Abstract
Purpose :
Coordination of molecular pathways regulating retinal pigment epithelium (RPE) phagocytosis for outer segment renewal is not fully understood. The Hippo pathway influences retinal tissue development and homeostasis through yes-associated protein (YAP). We found recently that silencing Yap in primary rat RPE cells in culture increases phagocytosis of photoreceptor outer segment fragments (POS). Here, we test if this effect of Yap silencing is related to changes in glucose metabolism.
Methods :
Primary wild-type rat RPE cultures were transfected with Yap-targeting (siYAP) or non-targeting (siNT) siRNA followed by immunoblotting of YAP, RPE marker proteins, and proteins relevant for energy metabolism. Phagocytosis assays with fluorescent purified POS were conducted in assay media with and without glucose or pyruvate, the ATP synthase inhibitor oligomycin, or the glucose transporter 1 (GLUT1) inhibitor STF-31, followed by rhodopsin immunofluorescence of surface-bound POS. POS binding and internalization were quantified by confocal microscopy and ImageJ analysis. Two-tailed Student’s t-test or ANOVA as appropriate was applied to 3 or more independent experiments.
Results :
siYAP RPE cells with reduction of YAP protein by 74% on average showed a 1.5-fold increase in GLUT1 steady-state protein levels compared to siNT RPE cells (p<0.05), but no change in mitochondrial proteins. Unlike in media with glucose, siYAP RPE cells did not differ from siNT cells in POS phagocytosis in media with pyruvate. In media with both glucose and pyruvate, GLUT1 inhibitor reduced POS by siYAP, siNT, and untransfected RPE cells to the same low level. In synchronized phagocytosis assays in media with glucose and pyruvate, GLUT1 inhibitor did not affect POS binding or subsequent internalization if applied only during POS binding. In contrast, applying GLUT1 inhibitor to RPE cells with pre-bound POS inhibited POS internalization (p<0.05).
Conclusions :
RPE cells upregulate GLUT1 in response to Yap silencing, and GLUT1 inhibition eliminates the effect of Yap silencing on RPE phagocytosis. Together, these results support the conclusion that the effect of Yap silencing on RPE phagocytosis depends on GLUT1 activity. These data provide novel insight into YAP and GLUT1’s functional relationship and the energy metabolism that is needed to sustain the phagocytic activity of RPE cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.