Purchase this article with an account.
Dongjoon Kim, Vijay P Sarthy, Sayon Roy; HG-induced upregulation of rab20 expression disrupts gap junctional intercellular communication and promotes retinal Müller cell (rMC) death. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):408.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate whether high glucose (HG) alters rab20 protein expression and consequently compromise gap junction intercellular communication (GJIC) and cell survival of rMCs through disruption of intracellular connexin 43 (Cx43) trafficking.
To examine whether HG alters rab20 protein expression, rMCs were grown in normal (N; 5mM glucose) or HG (30 mM glucose) medium for 7 days. In parallel, cells were grown in HG medium and transfected with rab20 siRNA or scrambled siRNA as control. Rab20 protein expression was analyzed using immunoprecipitation and Western Blot (WB) analysis, and changes in Cx43 localization and distribution were assessed via immunostaining. GJIC activity was determined by scrape loading dye transfer (SLDT) assay, and apoptosis was identified using a differential staining method.
WB analysis showed significant rab20 upregulation in cells grown in HG medium compared to cells grown in N medium (131±4% of control; p<0.05), and significant rab20 downregulation to near normal level in cells transfected with rab20 siRNA compared to untransfected cells. Immunostaining assay indicated that the relative number of Cx43 plaques was significantly reduced in cells grown in HG condition compared to those grown in N medium. Importantly, cells grown in HG medium and transfected with rab20 siRNA showed increased number of Cx43 plaques. In addition, SLDT analysis showed that HG-induced decrease in GJIC activity was improved in cells grown in HG and transfected with rab20 siRNA compared to those grown in HG medium only. As expected, cells grown in HG showed significant increase in the number of apoptotic cells (202±29% of control; p<0.05). Interestingly, reducing HG-induced overexpression of rab20 in the siRNA-transfected cells grown in HG medium resulted in rescue of rMCs from HG-induced apoptosis (114±36% of control; p<0.05).
HG-induced overexpression of rab20 compromises GJIC activity by reducing Cx43 localization and distribution at the cell surface where they are needed to form gap junctions. Rab20 downregulation via siRNA strategy improves GJIC activity and ultimately rescues rMCs from HG-induced rMC death associated with diabetic retinopathy.
This PDF is available to Subscribers Only