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-4mediated 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.

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

“…Inversely, the IgV domain of Tim-4 co-precipitated with the extracellular region of Mertk, while the mucin domain of Tim-4 did not co-precipitate ( Figure 3D and Figure S2). In agreement with a previous report [19], the expression level of the IgV domain of Tim-4 was exceptionally low. Thus, the IgV domain was undetectable in total cell lysates.…”

“…PS receptors have differing abilities to transduce signals into phagocytes. Some PS receptors can directly transduce signals into phagocytes after binding to apoptotic cells, while other receptors secure apoptotic cells to phagocytes without directly initiating signal transduction; these latter receptors are called tethering receptors [18][19][20]. Tethering receptors are thought to facilitate the recognition and ingestion of apoptotic cells by other engulfment receptors capable of mediating direct signaling [19,[21][22][23][24].…”

“…Tim-4, however, does not mediate direct signaling in efferocytosis because its cytoplasmic tail, which is about 40 amino acids long, is too short to contain a signaling motif [29]. Moreover, efferocytosis mediated by Tim-4 mutants lacking the cytoplasmic tail or transmembrane domain is unimpaired and comparable with that mediated by the full-length protein [18,19,23]. Thus, current understanding is that Tim-4 secures apoptotic cells to phagocytes by directly binding to PS on apoptotic cells, and that other engulfment receptors such as α v β 3 and Mertk that are capable of direct signaling thereafter efficiently recognize apoptotic cells and mediate their ingestion.…”

“…The IgV domain of Tim-4 interacts with the fibronectin type-III (FnIII) domain of Fn1, and thus Fn1 forms a complex with Tim-4 and integrins. The formation of this complex promotes synergistic cooperation of these two types of engulfment receptors, leading to efficient binding to and ingestion of apoptotic cells [19]. However, disrupting the complex or inhibiting integrins partially inhibits Tim-4-mediated efferocytosis.…”

Tim-4 in Health and Disease: Friend or Foe?

“…As expected above, Flannagan et al found that integrin proteins bound to Tim-4 as a co-receptor to induce the signal transduction cascade required for phagocytosis of apoptotic cells (43). Recently, Lee et al identified Fibronectin (Fn1) as a novel Tim-4-associating protein, and found that Fn1 acted as a scaffold to form the complex composed of Tim-4 and integrin to mediate efferocytosis (44).…”