ANGPTL7 is transcriptionally regulated by SP1 and modulates glucocorticoid-induced cross-linked actin networks in trabecular meshwork cells via the RhoA/ROCK pathway

Mengsha Sun,Wenjia Liu,Minwen Zhou

doi:10.1038/s41420-022-00847-3

Abstract

Glaucoma is one of the leading causes of worldwide irreversible blindness. Lowering elevated intraocular pressure (IOP) is currently the only effective approach for controlling the progress of glaucoma. Angiopoietin-like 7 (ANGPTL7) takes a key part in elevated outflow resistance of aqueous humor in dysfunctional trabecular meshwork (TM), along with the formation of cross-linked actin networks (CLANs), leading to high IOP. In this study, we explored the role of the ANGPTL7 signaling pathway in CLAN formation. We detected the expression of ANGPTL7 in cultured primary TM cells treated with dexamethasone (DEX) and ethanol as a control using qRT-PCR and western blotting. Actin filaments were revealed by phalloidin staining. ANGPTL7 short hairpin RNA (shRNA) was applied to TM cells to examine the effect of ANGPTL7 on DEX-induced CLAN formation. Western blotting was used to assess the effect of ANGPTL7 on the RhoA/Rho-associated kinase (Rho-kinase/ROCK) signaling pathway. Bioinformatics, dual-luciferase reporter assays, and chromatin immunoprecipitation were employed to identify the transcription factors of ANGPTL7. Transcription factor specificity protein 1 (SP1) overexpression and silencing were performed to determine their roles in the modulation of ANGPTL7 expression. We found DEX-induced ANGPTL7 expression and stress fiber rearrangement in TM cells. ANGPTL7 knockdown effectively inhibited the formation of CLANs. Moreover, it was involved in the regulation of the RhoA/ROCK signaling pathway, further affecting DEX-induced CLAN formation. SP1 was identified as a transcription factor of ANGPTL7 which regulated ANGPTL7 level to mediate CLAN formation through the RhoA/ROCK signaling pathway. This study contributes to revealing the molecular mechanisms of ANGPTL7 in CLAN formation, which is involved in TM dysfunction and glaucoma pathogenesis.

Highlights

Glaucoma is a leading cause of worldwide irreversible vision loss, characterized by progressive optic neuropathy [1]
The messenger RNA expression of Angiopoietin-like 7 (ANGPTL7) was tested by Quantitative reverse transcription–polymerase chain reaction (qRT-PCR), and the protein expression of ANGPTL7 was examined by western blotting (WB)
ANGPTL7 regulated DEX-induced cross-linked actin networks (CLANs) formation To study the effect of ANGPTL7 on CLAN formation, we first identified the transfection effect of ANGPTL7 short hairpin RNA (shRNA) by measuring the messenger RNA (mRNA) and protein expressions of ANGPTL7 in trabecular meshwork (TM) cells which were transfected with scr shRNA and ANGPTL7 shRNAs before DEX treatment

Summary

Introduction

Glaucoma is a leading cause of worldwide irreversible vision loss, characterized by progressive optic neuropathy [1]. The most common form of glaucoma is primary open-angle glaucoma (POAG), which is always accompanied by high intraocular pressure (IOP), the key risk factor for the pathogenesis of POAG [2]. Prolonged use of dexamethasone (DEX) poses a high risk of elevated IOP and results in secondary glaucoma, which has many common characteristics with POAG [3, 4]. The pathogenesis of POAG can be deduced from the mechanisms underlying DEX-induced ocular hypertension. Understanding the DEX-induced molecular mechanisms may assist in developing therapies for glucocorticoid-induced glaucoma and POAG. High IOP is caused by increased outflow resistance of aqueous humor (AH) [5]. There is inadequate knowledge of the molecular mechanisms underlying CLAN formation.

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