Roles of KLF4 and AMPK in the inhibition of glycolysis by pulsatile shear stress in endothelial cells

Abstract

Vascular endothelial cells (ECs) sense and respond to hemodynamic forces such as pulsatile shear stress (PS) and oscillatory shear stress (OS). Among the metabolic pathways, glycolysis is differentially regulated by atheroprone OS and atheroprotective PS. Studying the molecular mechanisms by which PS suppresses glycolytic flux at the epigenetic, transcriptomic, and kinomic levels, we have demonstrated that glucokinase regulatory protein (GCKR) was markedly induced by PS in vitro and in vivo, although PS down-regulates other glycolysis enzymes such as hexokinase (HK1). Using next-generation sequencing data, we identified the binding of PS-induced Krüppel-like factor 4 (KLF4), which functions as a pioneer transcription factor, binding to the GCKR promoter to change the chromatin structure for transactivation of GCKR. At the posttranslational level, PS-activated AMP-activated protein kinase (AMPK) phosphorylates GCKR at Ser-481, thereby enhancing the interaction between GCKR and HK1 in ECs. In vivo, the level of phosphorylated GCKR Ser-481 and the interaction between GCKR and HK1 were increased in the thoracic aorta of wild-type AMPKα2^+/+ mice in comparison with littermates with EC ablation of AMPKα2 (AMPKα2^-/-). In addition, the level of GCKR was elevated in the aortas of mice with a high level of voluntary wheel running. The underlying mechanisms for the PS induction of GCKR involve regulation at the epigenetic level by KLF4 and at the posttranslational level by AMPK.

Keywords: AMPK; GCKR; KLF4; epigenetics; glycolysis.

Fig. 1.

PS inhibits the expression of glycolysis genes in ECs. (A) Pathway diagram generated from RNA-seq data (GSE103672) analyzing HUVECs exposed to PS (12 ± 4 dyn/cm²) or OS (1 ± 4 dyn/cm²) for 0 to 24 h. The PS/OS fold changes of the mRNAs are shown with colors indicated in the scale on the Upper Right. Data are represented as the average fold changes at 12-, 16-, and 24-h time points. (B) Relative PS/OS mRNA levels of indicated glycolytic genes (detected by qPCR) in HUVECs exposed to PS or OS for 16 h. (C) ATAC-seq analysis of HUVECs exposed to PS or OS for 16 h in three biological repeats. The promoter regions of the glycolysis genes exhibited ATAC peak enrichment under PS (area in blue) or OS (area in red). Data are mean ± SEM from three independent experiments.

Fig. 2.

KLF4 regulates the expression of GCKR in response to PS. (A) Time course of PS/OS fold changes in mRNA level with a “glycolysis” gene ontology categorization in ECs exposed to PS or OS for 24 h. (B) qPCR analysis of GCKR mRNA expression in HUVECs subjected to PS or OS at 4, 8, and 16 h. (C) Level of H3K27ac in the GCKR promoter, which is annotated with the regions screened for TF binding sites. (D) RNA-seq data showing PS/OS fold change of TFs identified in the GCKR promoter as illustrated in C. (E) GCKR fold change and significance in RNA-seq data from HUVECs infected with Ad-null or Ad-KLF4 for 48 h. (F–H) HUVECs were infected with Ad-KLF4 for 48 h. Level of KLF4 binding to the GCKR promoter is shown in F, GCKR mRNA abundance in G, and GCKR protein abundance in H. (I) Levels of GCKR mRNA in HUVECs transfected with control (Ctrl) siRNA or KLF4 siRNA and subjected to PS or OS, respectively. (J) Levels of GCKR protein in HUVECs transfected with Ctrl siRNA or KLF4 siRNA and subjected to PS. (K and L) HUVECs were subjected to PS or OS. KLF4 binding to the GCKR promoter is shown in K and GCKR protein abundance in L. (M) GCKR mRNA abundance in the TA and AA from 7-wk-old C57BL/6 mice. *P < 0.05. Data are mean ± SEM from three independent experiments.

Fig. 3.

AMPK phosphorylates and regulates GCKR. (A) Number of species that contain GCKR phosphorylation sites phosphorylated by various kinases as indicated. (B) In vitro kinase assays with immunopurified GCKR or GCKR S481A (dephosphomimetic) with or without recombinant AMPK. (C) Compared with OS, PS increased the phosphorylation of GCKR S481 in AMPK^+/+ MEFs at 10 and 60 min. In contrast, AMPK^−/− MEFs showed little phosphorylation of GCKR S481. (D and E) HUVECs were transfected with GCKR S481, S481A, or S481D (phosphomimetic) expression plasmids for 2 d. GCKR coimmunoprecipitated with HK1 is shown in D and HK1 activity in E. (F and G) Aortic tissues were isolated from the TA parts of AMPKα2^+/+ and AMPKα2^−/− mice. Level of GCKR Ser-481 phosphorylation is shown in F and interaction with HK1 in G. *P < 0.05. Data are mean ± SEM from three independent experiments.

Fig. 4.

KLF4-AMPK/GCKR inhibition of glycolysis in mice with high level of voluntary wheel running. TAs were isolated and pooled from 7-wk-old mice with high level of voluntary wheel running (HR, three male) and nonselected control-line mice (C, three male), all of which had wheel access for 4 wk. (A) qPCR analysis of mRNA levels of glycolysis genes in the isolated TAs. (B) KLF4 binding to the GCKR promoter, detected by ChIP-PCR. (C) qPCR analysis of GCKR mRNA level. (D and E) Western blots of GCKR, KLF4 and phosphorylated GCKR Ser-481 and GCKR. (F) GCKR coimmunoprecipitated with HK1. (G) HK1 activity. Data are mean ± SEM from three mice. *P < 0.05.

Fig. 5.

Summary diagram of PS inhibition of glycolysis in ECs via KLF4 and AMPK.