Fluid shear stress inhibits TNF-alpha activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members

Abstract

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and IL-1 stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. TNF-alpha and IL-1 regulate gene expression in ECs, in part, by stimulating mitogen-activated protein kinases (MAPK), which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAPK in EC. To test this hypothesis, we determined the effects of flow (shear stress = 12 dynes/cm(2)) on TNF-alpha and IL-1-stimulated activity of three MAPK in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 and p38 activity but decreased JNK activity compared with static controls. TNF-alpha or IL-1 alone activated ERK1/2, p38, and JNK maximally at 15 min in HUVEC. Preexposing HUVEC for 10 min to flow inhibited TNF-alpha and IL-1 activation of JNK by 46% and 49%, respectively, but had no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, which inhibits flow-mediated ERK1/2 activation, prevented flow from inhibiting cytokine activation of JNK. Phorbol 12-myristate 13-acetate, which strongly activates ERK1/2, also inhibited TNF-alpha activation of JNK. These findings indicate that fluid shear stress inhibits TNF-alpha-mediated signaling events in HUVEC via the activation of the ERK1/2 signaling pathway. Inhibition of TNF-alpha signal transduction represents a mechanism by which steady laminar flow may exert atheroprotective effects on the endothelium.

Figure 1

Effect of flow, TNF-α, and IL-1 on ERK1/2, p38, and JNK activities. (A) HUVEC were maintained in static conditions or exposed to flow (shear stress = 12 dynes/cm²) for the indicated times. Cell lysates were prepared and analyzed for ERK1/2 activity by phospho-ERK Western blot, for p38 activity by phospho-p38 Western blot, and for JNK activity by in vitro kinase assay by using GST-jun as a substrate and anti-phospho-c-jun antibodies for detection. Error bars represent SEM (n = 5). HUVEC were stimulated with 10 ng/ml TNF-α (B) or 3 ng/ml IL-1 (C) for the indicated times and analyzed for MAPK activity. Analysis of different experiments was performed by normalizing autoradiographic intensity of control cells in each experiment to an arbitrary value of 1.0. Error bars represent SEM (n = 3).

Figure 2

Flow preexposure inhibits TNF-α and IL-1-mediated JNK activation, without effect on ERK1/2 and p38 activation. HUVEC were subjected to the following “preconditioning” protocol: maintained in static conditions for 25 min (Control), exposed to flow for 10 min and then held static for 15 min (Flow Alone), maintained in static conditions for 10 min followed by TNF-α (A) or IL-1 (B) stimulation for 15 min (TNF-α or IL-1 alone), or subjected to flow for 10 min followed by TNF-α (A) or IL-1 (B) stimulation for 15 min (Flow + TNF-α or IL-1). Cell lysates were prepared and analyzed for ERK1/2 activity by phospho-ERK Western blot, for p38 activity by phospho-p38 Western blot, and for JNK activity by in vitro kinase assay by using GST-jun as a substrate and anti-phospho-c-jun antibodies for detection. Densitometric analysis was performed as in Fig. 1. Error bars represent SEM (n = 5).

Figure 3

Flow preexposure inhibits TNF-α-mediated phosphorylation of c-jun. HUVEC were subjected to the same preconditioning protocol described in Fig. 2. Cell lysates were prepared and analyzed for c-jun phosphorylation by phospho-c-jun Western blot. Densitometric analysis was performed as in Fig. 1. Error bars represent SEM (n = 3).

Figure 4

Effect of PD98059 on ERK1/2 and JNK activation stimulated by flow and TNF-α. HUVEC were pretreated with 30 μM PD98059 for 30 min or vehicle and then subjected to the preconditioning protocol described in Fig. 2. Cells were then stimulated with TNF-α or subjected to flow followed by TNF-α stimulation in the presence or absence of 30 μM PD98059. In lanes 9 and 10, TNF-α was added for only 15 min in the absence (lane 9) or presence (lane 10) of PD98059 to show that ERK1/2 was inhibited. Assay of JNK activity was performed by using GST-c-jun as a substrate and anti-phospho-c-jun antibodies for detection and assay of ERK1/2 activity by phospho-ERK Western blot (A). Densitometry was performed as in Fig. 1. Error bars represent SEM (n = 3).

Figure 5

PMA activation of ERK1/2 inhibits TNF-α activation of JNK. HUVEC were treated with either vehicle for 25 min, 200 nM PMA for 25 min, vehicle for 10 min followed by TNF-α for 15 min, or 200 nM PMA for 10 min followed by TNF-α for 15 min. Cell lysates were prepared and analyzed for ERK1/2 activity by phospho-ERK Western blot and for JNK activity by in vitro kinase assay by using GST-jun as a substrate. Densitometric analysis was performed as in Fig. 1. Error bars represent SEM (n = 3).