Nrf2 upregulates ATP binding cassette transporter expression and activity at the blood-brain and blood-spinal cord barriers

Abstract

Activation of nuclear factor E2-related factor-2 (Nrf2), a sensor of oxidative stress, is neuroprotective in animal models of cerebral ischemia, traumatic brain injury, subarachnoid hemorrhage, and spinal cord injury. We show here that Nrf2 activation with sulforaphane (SFN) in vivo or in vitro increases expression and transport activity of three ATP-driven drug efflux pumps at the blood-brain barrier [P-glycoprotein, ATP binding cassette b1 (Abcb1); multidrug resistance-associated protein-2 (Mrp2), Abcc2; and breast cancer resistance protein (Bcrp), Abcg2]. Dosing rats with SFN increased protein expression of all three transporters in brain capillaries and decreased by 50% brain accumulation of the P-glycoprotein substrate verapamil. Exposing rat or mouse brain capillaries to SFN increased P-glycoprotein, Bcrp, and Mrp2 transport activity and protein expression; SFN increased P-glycoprotein activity in mouse spinal cord capillaries. Inhibiting transcription or translation abolished upregulation of P-glycoprotein activity. No such effects were seen in brain capillaries from Nrf2-null mice, indicating Nrf2 dependence. Nrf2 signaled indirectly to increase transporter activity/expression. The p53 inhibitor pifithrin abolished the SFN-induced increase in transporter activity/expression, and the p53-activator nutlin-3 increased P-glycoprotein activity. SFN did not alter P-glycoprotein transport activity in brain and spinal cord capillaries from p53-null mice. Inhibitors of p38 MAPK and nuclear factor κB (NF-κB) blocked the effects of SFN and nutlin-3 on P-glycoprotein activity. These results implicate Nrf2, p53, and NF-κB in the upregulation of P-glycoprotein, Bcrp, and Mrp2 at blood-CNS barriers. They imply that the barriers are tightened selectively (efflux transporter upregulation) by oxidative stress, providing increased neuroprotection, but also reduced penetration of many therapeutic drugs.

Keywords: ABC transporters; NF-κB; P-glycoprotein; blood-brain barrier; drug delivery; p53.

Figure 1.

Effect of SFN dosing (1–10 mg/kg, i.p., for 2 d) on ABC transporter expression and function at the rat blood–brain barrier. A, Western blots of brain capillary membranes (pooled tissue from 5 rats) showing that SFN dosing increases protein expression for three ABC transporters and the Nrf2-responsive gene HO-1. Pgp, P-glycoprotein. B, Representative confocal micrographs of brain capillaries from control and SFN-dosed rats (scale bar, 10 μm). Capillaries were isolated from control and SFN-dosed rats and incubated with 2 μm NBD-CSA without and with 5 μm PSC833 (middle; ex vivo assay). C, Quantitation of specific NBD-CSA luminal fluorescence in capillaries isolated from control and SFN-dosed rats (ex vivo assay). Shown are mean ± SEM for 8–12 capillaries from single preparations (pooled brains from 5 rats). ***p < 0.001, significantly higher than control. D, SFN dosing reduces brain accumulation of [³H]verapamil (P-glycoprotein substrate) as measured by in situ brain perfusion. Each point represents the value for a single rat. **p < 0.01, significantly higher than control; ***p < 0.001, significantly higher than control. E, SFN dosing does not change brain accumulation of [¹⁴C]sucrose (tight junction permeability marker). Data are from five rats per group.

Figure 2.

Effects of SFN exposure on ABC transporter transport activity in isolated rat brain capillaries (specific luminal accumulation of transporter-selective fluorescent drugs; see Results). A, Time course of SFN-induced increase of P-glycoprotein transport activity. Capillaries were incubated in medium with 5 μm SFN for the time shown and then incubated for 1 additional hour with SFN and 2 μm NBD-CSA. B, SFN dose response for P-glycoprotein activity. The exposure time is 4 h (3 h with SFN plus 1 h with SFN and 2 μm NBD-CSA). C, Increased P-glycoprotein transport activity in brain capillaries exposed to tBHQ. D, SFN exposure increases Mrp2 and Bcrp transport activity in brain capillaries. Shown are mean ± SEM for 10–15 capillaries from single preparations (pooled brains from 8–10 rats). *p < 0.05, **p < 0.01,***p < 0.001, significantly higher than control.

