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 ...
Genetic changes in HER2, PTEN, PIK3CA and AKT1 are all common in breast cancer and lead to the elevated phosphorylation of downstream targets of the PI3K/AKT signalling pathway. Changes in HER2, PTEN, PIK3CA and AKT have all been reported to lead to both enhanced proliferation and failures in hollow lumen formation in three dimensional epithelial culture models, but it is not clear whether these failures in lumen formation are caused by any failure in the spatial coordination of lumen formation (hollowing) or purely a failure in the apoptosis and clearance of luminal cells (cavitation). Here we use NMuMG mammary epithelial cells, which form a hollow lumen without significant apoptosis, to compare the transformation by these four genetic changes. We find that either mutant PIK3CA expression or PTEN loss, but not mutant AKT1 E17K, cause disrupted epithelial architecture, whereas HER2 over-expression drives strong proliferation without affecting lumen formation in these cells. We also show that PTEN requires both lipid and protein phosphatase activity, its extreme C-terminal PDZ binding sequence and probably Myosin 5A to control lumen formation through a mechanism that does not correlate with its ability to control AKT, but which is selectively lost through mutation in some tumours. These findings correlate AKT independent signalling activated by mutant PIK3CA or PTEN loss, but not strongly by HER2, with disrupted epithelial architecture and tumour formation.
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