do taxol and nocodazole affect the HSP90-eNOS
interaction? An explanation favored by the authors (27) is
that increased tubulin polymerization brings HSP90 physically closer to
eNOS, promoting their association. This is a plausible explanation,
although another possibility consistent with the data is that tubulin
polymerization alters the conformation of bound HSP90, increasing its
affinity for eNOS, which results in enhanced eNOS activity.
The findings of Su et al. (27) provide a mechanistic
explanation for earlier reports of the ability of
microtubule-depolymerizing agents to potentiate pressor responses to
vasoconstrictor agents (16) and reduce shear
stress-induced NO production in rabbit aortic rings (15).
These results could also have clinical relevance because cytoskeletal
integrity and disruption play an important role in signal transduction
cascades intimately related to cardiovascular diseases
(16). Further investigations into the relationship between
the cytoskeleton and eNOS will undoubtedly bring new insight into the
ways this important enzyme is regulated in the pulmonary and
cardiovascular systems in both normal and disease states.
ARTICLE
TOP
ARTICLE
REFERENCES
-subunits (24).
Although the functions of many of these interactions are unclear, HSP90
was recently shown to be an essential component of
G
12-induced serum response element activation,
cytoskeletal changes, and mitogenic response (28). HSP90
also plays an important role in the regulation of eNOS activity;
agonists such as VEGF, histamine, and shear stress increase the
association of HSP90 with eNOS, which enhances eNOS activation
(11). Further details regarding the regulation of eNOS by
HSP90 have since been revealed in a variety of studies (8). For example, eNOS, caveolin-1, and HSP90
coimmunoprecipitate in a macromolecular complex, and HSP90 enhances the
displacement of eNOS from caveolin-1 (12). In addition,
eNOS-HSP90 complexes are less sensitive to inhibition by a caveolin
scaffolding domain peptide (12). HSP90 also mediates the
VEGF-stimulated transition of eNOS from a calcium-dependent activation
state to phosphorylation-dependent potentiation through recruitment of
Akt to the eNOS/HSP90 complex to phosphorylate Ser1177
(1). Because HSP90 is not known to directly interact with caveolin, there have been two models proposed (8) to
explain the dynamic interactions between eNOS, caveolin, and HSP90:
1) Recruitment or activation of HSP90 and calmodulin to eNOS
results in weak displacement of eNOS from caveolin, but the complex
remains in caveolae; and 2) HSP90 and calcium-activated
calmodulin interact with eNOS and, while still bound to caveolin, cause
a conformational change in eNOS that allows for efficient
stimulation-response coupling.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. A. Skidgel, Dept. of Pharmacology (m/c 868), Univ. of Illinois College of Medicine, 835 S. Wolcott, Chicago, IL 60612 (E-mail: rskidgel{at}uic.edu).
10.1152/ajplung.00045.2002
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