Angiotensin II (Ang II) stimulates hypertrophy in vascular smooth muscle cells (VSMCs) primarily through the G protein-coupled receptor (GPCR) Ang II type 1 receptor (AT1R) at the cell membrane and after its internalization. Major outputs of AT1R leading to vascular hypertrophy are triggered by reactive oxygen species (ROS)-dependent transactivation of the EGF receptor (EGFR), followed by activation of p38MAPK, Akt, p70S6K, and ERK1/2. However, underlying regulatory mechanisms are largely unknown. The small GTPase Rab5 and its downstream effector early endosome antigen 1 (EEA1) regulate endosomal internalization of GPCR. We hypothesized that EEA1 could be a scaffold facilitating the multiple outputs of AT1R signaling including those involved in hypertrophy. We found that Ang II (0.1
μ
M, 5 min) and H2O2 (100
μ
M, 5 min) induce formation of a complex composed of EEA1, Rab5, AT1R and EGFR that is inhibited by PEG-catalase (100 U/ml) and PEG-superoxide dismutase (SOD, 200 U/ml), inferring the redox sensitivity of the process. To explore this possibility further, we showed that Ang II and H2O2 stimulate EEA1 threonine phosphorylation (3.0- and 2.0-fold, respectively) co-temporaneously with Ang II- and H2O2-induced complex formation. Inhibition of p38MAPK by inhibitors (SB202190, PD169316; 1
μ
M) or treatment with PEG-catalase and PEG-SOD prevents EEA1 phosphorylation and its recruitment to the complex. To gain further insight, we used EEA1 siRNA to knock down EEA1 (80%). EEA1 siRNA significantly inhibits (79% control) Ang II-stimulated [3H]leucine incorporation in VSMC and is associated with decrease in Akt phosphorylation and its downstream p70S6K (80% and 50%, respectively) without affecting ROS-independent Erk1/2 phosphorylation. EEA1 siRNA does not affect Ang II-induced EGFR phosphorylation.
Conclusion: ROS- and p38MAPK-dependent Ang II-induced EEA1 phosphorylation is involved in formation of a signaling complex (AT1R, EGFR, EEA1, Rab5) that is related to Ang II stimulated, AT1R- and EGFR-mediated vascular hypertrophy. Akt-dependent phosphorylation of the p70S6K kinase is also EEA1-dependent. These findings provide insight into a novel role for EEA1 in Ang II signaling in VSMC hypertrophy.
Nox1‐derived reactive oxygen species (ROS) mediate thrombin‐induced influx of calcium in vascular smooth muscle cells (SMC)
