Outer Segment Phagocytosis by Cultured Retinal Pigment Epithelial Cells Requires Gas6 (original) (raw)
Related papers
Experimental eye …, 2002
The phagocytosis of photoreceptor outer segments by retinal pigment epithelial (RPE) cells plays a critical role in preserving normal retinal function. Recently the receptor protein tyrosine kinase (RTK) Mer, has been shown to be necessary for this cellular process to take place. Gas6, the ligand for the Mer RTK, can speci®cally and selectively stimulate the phagocytosis of photoreceptor outer segments (OS) by normal cultured rat RPE cells, as we have previously reported. The Gas6 protein has been shown to associate with plasma membrane phosphatidylserine by its amino-terminal portion, while its carboxylterminal portion can bind and activate Mer and its related RTKs, Axl and Tyro-3. Given the capability of Gas6 to interact with more than one molecule, we have performed a series of experiments to further dissect the interactions of Gas6 with the OS and RPE and to determine the speci®c calcium requirements necessary for Gas6 to exert its stimulatory effect on phagocytosis. These experiments show that Gas6 must bind to OS before the stimulation of OS ingestion can occur and that this binding requires a Ca 2 concentration of 500±600 mM. The same Ca 2 concentration is required for the Gas6 mediated stimulation of OS ingestion. We further demonstrate that in order to bind to OS and to stimulate OS phagocytosis, Gas6 requires g-carboxylation in a vitamin K-dependent reaction. By analogy with other systems, we propose that Ca 2 mediates the linkage between the g-carboxyglutamic acid (Gla)-rich N-terminal region of Gas6 with phospholipids, presumably phosphatidylserine, in the plasma membrane of the OS. Only after this binding has occurred can Gas6 interact with receptor molecule(s) on the surface of the RPE, and activate RPE cell signaling pathways leading to OS ingestion. These studies further underscore the importance of Gas6 in the phagocytic function of the RPE and open new avenues of investigation to understand the molecular events mediated and triggered by Gas6, and its interaction with the OS and RPE.
Experimental Eye Research, 2002
The phagocytosis of photoreceptor outer segments by retinal pigment epithelial (RPE) cells plays a critical role in preserving normal retinal function. Recently the receptor protein tyrosine kinase (RTK) Mer, has been shown to be necessary for this cellular process to take place. Gas6, the ligand for the Mer RTK, can speci®cally and selectively stimulate the phagocytosis of photoreceptor outer segments (OS) by normal cultured rat RPE cells, as we have previously reported. The Gas6 protein has been shown to associate with plasma membrane phosphatidylserine by its amino-terminal portion, while its carboxylterminal portion can bind and activate Mer and its related RTKs, Axl and Tyro-3. Given the capability of Gas6 to interact with more than one molecule, we have performed a series of experiments to further dissect the interactions of Gas6 with the OS and RPE and to determine the speci®c calcium requirements necessary for Gas6 to exert its stimulatory effect on phagocytosis. These experiments show that Gas6 must bind to OS before the stimulation of OS ingestion can occur and that this binding requires a Ca 2 concentration of 500±600 mM. The same Ca 2 concentration is required for the Gas6 mediated stimulation of OS ingestion. We further demonstrate that in order to bind to OS and to stimulate OS phagocytosis, Gas6 requires g-carboxylation in a vitamin K-dependent reaction. By analogy with other systems, we propose that Ca 2 mediates the linkage between the g-carboxyglutamic acid (Gla)-rich N-terminal region of Gas6 with phospholipids, presumably phosphatidylserine, in the plasma membrane of the OS. Only after this binding has occurred can Gas6 interact with receptor molecule(s) on the surface of the RPE, and activate RPE cell signaling pathways leading to OS ingestion. These studies further underscore the importance of Gas6 in the phagocytic function of the RPE and open new avenues of investigation to understand the molecular events mediated and triggered by Gas6, and its interaction with the OS and RPE.
The Journal of biological chemistry, 2015
Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK rel...
Biochimica Et Biophysica Acta - General Subjects, 2015
Background: The apopto-phagocytic gene expression patterns during clearance of dying cells in the retina and the effect of triamcinolone (TC) upon these processes have relevance to development of age-related macular degeneration (AMD). Methods: ARPE-19 cells and primary human retinal pigment epithelium (hRPE) were induced to undergo cell death by anoikis and the clearance of these cells by living hRPE/ARPE-19 or human monocyte-derived macrophages (HMDMs) in the presence or absence of TC was quantified by flow cytometry. TaqMan low-density gene expression array determining known markers of phagocytosis and loss-of-function studies on selected apopto-phagocytic genes was carried out in HMDM engulfing anoikic cells. Results: The glucocorticoid TC had a profound phagocytosis-enhancing effect on HMDM engulfing anoikic ARPE-19 or hRPE cells, causing a selective upregulation of the Mer tyrosine kinase (MERTK) receptor, while decreasing the expression of the AXL receptor tyrosine kinase and thrombospondin-1 (THSB-1). The key role of the MERTK could be demonstrated in HMDM engulfing dying cells using gene silencing as well as blocking antibodies. Similar pathways were found upregulated in living ARPE-19 engulfing anoikic ARPE-19 cells. Gas6 treatment enhanced phagocytosis in TC-treated HMDMs. Conclusions: Specific agonists of the Mertk receptor may have a potential role as phagocytosis enhancers in the retina and serve as future targets for AMD therapy. General significance: The use of Gas6 as enhancer of retinal phagocytosis via the MerTK receptor, alone or in combination with other specific ligands of the tyrosine kinase receptors' family may have a potential role in AMD therapy.
