TRIM46 contributes to high glucose-induced ferroptosis and cell growth inhibition in human retinal capillary endothelial cells by facilitating GPX4 ubiquitination

Jingfa Zhang; Qinghua Qiu; Haiyan Wang; Chong Chen; Dawei Luo

doi:10.1016/j.yexcr.2021.112800

TRIM46 contributes to high glucose-induced ferroptosis and cell growth inhibition in human retinal capillary endothelial cells by facilitating GPX4 ubiquitination

Exp Cell Res. 2021 Oct 15;407(2):112800. doi: 10.1016/j.yexcr.2021.112800. Epub 2021 Sep 4.

Authors

Jingfa Zhang¹, Qinghua Qiu¹, Haiyan Wang¹, Chong Chen¹, Dawei Luo²

Affiliations

¹ Department of Ophthalmology, Shanghai General Hospital (Shanghai First People's Hospital), Shanghai Jiao Tong University, Shanghai, China; National Clinical Research Center for Eye Diseases, Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai Engineering Center for Precise Diagnosis and Treatment of Eye Diseases, Shanghai, China.
² Department of Ophthalmology, Shanghai General Hospital (Shanghai First People's Hospital), Shanghai Jiao Tong University, Shanghai, China; National Clinical Research Center for Eye Diseases, Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai Engineering Center for Precise Diagnosis and Treatment of Eye Diseases, Shanghai, China. Electronic address: dr-davie@yeah.net.

PMID: 34487731
DOI: 10.1016/j.yexcr.2021.112800

Abstract

Purpose: Increased permeability of retinal capillary endothelial cells is a key feature in the progression of diabetic retinopathy (DR). Precisely why and how diabetes causes dysfunction in retinal capillary endothelial cells is not well understood, making it challenging to explore more advanced therapeutics.

Methods: Cell proliferation was assessed by the Cell Counting Kit-8 assay. Ferroptosis was evaluated by measuring lipid reactive oxygen species levels by flow cytometry and determining malondialdehyde, superoxide dismutase, and glutathione peroxidase levels through biochemical assays. Western blot analysis and quantitative PCR were respectively used to check the expression of proteins and RNAs. Co-immunoprecipitation assays were used to confirm the interaction between TRIM46 and GPX4.

Results: High glucose (HG, 25 mM glucose) significantly suppressed cell growth, which could be reversed by the ferroptosis inhibitor, ferrostatin-1. HG treatment time-dependently induced ferroptosis in human retinal capillary endothelial cells (HRCECs) and induced TRIM46 expression. Lentiviral-mediated overexpression of TRIM46 decreased cell resistance against HG-induced ferroptosis, whereas knockdown showed the opposite effect. Administration of RSL3, a ferroptosis agonist, was able to reverse the protective effects of TRIM46 silencing. TRIM46 interacted with GPX4, an important enzyme that suppresses ferroptosis, and promoted GPX4 ubiquitination. Furthermore, lentiviral-mediated overexpression ofGPX4 ameliorated the effects of TRIM46 overexpression and conferred protection to cells against HG-induced ferroptosis.

Conclusion: TRIM46 and GPX4 form a regulatory pathway that controls HG-induced ferroptosis of HRCECs. Inhibiting this pathway or sustaining the expression of GPX4 enables cells to resist damage caused by HG. We provide new mechanistic insight into the pathology of DR and identified TRIM46 and GPX4 as two molecular targets for the development of effective drugs for DR treatment.

Keywords: Ferroptosis; TRIM46; Ubiquitination.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Death
Cell Proliferation
Endothelium, Vascular / drug effects
Endothelium, Vascular / metabolism
Endothelium, Vascular / pathology*
Ferroptosis*
Glucose / adverse effects*
Growth Inhibitors / adverse effects*
Humans
Phospholipid Hydroperoxide Glutathione Peroxidase / genetics
Phospholipid Hydroperoxide Glutathione Peroxidase / metabolism*
Reactive Oxygen Species
Retina / drug effects
Retina / metabolism
Retina / pathology*
Sweetening Agents / adverse effects
Tripartite Motif Proteins / metabolism*
Ubiquitination*

Substances

Growth Inhibitors
Reactive Oxygen Species
Sweetening Agents
Tripartite Motif Proteins
Phospholipid Hydroperoxide Glutathione Peroxidase
Glucose