A scaffold for signaling of Tim-4-mediated efferocytosis is formed by fibronectin.

Abstract

An essential step during clearance of apoptotic cells is the recognition of phosphatidylserine (PS) exposed on apoptotic cells by its receptors on phagocytes. Tim-4 directly binding to PS and functioning as a tethering receptor for phagocytosis of apoptotic cells has been extensively studied over the past decade. However, the molecular mechanisms by which Tim-4 collaborates with other engulfment receptors during efferocytosis remain elusive. By comparing efferocytosis induced by Tim-4 with that by Anxa5-GPI, an artificial tethering receptor, we found that Tim-4 possesses auxiliary machinery to induce a higher level of efferocytosis than Anxa5-GPI. To search for that, we performed a yeast two-hybrid screen and identified Fibronectin (Fn1) as a novel Tim-4-associating protein. Tim-4 directly associated with Fn1 and formed a complex with integrins via the association of Fn1. Through Tim-4-/- mice and cell-based assays, we found that modulation of the Fn1 level affected efferocytosis induced by Tim-4 and disruption of the interaction between Tim-4 and Fn1 abrogated Tim-4-mediated efferocytosis. In addition, Tim-4 depletion attenuated integrin signaling activation and perturbation of integrin signaling suppressed Tim-4-promoted efferocytosis. Taken together, the data suggest that Fn1 locates Tim-4 and integrins in close proximity by acting as a scaffold, resulting in synergistic cooperation of Tim-4 with integrins for efficient efferocytosis.