Regulatory Mechanisms of Shear Stress-induced Signal Transduction Pathway of Transcription Factor Nrf2 in Endothelial Cells

Nrf2 belongs to the Cap’n’Collar (CNC) family. When activated, Nrf2 can bind to the antioxidant response element (ARE) region and initiate the expression of antioxidant genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase-1 (NQO1), and then trigger the antioxidant protection mechanism.

In this study, we used the human umbilical vein endothelial cells (HUVECs) to probe into the regulation of shear-induced Nrf2 activation. After exposed to different shear stresses of 5, 12, 25 dyn/cm2, we found that cells exposed to shear stress of 5 dyn/cm2 had the greatest Nrf2 protein expression and nuclear translocation level while the mRNA level seemed to be greatest for cells exposed to shear stress of 12 dyn/cm2, indicating that HUVECs might need higher Nrf2 activation level for better antioxidant protection against low shear stress, also the regulation of Nrf2 stability might play an important role in this aspect. Previously it has been reported that shear stress can induce Nrf2 activation and the downstream antioxidant gene HO-1 expression through PI3K pathway, and in this study we tried to elucidate the detailed mechanism. We discovered that the MAPK family members, JNK and p38, as well as the PI3K downstream targets, Akt, eNOS, and PKA, might not be directly involved in the signaling pathway of shear-induced Nrf2 activation. When applying long-effect NO donor (NOC18) and short-effect NO donor (SNAP) as stimulators, Nrf2 would be activated and the activation level was dependent of the amount of NO released by NO donors. However, the NO scavenger PTIO and eNOS inhibitor L-NMMA exerted no effect on the shear-induced Nrf2 nuclear translocation. With the above information, we speculate that perhaps the amount of NO released by HUVECs exposed to shear stress is still too low in comparison to NO donors, which may not be enough for activation of Nrf2. Moreover, when HUVECs were pretreated with the reactive oxygen species (ROS) scavenger NAC, the shear-induced Nrf2 protein and mRNA level would be both inhibited, which indicates that by increasing the cellular ROS level, shear stress could increase the transcriptional Nrf2 mRNA level, and in turn activate Nrf2. When trying to investigate the effect of NADPH oxidase on shear-induced Nrf2 activation, it was found that the NADPH oxidase inhibitor DPI exhibited some undesirable side-effects, therefore we could not identify the role of NADPH oxidase yet. In addition, PMA was also chosen as an inducer in this study. We demonstrated that PMA could induce Nrf2 nuclear-tranlocation, and PI3K, PKC, and NADPH oxidase might be involved in the PMA-induced Nrf2 activation mechanism.

Taken together, we proposed a hypothetical mechanism of shear-induced Nrf2 activation: when HUVECs were exposed to oxidative stress, the membrane-bound PI3K would be first activated, and the PI3K product PI(3,4,5)P3 might in turn activate NADPH oxidase through a series activation of proteins, which could alter the redox-status in cells through the increase of ROS, thus causing Nrf2 to translocate into nucleus and form a heterodimer with small Maf protein. This heterodimer would finally bind to the ARE region, initiating downstream antioxidant genes and hence providing the athero-protective effects.