Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca 2+ /calmodulin-dependent protein kinase that mainly regulates protein translation. We have recently demonstrated that eEF2K protein increases in mesenteric artery from spontaneously hypertensive rats (SHR) compared with Wistar Kyoto rats. Pathogenesis of hypertension is modulated in part by vascular inflammation. We examined whether eEF2K mediates vascular inflammatory responses and development of hypertension. In human umbilical vein endothelial cells (HUVECs) and rat mesenteric arterial smooth muscle cells (SMCs), eEF2K phosphorylation was increased by TNF-α (48% increase in HUVECs and 46% increase in SMCs, n=5, P<0.01). In HUVECs, small interfering RNA (siRNA) against eEF2K inhibited induction of VCAM-1 (62% inhibition, n=4) and e-selectin (29% inhibition, n=6) as well as monocyte adhesion (43% inhibition, n=4) by TNF-α (P<0.01). eEF2K siRNA inhibited phosphorylation of JNK (76% inhibition) and NF-κB (54% inhibition) as well as reactive oxygen species (ROS) production (69% inhibition) by TNF-α (n=4, P<0.01). In SMCs, eEF2K siRNA inhibited VCAM-1 induction (41% inhibition, n=5, P<0.05) and phosphorylation of JNK (65% inhibition, n=6, P<0.01) and NF-κB (83% inhibition, n=4, P<0.05) by TNF-α. In vivo, increased blood pressure (systolic blood pressure, SHR; 199.5 mmHg vs. SHR+NH125; 159.4 mmHg, n=4, P<0.01) and increased eEF2K phosphorylation (68% inhibition, n=4), induction of VCAM-1 (62% inhibition, n=4, P<0.05) and hypertrophy (70% inhibition, n=4) in SHR mesenteric artery was normalized by long-term treatment with NH125 (500 μg/kg/day for 6 weeks). In SHR mesenteric artery, impairment of acetylcholine (ACh)-induced endothelium-dependent relaxation was normalized by NH125 (relaxation induced by 30 nM ACh, SHR; 60.4% vs. SHR+NH125; 90.0%, n=4, P<0.01). The present results for the first time demonstrated in cultured ECs and SMCs that eEF2K mediates TNF-induced inflammatory responses via ROS-dependent mechanism. It is also suggested that eEF2K may mediate development of hypertension in SHR likely via inflammation, hypertrophy and endothelial dysfunction.

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Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

Journal of Clinical Investigation ◽

10.1172/jci75776 ◽

2014 ◽

Vol 124 (4) ◽

pp. 1869-1869

Author(s):

Bárbara González-Terán ◽

José R. Cortés ◽

Elisa Manieri ◽

Nuria Matesanz ◽

Ángeles Verdugo ◽

...

Keyword(s):

Elongation Factor ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor ◽

Tnf Α

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Eukaryotic elongation factor 2 kinase regulates the development of hypertension through oxidative stress-dependent vascular inflammation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00373.2013 ◽

2013 ◽

Vol 305 (5) ◽

pp. H756-H768 ◽

Cited By ~ 24

Author(s):

Tatsuya Usui ◽

Muneyoshi Okada ◽

Yukio Hara ◽

Hideyuki Yamawaki

Keyword(s):

Superior Mesenteric Artery ◽

Mesenteric Artery ◽

Vascular Inflammation ◽

Elongation Factor ◽

Ros Production ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor ◽

Tnf Α ◽

Stress Dependent

Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca2+/calmodulin-dependent protein kinase. We recently demonstrated that eEF2K protein increases in mesenteric artery from spontaneously hypertensive rats (SHR). Pathogenesis of hypertension is regulated in part by vascular inflammation. We tested the hypothesis whether eEF2K mediates vascular inflammatory responses and development of hypertension. In vascular endothelial cells, small interfering RNA (siRNA) against eEF2K inhibited induction of VCAM-1 and endothelial-selectin as well as monocyte adhesion by TNF-α (10 ng/ml). eEF2K siRNA inhibited phosphorylation of JNK and NF-κB p65 as well as reactive oxygen species (ROS) production by TNF-α. In vascular smooth muscle cells, eEF2K siRNA also inhibited VCAM-1 induction and phosphorylation of JNK and NF-κB by TNF-α. In vivo, increased blood pressure in SHR and ROS production, induction of inflammatory molecules, and hypertrophy in SHR superior mesenteric artery were reduced by an eEF2K inhibitor NH125 (500 μg·kg−1·day−1). In SHR superior mesenteric artery, impairment of ACh-induced relaxation was normalized by NH125. The present results for the first time demonstrate that eEF2K mediates TNF-α-induced vascular inflammation via ROS-dependent mechanism, which is at least partly responsible for the development of hypertension in SHR.

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Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and −insensitive pathways

Biochemical Journal ◽

10.1042/bj20020916 ◽

2002 ◽

Vol 367 (2) ◽

pp. 525-532 ◽

Cited By ~ 63

Author(s):

Axel KNEBEL ◽

Claire E. HAYDON ◽

Nick MORRICE ◽

Philip COHEN

Keyword(s):

Protein Kinase ◽

Elongation Factor ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Eukaryotic Elongation Factor ◽

Tnf Α ◽

High Concentrations ◽

Eef2 Kinase ◽

Sb 203580

Eukaryotic elongation factor 2 (eEF2) kinase, the enzyme that inactivates eEF2, is controlled by phosphorylation. Previous work showed that stress-activated protein kinase 4 (SAPK4, also called p38Δ) inhibits eEF2 kinase in vitro by phosphorylating Ser-359, while ribosomal protein S6 kinases inhibit eEF2 kinase by phosphorylating Ser-366 [Knebel, Morrice and Cohen (2001) EMBO J. 20, 4360—4369; Wang, Li, Williams, Terada, Alessi and Proud (2001) EMBO J. 20, 4370—4379]. In the present study we have examined the effects of the protein synthesis inhibitor anisomycin and tumour necrosis factor-α (TNF-α) on the phosphorylation of eEF2 kinase. We demonstrate that Ser-359, Ser-366 and two novel sites (Ser-377 and Ser-396) are all phosphorylated in human epithelial KB cells, but only the phosphorylation of Ser-359 and Ser-377 increases in response to these agonists and correlates with the dephosphorylation (activation) of eEF2. Ser-377 is probably a substrate of MAPKAP-K2/K3 (mitogen-activated protein kinase-activated protein kinase 2/kinase 3) in cells, because eEF2 kinase is phosphorylated efficiently by these protein kinases in vitro and phosphorylation of this site, induced by TNF-α and low (but not high) concentrations of anisomycin, is prevented by SB 203580, which inhibits SAPK2a/p38, their ‘upstream’ activator. The phosphorylation of Ser-359 induced by high concentrations of anisomycin is probably catalysed by SAPK4/p38Δ in cells, because no other stress-activated, proline-directed protein kinase tested phosphorylates this site in vitro and phosphorylation is insensitive to SB 203580. Interestingly, the phosphorylation of Ser-359 induced by TNF-α or low concentrations of anisomycin is suppressed by SB 203580, indicating that phosphorylation is also mediated by a novel pathway. Since the phosphorylation of Ser-377 does not inhibit eEF2 kinase in vitro, our results suggest that anisomycin or TNF-α inhibit eEF2 kinase via the phosphorylation of Ser-359.

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