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Experimental Biology 2010: P-Glycoprotein Does Not Contribute To Glucocorticoid Efflux In A Respiratory Epithelial Cell Model
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P-Glycoprotein Does Not Contribute To Glucocorticoid Efflux In A Respiratory Epithelial Cell Model
J. Cohen1, A.S. Benton2, F. Nwachukwu3, T. Ozedirne4, R.J. Freishtat1,2 1 Children's National Medical Center, Washington, DC 2 Children's Research Institute, Washington, DC 3 Temple University, Philadelphia, PA 4University of California-Davis, Davis, CA
Purpose of Study: Several mechanisms of glucocorticoid (GC) resistance in asthma have been identified, including GC-resistant genes and defective GC receptor binding and nuclear translocation. P-glycoprotein (P-gp), a ubiquitous ATP-dependent efflux transport protein expressed on airway epithelium, is known to transport GCs. High levels of P-gp expression have been found in patients with other GC-resistant inflammatory conditions, such as inflammatory bowel disease, lupus, and rheumatoid arthritis, suggesting that the role of P-gp as a steroid transporter contributes to GC resistance. To investigate whether P-gp can contribute to GC-resistance in asthma by testing whether decreasing P-gp expression in dexamethasone (DEX)-exposed respiratory epithelial cells in vitro results in higher intracellular DEX levels. Methods Used: We transfected A549 respiratory epithelial cells with small interfering (si)RNA targeted at mRNA for MDR1, the gene encoding P-gp. Transfected cells were exposed to 100nM fluorescently-labeled DEX for 15 minutes. siRNA transfection efficiency, surface P-gp, and intracellular DEX were measured by flow cytometry. Summary of Results: MDR1 siRNA transfection decreased P-gp expression by 20% when compared to nonsense siRNA transfection (geometric mean±SEM nonsense vs. MDR1=389±64 vs. 311±13). This decrease in P-gp expression resulted in decreased intracellular DEX. Specifically, transfected cells that were P-gp- were 3 times less likely to be DEX+ than P-gp+ cells (mean±SEM: 7±2 vs. 19±4% of transfected cells; P=0.02). Conclusions: These results are contrary to our hypothesis that decreasing P-gp expression in respiratory epithelial cells decreases cellular efflux of DEX. In fact, our results show a positive relationship between P-gp expression and intracellular DEX. There is some evidence that expression of P-gp may actually be enhanced by GCs. Therefore, our data do not support the concept that P-gp contributes to GC efflux in respiratory epithelium.
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