Differential Effect of Shear Stress on Extracellular Signal-regulated Kinase and N-terminal Jun Kinase in Endothelial Cells

Shear stress differentially regulates production of many vasoactive factors at the level of gene expression in endothelial cells that may be mediated by mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and N-terminal Jun kinase (JNK). Here we show, using bovine aortic endothelial cells (BAEC), that shear stress differentially regulates ERK and JNK by mechanisms involving Gi2 and pertussis toxin (PTx)-insensitive G-protein-dependent pathways, respectively. Shear activated ERK with a rapid, biphasic time course (maximum by 5 min and basal by 30-min shear exposure) and force dependence (minimum and maximum at 1 and 10 dyn/cm2 shear stress, respectively). PTx treatment prevented shear-dependent activation of ERK1/2, consistent with a Gi-dependent mechanism. In contrast, JNK activity was maximally turned on by a threshold level of shear force (0.5 dyn/cm2 or higher) with a much slower and prolonged time course (requiring at least 30 min to 4 h) than that of ERK. Also, PTx had no effect on shear-dependent activation of JNK. To further define the shear-sensitive ERK and JNK pathways, vectors expressing hemagglutinin epitope-tagged ERK (HA-ERK) or HA-JNK were co-transfected with other vectors by using adenovirus-polylysine in BAEC. Expression of the mutant αi2(G203), antisense Gαi2 and a dominant negative Ras (N17Ras) prevented shear-dependent activation of HA-ERK, while that of αi2(G204) and antisense αi3 did not. Expression of a Gβ/γ scavenger, the carboxyl terminus of β-adrenergic receptor kinase (βARK-ct), and N17Ras inhibited shear-dependent activation of HA-JNK. Treatment of BAEC with genistein prevented shear-dependent activation of ERK and JNK, indicating the essential role of tyrosine kinase(s) in both ERK and JNK pathways. These results provide evidence that 1) Gi2-protein, Ras, and tyrosine kinase(s) are upstream regulators of shear-dependent activation of ERK and 2) that shear-dependent activation of JNK is regulated by mechanisms involving Gβ/γ, Ras, and tyrosine kinase(s). Shear stress differentially regulates production of many vasoactive factors at the level of gene expression in endothelial cells that may be mediated by mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and N-terminal Jun kinase (JNK). Here we show, using bovine aortic endothelial cells (BAEC), that shear stress differentially regulates ERK and JNK by mechanisms involving Gi2 and pertussis toxin (PTx)-insensitive G-protein-dependent pathways, respectively. Shear activated ERK with a rapid, biphasic time course (maximum by 5 min and basal by 30-min shear exposure) and force dependence (minimum and maximum at 1 and 10 dyn/cm2 shear stress, respectively). PTx treatment prevented shear-dependent activation of ERK1/2, consistent with a Gi-dependent mechanism. In contrast, JNK activity was maximally turned on by a threshold level of shear force (0.5 dyn/cm2 or higher) with a much slower and prolonged time course (requiring at least 30 min to 4 h) than that of ERK. Also, PTx had no effect on shear-dependent activation of JNK. To further define the shear-sensitive ERK and JNK pathways, vectors expressing hemagglutinin epitope-tagged ERK (HA-ERK) or HA-JNK were co-transfected with other vectors by using adenovirus-polylysine in BAEC. Expression of the mutant αi2(G203), antisense Gαi2 and a dominant negative Ras (N17Ras) prevented shear-dependent activation of HA-ERK, while that of αi2(G204) and antisense αi3 did not. Expression of a Gβ/γ scavenger, the carboxyl terminus of β-adrenergic receptor kinase (βARK-ct), and N17Ras inhibited shear-dependent activation of HA-JNK. Treatment of BAEC with genistein prevented shear-dependent activation of ERK and JNK, indicating the essential role of tyrosine kinase(s) in both ERK and JNK pathways. These results provide evidence that 1) Gi2-protein, Ras, and tyrosine kinase(s) are upstream regulators of shear-dependent activation of ERK and 2) that shear-dependent activation of JNK is regulated by mechanisms involving Gβ/γ, Ras, and tyrosine kinase(s).