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

Abstract

Shear stress differentially regulates production of many vasoactive factors at the level of gene expression in endothelial cells that may be mediated by mitogenactivated 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 G i2 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/cm 2 shear stress, respectively). PTx treatment prevented shear-dependent activation of ERK1/2, consistent with a G i -dependent mechanism. In contrast, JNK activity was maximally turned on by a threshold level of shear force (0.5 dyn/ cm 2 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 cotransfected with other vectors by using adenoviruspolylysine in BAEC. Expression of the mutant ␣ i2 (G203), antisense G␣ i2 and a dominant negative Ras (N17Ras) prevented shear-dependent activation of HA-ERK, while that of ␣ i2 (G204) and antisense ␣ i3 did not. Expression of a G␤/␥ scavenger, the carboxyl terminus of ␤-adrenergic receptor kinase (␤ARK-ct), and N17Ras inhibited sheardependent 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) G i2 -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 G␤/␥, Ras, and tyrosine kinase(s).Endothelial cells lining the inner vessel wall are in direct contact with flowing blood, which generates a frictional force, hemodynamic shear stress, acting on the surface of the endothelium. Hemodynamic shear stress controls vascular tone, vessel wall remodeling, interaction of blood cells with endothelium, coagulation, and fibrinolysis (1). The focal pattern of atherosclerotic lesions in areas of low and/or unstable shear stress further highlights the importance of shear stress in the atherogenic process (2, 3). Endothelial cells play a key role in shear-dependent vascular changes, sensing shear stress by an unidentified mechanoreceptor(s) followed by production of autocrine and paracrine factors (1). For example, hemodynamic shear stress selectively and differentially regulates production of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, platelet-derived growth factor-B, basic fibroblast growth factor, transforming growth factor ␤-1, tissue plasminogen activator,...

Shear stress stimulates phosphorylation of eNOS at Ser635by a protein kinase A-dependent mechanism

“…Bovine aortic endothelial cells (BAEC) harvested from descending thoracic aortas were maintained (37°C, 5% CO 2) in a growth medium [Dulbecco's modified Eagle's medium (DMEM) containing 1 g/l glucose (GIBCO) and 20% fetal bovine serum (FBS, Atlanta Biologicals) without antibiotics] (26). BAEC used in this study were between passages 5 and 10.…”

“…Aliquots of cell lysates (20 g protein each) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore) (26). The membrane was incubated with a primary antibody overnight at 4°C and then with a secondary antibody conjugated with alkaline phosphatases (1 h at room temperature), which were detected by a chemiluminescence method (26).…”

“…Cell lysates were clarified by spinning at 14,000 g for 15 min at 4°C. Protein content of each sample was measured by using a Bio-Rad DC assay (26).…”

Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells

“…Animals were designated as "in labor" when at least one pup was delivered. After mating, myometrial samples were collected on days 15,17,19,21,22,23, or 1 day postpartum (1PP; n ϭ 3). At the time of tissue collection, the ligated empty horn was separated from the gravid horn.…”

Vascular Responses to Shear Stress: The Involvement of Mechanosensors in Endothelial Cells

“…Treatment of endothelial cells with anti-sense G q oligonucleotides inhibits shear stress-induced Ras-GTPase activity, while the transfection of G 1 2 enhances the shear stress-induced Ras activation [78]. It has also been reported that the shear stress-mediated activation of ERK1/2 is abolished by the treatment of endothelial cells with the G i inhibitor pertussis toxin [79]. In addition, shear stress increases the activity of G proteins reconstituted in liposomes in the absence of protein receptors [80].…”