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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

Exogenous CnB uptake is MD2-dependent.(a) Co-localization of CnB and MD2. The MD2-cherry-transfected Hek293 cells were co-incubated with CnB-GFP for 30 min, fixed, and visualized using a LSM700 confocal laser scanning microscope (63×, scale bar 10 μm). (b,c) Influence of MD2 knock down on CnB uptake. The influence of MD2 knock down on CnB uptake was analysed by western blot analysis (b) or FI (c). (d) Co-IP of CnB and MD2. The MD2-transfected HEK293 cells were lysed, and the lysates were co-incubated with CnB-GFP or GFP for 2 h at 4 °C. Next, rabbit anti-MD2 pAbs or rabbit IgG1 were added and incubated overnight. Protein A beads were used to capture the complex for 2 h, and the beads were then washed five times and boiled for 5 min. The interaction was detected by western blot analysis of anti-CnB antibody. Data represent mean ± s.e.m from two independent experiments. **P < 0.01 (t-test, two-tailed).
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f6: Exogenous CnB uptake is MD2-dependent.(a) Co-localization of CnB and MD2. The MD2-cherry-transfected Hek293 cells were co-incubated with CnB-GFP for 30 min, fixed, and visualized using a LSM700 confocal laser scanning microscope (63×, scale bar 10 μm). (b,c) Influence of MD2 knock down on CnB uptake. The influence of MD2 knock down on CnB uptake was analysed by western blot analysis (b) or FI (c). (d) Co-IP of CnB and MD2. The MD2-transfected HEK293 cells were lysed, and the lysates were co-incubated with CnB-GFP or GFP for 2 h at 4 °C. Next, rabbit anti-MD2 pAbs or rabbit IgG1 were added and incubated overnight. Protein A beads were used to capture the complex for 2 h, and the beads were then washed five times and boiled for 5 min. The interaction was detected by western blot analysis of anti-CnB antibody. Data represent mean ± s.e.m from two independent experiments. **P < 0.01 (t-test, two-tailed).

Mentions: MD2 is a secreted glycoprotein that primarily binds to the ectodomain of TLR4 to form TLR4-MD2 complex on the plasma membrane for LPS recognition, TLR4–MD-2 heterodimer is thought to form the complete recognition site for LPS, but MD2 is secreted in the absence of TLR4, and secreted MD2 (sMD2) was normally present in serum and body fluids. sMD2 probably impacts LPS recognition. MD2 is an essential accessory protein for TLR4 signaling35. To investigate the role of MD2 in CnB uptake, we incubated MD2-cherry-transfected Hek293 cells with CnB-GFP. The proteins clearly co-localized (Fig. 6a), but this co-localization was different from the that of TLR4/CnB or CD14/CnB. MD2-cherry and CnB-GFP partially overlapped, probably because there was not sufficient TLR4 for all of the over-expressed MD2 to form a complex. To further validate the importance of MD2 in CnB uptake, we knocked down MD2 with siRNA. Western blot (Fig. 6b) and fluorescence intensity analyses (Fig. 6c) showed that CnB uptake was significantly reduced in the MD2 siRNA cells compared with the negative siRNA control cells. The above results showed that MD2 was not only required, but was also dependent for CnB uptake, and it must form a complex with TLR4. Co-immuno-precipitation experiments revealed a direct interaction between CnB and MD2 in vitro (Fig. 6d). Hence we inferred that the role of MD2 in CnB uptake was to bind and internalize CnB together with TLR4. Concomitant with TLR4, CnB stimulation also induced surface MD2 clustering and an increase in surface expression of MD2 (Supplementary Fig. S7a). In SK-HEP-1 cells, treatment with CnB up-regulated MD2 gene expression (Supplementary Fig. S7c) and significantly increased the level of MD2 protein within 30 min (Supplementary Fig. S7b); this up-regulation was similar to the effect of CnB treatment on TLR4 expression.


