Abstract
The ATP-binding cassette (ABC) transporter ABCB1, encoded by the multidrug resistance gene MDR1, is expressed on brain microvascular endothelium and several types of epithelium, but not on endothelia outside the CNS. It is an essential component of the blood-brain barrier. The aim of this study was to identify cell-specific controls on the transcription of MDR1 in human brain endothelium. Reporter assays identified a region of 500 bp around the transcription start site that was optimally active in brain endothelium. Chromatin immunoprecipitation identified Sp3 and TFIID associated with this region and EMSA (electrophoretic mobility shift assays) confirmed that Sp3 binds preferentially to an Sp-target site (GC-box) on the MDR1 promoter in brain endothelium. This result contrasts with findings in other cell types and with the colon carcinoma line Caco-2, in which Sp1 preferentially associates with the MDR1 promoter. Differences in MDR1 transcriptional control between brain endothelium and Caco-2 could not be explained by the relative abundance of Sp1:Sp3 nor by the ratio of Sp3 variants, because activating variants of Sp3 were present in both cell types. However differential binding of other transcription factors was also detected in two additional upstream regions of the MDR1 promoter. Identification of cell-specific controls on the transcription of MDR1 indicates that it may be possible to modulate multi-drug resistance on tumours, while leaving the blood brain barrier intact.
Highlights
Microvascular endothelium in the brain is a key component of the blood-brain barrier, which controls the movement of nutrients into the CNS and excludes many toxic molecules from the CNS
In order to investigate how ABCB1 expression is controlled in brain endothelium and to compare different cell types, promoter reporter vectors were generated containing segments of the proximal MDR1 promoter region (Fig. 1)
The aim of the experiments was to detect whether the profile of activity of the reporter vectors was different in the two cell types, indicating that different sets of transcription factors were active on MDR1 in the two cells
Summary
Microvascular endothelium in the brain is a key component of the blood-brain barrier, which controls the movement of nutrients into the CNS and excludes many toxic molecules from the CNS. Brain endothelial cells are connected by continuous tight junctions that confer low permeability to ions and hydrophilic molecules [1] They express several members of the ATP-binding cassette (ABC) super family, of which the most important is ABCB1, encoded by the multi-drug resistance gene, MDR1 [2,3]. (ABCB1 is often referred to as p-gp1.) Additional multi-drug resistance proteins (MRP) are located at the blood-brain barrier, including MRP-1, -2 and -4 (ABCC1, 2, 4) as well as breast-cancer resistance protein, (ABCG2) [4]. For example ABCB1 is expressed on brain endothelium, but not on endothelia that lack barrier properties It is present on a variety of other cell-types, including intestinal epithelium and on cells from the proximal kidney tubules. The aim of this study was to identify transcription factors that control ABCB1 expression in human brain endothelium
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