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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

Uptake of exogenous CnB did not occur via binding to LPS, and CnB induced cytokine secretion.(a) The uptake of CnB could be inhibited by LPS. (b) The uptake of LPS could be inhibited by CnB. A 30-fold excess of LPS or CnB was mixed with CnB-GFP or rhodamine-labelled LPS and incubated with the SK-HEP-1 cells for 30 min. The results were visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×) and quantified using a microplate reader (lower panel). (c) CnB-GFP did not bind to LPS. To a black ELISA plate, 10 μg/ml LPS was immobilized and incubated with different concentrations of CnB-GFP, DyLight 488-labeled CD14 or GFP to evaluate the binding between CnB and LPS. (d) The CnB-induced cytokine production was not due to LPS contamination. The RAW264.7 cells were incubated with 1 μg/ml LPS, 100 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B or 100 μg/ml CnB in the presence of 100 μg/ml polymyxin B for 24 h and the levels of the secreted cytokines in the supernatant were measured by ELISA. (e) NF-κB was activated by CnB in the TLR4-transfected Hek293 cells. The Hek293 cells were transfected with the TLR4-pcDNA3.1 plasmid and incubated for 24 h. The cells were plated in 24-well plates (1 × 105/well) and co-transfected with the pNF-κB-luc and pRL--Renilla-luc plasmids. Twenty-four hours post-transfection, the cells were co-incubated with 1 μg/ml LPS, 400 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B, 400 μg/ml CnB in the presence of 100 μg/ml polymyxin B, 1 μg/ml proteinase K-treated LPS or 400 μg/ml proteinase K-treated CnB for 12 h. Luciferase activity was measured using a Dual-luciferase Reporter system. The data were normalized to the control. Data represent three independent experiments (mean ± s.e.m., n = 3). *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).
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f4: Uptake of exogenous CnB did not occur via binding to LPS, and CnB induced cytokine secretion.(a) The uptake of CnB could be inhibited by LPS. (b) The uptake of LPS could be inhibited by CnB. A 30-fold excess of LPS or CnB was mixed with CnB-GFP or rhodamine-labelled LPS and incubated with the SK-HEP-1 cells for 30 min. The results were visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×) and quantified using a microplate reader (lower panel). (c) CnB-GFP did not bind to LPS. To a black ELISA plate, 10 μg/ml LPS was immobilized and incubated with different concentrations of CnB-GFP, DyLight 488-labeled CD14 or GFP to evaluate the binding between CnB and LPS. (d) The CnB-induced cytokine production was not due to LPS contamination. The RAW264.7 cells were incubated with 1 μg/ml LPS, 100 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B or 100 μg/ml CnB in the presence of 100 μg/ml polymyxin B for 24 h and the levels of the secreted cytokines in the supernatant were measured by ELISA. (e) NF-κB was activated by CnB in the TLR4-transfected Hek293 cells. The Hek293 cells were transfected with the TLR4-pcDNA3.1 plasmid and incubated for 24 h. The cells were plated in 24-well plates (1 × 105/well) and co-transfected with the pNF-κB-luc and pRL--Renilla-luc plasmids. Twenty-four hours post-transfection, the cells were co-incubated with 1 μg/ml LPS, 400 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B, 400 μg/ml CnB in the presence of 100 μg/ml polymyxin B, 1 μg/ml proteinase K-treated LPS or 400 μg/ml proteinase K-treated CnB for 12 h. Luciferase activity was measured using a Dual-luciferase Reporter system. The data were normalized to the control. Data represent three independent experiments (mean ± s.e.m., n = 3). *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).

Mentions: LPS is a well-validated ligand of TLR4. Recently, a number of other endogenous TLR4 agonists have been identified; however, it remains a challenge to provide convincing evidence that they are direct TLR4 ligands. Previous research has shown that some endogenous ligands can function as mediators or chaperones by binding to LPS2632. To evaluate the ability of LPS to facilitate CnB uptake, we treated cells with excess LPS and CnB. We found that excess LPS completely inhibited the uptake of CnB (upper panel in Fig. 4a), and there was a significant difference between the LPS-untreated group and the LPS-treated group (lower panel in Fig. 4a). Conversely, excess CnB also partially inhibited LPS uptake under the present experimental conditions (Fig. 4b); its inability to completely inhibit LPS uptake was probably due to the higher affinity between LPS and the TLR4 receptor complexes. Next, we tested for direct binding between CnB and LPS by ELISA. The results indicated that CnB did not bind to LPS, but the positive control, CD14, bound to LPS in a concentration-dependent manner. CD14 is a known receptor of LPS and is essential for LPS-induced endocytosis of TLR4 (Fig. 4c).


