Abstract
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 (PPARα), 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 PPARα-selective ligands (i.e., clofibrate, GW7647) significantly induce BCRP mRNA and protein expression in a time- and concentration-dependent manner, whereas pharmacological inhibitors (i.e., MK886, GW6471) prevent this induction. Using [3H]mitoxantrone, 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 PPARα is down-regulated by small interfering RNA. Applying chromatin immunoprecipitation and quantitative real-time polymerase chain reaction, we observe that clofibrate treatment increases PPARα binding to the peroxisome proliferator response element within the ABCG2 gene promoter. This study provides the first evidence of direct BCRP regulation by PPARα in a human in vitro BBB model and suggests new targeting strategies for either improving drug brain bioavailability or increasing neuroprotection.
Footnotes
This work was supported by the Canadian Institutes of Health Research [Grant MOP56976] and the Ontario HIV Treatment Network, Ministry of Health of Ontario [Grant ROGB189] (to R.B.). R.B. is a career scientist of the Ontario HIV Treatment Network, Ministry of Health of Ontario. K.R. is a recipient of an Ontario HIV Treatment Network doctoral studentship award.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- BBB
- blood-brain barrier
- CNS
- central nervous system
- ABC
- ATP-binding cassette
- BCRP
- breast cancer resistance protein
- RXR
- retinoid X receptor
- P-gp
- P-glycoprotein
- PPARα
- peroxisome proliferator-activated receptor α
- PPRE
- peroxisome proliferator response element
- PPARγ
- peroxisome proliferator-activated receptor γ
- hCMEC/D3
- human cerebral microvessel endothelial cells
- DMSO
- dimethyl sulfoxide
- BBB-ECs
- human brain-derived microvascular endothelial cells
- siRNA
- small interfering RNA
- GW7647
- 2-methyl-2-[[4-[2-[[(cyclohexylamino)carbonyl](4-cyclohexylbutyl)amino]-ethyl]phenyl]thio]-propanoic acid
- GW6471
- [(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester
- MK886
- 1-[(4-chlorophenyl)methyl]-3-[(1,1-dimethylethyl)thio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoic acid, sodium salt
- MTT
- (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- Ko143
- (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester
- FBS
- fetal bovine serum
- PBS
- phosphate-buffered saline
- qPCR
- quantitative real-time PCR
- ChIP
- chromatin immunoprecipitation
- PCR
- polymerase chain reaction
- PAGE
- polyacrylamide gel electrophoresis
- PVDF
- polyvinylidene difluoride
- DAPI
- 4,6-diamidino-2-phenylindole
- ANOVA
- analysis of variance
- ChIP
- chromatin immunoprecipitation
- bp
- base pair(s).
- Received November 13, 2011.
- Accepted January 19, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
