Mrp4 confers resistance to topotecan and protects the brain from chemotherapy

Abstract

The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane of the endothelial cells of the brain capillaries. Microdialysis sampling of ventricular CSF demonstrated that localization of Mrp4 at the choroid epithelium is integral to its function in limiting drug penetration into the CSF. The topotecan resistance of cells overexpressing Mrp4 and the polarized expression of Mrp4 in the choroid plexus and brain capillary endothelial cells indicate that Mrp4 has a dual role in protecting the brain from cytotoxins and suggest that the therapeutic efficacy of central nervous system-directed drugs that are Mrp4 substrates may be improved by developing Mrp4 inhibitors.

FIG. 1.

Targeted disruption of the Mrp4 locus causes loss of renal Mrp4 expression. (a) Targeting construct design and locus. Xho, XhoI cleavage site; B, BamHI cleavage site; E, EcoRI cleavage site; Sal, SalI cleavage site; neo, position of neomycin phosphotransferase gene; TGA, stop codons engineered into exon 27 (see text). Exon numbers are shown. (b) Representative Mrp4 genotype analysis of Mrp4 wild-type, heterozygous, and nullizygous mice. (c) Immunoblot with anti-mouse Mrp4 shows Mrp4 expression in kidney membranes from Mrp4^+/+ and Mrp4^+/− but not Mrp4^−/− mice. (d) Immunohistochemical analysis of Mrp4 expression in paraffin-embedded kidneys from Mrp4^+/+ and Mrp4^−/− mice. The murine anti-Mrp4 polyclonal antiserum Mrp4-mp was used in panels 1 and 2. The monoclonal anti-Mrp4 antibody M₄I-10 was used in panels 3 and 5. The inset (panel 4) shows the proximal tubules at greater magnification.

FIG. 2.

Topotecan is an Mrp4 substrate. (a) Transport of the Mrp4 substrate estradiol-17β-glucuronide into membrane vesicles prepared from Saos-2 cells engineered to express Mrp4 (13) in the presence of various concentrations of topotecan. (b) The intracellular accumulation of topotecan was assayed in Saos-2 cells transfected with empty vector or stably expressing human MRP4 at the indicated times after treatment. (c) Topotecan sensitivity of the cells shown in panel d, shown as percent cell viability. Localization of Mrp4 in Saos-2 cells stably expressing Mrp4 (d) or transfected with empty vector (e). Bars represent 1 standard deviation.

FIG. 3.

Topotecan concentration in the brain is higher in the absence of Mrp4. (a) Mean (± standard deviation) concentration of topotecan in the brains of six Mrp4^−/− (open bars) and six Mrp4^+/+ (solid bars) mice 15 min and 6 h after intravenous injection of 2 mg of topotecan/kg. (b) Equal samples of total brain protein (10 μg) from Mrp4^+/+ and Mrp4^−/− animals were fractionated by polyacrylamide gel electrophoresis and treated with the monoclonal anti-Mrp4 antibody M4I-10.

FIG. 4.

Mrp4 is highly expressed in the basolateral membrane of the choroid plexus in multiple species. (a) Detection of Mrp4 in the choroid plexus (CP) and kidney of Mrp4^+/+ mice by immunoblot analysis with two different anti-Mrp4 antibodies. 3T3, NIH 3T3 cells transfected with the Mrp4 expression vector or empty vector served as positive and negative controls, respectively. (b) Mrp4 was more abundant in the choroid plexus than in the kidney of Mrp4^+/+ mice, as shown by immunoblot analysis with polyclonal anti-mouse Mrp4 (Mrp4-mp). (c) Immunohistochemical localization of Mrp4 in choroid plexus epithelial cells from Mrp4^+/+, Mrp4^+/−, and Mrp4^−/− animals with the murine polyclonal Mrp4 antiserum (Mrp4-mp). The magnification is indicated above each panel. (d) Mrp4 expression detected in the choroid plexus from an FVB mouse, rat, and human (panels 1 to 4, respectively) with the anti-Mrp4 monoclonal antibody (M₄I-10).

FIG. 5.

Mrp4 is expressed in brain capillaries. (a) Frozen tissue sections prepared from the brains of Mrp4^+/+ and Mrp4^−/− FVB mice were treated with anti-Mrp4 monoclonal antibody. (b) Panel 1 shows a cross-section of wild-type mouse capillary, revealing red blood cells (asterisk), surrounding capillary wall, and luminal Mrp4 staining (red); panel 2 shows a rat capillary, revealing interior red blood cells (asterisk). Arrows indicate the capillary wall and the luminal endothelial cell Mrp4 staining (red). Magnifications are indicated to the left of the panels.

FIG. 6.

Expression of Mrp4 in choroid plexus epithelium restricts the CSF uptake of systemically administered topotecan. (a) CSF concentration of topotecan in Mrp4^+/+ (solid circles) and Mrp4^−/− (open circles) mice at the indicated times after intravenous injection of 2 mg of topotecan/kg. (b, left) Model showing the role of Mrp4 in the basolateral choroid plexus in restricting topotecan (TPT) penetration into the CSF. BCB, blood-CSF barrier. (b, right) Model depicting the role of Mrp4 in capillary endothelial cells in restricting penetration of topotecan into the brain. BBB, blood-brain barrier.