Activation of β‐catenin signalling by GSK‐3 inhibition increases p‐glycoprotein expression in brain endothelial cells

Abstract

This study investigates involvement of β-catenin signalling in regulation of p-glycoprotein (p-gp) expression in endothelial cells derived from brain vasculature. Pharmacological interventions that enhance or that block β-catenin signalling were applied to primary rat brain endothelial cells and to immortalized human brain endothelial cells, hCMEC/D3, nuclear translocation of β-catenin being determined by immunocytochemistry and by western blot analysis to confirm effectiveness of the manipulations. Using the specific glycogen synthase kinase-3 (GSK-3) inhibitor 6-bromoindirubin-3′-oxime enhanced β-catenin and increased p-gp expression including activating the MDR1 promoter. These increases were accompanied by increases in p-gp-mediated efflux capability as observed from alterations in intracellular fluorescent calcein accumulation detected by flow cytometry. Similar increases in p-gp expression were noted with other GSK-3 inhibitors, i.e. 1-azakenpaullone or LiCl. Application of Wnt agonist [2-amino-4-(3,4-(methylenedioxy) benzylamino)-6-(3-methoxyphenyl)pyrimidine] also enhanced β-catenin and increased transcript and protein levels of p-gp. By contrast, down-regulating the pathway using Dickkopf-1 or quercetin decreased p-gp expression. Similar changes were observed with multidrug resistance protein 4 and breast cancer resistance protein, both known to be present at the blood-brain barrier. Supplementary materialThe following supplementary material is available for this article: Table S1 Primers used in the polymerase chain reaction. This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/j. 1471-4159.2008.05537.x. (This link will take you to the article abstract). Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Europe PMC Funders GroupAuthor Manuscript J Neurochem. Author manuscript; available in PMC 2015 January 23. et al. 2004). In this signalling pathway, interactions of Wnt proteins with the cell surface Frizzled receptors and associated membrane proteins lead to inactivation of glycogen synthase kinase-3 (GSK-3), resulting in stabilization of β-catenin. As a result, free β-catenin is allowed to accumulate and be translocated to the nucleus, binding to the transcription factor Tcf/Lef to alter the expression of target genes (Logan and Nusse 2004). Wnt proteins can also activate non-canonical pathways that do not involve β-catenin.There is evidence that Wnt signalling, particularly via the canonical pathway plays a role in vascular endothelial survival and proliferation (Wright et al. 1999;Masckauchan et al. 2005). Wnt ligands and Wnt ligand receptors have been identified in different types of vascular endothelial cells (Goodwin et al. 2006). Interactions between canonical and noncanonical pathways may be such that the one then modulates the ...

Overexpression and involvement of special AT‐rich sequence binding protein 1 in multidrug resistance in human breast carcinoma cells

“…(19) Also, the regulation of P-gp expression can be influenced by b-catenin signaling. (20) Our data confirmed that SATB1 could significantly elevate the level of ErbB2 (data not shown), which may be due to P-gp upregulation in cell lines in which SATB1 is highly expressed. The regulation of MDR1 expression in tumor cells involves a complex interplay of multiple factors.…”

Chrysin blocks topotecan-induced apoptosis in Caco-2 cells in spite of inhibition of ABC-transporters

“…The stabilization of ß-catenin might finally A c c e p t e d M a n u s c r i p t 19 be transmitted into enhanced expression of the investigated ABC-transporters as it was shown for P-gp in brain endothelial cells that its promoter can be activated by ß-catenin signaling [43]. Since expression of BCRP and MRP-2 increased under activation of canonical Wnt/ß-catenin signaling as well [43] it can be suggested that stabilized ß-catenin activates a number of ABC-transporters by promoter activation.…”

The Cross-Talk between Canonical and Non-Canonical Wnt-Dependent Pathways Regulates P-Glycoprotein Expression in Human Blood–Brain Barrier Cells

“…The activation of Wnt/GSK3/b-catenin axis is known to increase the expression of Pgp in hCMEC/D3 cells. 2,3,6 The activation of RhoA and RhoAK is known to enhance the Pgp activity in BBB cells. 11 It is not known: (1) whether the Wnt/RhoA/ RhoAK axis controls also the Pgp expression; (2) whether the canonical and non-canonical Wnt pathways cooperate in regulating the Pgp levels in BBB cells.…”

“…1 P-glycoprotein (Pgp) is one of the ATP binding cassette transporters present on the luminal side of BBB cells: it effluxes back into the bloodstream several chemotherapeutic drugs (e.g., anthracyclines, taxanes, Vinca alkaloids, epipodophyllotoxins, topotecan, methotrexate, imatinib, dasatinib, lapatinib, gefitinib, sorafenib, erlotinib), analgesics, anti-epileptics, anti-retrovirals, and antibiotics. 1 The Wnt signaling has a central role in regulating the expression of Pgp in BBB cells 2,3 and relies on the simultaneous activation of different intracellular transducers. In the so-called 'Wnt canonical pathway', the soluble Wnt proteins bind to the Frizzled receptor and the low-density lipoprotein receptor-related protein-5 and -6 co-receptors, reduces the activity of glycogen synthase kinase 3 (GSK3), allowing the release of b-catenin from the cytosolic adenomatous polyposis coli-axin complex and its translocation into the nucleus.…”