TI - Discussion . AB - In the present study , we examined the regulatory mechanisms of shear stress on the Nrf2 transcription factor and its downstream target , HO-1 , in HUVECs . It was found that shear stress activated Nrf2 nuclear translocation and increased the amount of Nrf2 protein and the level of mRNA transcription ( Figure 1 ) . The translocation of Nrf2 occurred after 10 min of shear stress , suggesting that Nrf2 translocation which induces downstream gene regulation is a rapid response . However , the mRNA level of Nrf2 increased obviously after 30 min of shear stress and reached a maximum at around 60 min . Hong et al .and Eggler et al .reported that under a basal condition , Nrf2 undergoes Cul3 -dependent ubiquitination and proteosomal degradation , but under various stimuli , Keap1 is ubiquitinated to release Nrf2 into the nucleus [60,61] . It is likely that when HUVECs are exposed to shear stress , the early response is ubiquitination of Keap1 which induces Nrf2 translocation . Nuclear translocation may decrease the concentration of Nrf2 in the cytosol thus triggering the expression of Nrf2 at both the mRNA and protein levels . But by 2 h later , the cells may have adapted to the shear stress stimulation , and thus the transcription level of Nrf2 began to decline . However , the amount of Nrf2 protein was still higher than the basal condition ( Figure 1A ) . We also found that PI3K played a crucial role in shear-induced Nrf2 translocation ( Figure 2 ) . Kang et al .revealed that before entering nuclei , it is necessary for Nrf2 to be translocated to plasma membranes where it is PHOSphorylated by PI3K or PKC [36 , 42] . Based on our results , we found that Nrf2 translocation and activation were PI3K -dependent . But PI3K is a lipid kinase , and the SUBstrate of PI3K should be a lipid like PIP2 , not Nrf2 . This implies that the mechanism of Nrf2 translocation is indirectly mediated by PI3K . There are likely more protein kinases involved in Nrf2 activation and PKC may be one candidate . When shear stresses activate PI3K , it PHOSphorylates PIP2 to PIP3 , and at this time , phosphoinositide-dependent kinase 1 ( PDK1 ) may bind to PIP3 through its PH domain . PKC , one of the downstream subSTRates of PDK1 , can be PHOSphorylated and activated . Activation of Nrf2 by PKC possibly occurs through the PHOSphorylation of Ser40 at the Neh2 domain of Nrf2 , which interacts with Keap1 [38,39] . The PHOSphorylation of Ser40 triggers a conformational change in Nrf2 , and thus Nrf2 is released from Keap1 . Free Nrf2 in the cytosol is translocated into nuclei or interacts with other protein kinases . We speculate that nuclear translocation of Nrf2 requires either PHOSphorylation by PKC or another serine or threonine kinase at Ser40 of the Neh2 domain , which disrupts the interactions of Nrf2 and Keap1 , leading to Nrf2 separating from Keap1 . With the exception of PI3K , ROS also participate in the nuclear translocation of Nrf2 ( Figure 3 ) . However , the detailed mechanism is not clear at the present time . ROS may change the intracellular redox state of HUVECs , triggering the formation of disulfide bonds between the SH groups of cysteine residues within protein molecules , thus altering the activities of those proteins ( see discussion below ) . In a previous study , we showed that shear stresses increase the amount of ROS in endothelial cells [45] . The generation of ROS may participate in many cellular responses and signal transduction . This study verified that ROS are important regulators of the nuclear translocation of Nrf2 . The intracellular redox state of HUVECs can be altered by ROS . On the other hand , many previous studies reported that shear stresses activate eNOS and increases the amount of endogenous NO [57,58] , which also affects the intracellular redox state of HUVECs . However , we found that shear-increased NO had no influence on Nrf2 translocation ( Figure 4 ) . The detailed mechanism of the regulation of Nrf2 translocation by the intracellular redox state remains to be elucidated . Wakabayashi et al .showed that both C273 and C288 residues are necessary for Keap1 to repress Nrf2 [62] . Based on our data in Figs. 3 &4 , we speculated that the redox state of HUVECs may influence Nrf2 translocation by changing the redox state of the C273 and C288 residues of Keap1 . There might be several possible mechanisms that alter the C273 and C288 residues of Keap1 and thus release Nrf2 . For example , it is possible that when the amount of ROS is increased , the SH groups on C273 and C288 may be oxidized to form disulfide bonds , thus triggering Keap1 to release Nrf2 and initiating Nrf2 nuclear translocation . It was reported that an elevated concentration of xenobiotics in the cytosol causes oxidative stress and induces the dissociation of Nrf2 from Keap1 through the above-mentioned ROS -mediated mechanism [31] . Another possibility is the nitrosylation of C273 and C288 residues of Keap1 which causes the release of Nrf2 , and this mechanism is carried out only when the concentration of NO in the cytosol is much higher than the basal condition . We speculated that the concentration of shear-induced NO was not high enough to trigger Nrf2 translocation . Thus , it is likely that the ROS -mediated mechanism plays a relatively more important role than the NO -mediated mechanism in regulating Nrf2 translocation . As for the intracellular sources of ROS , Li et al .and Hancock et al .showed that NADPH oxidases ( NOXs ) are a major source of ROS [63,64] , and in our preliminary study , we found that the use of small interfering ( si ) RNA against NOX2 repressed Nrf2 translocation ( data not shown ) . This result also supports our deduction that a change in the redox state of HUVECs triggers Nrf2 translocation . Results obtained from the gel shift assay further revealed that shear stress enhanced the ARE -binding activity of Nrf2 through the involvement of ROS ( Figure 5 ) . Shear-induced HO-1 protein expression was also suppressed by a PI3K inhibitor ( Figure 6 ) . Parallel experiments were conducted using H2O2 , a major ROS , as the stimulus , and similar results were obtained . These results provide evidence that shear-induced Nrf2 regulates HO-1 expression via binding to the ARE in the promoter region , and this regulation involves PI3K and ROS .