Analysing the factors that regulate expression of blood-brain barrier drug transporter proteins

  • Yu Siong Ho

Student thesis: Phd


The blood-brain barrier (BBB) is a highly selective physical and enzymatic barrier formed by the endothelial cells of cerebral microvessels. It helps to regulate and restrict the entry of endogenous and exogenous compounds into the central nervous system. A number of ATP-binding cassette (ABC) transporters, including ABCB1, ABCG2 and ABCC5, are expressed in BBB brain endothelial cells and efflux an array of substances into the systemic circulation, impairing the pharmacotherapy of brain disorders. Regulation of ABC transporter expression and activity is highly complex and little is known of the precise regulatory mechanisms in BBB endothelial cells compared to other systems, for example liver, with expression and activity influenced by therapeutic drugs, xenobiotics and pathophysiological conditions, for example inflammation. The nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), glucocorticoid receptor (GR) and retinoid x receptor (RXR) are major regulators of ABC efflux transporter activities yet their roles in regulating ABC transporters in BBB is not well understood. Therefore, the objectives of this thesis were to: (i) identify optimal culture conditions for an in vitro BBB model comprising primary porcine brain endothelial cells (PBECs) (ii) characterise ABCB1, ABCG2 and ABCC5 expression and activity in PBECs (iii) determine the expression profile of GR, PXR, CAR and RXR nuclear receptors in PBECs (iv) determine the role of GR, PXR, CAR, RXR nuclear receptor signalling pathways on the expression and activity of ABCB1, ABCG2 and ABCC5 in PBECs (v) determine the effects of the inflammatory mediator interleukin-1β (IL-1β) on expression and activity of ABCB1, ABCG2 and ABCC5 in PBECs and (vi) determine the effects of anti-inflammatory glucocorticoids on expression and activity of ABCB1, ABCG2 and ABCC5 in PBECs. Western blotting confirmed expression of ABCB1, ABCG2 and ABCC5 transporters and PXR, CAR, RXR and GR receptors in PBECs. PBECs possessed higher levels of ABCB1, ABCG2 and ABCC5 activities when maintained in Dulbecco’s Modified Eagle medium (DMEM) compared to DMEM:Ham’s F12 (1:1) or M199 medium. Activation of PXR with rifampicin or GR with dexamethasone and hydrocortisone significantly up-regulated ABCB1, ABCG2 and ABCC5 transport activities and expression, which were abrogated by co-treatment with PXR agonist (rifampicin) and antagonist (L-sulforaphane) or GR agonist (dexamethasone, hydrocortisone) and antagonist (mifepristone). Both CAR agonist (CITCO) and inverse agonist (meclizine) significantly down-regulated ABCB1, ABCG2 and ABCC5 transporter activities and expression. Expression of PXR, CAR and RXR at the protein level was not significantly affected by treatment of PBECs with PXR and CAR agonists and antagonists/inverse agonist, but was significantly up-regulated by treatment with GR agonists, demonstrating distinct PXR/CAR and GR pathways are involved in the regulation of ABC transporter activity and expression. Interleukin-1β significantly up-regulated the protein expression of PXR, CAR and RXR and the activity and expression of ABCB1, ABCG2 and ABCC5. The use of inhibitors of the NF-κB pathway, namely caffeic acid phenethyl ester, honokiol and SN50 failed to revert the IL-1β-induced up-regulation of ABCB1 ABCG2 and ABCC5 transporter activities, but significantly down-regulated the protein expression of ABCB1, ABCG2, ABCC5 and of the nuclear receptors PXR, CAR, RXR and GR. Furthermore, inhibition of the MAPK JNK and ERK1/2 with SP600125 and FR180204 respectively, significantly abrogated the IL-1β-mediated upregulation of ABCB1, ABCG2 and ABCC5 transport activity. In summary, this thesis has shed light, for the first time in some cases, on the signalling pathways involved in regulating BBB transporter (ABCB1, ABCG2 and ABCC5) and nuclear receptor (PXR, RXR, CAR and GR) expression and activity, and demonstrates PBECs can prove valuable in deciphering how xenobiotics,
Date of Award1 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJeffrey Penny (Supervisor) & Costas Demonacos (Supervisor)


  • PXR
  • RXR
  • CAR
  • GR
  • blood brain barrier
  • PBEC
  • BBB
  • brain endothelial cell

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