After cell death: the molecular machinery of efferocytosis.

Cells constituting a multicellular organism die in a variety of ways throughout life, and most of them die via apoptosis under normal conditions. The occurrence of apoptosis is especially prevalent during development and in tissues with a high cellular turnover rate, such as the thymus and bone marrow. Interestingly, although the number of apoptotic cells produced daily is known to be innumerable in a healthy adult human body, apoptotic cells are rarely observed. This absence is due to the existence of a cellular process called efferocytosis that efficiently clears apoptotic cells. Studies over the past decades have focused on how phagocytes are able to remove apoptotic cells specifically, swiftly, and continuously, resulting in defined molecular and cellular events. In this review, we will discuss the current understanding of the clearance of apoptotic cells at the molecular level.

Internalization of apoptotic cells during efferocytosis requires Mertk-mediated calcium influx.

Phagocytosis of apoptotic cells, called efferocytosis, requires calcium inside and outside of phagocytes. Due to its necessity, calcium flux is sophisticatedly modulated, and the level of intracellular calcium in phagocytes is ultimately elevated during efferocytosis. However, the role of elevated intracellular calcium in efferocytosis remains elusive. Here, we report that Mertk-mediated intracellular calcium elevation is necessary for internalization of apoptotic cells during efferocytosis. Drastic depletion of intracellular calcium abrogated the internalization step of efferocytosis by delaying phagocytic cup extension and closure. Especially, the defect of phagocytic cup closure for internalization of apoptotic cells was caused by impaired F-actin disassembly and the attenuated interaction of Calmodulin with myosin light chain kinase (MLCK), leading to diminished myosin light chain (MLC) phosphorylation. Genetic and pharmacological impairment of the Calmodulin-MLCK-MLC axis or Mertk-mediated calcium influx also resulted in inefficient efferocytosis due to a defect in internalization of the targets. Taken together, our observations imply that intracellular calcium elevation through Mertk-mediated calcium influx facilitates efferocytosis by inducing myosin II-mediated contraction and F-actin disassembly required for internalization of apoptotic cells.

Phagocyte Chemoattraction Is Induced through the Mcp-1-Ccr2 Axis during Efferocytosis.

Apoptotic cells generated during development and for tissue homeostasis are swiftly and continuously removed by phagocytes via a process called efferocytosis. Efficient efferocytosis can be achieved via transcriptional modulation in phagocytes that have engulfed apoptotic cells. However, such modulation and its effect on efferocytosis are not completely understood. Here, we report that phagocytes are recruited to apoptotic cells being cleared through the Mcp-1-Ccr2 axis, which facilitates clearance of apoptotic cells. We identified Mcp-1 as a modulated transcript using a microarray and found that Mcp-1 secretion was augmented in phagocytes engulfing apoptotic cells. This augmented Mcp-1 secretion was impaired by blocking phagolysosomal degradation of apoptotic cells. Conditioned medium from wild type (WT) phagocytes promoted cell migration, but that from Mcp-1-/- phagocytes did not. In addition, blockade of Ccr2, the receptor for Mcp-1, abrogated cell migration to conditioned medium from phagocytes incubated with apoptotic cells. The intrinsic efferocytosis activity of Mcp-1-/- and Ccr2-/- phagocytes was unaltered, but clearance of apoptotic cells was less efficient in the peritoneum of Mcp-1-/- and Ccr2-/- mice than in that of WT mice because fewer Ccr2-positive phagocytes were recruited. Taken together, our findings demonstrate a mechanism by which not only apoptotic cells but also phagocytes induce chemoattraction to recruit phagocytes to sites where apoptotic cells are cleared for efficient efferocytosis.

Apoptotic Cells Trigger Calcium Entry in Phagocytes by Inducing the Orai1-STIM1 Association.

