Overexpression of Brg1 alleviates high glucose-induced retinal ganglion cell apoptosis though regulating Notch/Hes1 signaling

https://doi.org/10.1016/j.bbrc.2019.05.069Get rights and content

Highlights

  • Brg1 expression is decreased in RGCs exposed to HG.

  • Brg1 overexpression represses HG-induced apoptosis in RGCs.

  • Brg1 enhances Notch1 signaling by upregulating Notch ligands.

  • Brg1 inhibits HG-induced apoptosis through Notch signaling.

Abstract

High glucose (HG)-caused damage of retinal ganglion cells (RGCs) plays an important role in the pathogenesis of diabetic retinopathy, a common and severe complication of diabetes mellitus. Accumulating evidence has reported that brahma-related gene 1 (Brg1) exerts a cytoprotective role in protection of cells from various injuries. In this study, we investigated the role of Brg1 in regulating HG-induced injury of RGCs. We found that RGCs treated with HG exhibited a low expression level of Brg1 compared with untreated RGCs. Gain-of-function experiments showed that Brg1 overexpression significantly improved the viability and reduced the apoptosis of RGCs exposed to HG. Notably, our data revealed that Brg1 overexpression increased the expression of Notch ligands, including Jagged 1, Jagged 2, Delta-like 1, and Delta-like 4. Moreover, Brg1 overexpression upregulated the expression of Notch1 intracellular domain (NICD) and Hes1, which resulted in activation of Notch signaling. Blockade of Notch signaling partially reversed Brg1-mediated protection effect in HG-treated RGCs, while overexpression of NICD significantly attenuated the promotion effect of Brg1 silencing on HG-induced injury in RGCs. Taken together, these results suggest that Brg1 protects RGCs against HG-induced injury through enhancing the activation of Notch signaling, suggesting a potential target for preserving RGCs in diabetic retinopathy.

Introduction

Diabetic retinopathy (DR), a common complication of diabetes, is a leading cause of vision impairment and blindness [1]. Retinal ganglions cells (RGCs) located in the inner retinas are the retinal neurons that play a critical role in high glucose (HG)-induced retinal neurodegeneration [2]. HG-induced RGC loss results in a reduction in the thickness of retinal nerve fiber layer, which contributes to vision dysfunction during early diabetes [2]. However, the molecular mechanism underlying HG-induced RGC loss remains poorly understood. Therefore, it is imperative to uncover the molecular mechanism of HG-induced injury in RGCs, which may help to develop promising strategies that protect from diabetes-induced visual dysfunction.

Brahma-related gene 1 (Brg1), the central catalytic subunit of chromatin-modifying enzymatic complexes, has been reported as a critical transcriptional co-regulator that plays an important role in diverse biological functions [3]. The dysregulation of Brg1 is implicated in various pathological processes [4,5]. Of interest, Brg1 has emerged as a cytoprotective protein. Brg1 protects cancer cells against radiotherapy- and chemotherapy-induced apoptosis by enhancing the pro-survival signaling [[6], [7], [8]]. Brg1 overexpression alleviates hepatic ischemia-reperfusion injury and acute lung injury [9,10]. Moreover, Brg1 exerts neuroprotective effect by reducing oxygen-glucose deprivation/re-oxygenation-induced apoptosis [11,12]. Notably, accumulating evidence has shown that Brg1 plays an important role in regulating HG-induced cell injury in diabetes [[13], [14], [15]]. These findings suggest that Brg1 may serve as a promising target for providing cellular protection.

Notch signaling is a multifunctional signaling pathway that plays an important role in various physiological and pathological processes [16]. Upon binding with Notch ligands, Notch receptor undergoes sequential proteolytic cleavage to release Notch intracellular domain (NICD), which can then translocate into nucleus to activate the expression of Notch target gene [17]. Accumulating evidence has revealed an important role of Notch signaling in the pathogenesis of various diabetic complications [[18], [19], [20]]. Notably, Notch1 expression is found to be decreased in retina of diabetic mice, and Notch signaling activation, as mediated by Notch1 overexpression, protects retinal vascular endothelial cells from HG-induced apoptosis in vitro [21]. Moreover, NICD nuclear translocation is reduced in diabetic mice, and Notch inhibition phenocopies diabetes-induced capillary abnormality in retina of normal mice [22]. Interestingly, our previous study has demonstrated that Notch signaling activation alleviates HG-induced apoptosis in RGCs [23]. These findings suggest that Notch signaling plays a crucial role in DR and may serve as potential therapeutic target.

To date, little is known about the role of Brg1 in DR. In this study, we aimed to investigate the role of Brg1 in regulating HG-induced apoptosis of RGCs in vitro. Our results demonstrated that Brg1 expression was significantly decreased in RGCs exposed to HG. Gain-of-function experiments revealed that Brg1 overexpression significantly improved the viability and reduced the apoptosis of RGCs exposed to HG. Notably, our data revealed that Brg1 overexpression increased the expression of Notch ligands, including Jagged 1 (Jag1), Jagged 2 (Jag2), Delta-like 1 (Dll1), and Delta-like 4 (Dll4). Moreover, Brg1 overexpression upregulated the expression levels of NICD and Hes1, which resulted in activation of Notch signaling. Notch inhibition partially reversed Brg1-mediated protection effect in HG-treated RGCs, while overexpression of NICD significantly attenuated the promotion effect of Brg1 silencing in HG-induced injury. Overall, these results demonstrate that Brg1 protects RGCs against HG-induced injury through enhancing the activation of Notch signaling, suggesting a potential relevance of Brg1 in DR.

Section snippets

Cell culture

Primary RGCs were isolated from newborn Sprague-Dawley (SD) rats as previously described [24]. Briefly, retinas were dissected from newborn rats on postnatal days 1–3 under sterile conditions. Then, retinas were digested in Hank's balanced salt solution (HBSS) containing 70 U/ml collagenase and 15 U/ml papain solution for 30 min at 37 °C. The tissues were triturated sequentially with Pasteur pipette in DMEM medium (Gibco, Rockville, MD, USA) containing 1 mg/ml bovine serum albumin (BSA),

HG exposure induces a decrease in Brg1 expression in RGCs

To investigate whether Brg1 is involved in HG-induced injury in RGCs, we detected the effect of HG exposure on Brg1 expression in RGCs in vitro. The RT-qPCR results showed that the mRNA expression of Brg1 was significantly decreased in RGCs in response to HG exposure (Fig. 1A). Moreover, HG exposure resulted in a significant decrease in Brg1 protein expression in RGCs (Fig. 1B). These data indicate that Brg1 expression was downregulated in RGCs with HG exposure.

Overexpression of Brg1 alleviates HG-induced apoptosis in RGCs

To investigate the biological

Discussion

In this study, we reveal an important role of Brg1 in regulating HG-induced apoptosis of RGCs. Results demonstrated that Brg1 overexpression rescued HG-impaired cell viability and protected against HG-induced apoptosis. The underlying mechanism is associated with the promotion effect of Brg1 on Notch signaling activation. Therein, results showed that Brg1 contributed to the upregulation of Notch ligands, leading to the activation of Notch signaling which antagonizes HG-induced injury in RGCs (

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This study was supported by Key Research Program of Shaanxi Province (2018SF-202).

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