1Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University and 2Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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shear stress; nitric oxide; endothelial cells; protein kinases
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NITRIC OXIDE IN VASCULAR BIOLOGY |
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NO production from endothelial cells is stimulated by a variety of mechanical forces such as shear stress and cyclic strain (2, 3, 19) and humoral factors ranging from growth factors to peptide hormones, including acetylcholine, vascular endothelial growth factor (VEGF), bradykinin, estrogen, sphingosine 1-phosphate, H2O2, and angiotensin II (12, 29, 42, 43, 55, 75, 78). It has become clear that the mechanisms by which NO is produced from eNOS in response to a number of different stimuli are quite variable. We summarize general mechanisms first and then present discussion of specific mechanisms regulating eNOS in response to shear stress in endothelial cells.
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SHEAR STRESS, ENDOTHELIUM, AND VASCULAR PHYSIOLOGY AND PATHOPHYSIOLOGY |
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ROLE OF NO IN SHEAR STRESS ACTION IN ENDOTHELIAL CELLS |
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REGULATION OF ENOS ACTIVITY |
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The eNOS is controlled 1) chronically by inducing its expression levels and 2) acutely by regulating its enzyme activity as summarized in Fig. 1. For example, chronic exposure of endothelial cells to certain conditions such as shear stress has been shown to increase eNOS expression level by both transcriptional induction and stabilization of mRNA (23, 24, 32). The eNOS is composed of two identical monomers, and each monomer contains the amino-terminal oxidase domain and carboxy-terminal reductase domain (1, 43). For NO to be produced from substrates O2 and L-arginine, electron flux has to occur from the reductase domain of one monomer to the oxygenase domain of the other monomer. Ca2+/CaM binding to eNOS facilitates the electron transfer from NADPH to the reductase domain flavins or that from the flavins to the oxygenase domain heme iron (45).
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Acutely, eNOS is regulated by several different mechanisms involving
eNOS-interacting proteins such as Ca2+/CaM, caveolin-1,
and Hsp90; posttranslational regulations (phosphorylation, acylation);
cofactors and substrates; and subcellular localization (plasma membrane
caveolae, Golgi, and cytosolic compartments). Recent papers by Fleming and
Busse (38), Fulton et al.
(43), and Shaul
(92) have reviewed these
topics elegantly and comprehensively, with a focus on the mechanisms activated
by humoral factors such as growth factors and hormone. In basal conditions,
eNOS activity is kept inactive by several independent mechanisms. First, the
majority of eNOS appears to be bound to caveolin-1 with its enzyme activity
repressed in caveolae (60,
72). This tonic inhibition of
eNOS can be released by displacing caveolin-1 with
Ca2+/CaM in response to
Ca2+-mobilizing agonists, including acetylcholine and
ATP (60,
73). Second, a putative
"autoinhibitory element," an 50-amino acid residue segment
present in the flavin mononucleotide-binding domain of eNOS (residue
596647 based on the bovine sequence), impedes CaM binding to eNOS
(15,
80,
99). Third, eNOS has been
shown to be inhibited by the interaction with some G protein-coupled receptors
such as bradykinin B2 receptor, angiotensin II AT1
receptor, and endothelin-1 ETB receptor
(59,
70). It was shown that
bradykinin stimulates tyrosine phosphorylation of the B2 receptor,
and this was accompanied by a transient dissociation of eNOS from the receptor
and increase in NO production.
(70). Fourth, eNOS activity is
also suppressed by interaction with NO-interacting protein (NOSIP), which has
been shown to negatively regulate intracellular translocation and activity of
eNOS (26).
Even though Ca2+/CaM binding largely controls eNOS activation, there are several other proteins interacting with eNOS and regulating its activity positively. Hsp90, which was first found as a 90-kDa tyrosine-phosphorylated eNOS-associated protein (ENAP), positively regulates eNOS activity (9, 37, 47, 87, 100). Its interaction with eNOS is stimulated by both humoral (VEGF and histamine) and physical factors (shear stress) and leads to activation of eNOS, producing NO (47). Dynamin-2 (a GTP-binding protein) and porin (a voltage-dependent anion channel) have also been shown to colocalize and directly interact with eNOS (13, 93). Their interactions with eNOS are stimulated by intracellular Ca2+ and lead to eNOS activation (13, 93).
Although shear stress is one of the most potent and physiologically important regulators of eNOS activity, the underlying mechanisms are quite unique from those of humoral stimuli. In addition, there have been recent conflicting reports as to the role of protein kinases in shear-dependent eNOS regulation. Moreover, the functional significance of each phosphorylation site is still unclear. Therefore, we have summarized below the emerging concept in phosphorylation-dependent regulation of eNOS with a focus on shear stress as a stimulus.
