Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

Abstract

The daily phagocytosis of shed photoreceptor outer segments by pigment epithelial cells is critical for the maintenance of the retina. In a subtractive polymerase chain reaction analysis, we found that functional differentiation of human ARPE19 retinal pigment epithelial (RPE) cells is accompanied by up-regulation of annexin (anx) A2, a major Src substrate and regulator of membrane-cytoskeleton dynamics. Here, we show that anx A2 is recruited to the nascent phagocytic cup in vitro and in vivo and that it fully dissociates once the phagosome is internalized. In ARPE19 cells depleted of anx A2 by using small interfering RNA and in ANX A2 ؊/؊ mice the phagocytosis of outer segments was impaired, and in ANX A2 ؊/؊ mice there was an accumulation of phagocytosed outer segments in the RPE apical processes, indicative of retarded phagosome transport. We show that anx A2 is tyrosine phosphorylated at the onset of phagocytosis and that the synchronized activation of focal adhesion kinase and c-Src is abnormal in ANX A2 ؊/؊ mice. These findings reveal that anx A2 is involved in the circadian regulation of outer segment phagocytosis, and they provide new insight into the protein machinery that regulates phagocytic function in RPE cells. INTRODUCTIONThe retinal pigment epithelium (RPE) performs a range of functions that directly support the retina (Strauss, 2005), including the daily phagocytosis and digestion of shed photoreceptor outer segments (POS). The importance of this activity is exemplified in the dystrophic Royal College of Surgeons rat, which due to a failure in phagocytosis undergoes a progressive and rapid retinal degeneration characterized by accumulation of cellular debris and photoreceptor cell death. The gene responsible for this pathology was identified by positional cloning as the Mer tyrosine kinase (MerTK), a key signaling intermediate that is expressed in RPE cells and plays an essential role in the internalization of bound POS (Chaitin and Hall, 1983;D'Cruz et al., 2000). Recent studies have provided insight into other RPE cell proteins that have distinct roles either in the binding or the internalization of shed POS. For example, the apically expressed ␣v␤5 integrin is required for efficient binding of POS, and RPE cells from ␤5 Ϫ/Ϫ mice show markedly reduced binding of POS in vitro (Nandrot et al., 2004). Intriguingly, engagement of the ␣v␤5 integrin by its cognate ligand, milk fat globule-EGF8 (MFG-E8), is essential for the circadian synchronization of phagocytosis (Nandrot and Finnemann, 2006;Nandrot et al., 2007), and although phagocytosis of POS occurs in MFG-E8 Ϫ/Ϫ and ␤5 Ϫ/Ϫ mice, in both mutants the daily rhythm is absent. In contrast, focal adhesion kinase (FAK) and MerTK are not required for the initial binding of POS to the apical surface of the RPE, but they are sequentially activated by ␣v␤5 receptors and are essential for phagosome internalization (Feng et al., 2002;Finnemann, 2003).Between the pigment epithelium and neuroretina RPE cells extend apical processes into the interphotorecept...

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

“…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 .…”

The 1D4 Antibody Labels Outer Segments of Long Double Cone But Not Rod Photoreceptors in Zebrafish

“…10,11 Given its high specificity, it has been widely used in studies addressing structure and function of rhodopsin, 12 as well as a reliable fusion tag for purification of nonrelated receptors. 13,14 In mice, it has been used for the detection of rhodopsin 15 and its subcellular distribution 16 during retinal degeneration. This antibody is commercially available (ab5417; Abcam, Cambridge, UK; R5403; Sigma-Aldrich, St. Louis, MO; sc-57,432; SCBT, Santa Cruz, CA; MAB5356; Millipore, Billerica, MA) and according to the supplier is used to detect rhodopsin also in zebrafish.…”

“…9 It binds to the C-terminal eight amino acids of rhodopsin and labels rod outer segments in mice, cows, and humans. 15,16 Recently, two zebrafish reports used this antibody as a predicted rod outer segment marker for functional studies on retinitis pigmentosa (RP) related genes. In the first study, the protein ZFPRGP was examined in the presumptive rod cilium region using 1D4 as a co-localizing marker.…”

Receptor role of the annexin A2 in the mesothelial endocytosis of crocidolite fibers

“…44 Intracellularly, ANXA2 is present on the plasma membrane and early endosome and interacts with actin to act as a regulator of membra-cytoskeleton dynamics. [45][46][47] It was reported that ANXA2 is involved in the phagocytosis of the shed photoreceptor outer segment by pigment epithelial cells in the retina. 47 Furthermore, ANXA2 is known to be present in various cells such as endothelial cells, monocytes, macrophages, and most cancer cells.…”

“…[45][46][47] It was reported that ANXA2 is involved in the phagocytosis of the shed photoreceptor outer segment by pigment epithelial cells in the retina. 47 Furthermore, ANXA2 is known to be present in various cells such as endothelial cells, monocytes, macrophages, and most cancer cells. 39,48,49 Asbestos is neither an intracellular pathogen that has developed specific ligand proteins to enter nonprofessional phagocytes nor an apoptotic body that arises during the normal physiological state and has a phagocytic removal system through nonprofessional phagocytes.…”