Swift and continuous phagocytosis of apoptotic cells can be achieved by modulation of calcium flux in phagocytes. However, the molecular mechanism by which apoptotic cells modulate calcium flux in phagocytes is incompletely understood. Here, using biophysical, biochemical, pharmaceutical, and genetic approaches, we show that apoptotic cells induced the Orai1-STIM1 interaction, leading to store-operated calcium entry (SOCE) in phagocytes through the Mertk-phospholipase C (PLC) γ1-inositol 1,4,5-triphosphate receptor (IP3R) axis. Apoptotic cells induced calcium release from the endoplasmic reticulum, which led to the Orai1-STIM1 association and, consequently, SOCE in phagocytes. This association was attenuated by masking phosphatidylserine. In addition, the depletion of Mertk, which indirectly senses phosphatidylserine on apoptotic cells, reduced the phosphorylation levels of PLCγ1 and IP3R, resulting in attenuation of the Orai1-STIM1 interaction and inefficient SOCE upon apoptotic cell stimulation. Taken together, our observations uncover the mechanism of how phagocytes engulfing apoptotic cells elevate the calcium level.

EphA2 Interacts with Tim-4 through Association between Its FN3 Domain and the IgV Domain of Tim-4.

Tim-4 promotes the engulfment of apoptotic cells or exogenous particles by securing them on phagocytes. It is unable to transduce signals by itself but helps other engulfment receptors sense and internalize them. However, the identity of the engulfment receptors collaborating with Tim-4 is still incompletely understood. In this study, we searched for a candidate transmembrane protein with a FN3 domain, important for interaction with Tim-4, in silico and investigated whether it indeed interacts with Tim-4 and is involved in Tim-4-mediated phagocytosis. We found that EphA2 containing a FN3 domain in the extracellular region interacted with Tim-4, which was mediated by the IgV domain of Tim-4 and the FN3 domain of EphA2. Nevertheless, we found that EphA2 expression failed to alter Tim-4-mediated phagocytosis of apoptotic cells or polystyrene beads. Taken together, our findings suggest that EphA2, a new Tim-4 interacting protein, may intervene in a Tim-4-mediated cellular event even if it is not phagocytosis of endogenous or exogenous particles and vice versa.

The Peroxisomal Localization of Hsd17b4 Is Regulated by Its Interaction with Phosphatidylserine.

Phosphatidylserine (PS), a negatively charged phospholipid exclusively located in the inner leaflet of the plasma membrane, is involved in various cellular processes such as blood coagulation, myoblast fusion, mammalian fertilization, and clearance of apoptotic cells. Proteins that specifically interact with PS must be identified to comprehensively understand the cellular processes involving PS. However, only a limited number of proteins are known to associate with PS. To identify PS-associating proteins, we performed a pulldown assay using streptavidin-coated magnetic beads on which biotin-linked PS was immobilized. Using this approach, we identified Hsd17b4, a peroxisomal protein, as a PS-associating protein. Hsd17b4 strongly associated with PS, but not with phosphatidylcholine or sphingomyelin, and the Scp-2-like domain of Hsd17b4 was responsible for this association. The association was disrupted by PS in liposomes, but not by free PS or the components of PS. In addition, translocation of PS to the outer leaflet of the plasma membrane enriched Hsd17b4 in peroxisomes. Collectively, this study suggests an unexpected role of PS as a regulator of the subcellular localization of Hsd17b4.

Crbn modulates calcium influx by regulating Orai1 during efferocytosis.

Calcium flux regulating intracellular calcium levels is essential and modulated for efficient efferocytosis. However, the molecular mechanism by which calcium flux is modulated during efferocytosis remains elusive. Here, we report that Orai1, a Crbn substrate, is upregulated via its attenuated interaction with Crbn during efferocytosis, which increases calcium influx into phagocytes and thereby promotes efferocytosis. We found that Crbn deficiency promoted phagocytosis of apoptotic cells, which resulted from facilitated phagocytic cup closure and was nullified by a CRAC channel inhibitor. In addition, Orai1 associated with Crbn, resulting in ubiquitination and proteasomal degradation of Orai1 and alteration of SOCE-mediated calcium influx. The association of Orai1 with Crbn was attenuated during efferocytosis, leading to reduced ubiquitination of Orai1 and consequently upregulation of Orai1 and calcium influx. Collectively, our study reveals a regulatory mechanism by which calcium influx is modulated by a Crbn-Orai1 axis to facilitate efferocytosis.

