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Ayelen Bulloj, Silvia C Finnemann; Semaphorin 4D inhibits photoreceptor outer segment phagocytosis by retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):236.
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© 2017 Association for Research in Vision and Ophthalmology.
Daily clearance phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segment fragments (POS) is critical for photoreceptor function and longevity. In this process, POS bind to ανβ5 integrin to stimulate signaling via Mertk and AKT kinases, which ultimately target Rac1-dependent F-actin reorganization that is required for POS engulfment. Semaphorins belong to a diverse family of secreted or membrane associated glycoproteins that are ligands for the plexin receptor family. The receptor for semaphorin 4D (Sema4D) is plexinB1 (PlB1). PlB1 can reorganize the cytoskeleton by interacting with active Rac1 in a ligand dependent manner and/or by promoting integrin-dependent activation of AKT through RhoA/Rho kinase (ROCK). Here, we set out to study if Sema4D and its receptor PlB1 contribute to the molecular mechanism of RPE phagocytosis.
Bound and internalized POS were quantified by feeding primary fetal human RPE cells with POS with or without recombinant Sema4D followed by confocal microscopy (CM) analysis. RhoA and Rac1 activity were measured by ELISA. Retinal Sema4D protein levels before and after light onset were studied by immunoblotting (IB) and by live tissue staining followed by CM in wild-type (wt) and Mertk null (RCS) rat eyes. Phosphorylation of PlB1 in rat eyes were analyzed by anti-phosphotyrosine immunoprecipitation followed by IB. Phagosome quantification and retina structure analysis were performed on sections of wt, Sema4D and PlB1 null mouse retina.
Added Sema4D abolished POS internalization by RPE cells in culture without affecting binding. ROCK inhibition or Rac1 activation restored POS internalization blocked by Sema4D. PlB1 phosphorylation and Sema4D levels were reduced during the peak of phagocytosis in wt rat eyes. This pattern was altered in RCS rat eyes, in which POS internalization does not occur. In mice with Sema4D or PlB1 gene deletion the RPE showed increased POS phagosome content, but otherwise their retina lacked obvious structural defects.
Our findings identify a new function for the ligand-receptor pair Sema4D/PlB1 in the retina and add new players to the photoreceptor outer segment renewal mechanism that is essential to maintain health and functionality of the retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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