β-Integrins traffic with system xc- in confluent and non-confluent cultures of human glioma (U138MG) cells
Dr. Leah Chase, Hope College
System xc- is a plasma membrane transport system that is comprised of two proteins, xCT and 4F2HC. The transporter catalyzes the exchange of cystine and glutamate across the membrane glia and some neurons. Previous research in the Chase lab demonstrated that differences in basal transporter activity in confluent and non-confluent cultured human glioma (U138-MG) cells are a result of differential membrane localization of xCT. We hypothesized that the changes in membrane localization of the transporter is mediated by its association with cell-surface adhesion molecules, b1-integrins, as one of the protein components of System xc-, 4F2HC, forms a dimer with b1-integrins. Specifically, we hypothesized that the decrease in transporter activity observed with increased cell density is a result of internalization of a 4F2HC: xCT: β1-integrin complex. To test this hypothesis, we measured relative membrane expression and internalization of xCT, CD98, and β-integrin in cultures that were 95% confluent compared with cultures that were 50% confluent. We first confirmed that xCT and 4F2HC form complexes with b1-integrins in both confluent and non-confluent cultures. In addition, we confirmed that the localization of β1-Integrins on the plasma membrane decreases with increased cell culture density. We also explored the functional implications of these findings. We hypothesized that the differences in membrane trafficking of System xc- in confluent and non-confluent cultures would result in a difference in their susceptibility to H2O2 –mediated damage. Using a trypan blue cell death assay, we demonstrated that confluent cultures were less susceptible to H2O2 –mediated cell death in comparison to non-confluent cell cultures (p <0.01). These data provide further evidence that the trafficking of System xc- plays an important role in the acute antioxidant defense pathway of glioma cells.
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