Abstract
Purpose :
The retinal pigment epithelium (RPE) is responsible for the daily shedding of photoreceptor outer segments (POS).The RPE cytoskeleton plays an instrumental role during this process through activation of signalling cascades leading to its re-organisation and the uptake of the POS.The role of gap junctions has not been explored.We performed in vitro studies to test the hypothesis that gap junction communication participates in the phagocytosis,investigating the role of Connexin 43 (Cx43) and PKCδ as mediators.
Methods :
Freshly enucleated bovine eyes were used to isolate the RPE cells and the POS.In vitro studies were performed using primary RPE cells (pRPE),cultured RPE cells (ARPE-19) and primary POS.Western Blots examined the effect of phagocytosis on Cx43 and PKCδ phosphorylation at different time points.PKCδ was suppressed with small interference RNA (siRNA).Immunofluorescence studies in ARPE-19 cells were performed to monitor the effect of gap junction inhibition (using 18β-glycyrrhetinic acid, 18β-GA) and PKCδ knock-down in POS binding and internalisation.Experiments were repeated three times.Two-tailed Student’s t-test was used for statistical analysis.Statistical significance was set at P<0.05.
Results :
A time-dependent phosphorylation of Cx43 (pS368-Cx43) and PKCδ (pY311-PKCδ) occurs during phagocytosis in the control condition and in presence of 18β-GA and PKCδ siRNA respectively.Immunofluorescence experiments showed that PKCδ knock-down led to a 41% statistically significant decrease in POS binding and internalisation,while PKCδ activation by phorbol-12-myristate-13-acetate (PMA) led to a non-significant increase in phagocytosis.PMA was unable to abolish the down-regulatory effect of PKCδ suppression (PKCδ siRNA+PMA treatment). Use of 18β-GA was found to have a 1.5 fold increased impact on internalisation and binding of the POS.The combination of the two treatments (PKCδ siRNA+18β-GA) recoved phagocytosis.
Conclusions :
Our results are consistent with the hypothesis that gap junction communication particiates in phagocytosis.Cx43 and PKCδ participate in the same signalling cascade up-regulating POS binding and internalisation. In response to outer segments,PKCδ phosphorylates Cx43 leading to gap junction disassembly which promotes phagocytosis.Src is likely to be the upstream kinase regulating PKCδ activity,while morphologic experiments will be needed to understand the structural changes accounting.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.