Limits...
Basigin-mediated redistribution of CD98 promotes cell spreading and tumorigenicity in hepatocellular carcinoma.

Wu B, Wang Y, Yang XM, Xu BQ, Feng F, Wang B, Liang Q, Li Y, Zhou Y, Jiang JL, Chen ZN - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: Dysregulated endocytosis of membrane proteins contributes significantly to several hallmarks of cancer.Internalization of basigin and CD98 was flotillin-1 regulated the and their recycling was mediated by Arf6.Basigin, as a redistribution chaperone of CD98, plays a critical role in promoting cell spreading and the progression of hepatocellular carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Cell Engineering Research Centre & Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, 169 Changle West Road, Xi'an, 710032, P. R. China. soldier2158wubo@163.com.

ABSTRACT

Background: Dysregulated endocytosis of membrane proteins contributes significantly to several hallmarks of cancer. Basigin can enhance cancer progression, but its precise mechanism remains unclear. CD98 promotes cell spreading and tumorigenicity by triggering integrin clustering and enhancing cell adhesion to the extracellular matrix. The endocytosis and recyle of basigin and CD98 might play critical roles in cancer.

Methods: The role of CD98 was confirmed in liver cancer cells by cell spreading in vitro and tumorigenicity by nude mice xenograft tumor assay in vivo; membrane expression of basigin and CD98 in SMMC-7721 was measured by FCAS; pull down and SPR analysis were uses to reveal the direct association between basigin and CD98; DsRed1 tagged CD98 was blocked in the cytoplasm in K7721 (whose basigin was knockn out) and had a well colocalization with ER and Rab5a positive recycling endosomes under co-focal; finally, by FRET imaging and FCAS we observed the internalization of basigin and CD98 was flotillin-1-regulated, and their recycle at early steps was Arf6-mediated.

Results: Basigin and CD98 were highly expressed and co-localized on the human hepatocellular carcinoma (HCC) cell membrane; basigin can directly bind to CD98, mediating CD98 redistribution on the HCC cell membrane and activating the downstream integrin signaling pathway. Internalization of basigin and CD98 was flotillin-1 regulated the and their recycling was mediated by Arf6. This recycling process for basigin and CD98 promotes cell spreading and tumor growth in liver cancer xenografts.

Conclusion: Basigin, as a redistribution chaperone of CD98, plays a critical role in promoting cell spreading and the progression of hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus

CD98 directly interacts with basigin in HCC cells. a Co-localization of basigin and CD98 in SMMC7721, HepG2 and Huh7 HCC cell lines. The cells were grown on coverslips for 24 h, fixed and stained with Dylight488-conjugated goat-anti-rabbit antibodies (basigin, Green) and Dylight594-conjugated goat-anti-mouse antibodies (CD98, Red). Huh7 cells were treated with 0.2 % Triton X-100 after fixation to visualize the intracellular distribution of basigin and CD98. Bar, 10 μm. b CD147-ED pulls down CD98-ED in vitro. HAb18 (antibody against CD147-ED; 10 μg) was used as a coupling antibody by immobilizing it onto the coupling resin. Then, the indicated amount of the antigen was added and eluted. The collected samples were analyzed by western blotting. c Biophysical analysis of the interaction of CD98-ED with CD147-ED using SPR. The indicated concentrations of purified CD98-ED were injected over immobilized CD147-ED, and the biophysical parameters were derived from a 1:1 binding model (red line). RU, response units
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4594993&req=5

Fig3: CD98 directly interacts with basigin in HCC cells. a Co-localization of basigin and CD98 in SMMC7721, HepG2 and Huh7 HCC cell lines. The cells were grown on coverslips for 24 h, fixed and stained with Dylight488-conjugated goat-anti-rabbit antibodies (basigin, Green) and Dylight594-conjugated goat-anti-mouse antibodies (CD98, Red). Huh7 cells were treated with 0.2 % Triton X-100 after fixation to visualize the intracellular distribution of basigin and CD98. Bar, 10 μm. b CD147-ED pulls down CD98-ED in vitro. HAb18 (antibody against CD147-ED; 10 μg) was used as a coupling antibody by immobilizing it onto the coupling resin. Then, the indicated amount of the antigen was added and eluted. The collected samples were analyzed by western blotting. c Biophysical analysis of the interaction of CD98-ED with CD147-ED using SPR. The indicated concentrations of purified CD98-ED were injected over immobilized CD147-ED, and the biophysical parameters were derived from a 1:1 binding model (red line). RU, response units

Mentions: To explore how basigin mediates the membrane localization of CD98 in HCC cells, the interaction of these two molecules was analyzed. The immunofluorescence staining results showed that there was a significant colocalization of CD147 with CD98 in HCC cells (SMMC-7721, HepG2 and Huh7; Fig. 3a). The immunoprecipitation results in our previous report showed that CD147 and CD98 could form a complex in vitro [21]. To determine whether the two molecules directly interact, we then expressed the extracellular domain of basigin (CD147-ED) [33] and the extracellular domain of CD98 (CD98-ED) in Escherichia coli BL21 (DE3) and purified the proteins (Additional file 2: Supplementary material Fig. S3) to further explore their interaction. As shown in Fig. 3b, CD147-ED could pull down CD98-ED in a concentration-dependent manner. Furthermore, surface plasmon resonance (SPR) assays obtained a similar result, in which the SPR data fit well to a one-shot kinetic binding model (chi-squared value 3.73) and revealed an average binding affinity (dissociation equilibrium constant, KD) of approximately 0.187 μM (see table in Fig. 3c). These results were also consistent with our previous report [21] and indicated that basigin directly interacted with CD98.Fig. 3


Basigin-mediated redistribution of CD98 promotes cell spreading and tumorigenicity in hepatocellular carcinoma.