Journal of Biological Chemistry, 2016
Accumulation of bis-retinoids in the retinal pigmented epithelium (RPE) is a hallmark of aging and retinal disorders such as Stargardt disease and age-related macular degeneration. These aberrant fluorescent condensation products, including di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primarily through phagocytosis of the photoreceptor outer segments. However, we observed by twophoton microscopy that mouse retinas incapable of phagocytosis due to a deficiency of the c-Mer proto-oncogene tyrosine kinase (Mertk) nonetheless contained fluorescent retinoid condensation material in their RPE. Primary RPE cells from Mertk ؊/؊ mice also accumulated fluorescent products in vitro. Finally, quantification of A2E demonstrated the acquisition of retinal condensation products in Mertk ؊/؊ mouse RPE prior to retinal degeneration. In these mice, we identified activated microglial cells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of the dying photoreceptor cells. These observations demonstrate a novel transport mechanism between photoreceptor cells and RPE that does not involve canonical Mertk-dependent phagocytosis.
Neurobiology of Disease, 2003
Shed photoreceptor outer segments (POS) are phagocytosed by RPE cells in a circadian manner. The homozygous deletion of the c-mer gene abolishes the ingestion phase of this phagocytosis in the Royal College of Surgeons (RCS) rat strain, which in turn leads to the death of photoreceptor cells. We identified RPE transcripts for which the expression is modulated by the abrogation of POS phagocytosis. A microarray approach and the differential display (DDRT–PCR) technique revealed 116 modulated known genes, 4 modulated unknown genes, and 15 expressed sequenced tags (ESTs) corresponding to unknown genes. The microarray and DDRT–PCR analyses detected alterations in signaling pathways such as the phosphatidylinositol 3-kinase–Akt–mTOR pathway and the DLK/JNK/SAPK pathway. The abrogation of POS phagocytosis caused a decrease in endomembrane biogenesis and altered endocytosis, exocytosis, transcytosis, and several metabolic and signaling pathways in RCS RPE cells. We also found differential levels of transcripts encoding proteins involved in phagocytosis, vesicle trafficking, the cytoskeleton, retinoic acid, and general metabolism.
TAM receptor function in the retinal pigment epithelium
Molecular and Cellular …, 2006
The TAM receptor tyrosine kinase Mer is expressed by cells of the retinal pigment epithelium (RPE), and genetic studies have demonstrated that Mer is essential for RPE function. RPE cells that lack Mer exhibit a severely compromised ability to phagocytose the distal ends of photoreceptor (PR) outer segments, which leads to the complete postnatal degeneration of photoreceptors and to blindness. Although in vitro experiments have implicated Gas6 as the critical TAM ligand for this process, we find that Gas6 mutant mice have a histologically intact retina with no photoreceptor degeneration. We further find that, in addition to Mer, RPE cells also express another TAM receptor -Tyro 3and that both of these receptors are instead activated independently by the Gas6-related ligand Protein S. This protein is also expressed by RPE cells. Finally, we demonstrate that loss of Mer function is accompanied by a substantial down-regulation in Tyro 3 as well. These observations indicate that both Mer and Tyro 3 act in mouse RPE cells and suggest that their biologically relevant ligand in these cells is Protein S.
Loss of Synchronized Retinal Phagocytosis and Age-related Blindness in Mice Lacking v 5 Integrin
Journal of Experimental Medicine, 2004
Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor ␣ v  5 for particle binding. Here, we tested RPE phagocytosis and retinal function in  5 integrin-deficient mice, which specifically lack ␣ v  5 receptors. Retinal photoresponses severely declined with age in  5 Ϫ / Ϫ mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease.  5 Ϫ / Ϫ RPE in culture failed to take up isolated photoreceptor particles.  5 Ϫ / Ϫ RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in  5 Ϫ / Ϫ retina. These results demonstrate an essential role for ␣ v  5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the  5 Ϫ / Ϫ integrin mouse strain as a new animal model of age-related retinal dysfunction.