MFG-E8 in the retina and retinal pigment epithelium of rat and mouse.

PURPOSE: To study the distribution of milk fat globule epidermal growth factor E8 (MFG-E8) in the rodent eye and to investigate a potential role for this molecule in the phagocytosis of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE). METHODS: We have used immunohistochemistry, in situ hybridization, Northern and Western blotting to demonstrate the presence and distribution of MFG-E8 in the rat and mouse retina. siRNA technology was used to knock down MFG-E8 mRNA and to study the effect of such knockdown on the phagocytosis of POS by the RPE. RESULTS: We identified a novel long form of this protein (MFG-E8L) in rat tissues, which contains a 56 amino acid insert that is rich in proline and threonine. This is the first demonstration that MFG-E8L is present in a species other than the mouse. Immunohistochemistry and in situ hybridization demonstrate that MFG-E8 is present in the retina and RPE. Northern blotting and PCR show that the short form of MFG-E8 (MFG-E8S) is present in both the retina and RPE, but MFG-E8L is found only in the RPE. Our results do not demonstrate a role for MFG-E8 in POS phagocytosis by cultured RPE cells. CONCLUSIONS: In all tissues in which MFG-E8 has been localized, it has been shown to perform an important role in cell-cell binding, or in promoting phagocytosis. The localization of this glycoprotein in the retina and RPE, and particularly the specific localization of MFG-E8L in the RPE, suggests that this molecule may play an important, but as yet unknown role in retinal function.

Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells.

Survival of the retina requires the daily phagocytosis of photoreceptor outer segments (OS) by the overlying retinal pigment epithelium (RPE). OS phagocytosis by cultured RPE requires serum and we have recently shown that the vitamin K-dependent serum protein, Gas6, can completely replace serum in this process. Surprisingly, however, we show here that 4-month-old Gas6 knockout mice have normal appearing retinas, except for a reduced ratio of outer segment to inner segment length. We also show that removal of Gas6 from serum does not abrogate the ability of serum to support OS phagocytosis by rat RPE. Both of these findings suggest the presence of an additional serum ligand that is able to support OS phagocytosis by RPE cells. Protein S (PS) is a vitamin K-dependent serum protein with a high degree of structural similarity to Gas6, and a well characterized role in blood coagulation. We report here that recombinant rat PS is able to stimulate OS phagocytosis, and similar to Gas6, it does so through a Mer-dependent mechanism. This is the first demonstration of a common role for Gas6 and PS in any biological process. The existence of redundant ligands for Mer-dependent OS phagocytosis underscores the critical role of this process in the maintenance of retinal function.

Integrin alphavbeta5 is not required for the phagocytosis of photoreceptor outer segments by cultured retinal pigment epithelial cells.

The phagocytosis of photoreceptor outer segments (OS) by the retinal pigment epithelium (RPE) is a receptor mediated process. A key component of this process is the receptor tyrosine kinase, Mer. RPE cells from the RCS rat, which lacks a functional mer gene, and do not express Mer protein, are able to bind OS, but are unable to ingest them, suggesting that both a binding receptor and an ingestion receptor (Mer) are required for phagocytosis to occur. These rats become blind shortly after birth. To date the binding receptor has not been identified. Recent studies, using an SV40 transformed rat RPE cell line, RPE-J, or cultured human RPE cells, have suggested that the receptor for OS binding is the integrin alphavbeta5. However, the results presented here clearly show that this integrin plays at most a minor role in the phagocytosis of OS by primary cultures of rat RPE cells. OS phagocytosis by normal RPE cells is not affected by a function-blocking antibody to alphavbeta5 integrin, nor by the integrin-specific blocking peptide GRGDSP. Additionally, RPE-J cells do not express the Mer receptor protein, which has been shown to be obligatory for OS phagocytosis, or RPE65, a specific marker for RPE cells. We suggest that the RPE-J cell line is not a valid model with which to study the complex process of OS phagocytosis.

Gas6 binding to photoreceptor outer segments requires gamma-carboxyglutamic acid (Gla) and Ca(2+) and is required for OS phagocytosis by RPE cells in vitro.

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 specifically 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 carboxyl-terminal 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 specific 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 microM. 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 gamma-carboxylation in a vitamin K-dependent reaction. By analogy with other systems, we propose that Ca(2+) mediates the linkage between the gamma-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.