Abstract
The efficient engulfment of apoptotic cells by professional or nonprofessional phagocytes is critical to maintain mammalian homeostasis. To identify molecules involved in the engulfment of apoptotic cells, we established a retrovirus-based expression cloning system coupled with the engulfment assay. By screening a cDNA library of a mouse macrophage cell line, we identified two small GTPase family members (RhoG and Rab5) that enhanced the engulfment of apoptotic cells. By examining other small GTPase family members, we found that Rac1 enhanced the engulfment of apoptotic cells, whereas RhoA inhibited the process. Accordingly, the expression of a dominant-negative form of RhoG or Rac1 in primary macrophage cultures severely reduced the ability of the macrophages to engulf apoptotic cells, and a dominant-negative form of RhoA enhanced the process. These results indicated that the efficient engulfment of apoptotic cells requires the concerted action of small GTPase family members. We demonstrated previously that NIH3T3 cells expressing the alphav beta3 integrin efficiently engulf apoptotic cells in the presence of milk fat globule epidermal growth factor 8 via a phosphatidylserine-dependent mechanism. The dominant-negative form of RhoG or Rac1 inhibited this process, which suggested RhoG and Rac1 are also involved in the integrin-mediated engulfment.
Highlights
Apoptotic cells are rapidly engulfed by professional phagocytes or less efficiently by nonprofessional phagocytes such as fibroblasts and epithelial cells [7, 8]
Identification of cDNAs that Enhance Engulfment of Apoptotic Cells— To identify molecules involved in the engulfment of apoptotic cells by macrophages, we used a retrovirus-mediated expression cloning system (Fig. 1A)
Plasmid DNA from each pool was introduced into PLAT-E packaging cells, and the retrovirus produced by the packaging cells was used to infect mouse NIH3T3 cells
Summary
The recognition of apoptotic cells seems to activate a cascade of intracellular molecules in phagocytes, leading to a rearrangement of the cytoskeleton that permits the efficient engulfment of apoptotic cells. Genetic analysis of programmed cell death in Caenorhabditis elegans has identified a set of genes that are involved in removal of cell corpses [14] Of these genes, ced-2, ced-5, ced-10, and ced-12 are on the same signaling pathway and encode homologs of mammalian CrkII, DOCK180, Rac, and Elmo [15,16,17]; these mammalian proteins are thought to be involved in the engulfment of apoptotic cells (18 –21). To identify the phagocyte receptors and their downstream molecules involved in the engulfment of apoptotic cells, we developed a functional screening strategy using a retrovirus cDNA library With this assay, we isolated cDNA clones encoding Rab and RhoG that enhanced the engulfment of apoptotic cells. By expressing the wild-type or dominantnegative forms of Rho family GTPases, we showed that Rac1/RhoG and RhoA had opposite effects on the engulfment of apoptotic cells
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