In the present study we demonstrate that Rho/ROCK not only downregulates eNOS expression but it also inhibits eNOS phosphorylation and its activity. Moreover, we provide evidence that inactivation of PKB by Rho/ROCK is responsible for the inhibition of eNOS phosphorylation but not the downregulation of eNOS gene expression. Furthermore, these conclusions are reinforced by the results obtained with thrombin. Concomitant with its ability to activate Rho/ROCK, thrombin suppressed the phosphorylation of PKB and eNOS, in addition to its downregulatory effect on eNOS expression. Although the inhibition of both expression and phosphorylation of eNOS by thrombin is mediated by Rho/ROCK pathway, only the suppression of eNOS phosphorylation could be attributed to inactivation of PKB by thrombin.
Rho GTPase has been shown to regulate a wide spectrum of cellular function (48
). Increasing evidence suggests a role of Rho-regulated cellular function in mediating cardiovascular pathologies, such as smooth muscle hypercontraction (47
), SMC growth and migration that account for neointimal formation of stenosis (13
), and platelet aggregation (40
). Moreover, in recent years, much has been learned about the role of Rho in modulating endothelial function. In the context of endothelial dysfunction, Rho has been reported to downregulate eNOS expression (12
) and is essential for the basal production of endothelin-1 (23
), a potent vasoconstrictor and mitogen that regulates vascular tone and remodeling (33
). In agreement with previous reports, we demonstrate here that activation of Rho is sufficient to downregulate eNOS expression and is required for thrombin-mediated decrease in eNOS expression (Fig. ). Moreover, in the present study, we provide additional evidence for an important role of Rho in modulating PKB/eNOS pathway. We show that the activation of Rho, either by ectopic expression of active mutant of Rho or by a pathological stimulus thrombin dramatically inhibited phosphorylation of eNOS in parallel to that of PKB (Fig. and ). Of interest, whereas thrombin-induced dephosphorylation of eNOS occurs within 15 min (Fig. ), it takes hours to days to achieve downregulation of eNOS expression (Fig. ) (12
). The same is true with the constructs expressing active Rho or ROCK. Due to the fact that it takes at least more than 8 h to express the transgenes, we do not know exactly how rapidly the dephosphorylation was induced upon Rho63 expression. However, eNOS dephosphorylation could be already observed at 18 h p.i., at a time point when eNOS expression was not yet significantly affected (Fig. and ). In this respect, signals such as thrombin would cause endothelial dysfunction via the activation of Rho that in turn leads to dephosphorylation or inactivation of eNOS and suppression of its gene expression. This mechanism may play an important role in acute coronary syndromes in patients with heart disease.
To date, a number of downstream effector proteins of Rho have been identified (5
). Among them, ROCK is the most extensively studied. Many of the Rho-regulated cellular functions have been shown to be mediated by ROCK, such as smooth muscle contraction (2
), cytoskeletal rearrangement and c-fos expression (7
), and cytokinesis (52
). We show here that Rho-induced dephosphorylation of PKB and eNOS is also mediated by ROCK. This conclusion is based on the observations that the active ROCK led to dephosphorylation of both PKB and eNOS (Fig. and ), the dominant-negative ROCK prevented Rho63-mediated inhibition of PKB and eNOS phosphorylation (Fig. ).
Another important evidence we provide in this report is the cross talk between Rho/ROCK and PKB. The importance of this finding should be underscored by the fact that PKB serves as a multifunctional regulator of cell survival, growth, and glucose metabolism (9
). With respect to its cardiovascular functions, PKB plays a critical role in endothelial cell survival (16
) and promotes angiogenesis (11
) mediated by vascular endothelial growth factor, in addition to its involvement in controlling vasomotor activity via regulating eNOS pathway as mentioned above. Here we demonstrate that the cross talk between Rho/ROCK and PKB pathways is responsible for Rho-mediated eNOS dephosphorylation. Two lines of evidence supported the involvement of PKB in Rho/ROCK-mediated inhibition of eNOS phosphorylation. The first evidence came from experiments with constitutively active mutants. The data in Fig. and show that dephosphorylation of PKB and eNOS by active Rho or ROCK closely parallel one another. The second piece of evidence is provided by the rescue experiment in which an active PKB, but not an inactive PKB, restored phosphorylation of eNOS in the presence of active Rho (Fig. ). Since evidence has been presented that eNOS phosphorylation by PKB led to increased NO production, we wish to point out that active PKB restored eNOS phosphorylation in the presence of active Rho mutant without restoring cellular NO production (Fig. ). Taking into account that eNOS activity was monitored by measuring the cellular NO production in living cells with l-
C]arginine and that the effects of Rho and ROCK mutants on eNOS phosphorylation and cellular NO production parallel each other (Fig. ), a probable explanation would be that Rho/ROCK pathway may also downregulate other factors such as the availability of the substrate arginine or eNOS cofactor that are necessary for a normal cellular NO production but through other pathway(s) independent of PKB.
Although the role of PKB in eNOS phosphorylation leading to increased NO production is well documented and established, little is know about an involvement of PKB in eNOS gene expression. In view of the fact that PI 3-kinase was reported to be involved in eNOS gene expression (8
), and PKB is a well-characterized downstream signaling of PI 3-kinase, it is important to point out that active PKB did not rescue Rho63- or thrombin-mediated eNOS gene downregulation (Fig. and ), indicating that PKB is not required for Rho63 or thrombin to downregulate eNOS expression. However, this does not rule out a role of PKB in the regulation of eNOS gene expression. One possibility could be that PKB does not play a role in the regulation of eNOS mRNA stability by thrombin or Rho and yet is involved in eNOS transcriptional regulation as reported in one study regarding regulation of eNOS gene expression by PI 3-kinase (8
Regarding a role of PI 3-kinase in Rho/ROCK-mediated downregulation of PKB phosphorylation and activity by active mutants of Rho/ROCK, our data suggest that the inhibition of PKB activity by Rho/ROCK is unlikely to result from inhibition of PI 3-kinase activity since the PKB activation upon adenoviral infection or the inhibition of PKB activation by active Rho/ROCK is not accompanied by corresponding changes in PI 3-kinase activity (Fig. ). This is noteworthy in view of the fact that PI 3-kinase is strongly activated upon adenoviral infection in the human colon carcinoma cell line SW480 (34
). Thus, although the basal PI 3-kinase activity is required for PKB phosphorylation and activity in HUVECs under our experimental conditions (Fig. ), the PKB activation probably did not result from PI 3-kinase activation but may be from other mechanism such as inactivation of PTEN, inactivation of phosphatase 2A, or even some other unknown novel mechanism. What mechanism accounts for the PKB activation and would be therefore the target of Rho/ROCK to negatively regulate PKB activation reported in this study remains to be shown.
The data presented here are summarized as follows: a stimulus that activates Rho/ROCK pathway would lead to dephosphorylation of eNOS while simultaneously suppressing its expression by decreasing its mRNA stability to achieve rapid and long-term decrease in NO production. Whereas inhibition of PKB by Rho/ROCK is responsible for the negative regulation of eNOS phosphorylation, the downregulation of eNOS expression is attributed to another yet-to-be-identified pathway. An important task will be to further elucidate the mechanism by which Rho/ROCK inactivates PKB. In addition, the impact of the cross talk between the two pathways on other PKB-regulated cellular functions should be a worthwhile goal for future research.