Abstract
Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug.
Highlights
Calcineurin B (CnB) is a regulatory subunit of calcineurin, and its basic function is to regulate the activity of calcineurin A (CnA)[1]
LPS is derived from the cell wall of Gram-negative bacteria, and it is recognized by a cascade of receptors, including CD14, TLR4 and MD2, that are known as TLR4 receptor complexes
We evaluated the interaction between soluble CD14 and CnB by ELISA, and the results revealed that CnB-GFP could bind to sCD14, which in turn could bind to CnB; GFP was unable to bind to either molecule (Fig. 5d)
Summary
Calcineurin B (CnB) is a regulatory subunit of calcineurin, and its basic function is to regulate the activity of calcineurin A (CnA)[1]. The activation of TRIF-dependent signalling requires TLR4 internalization and the transport of ligand-receptor complexes. This process is controlled by the co-receptor CD14 and culminates in the production of type I interferons (such as IFNβ ) and CCL513,14. LPS (endotoxin) was the first validated natural ligand of TLR4, and it functions as an agonist. LPS is derived from the cell wall of Gram-negative bacteria, and it is recognized by a cascade of receptors, including CD14, TLR4 and MD2, that are known as TLR4 receptor complexes. Followed by their transfer to CD14, which is found either in a soluble form or linked to the cell surface by a GPI anchor, CD14 presents LPS to the TLR4-MD2 complex. The safety and toxicity of these agents remain to be determined[20,21,22]
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