Regulation of Breast Cancer Resistant Protein by Peroxisome Proliferator-Activated Receptor α in Human Brain Microvessel Endothelial Cells

Breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) membrane-associated drug efflux transporter, is known to localize at the blood-brain barrier (BBB) and can significantly restrict xenobiotic permeability in the brain. The objective of this study is to investigate the regulation of BCRP functional expression by peroxisome proliferator-activated receptor alpha (PPAR alpha), a ligand-activated transcription factor primarily involved in lipid metabolism, in a cerebral microvascular endothelial cell culture system (hCMEC/D3), representative of human BBB. We demonstrate that PPAR alpha-selective ligands (i.e., clofibrate, GW7647) significantly induce BCRP mRNA and protein expression in a time-and concentrationdependent manner, whereas pharmacological inhibitors (i.e., MK886, GW6471) prevent this induction. Using [3H] mitoxan-trone, an established BCRP substrate, we observe a significant reduction in its cellular accumulation by monolayer cells treated with clofibrate, suggesting increased BCRP efflux activity. In addition, we show a significant decrease in BCRP protein expression and function when PPAR alpha is down-regulated by small interfering RNA. Applying chromatin immunoprecipitation and quantitative real-time polymerase chain reaction, we observe that clofibrate treatment increases PPAR alpha binding to the peroxisome proliferator response element within the ABCG2 gene promoter. This study provides the first evidence of direct BCRP regulation by PPAR alpha in a human in vitro BBB model and suggests new targeting strategies for either improving drug brain bioavailability or increasing neuroprotection.