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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

The uptake of exogenous CnB is CD14-dependent.(a) Co-localization of exogenous CnB and CD14. The CD14-cherry-transfected Hek293 cells were co-incubated with CnB-GFP for 30 min, fixed, and visualized using a confocal laser scanning microscope (63×, scale bar 10 μm). (b,c) Influence of CD14 knock down on CnB uptake. The influence of CD14 knock down on CnB uptake was analysed by western blot analysis (b) or FI (c). (d) The binding of CnB to sCD14 or sCD14 to CnB. To black ELISA plates, 10 μg/ml CnB or sCD14 was immobilized, and CnB-GFP or DyLight 488-labeled CD14 were incubated with the corresponding ELISA plates. Data represent mean ± s.e.m. from three independent experiments. *P < 0.05 (t-test, two-tailed).
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f5: The uptake of exogenous CnB is CD14-dependent.(a) Co-localization of exogenous CnB and CD14. The CD14-cherry-transfected Hek293 cells were co-incubated with CnB-GFP for 30 min, fixed, and visualized using a confocal laser scanning microscope (63×, scale bar 10 μm). (b,c) Influence of CD14 knock down on CnB uptake. The influence of CD14 knock down on CnB uptake was analysed by western blot analysis (b) or FI (c). (d) The binding of CnB to sCD14 or sCD14 to CnB. To black ELISA plates, 10 μg/ml CnB or sCD14 was immobilized, and CnB-GFP or DyLight 488-labeled CD14 were incubated with the corresponding ELISA plates. Data represent mean ± s.e.m. from three independent experiments. *P < 0.05 (t-test, two-tailed).

Mentions: CD14 is a (GPI)-linked membrane glycoprotein that can bind directly to LPS, and has two roles in LPS-induced endocytosis: recognizing and chaperoning LPS to the TLR4/MD2 receptor complex, and transporting TLR4 receptor into the cell14. Interestingly, CD14 is also present as a soluble protein in large quantities in serum, soluble CD14 (sCD14) as well as cell-associated GPI-anchored protein are capable of enhancing the cellular cytokine response to LPS34. To investigate the role of CD14 in the recognition and transport of CnB, CD14-cherry transfected 293 cells were co-incubated with CnB-GFP for 30 min. CD14 and CnB were clearly co-localized on the plasma membrane and endosome (Fig. 5a), subsequently, we also knocked down CD14 in SK-HEP-1 cells by siRNA (Supplementary Fig. S5a) and found that CnB uptake (demonstrated by fluorescence intensity and western blot analysis) was significantly reduced in comparison to the negative siRNA control group (Fig. 5b,c and Supplementary Fig. S5b). These data suggested the uptake of CnB required CD14 and is CD14-dependent. Next, 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; however, GFP was unable to bind to either molecule (Fig. 5d). This interaction was also confirmed by co-immunoprecipitation experiments. We co-incubated CnB-GFP or GFP with sCD14 and then used rabbit anti-GFP antibodies or rabbit IgG to pull down the complexes. Anti-CD14 antibodies were used to detect the proteins by western blot analysis. The results revealed a specific and positive band for the Co-IP samples with CnB-GFP, but no positive bands were identified for the GFP or rabbit IgG samples (Supplementary Fig. S5c). These data suggested recognition of CnB required CD14, and CD14 mediated the uptake of CnB. We also detected an influence of CnB treatment on cell surface CD14 expression and showed that CnB stimulation induced CD14 clustering at the plasma membrane; however, it did not increase the surface levels of CD14 (Supplementary Fig. S6a). The same results were obtained in SK-HEP-1 cells using qPCR (Supplementary Fig. S6b) and western blot analyses (Supplementary Fig. S6c).


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)

The uptake of exogenous CnB is CD14-dependent.(a) Co-localization of exogenous CnB and CD14. The CD14-cherry-transfected Hek293 cells were co-incubated with CnB-GFP for 30 min, fixed, and visualized using a confocal laser scanning microscope (63×, scale bar 10 μm). (b,c) Influence of CD14 knock down on CnB uptake. The influence of CD14 knock down on CnB uptake was analysed by western blot analysis (b) or FI (c). (d) The binding of CnB to sCD14 or sCD14 to CnB. To black ELISA plates, 10 μg/ml CnB or sCD14 was immobilized, and CnB-GFP or DyLight 488-labeled CD14 were incubated with the corresponding ELISA plates. Data represent mean ± s.e.m. from three independent experiments. *P < 0.05 (t-test, two-tailed).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f5: The uptake of exogenous CnB is CD14-dependent.(a) Co-localization of exogenous CnB and CD14. The CD14-cherry-transfected Hek293 cells were co-incubated with CnB-GFP for 30 min, fixed, and visualized using a confocal laser scanning microscope (63×, scale bar 10 μm). (b,c) Influence of CD14 knock down on CnB uptake. The influence of CD14 knock down on CnB uptake was analysed by western blot analysis (b) or FI (c). (d) The binding of CnB to sCD14 or sCD14 to CnB. To black ELISA plates, 10 μg/ml CnB or sCD14 was immobilized, and CnB-GFP or DyLight 488-labeled CD14 were incubated with the corresponding ELISA plates. Data represent mean ± s.e.m. from three independent experiments. *P < 0.05 (t-test, two-tailed).
Mentions: CD14 is a (GPI)-linked membrane glycoprotein that can bind directly to LPS, and has two roles in LPS-induced endocytosis: recognizing and chaperoning LPS to the TLR4/MD2 receptor complex, and transporting TLR4 receptor into the cell14. Interestingly, CD14 is also present as a soluble protein in large quantities in serum, soluble CD14 (sCD14) as well as cell-associated GPI-anchored protein are capable of enhancing the cellular cytokine response to LPS34. To investigate the role of CD14 in the recognition and transport of CnB, CD14-cherry transfected 293 cells were co-incubated with CnB-GFP for 30 min. CD14 and CnB were clearly co-localized on the plasma membrane and endosome (Fig. 5a), subsequently, we also knocked down CD14 in SK-HEP-1 cells by siRNA (Supplementary Fig. S5a) and found that CnB uptake (demonstrated by fluorescence intensity and western blot analysis) was significantly reduced in comparison to the negative siRNA control group (Fig. 5b,c and Supplementary Fig. S5b). These data suggested the uptake of CnB required CD14 and is CD14-dependent. Next, 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; however, GFP was unable to bind to either molecule (Fig. 5d). This interaction was also confirmed by co-immunoprecipitation experiments. We co-incubated CnB-GFP or GFP with sCD14 and then used rabbit anti-GFP antibodies or rabbit IgG to pull down the complexes. Anti-CD14 antibodies were used to detect the proteins by western blot analysis. The results revealed a specific and positive band for the Co-IP samples with CnB-GFP, but no positive bands were identified for the GFP or rabbit IgG samples (Supplementary Fig. S5c). These data suggested recognition of CnB required CD14, and CD14 mediated the uptake of CnB. We also detected an influence of CnB treatment on cell surface CD14 expression and showed that CnB stimulation induced CD14 clustering at the plasma membrane; however, it did not increase the surface levels of CD14 (Supplementary Fig. S6a). The same results were obtained in SK-HEP-1 cells using qPCR (Supplementary Fig. S6b) and western blot analyses (Supplementary Fig. S6c).

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