Figure 3.

The SFN-induced increase in P-glycoprotein activity requires transcription and translation (rat brain capillaries). A, Inhibition of transcription [1 μm actinomycin D (ActD)] or translation [100 μg/mL cycloheximide (CHX)] abolishes the increase in transport activity caused by exposure to 5 μm SFN. Shown are mean ± SEM for 8–12 capillaries from a single preparation (pooled brains from 10 rats). ***p < 0.001, significantly higher than control. B, C, Western blots of brain capillary membranes showing that 4 h exposure of capillaries to SFN exposure increases P-glycoprotein (Pgp) expression in a concentration-dependent manner.

Figure 4.

Effects of Nrf2 (SFN) and AhR (TCDD) ligands and p53 activation (nutlin-3) on P-glycoprotein transport activity in brain capillaries from wild-type (A, B) and Nrf2-null (C, D) mice. Shown are mean ± SEM for 8–12 capillaries from single preparations (pooled brains from 10 mice). **p < 0.01, ***p < 0.001, significantly higher than control.

Figure 5.

Involvement of p53 in SFN induction of ABC transporter expression and activity in rat brain capillaries. A, Nutlin-3, a p53 activator, increases P-glycoprotein activity in a concentration-dependent manner. B, Pifithrin, a p53 inhibitor, blocks the SFN-induced increase in P-glycoprotein activity. Shown are mean ± SEM for 8–12 capillaries from single preparations (pooled brains from 10 rats). **p < 0.01, ***p < 0.001, significantly higher than control. C, Western blots showing increased P-glycoprotein (Pgp), Bcrp, and Mrp2 expression in membranes from SFN-exposed capillaries. The increase in transporter expression is abolished when p53 is inhibited by pifithrin; pooled capillary membranes from 10 rats. Con, Control.

Figure 6.

Effects of Nrf2 (SFN), AhR (TCDD), and PXR (PCN) ligands on P-glycoprotein transport activity in brain capillaries from wild-type (A) and p53-null (B) mice. Shown are mean ± SEM for 8–12 capillaries from single preparations (pooled brains from 10 mice). ***p < 0.001, significantly higher than control.

Figure 7.

Effects of a p38 MAPK inhibitor (SB203580) on the increase in P-glycoprotein transport activity caused by exposure to SFN (A) and nutlin-3 (B). Shown are mean ± SEM for 10–12 capillaries from single preparations (pooled brains from 6 rats). ***p < 0.001, significantly higher than control.

Figure 8.

A, An NF-κB inhibitor (QNZ) blocks the increases in P-glycoprotein transport activity caused by exposure to SFN and nutlin-3. B, A second NF-κB inhibitor (SN50) blocks the increases in P-glycoprotein transport activity caused by exposure to SFN. Shown are mean ± SEM for 8–12 capillaries from single preparations (pooled brains from 6 rats). ***p < 0.001, significantly higher than control.

Figure 9.

A, EMSAs show nuclear translocation of Nrf2, p53, and NF-κB in response to exposure of rat brain capillaries to 5 μm SFN for 4 h. Con, Control. B, Immunostaining for p65 shows nuclear translocation of NF-κB after 30 min exposure to 5 μm SFN. Arrows indicate nuclei selected for pixel intensity profile analysis. C, Pixel intensity profiles for immunostained (p65) nuclei in control and SFN-exposed capillaries. There is no overlap in the intensity profiles. D, Mean nuclear pixel intensities in immunostained (p65) capillaries. SFN exposure more than doubles pixel intensity and inhibitors of p53, p38, and NF-κB activation and translocation essentially block this increase. Shown are mean ± SEM for 40–60 nuclei (15–25 capillaries) from two capillary preparations. *p < 0.05, ***p < 0.001, significantly higher than control. E, The sequence of events through which Nrf2 ligands increase P-glycoprotein expression. Activators are shown in red, and inhibitors are in blue.

Figure 10.

Effects of Nrf2 (SFN), AhR (TCDD), and PXR (PCN) ligands on P-glycoprotein transport activity in spinal cord capillaries from wild-type (A) and p53-null (B) mice. Shown are mean ± SEM for 10–12 capillaries from single preparations (pooled brains from 10 mice). *p < 0.05, **p < 0.01, ***p < 0.001, significantly higher than control.