17β-Estradiol exposure leads to activation of ERK and p38 but not JNK in vascular endothelial cells

Abstract

Estrogen has been shown to increase endothelial cell (EC) production of nitric oxide (NO) by the rapid activation of endothelial nitric oxide synthase (eNOS). To better understand the mechanisms by which estrogen acutely increases endothelial NO production, proteins of the mitogen activated protein kinase (MAPK) family, ERK, JNK, and p38 were examined to determine their potential role in this process. Bovine aortic endothelial cells (BAEC) were grown to confluence in phenol red-free DMEM F-12, supplemented with 10% fetal calf serum, on 35 cm² dishes. After confluence, cells were starved for 12–24 hours and then exposed to 10 nM 17β-estradiol (E₂) for 1–60 minutes. The cells were then lysed and proteins resolved by SDS-PAGE followed by transfer to nitrocellulose membranes. Membranes were then probed with anti-phosphospecific antibodies for ERK, JNK, and p38. Antibodies specific for total ERK and JNK were used to assure even protein loading. The level of ERK phosphorylation rapidly increased in biphasic pattern after E2 exposure: 1 min, 4.3 fold; 5 min, 1 fold; and 10 min, 7.3 fold (max). However, the level of JNK phosphorylation did not increase throughout the time course. The level of p38 phosphorylation increased rapidly to 3.6 fold at 10 minutes. We demonstrate that E2 rapidly activates ERK in a biphasic manner and p38 in a monophasic manner, but has no effect on the activity of JNK. Our data suggest that rapid eNOS activation by estrogen in vascular endothelial cells is predominantly mediated through ERK and p38 pathway. Rapid activation of ERK and p38 resulting in NO production may substantially contribute to the atheroprotective effect of estrogen.