Regulatory effects of AT₁R-TRAF6-MAPKs signaling on proliferation of intermittent hypoxia-induced human umbilical vein endothelial cells

Abstract

Endothelial dysfunction induced by intermittent hypoxia (IH) participates in obstructive sleep apnea syndrome (OSAS)-associated cardiovascular disorders. Myeloid differentiation primary response 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) regulate numerous downstream adaptors like mitogen-activated protein kinases (MAPKs) and the subsequent oxidative stress and inflammatory responses. This study aimed to characterize the role of MyD88/TRAF6 in IH-treated cell function and its associated signaling. Human umbilical vein endothelial cells (HUVECs) were randomly exposed to IH or normoxia for 0, 2, 4 and 6 h. Western blotting was used to detect the expression pattern of target gene proteins [angiotensin 1 receptor (AT₁R), p-ERK1/2, p-p38MAPK, MyD88 and TRAF6], and the relationships among these target genes down-regulated by the corresponding inhibitors were studied. Finally, the influence of these target genes on proliferation of HUVECs was also assessed by EdU analysis. Protein levels of AT₁R, TRAF6 and p-ERK1/2 were increased after IH exposure, with a slight rise in MyD88 and a dynamic change in p-p38MAPK. The down-regulation of TRAF6 by siRNA reduced ERK1/2 phosphorylation during IH without any effects on AT₁R. Blockade of AT₁R with valsartan decreased TRAF6 and p-ERK1/2 protein expression after IH exposure. ERK1/2 inhibition with PD98059 suppressed only AT₁R expression. IH promoted HUVECs proliferation, which was significantly suppressed by the inhibition of TRAF6, AT₁R and ERK1/2. The findings demonstrate that TRAF6 regulates the proliferation of HUVECs exposed to short-term IH by modulating cell signaling involving ERK1/2 downstream of AT₁R. Targeting the AT₁R-TRAF6-p-ERK1/2 signaling pathway might be helpful in restoring endothelial function.

Keywords

intermittent hyopxia / angiotensin 1 receptor / myeloid differentiation primary response 88 / tumor necrosis factor receptor-associated factor 6 / mitogen-activated protein kinases / cells proliferation

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