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ENOS PHOSPHORYLATION |
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Phosphorylation of eNOS may be largely determined by its specific subcellular locations such as the plasma membrane caveolae, Golgi and cytosolic compartments, and protein kinases and phosphatases associated with them. On the other hand, phosphorylation of eNOS may determine the fate of its intracellular locations (43, 73, 92). The importance of the relationship between phosphorylation and localization of eNOS has been suggested but needs further future study. We have described phosphorylation-dependent eNOS regulation in more detail below, first for protein kinases and phosphatases and second for each phosphorylation site of eNOS.
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PROTEIN KINASES AND PHOSPHATASES |
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Protein kinase C. Bredt et al. (8) found that nNOS protein purified from brain could be phosphorylated at distinct sites by protein kinase C (PKC), protein kinase A (PKA), and Ca2+/CaM protein kinase II (CaMKII) and that PKC-dependent phosphorylation decreased the enzyme activity. Similarly, PKC activation by phorbol 12-myristate 13-acetate (PMA) has been shown to inhibit eNOS activity in cultured cells (21, 54).
The PKC phosphorylation sites on eNOS were shown to be T497 or S116. It was shown that PKC activation by acute PMA treatments leads to phosphorylation of T497 (39, 74). Downregulation of PKC by incubating cells with PMA for 24 h attenuates T497 phosphorylation (39). The PKC inhibitor Ro-31-8220 decreases basal T497 phosphorylation, supporting the role of PKC in eNOS-T497 phosphorylation. Kou et al. (62) recently showed that eNOS phosphorylation at S116 is blocked by the PKC inhibitor calphostin but was not inhibited by the PI3K inhibitor wortmannin or the MAPK pathway inhibitor U0126.
Protein kinase B/Akt. The role of Akt in eNOS regulation was first demonstrated when several independent investigators reported phosphorylation of eNOS-S1179 in a PI3K- and Akt-dependent manner (29, 42, 46, 75). Several lines of evidence supported the notion that Akt was the protein kinase phosphorylating eNOS-S1179. For example, both wortmannin and LY-294002, specific inhibitors of PI3K (the upstream regulator of Akt), blocked eNOS-S1179 phosphorylation stimulated by various stimuli, including shear stress, VEGF, and insulin-like growth factor (29, 42, 46, 75). Expression of constitutively active Akt mutant alone was able to stimulate phosphorylation of eNOS-S1179 (29, 42, 46). In addition, overexpression of dominant negative Akt mutants (AktAA or AktAA mutants) blocked the eNOS-S1179 phosphorylation stimulated by ligands such as VEGF (29, 75). However, this critical direct evidence was not presented in early studies, which proposed a role for Akt in eNOS-S1179 phosphorylation in response to shear stress (see HOW DOES SHEAR STRESS STIMULATE NO PRODUCTION FROM ENOS?). More recently, an additional eNOS amino acid residue, S617, phosphorylated by Akt was identified by Michell et al. (76).
Protein kinase A and protein kinase G. Many protein kinases, including PKA and Akt, share similar consensus sequences of phosphorylation, resulting in phosphorylation of a substrate protein by multiple protein kinases. Therefore, it was not surprising when Butt et al. (11) showed that S1179 was phosphorylated by other kinases such as PKA and protein kinase G (PKG). In addition to S1179, PKA also phosphorylated eNOS at S635. PKA-dependent phosphorylation of eNOS at S1179 and S635 was demonstrated under a physiological stimulus, shear stress, by us in endothelial cells (4, 6). Shear-dependent phosphorylation of eNOS at S1179 and S635 can be inhibited by the PKA inhibitor H89 or by adenovirus-mediated expression of PKA inhibitor (4). Furthermore, treatment of cells with a cell-permeable cAMP analog or expression of constitutively active PKA catalytic subunit, Cqr, stimulates S1179 and S635 phosphorylation (5). Whereas PKA phosphorylates eNOS-S635 under physiological conditions, including shear stress, VEGF, and bradykinin (11, 76), Akt has been shown not to phosphorylate eNOS at S635 (29, 42, 76). Evidence for PKG-dependent eNOS regulation needs to be obtained in the future under physiological conditions. An additional site, S617, was also shown to be phosphorylated by PKA as well as Akt in an in vitro study (76). However, treatment of endothelial cells with isobutyl methylxanthine did not phosphorylate eNOS-S617, suggesting that PKA may not be a physiologically relevant kinase phosphorylating S617 (76).
