Endothelial cell senescence suppresses argininosuccinate synthetase 1 expression by promoter methylation while laminar shear stress rescues it by a mechanism involving KLF4

Abstract

Argininosuccinate synthetase 1 (ASS1) regulates the provision of L‐arginine to nitric oxide synthase 3 (NOS3). Previous studies demonstrated that ASS1 and NOS3 expressions were enhanced by laminar shear stress (LSS), and ASS1 but not NOS3 expression was suppressed by endothelial cell senescence, so that the impaired NO production in senescent cells could be attributed to the insufficient expression of ASS1, but not of NOS3. The regulation of ASS1 expression by LSS and cellular senescence was investigated in human umbilical vein endothelial cells (HUVECs), focusing on Kruppel‐like factors (KLFs) and promoter methylation. The basal and stimulated expression levels of KLF4 were similar in young versus senescent HUVECs. Regulatory roles of KLF4 in ASS1 expression were confirmed by siRNA‐mediated knockdown and over‐expression of KLF4. Bisulphite sequencing analysis revealed that cellular senescence caused ASS1 promoter hypermethylation. Treatment of senescent HUVECs with 5′‐aza‐2′‐deoxycytidine to inhibit DNA methylation led to increases of ASS1 expression levels under both static and LSS conditions. ASS1 promoter reporter activity was lowered by cellular senescence or DNA methylation and it was restored by LSS or KLF4 over‐expression. This study indicated that endothelial ASS1 expression levels were regulated not only by KLFs but also by promoter methylation in response to LSS and cellular senescence.