Atheroprotective Flow Upregulates ITPR3 (Inositol 1,4,5-Trisphosphate Receptor 3) in Vascular Endothelium via KLF4 (Krüppel-Like Factor 4)-Mediated Histone Modifications

Abstract

Objective— The topographical distribution of atherosclerosis in vasculature underscores the importance of shear stress in regulating endothelium. With a systems approach integrating sequencing data, the current study aims to explore the link between shear stress-regulated master transcription factor and its regulation of endothelial cell (EC) function via epigenetic modifications. Approach and Results— H3K27ac (acetylation of histone 3 lysine 27)-ChIP-seq (chromatin immunoprecipitation followed by high throughput sequencing), ATAC-seq (an assay for transposase-accessible chromatin-sequencing), and RNA-seq (RNA-sequencing) were performed to investigate the genome-wide epigenetic regulations in ECs in response to atheroprotective pulsatile shear stress (PS). In silico prediction revealed that KLF4 binding motifs were enriched in the PS-enhanced H3K27ac regions. By integrating PS- and KLF4-modulated H3K27ac, we identified 18 novel PS-upregulated genes. The promoter regions of these genes showed an overlap between the KLF4-enhanced assay for transposase-accessible chromatin signals and the PS-induced H3K27ac peaks. Experiments using ECs isolated from mouse aorta, lung ECs from EC-KLF4-TG versus EC-KLF4-KO mice, and atorvastatin-treated ECs showed that ITPR3 (inositol 1,4,5-trisphosphate receptor 3) was robustly activated by KLF4 and statins. KLF4 ATAC-qPCR (quantitative polymerase chain reaction) and ChIP-qPCR further demonstrated that a specific locus in the promoter region of the ITPR3 gene was essential for KLF4 binding, H3K27ac enrichment, chromatin accessibility, RNA polymerase II recruitment, and ITPR3 transcriptional activation. Deletion of this KLF4 binding locus in ECs by using CRISPR-Cas9 resulted in blunted calcium influx, reduced expression of endothelial nitric oxide synthase, and diminished nitric oxide bioavailability. Conclusions— These results from a novel multiomics study suggest that KLF4 is crucial for PS-modulated H3K27ac that allow the transcriptional activation of ITPR3. This novel mechanism contributes to the Ca 2+ -dependent eNOS (endothelial nitric oxide synthase) activation and EC homeostasis.

Epsin-mediated degradation of IP3R1 fuels atherosclerosis

“…IP3 receptors have also been implicated in playing an important role in atherosclerosis 28 , 29 . For instance, IP3R3 expression in human endothelial cells can be increased by atheroprotective pulsatile shear stress (PS) downstream of KLF4 48 . In support of this finding, we found IP3R1 mRNA and protein levels were significantly higher under a pulsatile shear stress of 12 dynes/cm 2 vs. oscillatory shear stress of 1 dynes/cm 2 using human aortic endothelial cells (Fig.…”

KLF4 Recruits SWI/SNF to Increase Chromatin Accessibility and Reprogram the Endothelial Enhancer Landscape under Laminar Shear Stress

“…6c). He and colleagues recently demonstrated that KLF4 regulates Inositol 1,4,5-trisphosphate receptor type 3 (IPTR3) expression by increasing chromatin accessibility and H3K27 acetylation at the ITPR3 promotor region 42 . In addition, many microRNAs are shear stress-regulated 43 , perhaps most notably, miR-92a, that targets KLF2 and KLF4 under conditions of disturbed flow 44,45 .…”

Angioplasty-induced epigenomic remodeling entails BRD4 and EZH2 hierarchical regulations

“…The histone acetylation reader BRD4 and H3K27me3 writer EZH2 are powerful epigenetic regulators, as reported for proliferative diseases 11,14,26 and implicated for IH. While rapidly growing, epigenetic studies concerning IH are mostly reliant on cell cultures 21,22 . Here we report in vivo genome-wide epigenetic survey in arteries that undergo injury-induced IH.…”

“…Epigenomic studies pertaining to IH have been limited and mostly confined to cell cultures 7,21,22 , which as oversimplified systems inevitably produce inaccurate information for interpreting in vivo pathogenic mechanisms 12,23 . Here we performed ChIPseq directly using rat carotid arteries that underwent angioplasty injury-induced IH.…”