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-κB (nuclear factor-kappa B) signaling. Indeed, luciferase assay shows TFEB suppresses NF-κB transcriptional activity. Mechanistically, TFEB suppresses IKK (IκB kinase) activity to protect IκB-α 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 IκBα level to suppress NF-κB activity. KLF2 mediates TFEB upregulation in response to laminar flow.
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-κB (nuclear factor-kappa B) signaling. Indeed, luciferase assay shows TFEB suppresses NF-κB transcriptional activity. Mechanistically, TFEB suppresses IKK (IκB kinase) activity to protect IκB-α 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 IκBα level to suppress NF-κB activity. KLF2 mediates TFEB upregulation in response to laminar flow.
| Original language | English |
|---|---|
| Pages (from-to) | 719-730 |
| Journal | Arteriosclerosis, Thrombosis, and Vascular Biology |
| Volume | 39 |
| Issue number | 4 |
| Online published | 28 Feb 2019 |
| DOIs | |
| Publication status | Published - Apr 2019 |
| Externally published | Yes |
Research Keywords
- Chemokines
- Diabetes mellitus
- Endothelial cells
- Monocytes
- Phosphorylation