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)

Uptake of exogenous CnB did not occur via binding to LPS, and CnB induced cytokine secretion.(a) The uptake of CnB could be inhibited by LPS. (b) The uptake of LPS could be inhibited by CnB. A 30-fold excess of LPS or CnB was mixed with CnB-GFP or rhodamine-labelled LPS and incubated with the SK-HEP-1 cells for 30 min. The results were visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×) and quantified using a microplate reader (lower panel). (c) CnB-GFP did not bind to LPS. To a black ELISA plate, 10 μg/ml LPS was immobilized and incubated with different concentrations of CnB-GFP, DyLight 488-labeled CD14 or GFP to evaluate the binding between CnB and LPS. (d) The CnB-induced cytokine production was not due to LPS contamination. The RAW264.7 cells were incubated with 1 μg/ml LPS, 100 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B or 100 μg/ml CnB in the presence of 100 μg/ml polymyxin B for 24 h and the levels of the secreted cytokines in the supernatant were measured by ELISA. (e) NF-κB was activated by CnB in the TLR4-transfected Hek293 cells. The Hek293 cells were transfected with the TLR4-pcDNA3.1 plasmid and incubated for 24 h. The cells were plated in 24-well plates (1 × 105/well) and co-transfected with the pNF-κB-luc and pRL--Renilla-luc plasmids. Twenty-four hours post-transfection, the cells were co-incubated with 1 μg/ml LPS, 400 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B, 400 μg/ml CnB in the presence of 100 μg/ml polymyxin B, 1 μg/ml proteinase K-treated LPS or 400 μg/ml proteinase K-treated CnB for 12 h. Luciferase activity was measured using a Dual-luciferase Reporter system. The data were normalized to the control. Data represent three independent experiments (mean ± s.e.m., n = 3). *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).
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f4: Uptake of exogenous CnB did not occur via binding to LPS, and CnB induced cytokine secretion.(a) The uptake of CnB could be inhibited by LPS. (b) The uptake of LPS could be inhibited by CnB. A 30-fold excess of LPS or CnB was mixed with CnB-GFP or rhodamine-labelled LPS and incubated with the SK-HEP-1 cells for 30 min. The results were visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×) and quantified using a microplate reader (lower panel). (c) CnB-GFP did not bind to LPS. To a black ELISA plate, 10 μg/ml LPS was immobilized and incubated with different concentrations of CnB-GFP, DyLight 488-labeled CD14 or GFP to evaluate the binding between CnB and LPS. (d) The CnB-induced cytokine production was not due to LPS contamination. The RAW264.7 cells were incubated with 1 μg/ml LPS, 100 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B or 100 μg/ml CnB in the presence of 100 μg/ml polymyxin B for 24 h and the levels of the secreted cytokines in the supernatant were measured by ELISA. (e) NF-κB was activated by CnB in the TLR4-transfected Hek293 cells. The Hek293 cells were transfected with the TLR4-pcDNA3.1 plasmid and incubated for 24 h. The cells were plated in 24-well plates (1 × 105/well) and co-transfected with the pNF-κB-luc and pRL--Renilla-luc plasmids. Twenty-four hours post-transfection, the cells were co-incubated with 1 μg/ml LPS, 400 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B, 400 μg/ml CnB in the presence of 100 μg/ml polymyxin B, 1 μg/ml proteinase K-treated LPS or 400 μg/ml proteinase K-treated CnB for 12 h. Luciferase activity was measured using a Dual-luciferase Reporter system. The data were normalized to the control. Data represent three independent experiments (mean ± s.e.m., n = 3). *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).
Mentions: LPS is a well-validated ligand of TLR4. Recently, a number of other endogenous TLR4 agonists have been identified; however, it remains a challenge to provide convincing evidence that they are direct TLR4 ligands. Previous research has shown that some endogenous ligands can function as mediators or chaperones by binding to LPS2632. To evaluate the ability of LPS to facilitate CnB uptake, we treated cells with excess LPS and CnB. We found that excess LPS completely inhibited the uptake of CnB (upper panel in Fig. 4a), and there was a significant difference between the LPS-untreated group and the LPS-treated group (lower panel in Fig. 4a). Conversely, excess CnB also partially inhibited LPS uptake under the present experimental conditions (Fig. 4b); its inability to completely inhibit LPS uptake was probably due to the higher affinity between LPS and the TLR4 receptor complexes. Next, we tested for direct binding between CnB and LPS by ELISA. The results indicated that CnB did not bind to LPS, but the positive control, CD14, bound to LPS in a concentration-dependent manner. CD14 is a known receptor of LPS and is essential for LPS-induced endocytosis of TLR4 (Fig. 4c).

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