Biologically active fragment of a human tRNA synthetase inhibits fluid shear stress-activated responses of endothelial cells

Abstract

Human tryptophanyl-tRNA synthetase (TrpRS) is active in translation and angiogenesis. In particular, an N-terminally truncated fragment, T2-TrpRS, that is closely related to a natural splice variant is a potent antagonist of vascular endothelial growth factor-induced angiogenesis in several in vivo models. In contrast, full-length native TrpRS is inactive in the same models. However, vascular endothelial growth factor stimulation is only one of many physiological and pathophysiological stimuli to which the vascular endothelium responds. To investigate more broadly the role of T2-TrpRS in vascular homeostasis and pathophysiology, the effect of T2-TrpRS on well characterized endothelial cell (EC) responses to flow-induced fluid shear stress was studied. T2-TrpRS inhibited activation by flow of protein kinase B (Akt), extracellular signal-regulated kinase 1/2, and EC NO synthase and prevented transcription of several shear stress-responsive genes. In addition, T2-TrpRS interfered with the unique ability of ECs to align in the direction of fluid flow. In all of these assays, native TrpRS was inactive, demonstrating that angiogenesis-related activity requires fragment production. These results demonstrate that T2-TrpRS can regulate extracellular signal-activated protein kinase, Akt, and EC NO synthase activation pathways that are associated with angiogenesis, cytoskeletal reorganization, and shear stress-responsive gene expression. Thus, this biological fragment of TrpRS may have a role in the maintenance of vascular homeostasis.

Fig. 1.

T2-TrpRS inhibits shear-dependent activation of ERK. BAECs were incubated with T2-TrpRS or TrpRS for 15 min at 37°C. Cells were then subjected to static control or shear stress (12 dyn/cm² for 10 min). Cell lysates were analyzed by Western blots with antibodies specific to total ERK1/2 or phospho-ERK1/2 (pERK1/2). Values are means ± SE (n = 3) obtained from densitometric quantitation of pERK1 bands at 10 min after shear stress.

Fig. 2.

Effect of T2-TrpRS on phosphorylation of eNOS at Ser-1179 (A) and Akt activation (B) in response to shear stress. BAECs were incubated with T2-TrpRS or TrpRS for 15 min at 37°C. Cells were then subjected to static control or shear stress (12 dyn/cm² for 10 min). Cell lysates were analyzed by Western blots with antibodies specific to total eNOS or phospho-eNOS [p(Ser1172) eNOS] and total Akt or phospho-Akt [p(Ser473) Akt]. Values are means ± SE (n = 3).

Fig. 3.

T2-TrpRS blocks shear stress-induced cell alignment. BAECs were incubated with T2-TrpRS or TrpRS for 15 min at 37°C and subjected to shear stress for the indicated times or kept under static conditions. Cells were fixed and stained with rhodamine phalloidin. Direction of flow is indicated by arrow.

Fig. 4.

Transcriptional induction of SSRE- and TRE-luciferase vectors does not occur in the presence of T2-TrpRS. BAEC cultures were cotransfected with a vector containing the PDGF-A/SSRE regulating the expression of a firefly luciferase reporter gene and a Renilla luciferase regulated by a minimal promoter (A) or a vector containing the monocyte chemotactic protein 1/TRE regulating the expression of a firefly luciferase reporter gene and a Renilla luciferase regulated by a minimal promoter (B). The transfected cells were incubated under static conditions or exposed to laminar shear stress (12 dynes/cm², 60 min), after being treated with TrpRS or T2-TrpRS, and the expression of the firefly luciferase was normalized to the expression of the Renilla luciferase. Statistical analysis was carried out on three independent experiments in which samples were in duplicate (P < 0.05).