AMP-activated protein kinase. AMP-activated kinase (AMPK) also phosphorylates eNOS-S1179, especially in the presence of Ca2+/CaM (16). Binding of Ca2+/CaM to eNOS may induce a conformational change in eNOS allowing S1179 phosphorylation to occur by AMPK. Recently, peroxynitrite, a nitric oxide-derived oxidant, has been shown to stimulate eNOS-S1179 phosphorylation by an AMPK-dependent mechanism (101). In the absence of Ca2+/CaM, AMPK phosphorylates T497 as well (16).
CaM-dependent kinase II. The eNOS-S1179 phosphorylation can occur by the PI3K-independent manner in response to certain agonists such as bradykinin (39, 52). Although bradykinin stimulates eNOS-S1179 phosphorylation, it was not inhibited by wortmannin, a PI3K inhibitor. Further study showed that bradykinin-induced phosphorylation of eNOS-S1179 can be blocked by KN-93, a CaMKII inhibitor (39). These results suggest that CaMKII can phosphorylate S1179 in response to bradykinin or other stimuli.
Phosphatases. At least three different classes of Ser/Thr protein phosphatases (PP1, PP2A, and calcineurin) have been implicated in the regulation of eNOS dephosphorylation and activity. Dephosphorylation of eNOS-S1179 is regulated by PP2A, whereas dephosphorylation of T497 is controlled by PP1, as was demonstrated using the PP2A-specific inhibitor okadaic acid and PP1-specific inhibitor calyculin A (39, 74). The Ca2+/CaM-dependent protein phosphatase calcineurin appears to regulate dephosphorylation of eNOS-S116, because VEGF-dependent dephosphorylation of S116 can be blocked by cyclosporin A (62). The role of calcineurin in T497 dephosphorylation is not clear because conflicting results have been reported (39, 52). Which protein phosphatases may regulate dephosphorylation of S635 or S617 is not known yet.
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ENOS PHOSPHORYLATION SITES |
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Bovine S635 (human S633). It has been recognized that a putative autoinhibitory element impedes CaM binding to eNOS and electron flux between the two eNOS monomers (15, 80, 99). Deletion of this element from eNOS decreases the Ca2+/CaM requirement for enzyme activation and enhances maximal activity (15, 80). The phosphorylation of eNOS-S635 located within the autoinhibitory element domain suggests its potential role in regulation of the enzyme activity.
Butt et al. (11) have shown that eNOS purified from the cells stimulated with a PKA agonist retains enzyme activity even in the presence of the calcium chelator EDTA, indicating that the PKA-dependent phosphorylation of either S1179 or S635 could activate eNOS in a Ca2+-independent mechanism. Controversy exists regarding the activity of eNOS-S635D mutant mimicking the continuously phosphorylated state. Dimmeler et al. (29) showed that eNOS-S635D mutant contains enzyme activity identical to that of wild-type eNOS, whereas Michell et al. (76) reported a twofold higher activity than that of wild type in in vitro assays carried out using a saturated concentration of Ca2+.
Recently, we have shown that shear stress stimulates eNOS-S635 phosphorylation in a PKA-dependent mechanism (4). It was also shown that treatment of cells with VEGF and PKA activators such as 8-bromo-cAMP (8-BrcAMP) and isobutyl methylxanthine stimulates eNOS-S635 phosphorylation (4, 76). More recently, using both endothelial cells and HEK-293 cells transfected with eNOS-635D mutants, we showed that phosphorylation of eNOS-S635 renders the enzyme active even when intracellular Ca2+ is virtually depleted by the calcium chelator BAPTA-AM (5). This result suggests that PKA-dependent phosphorylation of eNOS-S635 may play an important role in regulating vascular biology and pathophysiology by maintaining basal NO production in a Ca2+-independent manner. However, the discrepancy regarding the role of S635 phosphorylation in eNOS activity reported by the different laboratories needs further clarification.
Bovine S617 (human S615). Recently, eNOS-S617 has been shown to be phosphorylated in response to bradykinin, ATP, and VEGF (76). Although it can be phosphorylated by both PKA and Akt in vitro, treatment of endothelial cells with isobutyl methylxanthine phosphorylated S635 but not S617 (76). These results suggest that Akt may be the protein kinase responsible for S617 phosphorylation under physiological conditions. Michell et al. (76) have shown that phosphorylation of S617, using phosphomimicking eNOS constructs, increases Ca2+/CaM sensitivity of the enzyme without altering maximum activity. Transfection of endothelial cells with dominant negative Akt constructs has been shown to block shear-dependent NO production without inhibiting eNOS-S1179 phosphorylation (50), which raises a question as to what is the mechanism by which Akt regulates eNOS activity in response to shear stress. We now suggest that S617 phosphorylation by Akt in response to shear stress may be a potential mechanism.
