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

Abstract

This study investigates involvement of beta-catenin signalling in regulation of p-glycoprotein (p-gp) expression in endothelial cells derived from brain vasculature. Pharmacological interventions that enhance or that block beta-catenin signalling were applied to primary rat brain endothelial cells and to immortalized human brain endothelial cells, hCMEC/D3, nuclear translocation of beta-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 beta-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 beta-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. These results suggest that regulation of p-gp and other multidrug efflux transporters in brain vasculature can be influenced by beta-catenin signalling.

Fig. 1

Effect of pan Wnt agonist and GSK-3β inhibitor, BIO on stabilization and localization of β-catenin in human brain endothelial cells. Staining for β-catenin is shown in (a) untreated and (b) cells treated with 1 μM BIO for 16 h. The percentage of cells showing nuclear/perinuclear staining for β-catenin 16 h following treatment with (c) pan Wnt agonist or (d) GSK-3β inhibitor, BIO at the concentrations shown was calculated relative to the total number of DAPI-stained nuclei; in each case, values shown as the mean ± SEM are significantly higher than those of untreated control cells (**p < 0.01, n = 5). (e and f) Western blots of (e) whole cell lysates and (f) nuclear fractions from untreated cells and from cells treated with 1 μM BIO for 16 h showing increased intensity of the band corresponding to β-catenin in the treated cells. In each case, the blot is representative of immunoblots resulting from three separate experiments.

Fig. 2

Effect of GSK-3β inhibitor, BIO, on p-gp expression in (a) rat brain endothelial cells and (b) human immortalized brain endothelial cells. Cells were exposed to 1 μM BIO for 16 h (hCMEC/D3) or to 5 μM BIO for 5 days (rat) and then harvested for analysis at transcript level (a and b) by qRT-PCR. Expression was firstly estimated relative to the geometric mean of three most stable reference genes using geNorm analysis. Values shown are the mean ± SEM of these amounts expressed relative to the expression in control untreated cells for mdr1a or MDR1, mdr1b and cyclin D1. Significant increases over control are shown (a) for mdr1a (*p < 0.05, n = 6), cyclin D1 (*p < 0.05, n = 6) but not mdr1b in the rat cells and (b) for MDR1 (**p < 0.01, n = 4) and cyclin D1 (*p < 0.05, n = 4) in the human cells. (c) Activation of MDR1 transcription after 24 h or of TCF/LEF-dependent transcription after 16 h following exposure to 1 μM BIO. hCMEC/D3 cells were transfected with pMDR-Luc and pCMV-SEAP or with Topflash DNA and pCMV-SEAP together with empty vector or dnTCF, treated the next day with BIO and later harvested for luciferase and alkaline phosphatase activity. Values shown are the fold increases in luciferase/alkaline phosphatase activity ratio over control untreated cells and are given as mean ± SEM. Number of experiments and statistical significance (paired t-test on the log of the ratios) are shown above each bar. (d) Western blots of brain endothelial cell lysates from rat (RBEC) or human (hCMEC/D3) cells either untreated or exposed to BIO at the concentrations shown for 5 days (rat) or for 24 h (human). The blots were probed with anti-p-gp monoclonal antibody C219. Equal protein loading was verified by probing with anti-β-actin antibody (shown) and Ponceau staining (not shown). Images shown are representative of three independent experiments performed.

Fig. 3

Effect of GSK-3β inhibitor, BIO, on p-gp efflux activity in human immortalized brain endothelial cells. (a and b) Show fluorescence histograms of cells (a) untreated or (b) pre-treated with 1 μM BIO for 24 h and exposed either to vehicle for 30 min followed by 0.5 μM calcein-AM for a further 30 min (solid line) or the same procedure but with 20 μM verapamil present throughout (dotted line). Note in the presence of verapamil i.e. with p-gp activity blocked, accumulation of fluorescent calcein, as seen by the median values of the dotted line histograms, is similar in (a) and (b). In the absence of verapamil i.e. with p-gp activity still present, accumulation is significantly less in the BIO-treated cells as seen by comparing the median values of the solid-line histograms in (a) and (b). Median values of background fluorescence (histograms not shown) were very low i.e. 1.4 ± 0.4. (c) Show the increases in accumulation of calcein in the presence of 20 μM verapamil in untreated cells and in cells exposed to 0.5 or 1 μM BIO. Values shown are the mean ± SEM of results from four separate experiments and indicate significant differences from untreated controls for cells treated with 0.5 μM BIO (*p < 0.05) and with 1 μM BIO (**p < 0.01). Median values from fluorescence histograms as shown in (a and b) were used to derive these data.

Fig. 4

Effect of GSK-3β inhibitor BIO on expression of other ABC transporters in human immortalized brain endothelial cells. Cells were exposed to 1 μM BIO for 16 h and then analysed (a) by qRT-PCR for expression of MRP1, MRP2, MRP4, MRP5 and BCRP. As described in legend to Fig. 2, expression was firstly estimated relative to reference genes. Values shown are the mean ± SEM of these levels of expression relative to that of control untreated cells. Significant increases over control are shown for MRP2 (**p < 0.01, n = 4), MRP4 (**p < 0.01, n = 4) and BCRP (**p < 0.01, n = 4) but not for MRP1 or MRP5. (b and c) Western blots showing expression of (b) MRP4 and (c) BCRP 24 h after exposure to BIO at the concentrations shown. Equal protein loading was verified by probing with anti-β-actin antibody (shown) and Ponceau staining (not shown). Images shown are representative of those obtained from three independent experiments.

Fig. 5

Effect of quercetin on expression of ABC transporters, p-gp, MRP4 and BCRP in human immortalized brain endothelial cells. Cells were harvested 8 h following exposure to 50 μM quercetin and analysed at transcript level (a) for expression of MDR1, MRP4 and BCRP (**p < 0.01, n = 4 in each case). (b) Western blot analysis of lysates from cells 72 h following exposure to quercetin at the concentrations shown demonstrating decreases at the protein level in expression of p-gp, MRP4 and BCRP. The blots were probed sequentially with anti-p-gp monoclonal antibody C219, anti-MRP4 antibody, M41-10 and anti-BCRP antibody, BXP-21. Equal protein loading was verified by probing with anti-β-actin antibody (shown) and Ponceau staining (not shown). Images shown are representative of three independent experiments performed.

Fig. 6

Effect of Dkk-1 on expression of p-gp in rat brain endothelial cells (RBEC) and human immortalized brain endothelial cells. (a) Western blots showing decreased expression of p-gp in RBEC and in human immortalized brain endothelial cells (hCMEC/D3) following treatment with 0.2 μg/mL Dkk-1 for 24 h. The blots were probed with anti-p-gp monoclonal antibody C219 and then with anti-β-actin antibody to confirm equal protein loading per well. Images shown are representative of three independent experiments performed. (b) Immunocytochemical staining for β-catenin in human brain endothelial cells to show reduction in the presence of 0.2 μg/mL Dkk-1 of the nuclear/perinuclear localization brought about by 16 h exposure to 20 μM pan Wnt agonist.