Recent progress has been in 3 areas: identification of signals that rapidly reduce basal P-glycoprotein activity without changing expression (non-genomic signaling), identification of ligand-activated nuclear receptors that upregulate transporter expression and mapping of a signaling pathway by which DNA damage induces P-glycoprotein expression. Non-Genomic Signaling: P-glycoprotein is a major obstacle to the delivery of small molecule drugs across the blood-brain barrier and into the CNS. We have tested a novel, signaling-based strategy to overcome this obstacle. We previously mapped an extended, non-genomic signaling pathway that rapidly (minutes) and reversibly reduced P-glycoprotein transport activity without altering transporter protein expression;the defined pathway encompasses elements of proinflammatory, sphingolipid and protein kinase-based signaling. Central to this pathway is signaling through sphingosine-1-phosphate receptor 1 (S1PR1). S1P, the S1P analog, fingolimod (FTY720), currently in clinical trials for treatment of multiple sclerosis, and its active, phosphorylated metabolite (FTY720P) acted through S1PR1 to reduce P-glycoprotein transport activity. We validated these findings in vivo using in situ brain perfusion in rats. Recent transport experiments with isolated brain capillaries have extended the signaling pathway downstream of S1PR1 to include PI-3K, Akt and mTOR. Western blots show rapid, transient phosphorylation (activation) of Akt following exposure to S1P. Consistent with a role for mTOR in signaling, micromolar concentrations of L-leucine rapidly reduce P-glycoprotein transport activity;these effects are blocked by the mTOR inhibitor, rapamycin. To determine whether similar signaling to P-glycoprotein occurs in another intact tissue, we used an established comparative renal model, isolated killifish renal proximal tubules. This model permits direct measurements of P-glycoprotein transport activity. Isolated killifish tubules exposed to 0.01-1.0 μM sphingosine-1-phosphate (S1P) exhibited a profound decrease in P-glycoprotein transport activity, measured as specific accumulation of a fluorescent cyclosporine A derivative in the tubule lumen. Loss of activity had a rapid onset and was fully reversible when the S1P was removed. S1P effects were blocked by a specific S1P receptor 1 (S1PR1) antagonist and mimicked by a S1PR agonist. Sphingosine also reduced P-glycoprotein transport activity and those effects were blocked by an inhibitor of sphingosine kinase and by the S1PR1 antagonist. These results for a comparative renal model suggest that sphingolipid signaling to P-glycoprotein is not just restricted to the blood-brain and blood-spinal cord barriers, but occurs in other excretory and barrier tissues. Nuclear Receptor Regulation of Transporter Expression: Progress in this area involved defining the role of 2 ligand-activated nuclear receptors (PPAR-alpha and Nrf2) in ABC transporter regulation. Peroxisome Proliferator Activated Receptor alpha: PPAR-αis a master regulator of lipid metabolism. Endogenous free fatty acids act as natural PPAR-αligands whereas anti-hyperlipidemic fibrate drugs are synthetic ligands. In addition, recent work has shown that environmental toxicants, like the flame retardant, perfluorooctanesulfonic acid (PFOS), can activate PPAR-α. PPAR-αregulates β-oxidation of fatty acids. It also plays a role in regulation of drug metabolism by inducing expression of drug metabolizing enzymes and some ATP binding cassette (ABC) transporters in liver, kidney and skeletal muscle. We have found that clofibrate at μM concentrations and PFOS, at nM concentrations, increase transport activity and expression of P-glycoprotein, MRP2 and BCRP in isolated rat brain capillaries. All effects on transporter activity and expression were blocked by the PPAR-αantagonist, GW6471. The extent to which clofibrate and PFOS dosing as well as high fat diet alters blood-brain barrier transporter expression and drug delivery to the CNS remains to be determined. Nrf2, a Cellular Redox Sensor: Our recent studies show that complex signaling follows activation of Nrf2, a redox-sensor and ligand-activated transcription factor that plays a critical role in cellular defenses against oxidative and electrophilic stress. Importantly, Nrf2 had been proposed as a therapeutic target in stroke and traumatic brain injury. In addition, recent phase 3 clinical trials show that dimethyl fumarate, a Nrf2 ligand, reduced relapse rates and improved neuroradiologic outcomes in patients with relapsing-remitting multiple sclerosis. Given the potential for Nrf2 activation during treatment of CNS disease, we thought it important to understand the full range of consequences of intentionally activating Nrf2 for neuroprotection. We recently showed that 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, 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 through p53, p38 and NF-kB to increase transporter activity/expression. These results implicate Nrf2, p53 and NF-kB 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. We suggest caution when intentionally activating Nrf2 for neuroprotection, since increased drug efflux transporter expression and possibly drug metabolizing enzyme activity would impair subsequent CNS pharmacotherapy. Increased Transporter expression in response to DNA damage: Involvement of p53 in the mechanism by which Nrf2 signals increased ABC transporter expression at the blood-brain barrier led us to investigate whether genotoxic stress, which also activates p53, had similar effects. Radiation-induced DNA damage activates a signaling pathway that can promote DNA repair and in the canonical pathway p53 activation occurs downstream of repair signaling in the nucleus. We have activated p53 signaling through low level (1-4 Gy) radiation-induced DNA damage in isolated rat and mouse brain capillaries. In irradiated capillaries, P-glycoprotein expression and transport activity increase in an ATM-, p53- and NF-kB-dependent manner. Alkaline comet assays show dose-dependent DNA damage in rat brain micro-capillaries exposed to 1-6 Gy radiation. Capillaries from Nrf2-null mice respond to ionizing radiation in the same manner as capillaries from wild-type mice. Thus DNA damage, not oxidative stress is the initiating event. Initial experiments with rats given 4 Gy to the head show increased expression and transport activity of P-glycoprotein in brain capillaries 24 hours later. Taken together, these results suggest that genomic stress through radiation-induced DNA damage initiates signaling that upregulates blood-brain barrier drug efflux transporter expression. These results have important implications for CNS therapy, since radiation is often combined pharmacotherapy to treat CNS tumors.
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