Wu B, Wang Y, Yang XM, Xu BQ, Feng F, Wang B, Liang Q, Li Y, Zhou Y, Jiang JL, Chen ZN - J. Exp. Clin. Cancer Res. (2015)

CD98 directly interacts with basigin in HCC cells. a Co-localization of basigin and CD98 in SMMC7721, HepG2 and Huh7 HCC cell lines. The cells were grown on coverslips for 24 h, fixed and stained with Dylight488-conjugated goat-anti-rabbit antibodies (basigin, Green) and Dylight594-conjugated goat-anti-mouse antibodies (CD98, Red). Huh7 cells were treated with 0.2 % Triton X-100 after fixation to visualize the intracellular distribution of basigin and CD98. Bar, 10 μm. b CD147-ED pulls down CD98-ED in vitro. HAb18 (antibody against CD147-ED; 10 μg) was used as a coupling antibody by immobilizing it onto the coupling resin. Then, the indicated amount of the antigen was added and eluted. The collected samples were analyzed by western blotting. c Biophysical analysis of the interaction of CD98-ED with CD147-ED using SPR. The indicated concentrations of purified CD98-ED were injected over immobilized CD147-ED, and the biophysical parameters were derived from a 1:1 binding model (red line). RU, response units
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4594993&req=5

Fig3: CD98 directly interacts with basigin in HCC cells. a Co-localization of basigin and CD98 in SMMC7721, HepG2 and Huh7 HCC cell lines. The cells were grown on coverslips for 24 h, fixed and stained with Dylight488-conjugated goat-anti-rabbit antibodies (basigin, Green) and Dylight594-conjugated goat-anti-mouse antibodies (CD98, Red). Huh7 cells were treated with 0.2 % Triton X-100 after fixation to visualize the intracellular distribution of basigin and CD98. Bar, 10 μm. b CD147-ED pulls down CD98-ED in vitro. HAb18 (antibody against CD147-ED; 10 μg) was used as a coupling antibody by immobilizing it onto the coupling resin. Then, the indicated amount of the antigen was added and eluted. The collected samples were analyzed by western blotting. c Biophysical analysis of the interaction of CD98-ED with CD147-ED using SPR. The indicated concentrations of purified CD98-ED were injected over immobilized CD147-ED, and the biophysical parameters were derived from a 1:1 binding model (red line). RU, response units
Mentions: To explore how basigin mediates the membrane localization of CD98 in HCC cells, the interaction of these two molecules was analyzed. The immunofluorescence staining results showed that there was a significant colocalization of CD147 with CD98 in HCC cells (SMMC-7721, HepG2 and Huh7; Fig. 3a). The immunoprecipitation results in our previous report showed that CD147 and CD98 could form a complex in vitro [21]. To determine whether the two molecules directly interact, we then expressed the extracellular domain of basigin (CD147-ED) [33] and the extracellular domain of CD98 (CD98-ED) in Escherichia coli BL21 (DE3) and purified the proteins (Additional file 2: Supplementary material Fig. S3) to further explore their interaction. As shown in Fig. 3b, CD147-ED could pull down CD98-ED in a concentration-dependent manner. Furthermore, surface plasmon resonance (SPR) assays obtained a similar result, in which the SPR data fit well to a one-shot kinetic binding model (chi-squared value 3.73) and revealed an average binding affinity (dissociation equilibrium constant, KD) of approximately 0.187 μM (see table in Fig. 3c). These results were also consistent with our previous report [21] and indicated that basigin directly interacted with CD98.Fig. 3

Bottom Line: Dysregulated endocytosis of membrane proteins contributes significantly to several hallmarks of cancer.Internalization of basigin and CD98 was flotillin-1 regulated the and their recycling was mediated by Arf6.Basigin, as a redistribution chaperone of CD98, plays a critical role in promoting cell spreading and the progression of hepatocellular carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Cell Engineering Research Centre & Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, 169 Changle West Road, Xi'an, 710032, P. R. China. soldier2158wubo@163.com.

ABSTRACT

Background: Dysregulated endocytosis of membrane proteins contributes significantly to several hallmarks of cancer. Basigin can enhance cancer progression, but its precise mechanism remains unclear. CD98 promotes cell spreading and tumorigenicity by triggering integrin clustering and enhancing cell adhesion to the extracellular matrix. The endocytosis and recyle of basigin and CD98 might play critical roles in cancer.

Methods: The role of CD98 was confirmed in liver cancer cells by cell spreading in vitro and tumorigenicity by nude mice xenograft tumor assay in vivo; membrane expression of basigin and CD98 in SMMC-7721 was measured by FCAS; pull down and SPR analysis were uses to reveal the direct association between basigin and CD98; DsRed1 tagged CD98 was blocked in the cytoplasm in K7721 (whose basigin was knockn out) and had a well colocalization with ER and Rab5a positive recycling endosomes under co-focal; finally, by FRET imaging and FCAS we observed the internalization of basigin and CD98 was flotillin-1-regulated, and their recycle at early steps was Arf6-mediated.

Results: Basigin and CD98 were highly expressed and co-localized on the human hepatocellular carcinoma (HCC) cell membrane; basigin can directly bind to CD98, mediating CD98 redistribution on the HCC cell membrane and activating the downstream integrin signaling pathway. Internalization of basigin and CD98 was flotillin-1 regulated the and their recycling was mediated by Arf6. This recycling process for basigin and CD98 promotes cell spreading and tumor growth in liver cancer xenografts.

Conclusion: Basigin, as a redistribution chaperone of CD98, plays a critical role in promoting cell spreading and the progression of hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus