Study on calcium influx during efferocytosis and phagocytosis of colorectal cancer

Abstract

Efferocytosis, phagocytosis of apoptotic cells, is important to regulate cellular homeostasis and immune tolerance in multicellular organisms. Efferocytosis is the sophistical process that various mechanisms and factors are included to complete it. Defective efferocytosis is associated with severe diseases such as autoimmune disease, atherosclerosis and cancer. However, the full mechanisms and understandings of efferocytosis remain elusive. Although calcium is important for efferocytosis, the mechanism of calcium influx into phagocytes during efferocytosis has not been understood. In part I, we found that apoptotic cells induced calcium influx into phagocytes activating store-operated calcium entry (SOCE) via Mertk/phospholipase C (PLC) γ1-inositol 1,4,5-triphosphate receptor (IP3R) axis. Apoptotic cells enhanced the interaction between STIM1 and Orai1 leading calcium release from ER and calcium influx from extracellular to intracellular space. Especially, masking of PS on apoptotic cells inhibited the interaction STIM1-Orai1. In addition, Mertk-/- phagocytes decreased both phosphorylation of PLC1 and IP3R attenuating the association STIM1-Orai1. Apoptotic cells increased intracellular calcium level in WT BMDMs but not in Mertk-/- BMDMs. Taken together, we uncover the novel molecular mechanism that apoptotic cells induce calcium influx in phagocytes through PS-Mertk/PLC1/IP3R/STIM1-Orai1 leading SOCE pathway during efferocytosis. Phosphatidylserine (PS) is a representative eat-me ligand exposed on apoptotic cells, not live cells. However, PS is unusually expressed on outer leaflet in viable cancer cells and protects cancer cells from immune responses. In part II, we found that PS was externalized on human colorectal cancer cells. Especially, PS exposure increased on Dukes C, HCT15 and DLD-1 in Dukes types of colorectal cancer defined by the degree of metastasis. In addition, macrophages engulfed higher Dukes C, HCT15 and DLD-1, than Dukes B and D, SW480 and HCT116, respectively. The engulfment was dependent to PS on HCT15 and DLD-1 and PS masked by Mfge8D89E diminished clearance of HCT15 and DLD-1. Finally, TIMP1 expression increased in macrophages engulfing HCT15 and DLD-1, not SW480 and HCT116. Collectively, these data suggest that colorectal cancer cells differently externalize PS on outer leaflet and macrophages engulf colorectal cancer cells in PS-dependent manner leading elevation of TIMP1 expression. It will help to understand how macrophages regulate cancer progression in tumor microenvironment.