High-Glucose-Induced Rab20 Upregulation Disrupts Gap Junction Intercellular Communication and Promotes Apoptosis in Retinal Endothelial and Müller Cells: Implications for Diabetic Retinopathy.

Dongjoon Kim,Sayon Roy,Vijay P. Sarthy,Casey Stottrup Lewis

doi:10.3390/jcm9113710

Dongjoon Kim, Sayon Roy + Show 2 more

Open Access

https://doi.org/10.3390/jcm9113710

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Abstract

To investigate whether high glucose (HG) alters Rab20 expression and compromises gap junction intercellular communication (GJIC) and cell survival, retinal cells were studied for altered intracellular trafficking of connexin 43 (Cx43). Retinal endothelial cells (RRECs) and retinal Müller cells (rMCs) were grown in normal (N; 5 mM glucose) or HG (30 mM glucose) medium for seven days. In parallel, cells grown in HG medium were transfected with either Rab20 siRNA or scrambled siRNA as a control. Rab20 and Cx43 expression and their localization and distribution were assessed using Western Blot and immunostaining, respectively. Changes in GJIC activity were assessed using scrape load dye transfer, and apoptosis was identified using differential dye staining assay. In RRECs or rMCs grown in HG medium, Rab20 expression was significantly increased concomitant with a decreased number of Cx43 plaques. Importantly, a significant increase in the number of Cx43 plaques and GJIC activity was observed in cells transfected with Rab20 siRNA. Additionally, Rab20 downregulation inhibited HG-induced apoptosis in RRECs and rMCs. Results indicate HG-mediated Rab20 upregulation decreases Cx43 localization at the cell surface, resulting in compromised GJIC activity. Reducing Rab20 expression could be a useful strategy in preventing HG-induced vascular and Müller cell death associated with diabetic retinopathy.

Highlights

Diabetic retinopathy is the leading cause of blindness in the working-age population in Western countries [1]
Several studies suggest that hyperglycemia-induced disruption of gap junction intercellular communication (GJIC) plays a critical role in the pathogenesis of diabetic retinopathy [10,11,12,13,14,15,16,17,18,19,20]
Following immunoprecipitation for Rab20, the expression level for Rab20 was significantly elevated in rat retinal capillaries (RRECs) or retinal Müller cells (rMCs)-1 grown under HG condition compared to those grown in normal glucose (NG) condition (RRECs: 130 ± 3% of NG vs. 100 ± 1% of NG; p < 0.05; n = 6; Figure 1; rMC-1: 177 ± 17% of NG vs. 100 ± 15% of NG; p < 0.05; n = 6; Figure 1)

Summary

Introduction

Diabetic retinopathy is the leading cause of blindness in the working-age population in Western countries [1]. With respect to gap junctions, studies indicate that translocation and modification of connexins are critical in regulating GJIC activity [23,24], and Rab, a small GTPase, may impact the intracellular trafficking of connexin 43 (Cx43) [25]. It is currently unknown whether intracellular trafficking of Cx43 is altered by HG via regulation of Rab and whether such changes influence cell survival in the context of diabetic retinopathy. GTPases play an important role in regulating intracellular trafficking and facilitating membrane fusion and transport of proteins to the cell surface. The current study was undertaken to investigate whether HG alters Rab expression and subsequently affects Cx43 localization, GJIC activity, and cell survival in RRECs and rMC-1

Cell Culture

Transfection with Rab20 siRNA

Immunostaining

Differential Dye Staining

Statistical Analysis

High Glucose Upregulates Rab20 Protein Expression in RRECs and rMC-1