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 ...

ABCC6 mutations are responsible for the development of pseudoxanthoma elasticum, a rare recessive disease characterized by calcification of elastic fibers. Although ABCC6 is mainly expressed in the liver the disease has dermatologic, ocular, and cardiovascular symptoms. We investigated the transcriptional regulation of the gene and observed that hepatocyte growth factor (HGF) inhibits its expression in HepG2 cells via the activation of ERK1/2. Similarly, other factors activating the cascade also inhibited ABCC6 expression. We identified the ERK1/2 response element in the proximal promoter by luciferase reporter gene assays. This site overlapped with a region conferring the tissue-specific expression pattern to the gene and with a putative hepatocyte nuclear factor 4␣ (HNF4␣) binding site. We demonstrated that HNF4␣ regulates the expression of ABCC6, acts through the putative binding site, and determines its cell type-specific expression. We also showed that HNF4␣ is inhibited by the activation of the ERK1/2 cascade. In conclusion we describe here the first regulatory pathway of ABCC6 expression showing that the ERK1/2-HNF4␣ axis has an important role in regulation of the gene.

The identification of the multidrug resistance protein (MRP) as a conjugate export pump in several cell types suggested its involvement in the long-known glutathione-S-conjugate transport across erythrocyte membranes. We investigated the ATP-dependent transport of glutathione S-conjugates in human erythrocyte and erythroleukemia cell membrane vesicles using the endogenous conjugate leukotriene C, (LTC,), known to be a high-affinity substrate for MRP, in addition to S-(2,4-dinitrophenyl)glutathione. The kinetic parameters, including the K,,, value for LTC, of 11 8 2 5 nM and the inhibition constants for transport of both substrates for the quinoline-based inhibitor MK 571, were similar to those obtained for transport mediated by recombinant MRP. Direct photoaffinity labeling of human erythrocyte membranes with [3H]LTC, revealed a major binding protein of about 190 kDa which was immunoprecipitated by an anti-MRP serum. The radiolabeling of this protein was specifically suppressed by the transport inhibitor MK 571. Several additional anti-MRP sera detected the protein of about 190 kDa in human erythrocyte and erythroleukemia cell membranes. These data identify for the first time the glutathione-S-conjugate transporting protein in erythrocyte membranes.Keywords: ATP-dependent transport; erythrocytes ; glutathione conjugate ; multidrug-resistance protein ; photoaffinity labeling.The export of glutathione S-conjugates from erythrocytes has been functionally known for many years [1-61. Using insideout oriented vesicles of erythrocyte membranes, transport of glutathione S-conjugates and oxidized glutathione has been shown to be a primary-active ATP-dependent process [2, 6-91. Similar transport processes have been found in other tissues [lo-171, though erythrocytes are an especially convenient model system for studying this transport since they lack y-glutamyltransferase and they do not have intracellular membranes. Therefore, further metabolism of glutathione S-conjugates is avoided and preparation of pure inside-out plasma membrane vesicles is facilitatedThe most widely used glutathione S-conjugate is S-(2,4-dinitropheny1)glutathione (Dnp-SG). In several tissues, the endogenous mediator leukotriene C, (LTC,) was identified as the substrate with the highest affinity for this transport system [13, 16, 171, but until now its transport across the erythrocyte membrane has not been described. Attempts to identify the protein responsible for this transport process in erythrocytes yielded several candidate proteins with apparent molecular masses of 23-90 kDa [18-201. However, in none of these studies was a direct involvement of the respective protein in glutathione S-conjugate transport proven. PI.Correspondence to D. Keppler, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrm, Im Neuenheimer Feld 280, D-69120 Heidelberg, GermanyAbbreviations. MRP, multidrug resistance protein (multidrug resistance-associated protein); LTC,, leukotriene C,: Dnp-SG, S-(2,4-dinitro- Recently, MRP was found to function as an AT...