Bovine T497 (human T495). eNOS becomes less active if T497 is phosphorylated (16). eNOS-T497 phosphorylation may interfere CaM binding to the enzyme at low Ca2+ concentration. eNOS-T497A mutant has higher affinity to CaM and higher activity than T497D mutant at low Ca2+ concentration (39). eNOS is dephosphorylated when cells are stimulated with 8-BrcAMP (4), isobutyl methylxanthine (74), bradykinin (39, 52), and phosphorylated in response to PMA (74). The effect of VEGF on T497 phosphorylation is still controversial (4, 41, 52, 74).
Bovine S116 (human S114). It has been proposed that eNOS-S116 dephosphorylation may enhance eNOS activity by an uncharacterized mechanism (62). eNOS-S116D mutant showed higher enzyme activity in vitro compared with wild-type eNOS (62). Phosphorylation of eNOS-S116 has been shown to increase in response to shear stress (46) and decrease in response to VEGF (62). Another report published by us (4) showed that phosphorylation of eNOS-S116 did not change in response to shear stress or VEGF. Although the exact reasons are unclear, the discrepancy may be attributed to different experimental conditions such as cell confluency. Supporting this notion, eNOS-S116 becomes dephosphorylated as cells reach confluency (4).
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HOW DOES SHEAR STRESS STIMULATE NO PRODUCTION FROM ENOS? |
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In basal conditions, eNOS appears to be bound to caveolin-1 in caveolae and
kept inactive. Upon exposure to a step increase in shear stress, eNOS has been
shown to dissociate from caveolin-1 and associate with CaM, activating the
enzyme activity (47,
89). In addition, GTP-binding
proteins have been also shown to play a critical role during this early phase.
Whereas pertussis toxin-sensitive G proteins (Gi family)
have been shown to regulate shear-dependent NO production in BAEC, pertussis
toxin-insensitive G proteins (either heterotrimeric or small-molecular-weight
G proteins) have been shown to be involved in human umbilical vein endothelial
cells (65,
84). The exact identity of G
proteins may depend on the cell types. However, shear-dependent NO production
during the second phase was shown to be regulated in a G protein-independent
manner (65). In addition,
during this first phase of eNOS activation, phosphorylation of eNOS-S1179 also
occurs and plays a critical role in the early NO production, as inhibitors
blocking the phosphorylation also shut down NO production in response to shear
stress (6,
29). Once the
Ca2+ level drops back down to a basal level, changes in
the phosphorylation status of eNOS, especially S635, may then keep them active
during the second phase of NO production as long as shear stress is
maintained.
Initial evidence suggested that the phosphorylation of eNOS at S1179 by a sequential activation of PI3K and Akt pathway is the underlying mechanism by which shear stress stimulates NO production in a Ca2+/CaM-insensitive manner (28, 42). In the cases of VEGF, sphingosine 1-phosphate, and estrogen, strong evidence has been presented supporting the possibility that PI3K activates Akt, which in turn is responsible for regulating the phosphorylation and activation of eNOS (29, 42, 43, 55, 75, 78). However, the early reports (28, 42, 46) have not provided direct evidence as to whether Akt is indeed the protein kinase directly responsible for phosphorylation of eNOS-S1179 and its subsequent activation in response to shear stress. For example, it has not been reported whether expression of a dominant negative Akt constructs such as AktAA or AktAAA can block the shear-dependent phosphorylation of eNOS-S1179 and NO production in endothelial cells (28, 46).
This evidence is important because it is clear that Akt is not the only protein kinase that can phosphorylate eNOS-S1179. Other protein kinases including PKA, PKG, and AMPK have been also shown to phosphorylate eNOS-S1179 (6, 11, 16, 76). PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the plasma membrane. Elevation of PIP3 is believed to provide a surface to which the two phosphatidylinositol-dependent kinases (PDK1 and PDK2) bind (29, 96). In turn, PDK can activate several downstream targets, including PKA, PKC, serum-glucocorticoid-dependent kinase (SGK), and p70-S6 kinase (96).