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 uptake is MD2-dependent.(a) Co-localization of CnB and MD2. The MD2-cherry-transfected Hek293 cells were co-incubated with CnB-GFP for 30 min, fixed, and visualized using a LSM700 confocal laser scanning microscope (63×, scale bar 10 μm). (b,c) Influence of MD2 knock down on CnB uptake. The influence of MD2 knock down on CnB uptake was analysed by western blot analysis (b) or FI (c). (d) Co-IP of CnB and MD2. The MD2-transfected HEK293 cells were lysed, and the lysates were co-incubated with CnB-GFP or GFP for 2 h at 4 °C. Next, rabbit anti-MD2 pAbs or rabbit IgG1 were added and incubated overnight. Protein A beads were used to capture the complex for 2 h, and the beads were then washed five times and boiled for 5 min. The interaction was detected by western blot analysis of anti-CnB antibody. Data represent mean ± s.e.m from two independent experiments. **P < 0.01 (t-test, two-tailed).
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Related In: Results  -  Collection

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f6: Exogenous CnB uptake is MD2-dependent.(a) Co-localization of CnB and MD2. The MD2-cherry-transfected Hek293 cells were co-incubated with CnB-GFP for 30 min, fixed, and visualized using a LSM700 confocal laser scanning microscope (63×, scale bar 10 μm). (b,c) Influence of MD2 knock down on CnB uptake. The influence of MD2 knock down on CnB uptake was analysed by western blot analysis (b) or FI (c). (d) Co-IP of CnB and MD2. The MD2-transfected HEK293 cells were lysed, and the lysates were co-incubated with CnB-GFP or GFP for 2 h at 4 °C. Next, rabbit anti-MD2 pAbs or rabbit IgG1 were added and incubated overnight. Protein A beads were used to capture the complex for 2 h, and the beads were then washed five times and boiled for 5 min. The interaction was detected by western blot analysis of anti-CnB antibody. Data represent mean ± s.e.m from two independent experiments. **P < 0.01 (t-test, two-tailed).
Mentions: MD2 is a secreted glycoprotein that primarily binds to the ectodomain of TLR4 to form TLR4-MD2 complex on the plasma membrane for LPS recognition, TLR4–MD-2 heterodimer is thought to form the complete recognition site for LPS, but MD2 is secreted in the absence of TLR4, and secreted MD2 (sMD2) was normally present in serum and body fluids. sMD2 probably impacts LPS recognition. MD2 is an essential accessory protein for TLR4 signaling35. To investigate the role of MD2 in CnB uptake, we incubated MD2-cherry-transfected Hek293 cells with CnB-GFP. The proteins clearly co-localized (Fig. 6a), but this co-localization was different from the that of TLR4/CnB or CD14/CnB. MD2-cherry and CnB-GFP partially overlapped, probably because there was not sufficient TLR4 for all of the over-expressed MD2 to form a complex. To further validate the importance of MD2 in CnB uptake, we knocked down MD2 with siRNA. Western blot (Fig. 6b) and fluorescence intensity analyses (Fig. 6c) showed that CnB uptake was significantly reduced in the MD2 siRNA cells compared with the negative siRNA control cells. The above results showed that MD2 was not only required, but was also dependent for CnB uptake, and it must form a complex with TLR4. Co-immuno-precipitation experiments revealed a direct interaction between CnB and MD2 in vitro (Fig. 6d). Hence we inferred that the role of MD2 in CnB uptake was to bind and internalize CnB together with TLR4. Concomitant with TLR4, CnB stimulation also induced surface MD2 clustering and an increase in surface expression of MD2 (Supplementary Fig. S7a). In SK-HEP-1 cells, treatment with CnB up-regulated MD2 gene expression (Supplementary Fig. S7c) and significantly increased the level of MD2 protein within 30 min (Supplementary Fig. S7b); this up-regulation was similar to the effect of CnB treatment on TLR4 expression.

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