Limits...
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

Exogenous CnB was taken up by different cell lines in a concentration- and time-dependent manner.(a) Exogenous CnB was incorporated into different cell lines. (b) Comparison of the uptake of different concentrations of CnB in SK-HEP-1 cells. (c) Comparison of CnB uptake at different time points. The cells were co-incubated with CnB-GFP, fixed and visualized using a Zeiss LSM700 confocal laser scanning microscope. The data were quantified using ImageJ software. FI represents the fluorescence intensity. The scale bar represents 50 μm, and the data represent mean ± s.e.m. from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835703&req=5

f1: Exogenous CnB was taken up by different cell lines in a concentration- and time-dependent manner.(a) Exogenous CnB was incorporated into different cell lines. (b) Comparison of the uptake of different concentrations of CnB in SK-HEP-1 cells. (c) Comparison of CnB uptake at different time points. The cells were co-incubated with CnB-GFP, fixed and visualized using a Zeiss LSM700 confocal laser scanning microscope. The data were quantified using ImageJ software. FI represents the fluorescence intensity. The scale bar represents 50 μm, and the data represent mean ± s.e.m. from three independent experiments.

Mentions: Exogenous CnB-GFP was incubated with different cell lines for 10 min or 30 min, and the confocal images revealed differences in staining among the cells. The fluorescence intensity in SK-HEP-1 and HaCaT cells was higher after 30 min than after 10 min, although uptake by MDA-MB-231 cells exhibited almost no increase with the extended incubation time (Fig. 1a). Next, we examined uptake of CnB-GFP by SK-HEP-1 cells at different concentrations, and found that increasing concentrations resulted in greater CnB-GFP uptake (Fig. 1b). Unlinked GFP was not taken up by SK-HEP-1 cells. However, when CnB-GFP was incubated with the cells for different durations, rapid uptake and positive staining were observed within 1 min. In addition, longer incubation times resulted in greater uptake of CnB-GFP (Fig. 1c). Thus, the uptake of exogenous CnB was both time- and concentration-dependent.


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)

Exogenous CnB was taken up by different cell lines in a concentration- and time-dependent manner.(a) Exogenous CnB was incorporated into different cell lines. (b) Comparison of the uptake of different concentrations of CnB in SK-HEP-1 cells. (c) Comparison of CnB uptake at different time points. The cells were co-incubated with CnB-GFP, fixed and visualized using a Zeiss LSM700 confocal laser scanning microscope. The data were quantified using ImageJ software. FI represents the fluorescence intensity. The scale bar represents 50 μm, and the 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

f1: Exogenous CnB was taken up by different cell lines in a concentration- and time-dependent manner.(a) Exogenous CnB was incorporated into different cell lines. (b) Comparison of the uptake of different concentrations of CnB in SK-HEP-1 cells. (c) Comparison of CnB uptake at different time points. The cells were co-incubated with CnB-GFP, fixed and visualized using a Zeiss LSM700 confocal laser scanning microscope. The data were quantified using ImageJ software. FI represents the fluorescence intensity. The scale bar represents 50 μm, and the data represent mean ± s.e.m. from three independent experiments.
Mentions: Exogenous CnB-GFP was incubated with different cell lines for 10 min or 30 min, and the confocal images revealed differences in staining among the cells. The fluorescence intensity in SK-HEP-1 and HaCaT cells was higher after 30 min than after 10 min, although uptake by MDA-MB-231 cells exhibited almost no increase with the extended incubation time (Fig. 1a). Next, we examined uptake of CnB-GFP by SK-HEP-1 cells at different concentrations, and found that increasing concentrations resulted in greater CnB-GFP uptake (Fig. 1b). Unlinked GFP was not taken up by SK-HEP-1 cells. However, when CnB-GFP was incubated with the cells for different durations, rapid uptake and positive staining were observed within 1 min. In addition, longer incubation times resulted in greater uptake of CnB-GFP (Fig. 1c). Thus, the uptake of exogenous CnB was both time- and concentration-dependent.

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