When endothelial cells were transfected with dominant negative Akt constructs (AktAA or AktAAA), it was found that shear-dependent phosphorylation of eNOS-S1179 is mediated by an Akt-independent, but a PKA-dependent, mechanism (50). Expression of recombinant adenoviral constructs AktAA or AktAAA in endothelial cells inhibited phosphorylation of eNOS-S1179 if cells were stimulated by VEGF, but not when stimulated by shear stress (50). In the same study, dominant negative Akt constructs almost completely blocked shear-dependent NO production (50). This result suggested that there must be another unknown site that is phosphorylated by Akt. We propose that the Akt-dependent phosphorylation of S617 (76) may be a mechanism by which shear stress stimulates NO production from eNOS. Consistent with this hypothesis, we recently found that exposure of BAEC to shear stress for 1530 min significantly increased phosphorylation of eNOS-S617 (data not shown). Another possibility by which Akt can regulate eNOS includes an indirect pathway: Akt may phosphorylate eNOS-interacting proteins, which in turn regulate eNOS activity. In addition to S1179, PKA also phosphorylates eNOS-S635 (6, 11, 76). We have shown that shear stress stimulates eNOS-635 phosphorylation in the PKA-dependent manner and that it renders the enzyme able to produce NO continuously in the absence of any Ca2+ changes, providing its functional importance in vascular wall biology (4).
What is the mechanism by which shear stress maintains a low level of NO production for as long as shear is imposed on endothelial cells? We propose at least two mechanisms that may work in concert: 1) Hsp90 binding to eNOS and 2) eNOS-S635 phosphorylation. Whereas histamine and VEGF have been shown to induce a rapid association of Hsp90 to eNOS within 1 min, shear exposure induced a relatively slow binding of Hsp90 to eNOS, requiring more than 30 min for a discernible increase and 60 min for a maximum interaction (47). Therefore, it is tempting to suggest that Hsp90 plays a role in the second phase of NO production in response to shear stress. In addition, we also suggest that phosphorylation of eNOS-S635 provides, at least in part, a mechanism for the second phase of NO production under shear conditions. As we showed recently (4), compared with S1179, which becomes rapidly phosphorylated, reaching a maximum within 5 min, shear stress slowly stimulates phosphorylation of eNOS-S635, taking almost 30 min of shear exposure, in a PKA-dependent manner (4). Once phosphorylated at eNOS-S635, the enzyme produces NO in conditions even when intracellular Ca2+ is very low (5).
Much indirect evidence found with the use of PKA inhibitors has strongly
suggested a role for PKA in eNOS phosphorylation and activation by shear
stress. However, it remains to be directly demonstrated whether and how shear
stress stimulates PKA activity. In addition, it has been previously shown that
shear stress does not affect cAMP level in endothelial cells
(68). A typical activation of
PKA occurs when cAMP binds to the regulatory subunit, thereby releasing the
catalytic subunit and leading to activation of the released catalytic subunit.
Therefore, how would PKA be activated by shear stress without changing total
cAMP levels? First, cAMP level may rise in some subcellular domains without
changing global cAMP levels. For example, cAMP levels in eNOS residing regions
such as caveolae and the Golgi apparatus may increase in response to shear
stress. Second, accumulating evidence suggests that PKA can be regulated in a
cAMP-independent manner. One mechanism is phosphorylation of T197 in the
activation loop of C subunit by PDK1
(14,
17). Other mechanisms include
interactions of PKA with caveolin, A-kinase anchoring protein (AKAP110), and
inhibitory NF-B (I
B) protein.
(33,
82,
88). It is especially
interesting that the phosphorylation of the key regulatory site T197 is
mediated by PDK1 (17) because
shear stress has been shown to stimulate PI3K
(51), which in turn activates
PDK1. We speculate that shear stress activates PKA through a PI3K-PDK1 pathway
and that PKA then phosphorylates eNOS-S1179 either directly or indirectly.
In summary, shear stress stimulates NO production from endothelial cells by using a variety of protein regulators (including caveolin and CaM) and various protein kinases (PKA and Akt) in a time-dependent manner. The acute, robust NO production due to a step increase in shear stress may play a critical role in vessel relaxation, whereas the chronic, low level of NO production due to the steady laminar shear stress may play a critical role as an antiatherogenic and antiinflammatory molecule in cardiovascular biology and pathobiology.
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DISCLOSURES |
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Address for reprint requests and other correspondence: Wallace H. Coulter
Dept. of Biomedical Engineering at Georgia Tech and Emory Univ., 308D WMB,
Atlanta, GA 30322 (E-mail:
hanjoong.jo{at}bme.gatech.edu).
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