Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells

Abstract

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.

FIG. 1.

Effects of Rho/ROCK on eNOS gene expression. (A) HUVECs were infected with recombinant adenovirus (rAd) expressing HA-tagged LacZ, active Rho (Rho63), dominant-negative Rho (Rho19), active ROCK (CAT), and dominant-negative ROCK (RB) as indicated, at titers ranging from 100 to 150 MOI, and incubated in 0.2% FCS-RPMI 1640 supplemented with ECGS for 48 h. Cells were then extracted without any treatment, and the expression of the transgenes were analyzed by Western blotting with anti-HA antibody 12CA5. (B) The effect of the various transgenes on eNOS expression was examined by Western blotting with anti-eNOS antibody. (C) HUVECs were infected with recombinant adenovirus as indicated and serum-starved in 0.2% FCS culture medium for 24 h, followed by treatment with thrombin (4 U/ml) for 48 h. The expression of eNOS was detected by Western blotting. Actin in lower panels served as control for loading.

FIG. 2.

Active Rho or ROCK inhibits phosphorylation and activity of PKB without affecting p85-PI 3-kinase activity. (A) HUVECs were infected and extracted as described in Fig. 1A. Panel a shows the effect of Rho or ROCK mutants on PKB activity. A total of 30 μg of lysates was used for immunoprecipitation with immobilized Akt 1G1 monoclonal antibody and subjected to in vitro kinase assay with GSK-3 as substrate. Phosphorylation of GSK-3 was measured by Western blot with phospho-GSK-3α/β (Ser-21/9) antibody. In panel b, the extent of PKB phosphorylation was analyzed by Western blotting with antibodies specific for phosphorylated Thr-308 or Ser-473 of PKB as indicated, whereas total PKB expression was detected by Western blotting with antibodies that recognize PKB regardless of their phosphorylation status (lower panel). Shown are representative Western blots of five independent experiments. (B) HUVECs were either uninfected or infected, as indicated, and extracted at 18 h p.i. To inhibit PI 3-kinase, cells were treated with 250 nM wortmannin (WM) for 2 h before extraction. Cell lysates were assayed for PKB phosphorylation at Ser-473. (C) Lanes 1 to 6, HUVECs were either left uninfected or were infected as indicated and extracted at 18 h p.i. Cell lysates were assayed for PKB phosphorylation at Ser-473 (panel a) and in vitro PI 3-kinase activity in anti-phosphotyrosine (PY) immunoprecipitates (panel b) as described in Materials and Methods. Quantification of signal intensities by using the average of five independent experiments is shown in the right panels. Lanes 7 and 8, cell lysates prepared from untreated and PDGF-treated (50 ng/ml of PDGF-BB for 10 min) hSMCs were included as a positive control for PI 3-kinase activation.

FIG. 3.

Rho inhibits phosphorylation of PKB and eNOS in parallel via its downstream target ROCK. Cells were infected as indicated and extracted at 18 h p.i. A representative Western blot from five (A) or three (B) independent experiments is shown under various conditions as indicated. Quantification of the data is shown in the corresponding right panels. (A) The extent of eNOS phosphorylation was analyzed by Western blotting with antibodies specific for phosphorylated Ser-1177 of eNOS (upper panel), whereas eNOS expression was analyzed by Western blotting with antibodies that recognize eNOS regardless of its phosphorylation status (lower panel). In parallel, under the same experimental conditions, eNOS activity was monitored by analyzing the cellular NO production measured as the formation of its coproduct [¹⁴C]citrulline. (B) The extent of eNOS and PKB phosphorylation was analyzed as described in Fig. 3A and 2, respectively. (C) The expression of HA-tagged LacZ, RB, and Rho63 was detected by Western blotting with monoclonal anti-HA antibody 12CA5.

FIG. 4.

Active PKB reversed Rho63-mediated inhibition of phosphorylation but not of the expression of eNOS. Cells were infected as indicated, and the cell lysates were prepared 18 h p.i. (A and B) or 48 h p.i. (C). A representative Western blot from five independent experiments is shown under various conditions as indicated. Quantification of the data by using an average of five independent experiments is shown in the corresponding right panels. (A) Effect of PKB on Rho63-mediated dephosphorylation of eNOS and PKB and on cellular NO production measured as the formation of [¹⁴C]citrulline. (B) The expression of HA-tagged LacZ, active PKB (m/p), inactive PKB (KA), and Rho63 detected by Western blot with monoclonal anti-HA antibody 12CA5. (C) Effect of PKB on Rho63-mediated downregulation of eNOS gene expression.

FIG. 5.

Role of Rho and PKB in thrombin-mediated downregulation of eNOS phosphorylation and expression. (A) Time course of Rho activation by thrombin. HUVECs were serum starved for 24 h and then treated with 4 U of thrombin/ml for the indicated time. The cell lysates were subjected to pull-down assay of GTP-Rho as described in Materials and Methods. (B) Effect of dominant-negative Rho (Rho19) and active PKB (m/p) on thrombin-induced dephosphorylation of eNOS and PKB. Cells were infected as indicated and serum starved in 0.2% FCS culture medium without ECGS for 24 h. The cells were then either left untreated (−) or were treated with 4 U of thrombin/ml (+) for 15 min. (C) Effect of Rho19 and m/p-PKB on thrombin-mediated downregulation of eNOS gene expression. Cells were infected and serum starved as in panel B, except that cells were treated with thrombin for 48 h instead of 15 min. Shown are representative blots from three (panel A) or five (panels B and C) independent experiments. Quantification of the data under each condition is shown in the corresponding right panels.