We showed previously that the calcineurin B subunit (CnB) plays an important role in activation of peritoneal macrophage, but the underlying mechanism remained unknown. To examine whether there is a CnB receptor on peritoneal macrophages, we performed the radioligand binding assay of receptors. The receptor saturation binding curve demonstrated high-affinity and specific binding; the maximum binding was 1090 fmol/105 cells, and the Kd was 70.59 pM. Then, we used a CnB affinity resin to trap potential receptors from highly purified peritoneal macrophage membranes. Mass spectrometry analysis showed that the binding protein was mouse integrin αM. We next performed a competition binding experiment to confirm the binding of CnB to integrin αM. This showed that FITC-CnB bound specifically to peritoneal macrophages and that binding was blocked by the addition of integrin αM Ab. We observed that CnB could induce TRAIL gene expression in peritoneal macrophages in vitro and in vivo. Integrin αM Ab blocking, RNA interference, and ligand competition experiments demonstrated that CnB-induced TRAIL expression is dependent on integrin αM. Furthermore, the tumoricidal activity of CnB-activated peritoneal macrophages is partially dependent on TRAIL. In addition, CnB treatment significantly prolongs the survival of mice bearing H22 ascites tumors, which has a positive correlation with the induction level of TRAIL. These results reveal a novel function of the CnB in innate immunity and cancer surveillance. They also point to a new signaling pathway leading to induction of TRAIL and suggest a possible application of CnB in cancer therapy.

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SummaryCalcineurin is the only Ca 21 /calmodulin-dependent serine/ threonine protein phosphatase. The roles of the cytosolic calcineurin have been well researched; however, the roles of the serum calcineurin remain unknown. Here, we report that the recombinant human calcineurin B subunit (CnB) can bind to rabbit platelets and show an antiplatelet aggregation activity. Furthermore, CnB exerts an anticoagulant effect by prolonging the activated partial thromboplastin time and thrombin time and reducing the plasma fibrinogen concentration in a dose-dependent manner. We further reveal that the functional domain associated with the anticoagulant activity of CnB is located in the C-terminus. Hemolysis test and intravenous stimulation study show that the recombinant CnB does not cause obvious hemolysis and is safe for intravenous injection. These results reveal a new function of calcineurin B subunit. They also give an explanation for the roles of calcineurin B subunit in serum and point to a possible implication in antithrombotic therapy.

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