Endothelial TFEB (Transcription Factor EB) Restrains IKK (I&kgr;B Kinase)-p65 Pathway to Attenuate Vascular Inflammation in Diabetic db/db Mice

Abstract

Objective— TFEB (transcription factor EB) was recently reported to be induced by atheroprotective laminar flow and play an anti-atherosclerotic role by inhibiting inflammation in endothelial cells (ECs). This study aims to investigate whether TFEB regulates endothelial inflammation in diabetic db/db mice and the molecular mechanisms involved. Approach and Results— Endothelial denudation shows that TFEB is mainly expressed in ECs in mouse aortas. Western blotting shows TFEB total protein level decreases whereas the p-TFEB S142 (phosphorylated form of TFEB) increases in db/db mouse aortas, suggesting a decreased TFEB activity. Adenoviral TFEB overexpression reduces endothelial inflammation as evidenced by decreased expression of vascular inflammatory markers in db/db mouse aortas, and reduced expression of a wide range of adhesion molecules and chemokines in human umbilical vein ECs. Monocyte attachment assay shows TFEB suppresses monocyte adhesion to human umbilical vein ECs. RNA sequencing of TFEB-overexpressed human umbilical vein ECs suggested TFEB inhibits NF-&kgr;B (nuclear factor-kappa B) signaling. Indeed, luciferase assay shows TFEB suppresses NF-&kgr;B transcriptional activity. Mechanistically, TFEB suppresses IKK (I&kgr;B kinase) activity to protect I&kgr;B-&agr; from degradation, leading to reduced p65 nuclear translocation. Inhibition of IKK by PS-1145 abolished TFEB silencing-induced inflammation in human umbilical vein ECs. Lastly, we identified KLF2 (Krüppel-like factor 2) upregulates TFEB expression and promoter activity. Laminar flow experiment showed that KLF2 is required for TFEB induction by laminar flow and TFEB is an anti-inflammatory effector downstream of laminar flow-KLF2 signaling in ECs. Conclusions— These findings suggest that TFEB exerts anti-inflammatory effects in diabetic mice and such function in ECs is achieved by inhibiting IKK activity and increasing I&kgr;B&agr; level to suppress NF-&kgr;B activity. KLF2 mediates TFEB upregulation in response to laminar flow.