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A2903 - Endothelial Cell Barrier Enhancement Results in Differences in ITGB4 Variant Expression and Localization
Author Block: G. Kelly1, J. Gard2, J. G. Garcia2, A. Cress2, T. Wang1; 1University of Arizona, Phoenix, AZ, United States, 2University of Arizona, Tucson, AZ, United States.
Rationale: Integrin β4 (CD104, gene code: ITGB4) is an important transmembrane receptor protein involved in cellular signaling, cell-matrix communication, and endothelial cell barrier function. Recently we have shown that CD104 plays an important role in ventilator-induced lung injury (VILI), a clinical syndrome in the intensive care unit. Simvastatin, a potential therapeutic for VILI as well as acute respiratory distress syndrome (ARDS), upregulates both mRNA specific ITGB4 and the protein, which is critical for its protective effects. Multiple mRNA splice variants of CD104 exist, including the E variant (ITGβ4E), the only variant which has a unique 114 amino acid cytoplasmic domain. Our recent findings suggest that CD104E may be a key protective ITGB4 splicing variant against VILI, however, how this variant selectively offers protection remains unknown. Methods: In this study, we exposed human lung endothelial cells (ECs) to simvastatin and evaluated CD104E expression patterns compared to total CD104 using RT-PCR, western blotting, and imaging. Cells were also transfected with an CD104E expressing plasmid and analyzed by western blot. Results: Simvastatin upregulated all mRNA variants of the ITGB4 gene, with an initial spike in CD104E variant transcription. Additionally, this newly transcribed CD104, including the E variant, can heterodimerize with the expected binding partner integrin α6 (CD49f) and is on the cell surface. Since CD49f can also pair with an alternative beta subunit called β1 integrin (CD29), we tested if CD104E could influence α6β1 integrin pairing. Overexpression of CD104E decreased the abundance of α6β1 integrin on the cell surface, confirming its ability to be a dominant heterodimer combination. Confocal imaging results have confirmed that simvastatin upregulates CD104, with differing distributions of CD104E compared with other forms of CD104. Using an antibody specific for the extracellular domain that recognizes all variants of CD104, we observed a uniform distribution of CD104, with apparent cell surface clustering in about 50% of cells. Co-staining studies using an antibody that recognizes the cytoplasmic domain of CD104E indicates that CD104E does not participate in clustering, and has a distinct membrane domain distribution as compared to CD104. Conclusions: Our results indicate that exposure to simvastatin upregulates ITGB4 transcription and enhances production of CD104E, with variable distribution patterns between variants, suggesting alternative functions. Current work is underway to directly observe the dynamic distribution of these variants to determine their different roles in barrier protection.