Retinal pigment epithelial cells use a MerTK‐dependent mechanism to limit the phagocytic particle binding activity of αvβ5 integrin

Abstract

Background information αvβ5 integrin and Mer tyrosine kinase (MerTK) receptors reside at the apical surface of the retinal pigment epithelium (RPE) in the eye to promote the diurnal, synchronised phagocytosis of shed photoreceptor outer segment fragments (POS) that is critical for vision. Phagocytosis assays studying RPE cells in culture have defined roles for αvβ5 in POS surface binding and for MerTK in engulfment of bound POS. Both receptors have thus far only been studied separately. It is therefore unknown if αvβ5 integrin activity in POS binding is independent of the engulfment function of RPE cells. This study investigates how increasing αvβ5 receptor levels affect POS binding and internalisation by wild-type (wt), αvβ5- or MerTK-deficient RPE. Results β5 integrin–green fluorescent protein (β5–GFP) fusion proteins formed heterodimeric receptors with endogenous αv integrin subunits at the apical surface of mouse or rat RPE cells that co-immunoprecipitated focal adhesion kinase and redistributed with bound POS such as endogenous αvβ5 receptors. In β5−/− RPE cells, de novo formation of αvβ5–GFP receptors restored POS binding and internalisation up to, but not, above wt POS uptake levels. In wt RPE cells, increasing levels of αvβ5 surface receptors by over-expressing β5–GFP only moderately stimulated POS binding, even if POS internalisation was inhibited pharmacologically or by lowering incubation temperatures. In contrast, the same increase in αvβ5 receptor levels dramatically enhanced POS binding of RPE cells lacking MerTK. Furthermore, decreasing MerTK expression by RNA interference increased POS binding to endogenous αvβ5 receptors of wt RPE cells. Conclusions Expressing β5–GFP is sufficient to reverse phagocytic deficiencies of RPE cells derived from β5−/− mice, indicating that these cells do not irreversibly lose other components of the phagocytic machinery. RPE cells expressing the engulfment receptor MerTK control POS binding by limiting activity of endogenous αvβ5 and αvβ5–GFP integrins, although they reside at the apical, phagocytic surface. In contrast, RPE cells permanently or transiently losing MerTK expression lack this regulatory mechanism and bind excess POS via surface αvβ5 receptors. Taken together, these data reveal a novel feedback mechanism that restricts binding of POS to surface αvβ5 integrin receptors in RPE cells.

Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

“…We know that proper control of all steps of phagocytosis is necessary as deregulation of its completion can lead to various retinal disease phenotypes, some of them resembling age-related macular degeneration (14, 44 -46). In addition, we recently showed that the MerTK receptor also bears a role in controlling POS binding aside from its indispensable role for POS internalization (37). This study provides evidence of a negative feedback mechanism that down-regulates MerTK activity during RPE phagocytosis by generation of a soluble form of the receptor.…”

“…In addition to the sequential receptor-ligand activation steps undertaken by the ␣v␤5 integrin receptor, MerTK also controls the number of particles to be engulfed (36,37), thus acting as a negative feedback regulator. However, in contrast to macrophages, RPE cell phagocytosis follows a diurnal cyclic rhythm despite the permanent contact between POS and RPE cells (5), suggesting that a more sophisticated regulatory mechanism exists in RPE cells.…”

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

“…Isolated POS serve to test directly the phagocytic ability of mutant RPE cells; e.g., POS are readily phagocytosed by primary RPE cells from wild-type mice, whereas cells from beta5 integrin and MFG-E8 knockout mice phagocytose less POS in the same amount of time 6,12 . Quantification of POS phagocytosis can be done either by counting FITC-labeled POS manually on microscope pictures 6 ( Figure 3A), using equipment capable of reading fluorescence intensity 10,24,15,35 or a flow cytometer after cell trypsinization 36,27 . More recently, POS intake and degradation has been evaluated on immunoblots probed for opsins 37,38,27 .…”

“…Protein recruitment can be assessed by immunofluorescence co-localization assays [13][14][15][19][20][21]37,41 ( Figure 3B). Protein recruitment or activation can be validated on immunoblots after immunoprecipitation or by checking for phosphorylation 7,10,[12][13][14][15]17,19,20,27,35,37,41 ( Figure 3C). More open-ended studies on overall gene expression changes after different times of POS challenge have also been performed 42 .…”

“…Moreover, pre-labeling followed by freeze-thaw may not maintain POS surface characteristics and alter POS immunogenicity. Hence, for co-localization assays it is advisable to store unlabeled POS and label them after thaw immediately before using them for an experiment 10,[13][14][15]32 .…”