Characterization of Mcp-1 and Hsd17b4 in efferocytosis

Abstract

Programmed cell death, also known as apoptosis, occurs in human body for maintaining tissue homeostasis or eliminating unwanted cells. Apoptotic cells generated during development and for tissue homeostasis are not easily observed in vivo since they are swiftly removed by phagocytes. Elimination of apoptotic cells by phagocytes is called efferocytosis. When apoptosis is induced, cells expose phosphatidylserine (PS), a negatively charged phospholipid, to the outer leaflet of the membrane. Since asymmetric and limited distribution of phospholipids is one of the characteristics of plasma membrane, PS exposure can regulate various cellular processes including not only apoptotic cell clearance but also blood coagulation, myoblast fusion and mammalian fertilization. During efferocytosis, transcriptional modulation in apoptotic cell engulfed phagocytes occurs to more efficiently dispose of waste but such regulation is not fully understood. In part 1 of my research, I found that phagocytes incubated with apoptotic cells express increased amount of Mcp-1 and Mcp-3 in both transcriptional and translational level, which in turn recruits Ccr2 expressing cells and facilitates further efferocytosis. Expression of both chemokines during efferocytosis were abrogated when the phagolysosomal degradation is inhibited. Conditioned medium of apoptotic cell engulfing wild-type (WT) phagocytes promoted monocyte migration in vitro, whereas that of Mcp-1-/- phagocytes failed to induce cell migration. Blocking Ccr2 on monocytes using anti-Ccr2 antibody decreased the number of migrated cells towards conditioned medium, which suggests that Ccr2 is truly involved. When apoptotic cells were injected into the mice peritoneum, enrichment of Ccr2 expressing cells in peritoneum was observed in WT but not in either Mcp-1-/- or Ccr2-/- mice. Furthermore, higher number of apoptotic cells remaining in peritoneum was observed in Mcp-1-/- or Ccr2-/- mice than WT or Ccr2+/- control, despite of the comparable phagocytic ability of resident peritoneal macrophages between genotypes. To sum up, these data suggest that not only apoptotic cells but also phagocytes release signals to recruit additional phagocytes to sites of apoptosis through Mcp-1-Ccr2 axis and thus clear apoptotic cells more efficiently. In part 2, I focused on the identification of PS-binding proteins. Despite the importance of PS recognition in various situations, only a limited number of PS interacting proteins are reported so far and thus still remained to be elucidated. I performed a pulldown assay to identify PS-associating proteins by utilizing streptavidin-coated magnetic beads and biotin-linked PS. Hsd17b4, a peroxisomal protein, was identified as a PS-binding protein in this approach and further investigated. Interestingly, Hsd17b4 associates with PS regardless of calcium, unlike other PS binding proteins. Also, Hsd17b4 interacts with PS through its Scp-2-like domain. Hsd17b4 recognizes the topology of PS to associate with it, which is supported by the disrupted binding of Hsd17b4 with PS in the presence of liposomes, but not by free PS or the components of PS. Translocation of PS head from cytosolic space to the outer leaflet of plasma membrane affected the subcellular localization of Hsd17b4. Hsd17b4 enrichment in peroxisomes was observed when PS exposure was induced. To summarize, my study identified Hsd17b4 as a novel PS-binding protein and an unexpected ability of PS to regulate the subcellular localization of Hsd17b4.