Tim-4 functions as a scavenger receptor for phagocytosis of exogenous particles.

The phosphatidylserine (PS) receptor Tim-4 mediates phagocytosis of apoptotic cells by binding to PS exposed on the surface of these cells, and thus functions as a PS receptor for apoptotic cells. Some of PS receptors are capable of recognizing other molecules, such as LPS on bacteria, besides PS on apoptotic cells. However, it is unclear whether Tim-4 perceives other molecules like the PS receptors. Here, we report that Tim-4 facilitates the phagocytosis of exogenous particles as well as apoptotic cells. Similar to the process that occurs during Tim-4-mediated efferocytosis, the uptake of exogenous E. coli and S. aureus bioparticles was promoted by overexpression of Tim-4 on phagocytes, whereas phagocytosis of the bioparticles was reduced in Tim-4-deficient cells. A truncation mutant of Tim-4 lacking the cytoplasmic tail promoted phagocytosis of the particles, but a mutant lacking the IgV or the mucin domain failed to enhance phagocytosis. However, expression of Tim-4AAA (a mutant form of Tim-4 that does not bind phosphatidylserine and does not promote efferocytosis) still promoted phagocytosis. Tim-4-mediated phagocytosis was not blocked by expression of the phosphatidylserine-binding protein Anxa5. Furthermore, binding of lipopolysaccharide (LPS), which is found in the outer membrane of Gram-negative bacteria, was higher in Tim-4-overexpressing cells than in Tim-4-deficient cells. In summary, our study suggests that Tim-4 acts as a scavenger receptor and mediates phagocytosis of exogenous particles in a phosphatidylserine-independent manner.

Mertk Interacts with Tim-4 to Enhance Tim-4-Mediated Efferocytosis.

Apoptotic cells expressing phosphatidylserine (PS) on their cell surface are directly or indirectly recognized by phagocytes through PS-binding proteins. The PS-binding protein Tim-4 secures apoptotic cells to phagocytes to facilitate the engulfment of apoptotic cells. However, the molecular mechanism by which Tim-4 transduces signals to phagocytes during Tim-4-mediated efferocytosis is incompletely understood. Here, we report that Tim-4 collaborates with Mertk during efferocytosis through a biochemical interaction with Mertk. Proximal localization between the two proteins in phagocytes was observed by immunofluorescence and proximal ligation assays. Physical association between Tim-4 and Mertk, which was mediated by an interaction between the IgV domain of Tim-4 and the fibronectin type-III domain of Mertk, was also detected with immunoprecipitation. Furthermore, the effect of Mertk on Tim-4-mediated efferocytosis was abolished by GST-MertkFnIII, a soluble form of the fibronectin type-III domain of Mertk that disrupts the interaction between Tim-4 and Mertk. Taken together, the results from our study suggest that a physical interaction between Tim-4 and Mertk is necessary for Mertk to enhance efferocytosis mediated by Tim-4.

The Intermolecular Interaction of Ephexin4 Leads to Autoinhibition by Impeding Binding of RhoG.

Ephexin4 is a guanine nucleotide-exchange factor (GEF) for RhoG and is involved in various RhoG-related cellular processes such as phagocytosis of apoptotic cells and migration of cancer cells. Ephexin4 forms an oligomer via an intermolecular interaction, and its GEF activity is increased in the presence of Elmo, an Ephexin4-interacting protein. However, it is uncertain if and how Ephexin4 is autoinhibited. Here, using an Ephexin4 mutant that abrogated the intermolecular interaction, we report that this interaction impeded binding of RhoG to Ephexin4 and thus inhibited RhoG activation. Mutation of the glutamate residue at position 295, which is a highly conserved residue located in the region of Ephexin4 required for the intermolecular interaction, to alanine (Ephexin4E295A) disrupted the intermolecular interaction and increased binding of RhoG, resulting in augmented RhoG activation. In addition, phagocytosis of apoptotic cells and formation of membrane ruffles were increased more by expression of Ephexin4E295A than by expression of wild-type Ephexin4. Taken together, our data suggest that Ephexin4 is autoinhibited through its intermolecular interaction, which impedes binding of RhoG.