Shear stress increases Nrf2 protein expression and induces Nrf2 translocation into nuclei. (A) Shear stress increased the total amount of Nrf2 protein. Human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress at a magnitude of 12 dyne/cm² for the indicated times. Total cell lysates were recovered and subjected to Western blot analysis with anti-Nrf2 and anti-tubulin (internal control, used to indicate equal loading of protein in each lane) antibodies. (B) Shear stress triggered the translocation and accumulation of Nrf2 protein in nuclei. HUVECs were exposed to laminar shear stress (12 dyne/cm²) for the indicated times. Nuclear extracts were isolated and subjected to Western blotting with anti-Nrf2 and anti-nucleolin (internal control) antibodies. (C) Shear stress increased Nrf2 mRNA. HUVECs were exposed to laminar shear stress (12 dyne/cm²) for the indicated times. Total RNA was isolated, and Nrf2 and GAPDH (internal control) mRNA levels were detected by RT-PCR. Results are representative of three independent experiments.

ROS are necessary for Nrf2 nuclear translocation. HUVECs were pretreated with NAC, a reactive oxygen species (ROS) scavenger (10 mM), for 30 min and then kept as a static control or exposed to shear stress (12 dyne/cm²) in the presence of NAC for 30 min. Nuclear extracts were prepared and subjected to Western blotting with anti-Nrf2 and anti-nucleolin (internal control) antibodies. Similar results were obtained from repeated experiments.

Shear stress increases the antioxidant response element (ARE)-binding activity of Nrf2 through reactive oxygen species (ROS). HUVECs were kept in a static condition (Control), exposed to shear stress (SS) of 12 dyne/cm² for 2 h, incubated with H₂O₂ (200 μM) for 2 h (H₂O₂), or pretreated with NAC (10 mM) (NAC) and then incubated with H₂O₂ (200 μM) for 2 h (NAC+H₂O₂). Before applying the shear stress, HUVECs were pretreated with NAC (10 mM) for 60 min and then exposed to shear stress for 2 h (NAC+SS). Total nuclear extracts were prepared and analyzed by EMSA using a biotin-labeled oligonucleotide probe containing Nrf2 consensus binding sites corresponding to the HO-1 promoter region (5'-GGG ACT GGT GAC TCA GCA AAA TCT-3', within the promoter region lying about 4 kb upstream of the human HO-1 gene). The specificity of the Nrf2 binding was assessed by preincubating nuclear extracts with the biotin-labeled oligonucleotide probe in the presence of 100× unlabeled oligonucleotide probe to compete for Nrf2 binding (Competition). EMSA performed on the nuclear extracts preincubated with an Nrf2 antibody (SS+Nrf2 Ab) was also included. Results are representative of duplicate experiments with similar results.

PI3K is involved in shear-induced Nrf2 nuclear translocation. (A) PI3K is involved in shear-induced translocation of Nrf2 into nuclei. HUVECs were pretreated with LY294002, a PI3K inhibitor (50 μM), for 60 min and then kept as static controls or exposed to laminar shear stress (12 dyne/cm²) in the presence of LY294002 for 120 min. Nuclear extracts were prepared and subjected to Western blotting with an anti-Nrf2 antibody. The cytoplasmic fractions were subjected to Western blotting with an anti-tubulin (internal control) antibody. (B) PKC is involved in Nrf2 activation. HUVECs were pretreated with calphostin C, a PKC inhibitor (200 nM), or DMSO as a negative control for 30 min, and then exposed to laminar shear stress (12 dyne/cm²) or kept in a static condition for 2 h. After treatment, nuclear extracts were prepared and subjected to SDS-PAGE and immunoblotted with anti-Nrf2 and anti-nucleolin (internal control) antibodies. Similar results were obtained from repeated experiments. (C) p38 is not involved in shear-induced Nrf2 translocation. HUVECs were pretreated with SB203580, a p38 inhibitor (10 μM), for 30 min, and then kept as a static control or exposed to shear stress in the presence of inhibitors for 60 min. Nuclear extracts were prepared and subjected to Western blotting with anti-Nrf2 and anti-αC23 (internal control) antibodies. Similar results were obtained from repeated experiments. (D) JNK is not involved in shear-induced Nrf2 translocation. HUVECs were pretreated with SP600125, a JNK inhibitor (10 μM), for 60 min, and then kept as a static control or exposed to shear stress in the presence of the inhibitor for 60 min. Nuclear extracts were prepared and subjected to Western blotting with anti-Nrf2 and anti-αC23 (internal control) antibodies. Similar results were obtained from repeated experiments.

Shear-induced Nrf2 nuclear translocation is not affected by nitric oxide synthase (NOS) inhibitors. (A and B) Shear-induced NO is not involved in the Nrf2 nuclear translocation. HUVECs were (A) pretreated with L-NAME, an NOS inhibitor (300 μM), for 1 h or (B) pretreated with L-NNA, an NOS inhibitor (250 μM), for 1 h, and then kept as a static control or exposed to shear stress in the presence of the inhibitor for 2 h. Nuclear extracts were prepared and subjected to Western blotting with anti-Nrf2 and anti-nucleolin (internal control) antibodies. Similar results were obtained from repeated experiments.

Shear stress and hydrogen peroxide induce HO-1 expression via a PI3K-dependent pathway. (A) Shear stress increased HO-1 protein expression. HUVECs were exposed to laminar shear stress (12 dyne/cm²) for the indicated times. After cell lysis, total cell lysates were prepared and subjected to Western blotting with anti-HO-1 and anti-tubulin (internal control) antibodies. (B) PI3K is involved in shear-induced HO-1 protein expression. HUVECs were pretreated with LY294002, a PI3K inhibitor (50 μM) for 60 min and then kept as static controls or exposed to shear stress in the presence of LY294002 for 4 h. Total cell lysates were subjected to Western blotting with anti-HO-1, anti-phospho-Akt, and anti-Akt (internal control) antibodies. (C) Hydrogen peroxide (H₂O₂) increased HO-1 protein expression. HUVECs were incubated with H₂O₂ (200 μM) for the indicated times. Total cell lysates were subjected to Western blotting with anti-HO-1 and anti-tubulin (internal control) antibodies. (D) PI3K is involved in H₂O₂-induced HO-1 protein expression. HUVECs were either kept as a static control or treated with H₂O₂ (200 μM) for 4 h or pretreated with LY294002, a PI3K inhibitor (50 μM), for 60 min. Total cell lysates were subjected to Western blotting with anti-HO-1, anti-phospho-Akt, and anti-tubulin (internal control) antibodies. Results are representative of duplicate experiments with similar results.