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Semaphorin 3A Inhibits Endoplasmic Reticulum Stress Induced by High Glucose in Müller Cells
Mengyang Li1,2, Enzhong Jin1,2, Li Zhu1,2
1Department of Ophthalmology, Peking University People's Hospital, Beijing, China.
Purpose:
This study aimed to explore the effect of the Semaphorin3A (Sema3A)/Neuropilin-1 (Nrp-1) pathway on Müller cell activities and endoplasmic reticulum (ER) stress induced by high glucose (HG) in vitro.
Methods:
The primary Müller cells of C57BL/6J mice were isolated and cultured in normal or high glucose medium. The expression of endogenous Sema3A and its coreceptor Nrp-1 was measured by Western blot. Müller cells were incubated with exogenous recombinant Sema3A protein or transfected with lentiviral vectors expressing small hairpin RNA (shRNA) to knock down the expression of endogenous Sema3A. The proliferation of Müller cells was detected by CCK-8 assay and EdU staining. The migratory ability was detected by the Transwell migration assay. The level of endoplasmic reticulum (ER) stress was analyzed through the detection of GRP78/BiP, IRE1α, phosphorylated IRE1αS724 (p-IRE1αS724), and the splicing rate of XBP1 (XBP1s/XBP1) by using immunofluorescence, Western blot or quantitative polymerase chain reaction (qPCR).
Results:
HG induced the upregulation of endogenous Sema3A and Nrp-1 receptors in Müller cells. The expression of GRP78/BiP and IRE1α was upregulated by HG, with an increased splicing rate of XBP1. Exogenous Sema3A inhibited HG-induced Müller cell proliferation, migration, and GRP78/BiP-IRE1α-XBP1 axis activation. Knockdown of Sema3A promoted proliferation, migration, and ER stress induced by high glucose in Müller cells.
Conclusion:
Sema3A inhibited the increased proliferative and migratory activities induced by high glucose by attenuating ER stress in Müller cells.
Insights
Semaphorin3A (Sema3A) inhibits high glucose-induced Müller cell proliferation and migration by reducing endoplasmic reticulum (ER) stress. This pathway is crucial for understanding diabetic retinopathy pathogenesis.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Müller cells play a critical role in retinal homeostasis.
- High glucose (HG) conditions induce cellular stress, impacting Müller cell function.
- The Semaphorin3A (Sema3A)/Neuropilin-1 (Nrp-1) pathway's role in HG-induced Müller cell dysfunction is not fully understood.
Purpose of the Study:
- To investigate the effect of the Sema3A/Nrp-1 pathway on Müller cell activities under high glucose conditions.
- To determine the involvement of endoplasmic reticulum (ER) stress in this process.
- To elucidate the potential therapeutic implications of modulating this pathway.
Main Methods:
- Primary mouse Müller cells were cultured in normal or high glucose media.
- Sema3A and Nrp-1 expression were analyzed using Western blot.
- Cell proliferation, migration, and ER stress markers (GRP78/BiP, IRE1α, p-IRE1αS724, XBP1 splicing) were assessed.
- Sema3A was manipulated using recombinant protein and shRNA knockdown.
Main Results:
- High glucose upregulated endogenous Sema3A and Nrp-1 in Müller cells.
- HG induced ER stress, evidenced by increased GRP78/BiP, IRE1α, and XBP1 splicing.
- Exogenous Sema3A inhibited HG-induced proliferation, migration, and ER stress.
- Sema3A knockdown exacerbated HG-induced Müller cell proliferation, migration, and ER stress.
Conclusions:
- Sema3A attenuates high glucose-induced Müller cell proliferation and migration.
- The Sema3A/Nrp-1 pathway mitigates endoplasmic reticulum stress in Müller cells under high glucose conditions.
- Targeting the Sema3A pathway may offer a novel therapeutic strategy for diabetic retinopathy.
