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Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.

Yang J, Qin N, Zhang H, Yang R, Xiang B, Wei Q - Sci Rep (2016)

Bottom Line: 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.These properties of CnB support its potential for development as an anti-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.

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.

No MeSH data available.


Related in: MedlinePlus

CnB triggered cytokine secretion via the TLR4 signalling pathway in RAW264.7 macrophages, but not in SK-HEP-1 cells.(a) CnB induced the secretion of pro-inflammatory cytokines secretion through the MyD88-dependent pathway in the macrophage cell line. (b) CnB induced CCL5 and IFNβ secretion through the TRIF-dependent pathway in the macrophage cell line. The RAW264.7 cells were treated with 5, 25, and 100 μg/ml CnB for 24 h, and the SK-HEP-1 cells were treated with 200, 400, and 800 μg/ml CnB for 48 h. The amounts of secreted cytokines were determined by ELISA. Data represent mean ± s.e.m. from three independent experiments.
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f3: CnB triggered cytokine secretion via the TLR4 signalling pathway in RAW264.7 macrophages, but not in SK-HEP-1 cells.(a) CnB induced the secretion of pro-inflammatory cytokines secretion through the MyD88-dependent pathway in the macrophage cell line. (b) CnB induced CCL5 and IFNβ secretion through the TRIF-dependent pathway in the macrophage cell line. The RAW264.7 cells were treated with 5, 25, and 100 μg/ml CnB for 24 h, and the SK-HEP-1 cells were treated with 200, 400, and 800 μg/ml CnB for 48 h. The amounts of secreted cytokines were determined by ELISA. Data represent mean ± s.e.m. from three independent experiments.

Mentions: In RAW264.7 cells (a macrophage cell line), CnB stimulation induced the secretion and production of pro-inflammatory cytokines (Fig. 3a), and it also triggered the secretion of IFNβ and CCL5 (Fig. 3b). However, TNFα, IL-1β, CCL5 and IFNβ were not secreted by the SK-HEP-1 cells (a hepatocarcinoma cell line) (data not shown). The qPCR results revealed that CnB stimulation first up-regulated (Fig. S3a) and then down-regulated the expression of these genes in SK-HEP-1 cells (Fig. S3b). In RAW264.7 cells, rhCnB only up-regulated the expression of TNFα, CCL5 and IFNβ at the evaluated time points (Fig. S3c,d). Moreover, lower concentrations of CnB effectively stimulated cytokine production by the immune cells. However, higher concentrations of CnB were required to achieve similar stimulation in the tumour cells. In our previous studies, rhCnB was found to activate immune cells to secrete pro-inflammatory cytokines56710. These results reveal that CnB exerts different mechanisms in immune and non-immune cells.


Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.

Yang J, Qin N, Zhang H, Yang R, Xiang B, Wei Q - Sci Rep (2016)

CnB triggered cytokine secretion via the TLR4 signalling pathway in RAW264.7 macrophages, but not in SK-HEP-1 cells.(a) CnB induced the secretion of pro-inflammatory cytokines secretion through the MyD88-dependent pathway in the macrophage cell line. (b) CnB induced CCL5 and IFNβ secretion through the TRIF-dependent pathway in the macrophage cell line. The RAW264.7 cells were treated with 5, 25, and 100 μg/ml CnB for 24 h, and the SK-HEP-1 cells were treated with 200, 400, and 800 μg/ml CnB for 48 h. The amounts of secreted cytokines were determined by ELISA. Data represent mean ± s.e.m. from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835703&req=5

f3: CnB triggered cytokine secretion via the TLR4 signalling pathway in RAW264.7 macrophages, but not in SK-HEP-1 cells.(a) CnB induced the secretion of pro-inflammatory cytokines secretion through the MyD88-dependent pathway in the macrophage cell line. (b) CnB induced CCL5 and IFNβ secretion through the TRIF-dependent pathway in the macrophage cell line. The RAW264.7 cells were treated with 5, 25, and 100 μg/ml CnB for 24 h, and the SK-HEP-1 cells were treated with 200, 400, and 800 μg/ml CnB for 48 h. The amounts of secreted cytokines were determined by ELISA. Data represent mean ± s.e.m. from three independent experiments.
Mentions: In RAW264.7 cells (a macrophage cell line), CnB stimulation induced the secretion and production of pro-inflammatory cytokines (Fig. 3a), and it also triggered the secretion of IFNβ and CCL5 (Fig. 3b). However, TNFα, IL-1β, CCL5 and IFNβ were not secreted by the SK-HEP-1 cells (a hepatocarcinoma cell line) (data not shown). The qPCR results revealed that CnB stimulation first up-regulated (Fig. S3a) and then down-regulated the expression of these genes in SK-HEP-1 cells (Fig. S3b). In RAW264.7 cells, rhCnB only up-regulated the expression of TNFα, CCL5 and IFNβ at the evaluated time points (Fig. S3c,d). Moreover, lower concentrations of CnB effectively stimulated cytokine production by the immune cells. However, higher concentrations of CnB were required to achieve similar stimulation in the tumour cells. In our previous studies, rhCnB was found to activate immune cells to secrete pro-inflammatory cytokines56710. These results reveal that CnB exerts different mechanisms in immune and non-immune cells.

Bottom Line: 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.These properties of CnB support its potential for development as an anti-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.

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.

No MeSH data available.


Related in: MedlinePlus