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Celia Parinot, Jonathan Chatagnon, Emeline Nandrot; Investigation of protease candidates for the release of soluble MerTK during photoreceptor outer segments phagocytosis by retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2646. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Clearance of aged photoreceptor outer segment (POS) tips by retinal pigment epithelial (RPE) cells follows a circadian rhythm. We showed recently that the phagocytic internalization receptor MerTK is cleaved from the RPE cell membrane after POS challenge in vitro and rhythmically in vivo. We hypothesized that this proteolytic mechanism might participate in the fine timely regulation of this intricate function that occurs once a day despite the permanent contact between RPE cells and photoreceptors. Thus, we investigated the implication of various proteases in the release of soluble MerTK (sMerTK) in the interphotoreceptor matrix (IPM).
Candidates were chosen according to their potential implication in retinal disease and their expression on RPE cells or in the IPM. Expression of the proteases along the light:dark cycle was studied using qPCR and immunoblotting. Candidate’s potency to cleave MerTK was assessed by in vitro digestion assays using recombinant proteins or by using inhibitors on cultured cells challenged with medium or POS.
We first selected the HtrA1 serine protease, a single-nucleotide polymorphism being potentially related to increased AMD susceptibility. HtrA1 does not seem to follow a circadian-regulated pattern of expression. HtrA1 blocking peptides do not modify the pattern of sMerTK released by RPE-J cells after POS challenge. Similarly, recombinant HtrA1 proteins are not able to significantly cleave recombinant MerTK receptors in vitro. The second series of peptidases we explored was the ADAM’s family of metalloproteases. Recently, various ADAM members such as ADAM9, 10 and 12 have been linked to retinitis pigmentosa cases. We also included ADAM17, as it cleaves MerTK in macrophages. ADAM17 expression appears to increase just after the phagocytic peak, concomitant to augmented release of sMerTK in vivo. Additionally, ADAM17 is able to digest MerTK in vitro.
Our data imply that HtrA1 can be excluded as the main protease releasing MerTK in RPE cells. Our results suggest that ADAM17 can be considered as a strong candidate for MerTK cleavage during daily POS phagocytosis. Moreover, these results reinforce the hypothesis that MerTK activity is at least partially regulated by cleavage of its extracellular domain binding POS through specific ligands.
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