Influence of breast cancer resistance protein (Abcg2) and p-glycoprotein (Abcb1a) on the transport of imatinib mesylate (Gleevec) across the mouse blood-brain barrier

Imatinib, a protein tyrosine kinase inhibitor, may prevent the growth of glioblastoma cells. Unfortunately, its brain distribution is restricted by p-glycoprotein (p-gp or multidrug resistance protein Mdr1a), and probably by breast cancer resistance protein (Bcrp1), two efflux pumps expressed at the blood-brain barrier (BBB). We have used in situ brain perfusion to investigate the mechanisms of imatinib transport across the mouse BBB. The brain uptake of imatinib in wild-type mice was limited by saturable efflux processes. The inhibition of p-gp, by valspodar and zosuquidar, increased imatinib uptake (2.5-fold), as did the deficiency of p-gp in Mdr1a/1b(-/-) mice (5.5-fold). Perfusing imatinib with the p-gp/Bcrp1 inhibitor, elacridar, enhanced the brain uptake of imatinib in wild-type (4.1-fold) and Mdr1a/1b(-/-) mice (1.2-fold). However, the brain uptake of imatinib was similar in wild-type and Bcrp1(-/-) mice when it was perfused at a non-saturating concentration. The brain uptake of CGP74588, an active metabolite of imatinib, was low. It was increased by perfusion with elacridar (twofold), but not with valspodar and zosuquidar. CGP74588 uptake was 1.5 times greater in Bcrp1(-/-) mice than in wild-type mice. These data suggest that imatinib transport at the mouse BBB is limited by p-gp and probably by Bcrp1, and that CGP74588 transport is restricted by Bcrp1.     

High FFA-induced proliferation and apoptosis in human umbilical vein endothelial cell partly through Wnt/β-catenin signal pathway

Free fatty acids (FFA)-induced proliferation and apoptosis was studied in human umbilical vein endothelial cells (HUVECs). A recombinant adenovirus containing a RNAi cassette targeting the GSK-3β gene was produced and its silencing effect on GSK-3β gene was detected by Western blot analysis and immunohistochemistry assay in HUVECs. The effect of the RNAi on the protein level of β-catenin was explored by transfecting the RNAi adenovirus to inhibit the expression of GSK-3β protein. The subsequent effect on the Wnt/GSK-3β/β-catenin signal pathway and on proliferation and apoptosis of HUVECs cultured with FFAs, was analyzed by BrdU assay, Annexin V-FITC/PI Apoptosis Detection Kit, and 4′,6-diamidino-2- phenylindole(DAPI) to explore the possible connection between the signaling pathway and FFA-induced proliferation and apoptosis. The Western blot results showed that the expression of GSK-3β protein in HUVECs could be inhibited efficiently by the RNAi adenovirus, and that the protein level of β-catenin was increased by RNAi adenovirus transfection. The results of the BrdU assay suggested that knockdown of GSK-3β with the RNAi adenovirus may stimulate the proliferation of HUVECs. Apoptosis was observed in HUVECs exposed to FFAs (0.75 mmol/L) for 72 h, and this effect could be partly reversed when interfering with the RNAi adenovirus. It may be concluded that the RNAi adenovirus specific to GSK-3β may partly protect HUVECs from apoptosis induced by FFAs, probably through the up-regulation of the Wnt/β-catenin signal pathway.     

Regulation of major efflux transporters under inflammatory conditions at the blood-brain barrier in vitro

ATP-driven efflux transport proteins at the blood-brain barrier protect the healthy brain but impede pharmacotherapy of the disordered CNS. To investigate the question how ATP-binding cassette (ABC)-transporters are regulated during inflammation or infection we analysed the effects of the cytokines tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) on the expression of brain multidrug resistance proteins in primary cultures of porcine brain capillary endothelial cells. We found that TNF-α and IL-1β rapidly decrease Abcg2 ( BMDP/BCRP ) mRNA expression within 6 h. After 24 and 48 h the mRNA level came back to control values. The mRNA reduction at 6 h was counter-regulated by the anti-inflammatory glucocorticoid hydrocortisone. Abcg2 protein levels were suppressed at prolonged stimulations but not after 6 h of stimulation which correlates with Abcg2 specific substrate uptake measurements. Abcb1 (p-glycoprotein) protein expression was transiently increased after TNF-α addition within 6 h of incubation followed by a reduction after 24 and 48 h whereas the Abcb1 mRNA levels were not changed. IL-1β caused a continuous decrease in protein expression of both ABC-transporters. Long-term treatment with an assumed TNF-α-downstream agent, the vasoconstrictor endothelin-1, induced Abcg2 protein expression but suppressed Abcb1. Other efflux pumps like multidrug resistance-associated proteins/Abcc were rarely affected. The present results imply a complex regulation of the two most abundant ABC-transporters at the blood-brain barrier during early inflammation stages suggesting that Abcb1 (p-glycoprotein) is an early target of TNF-α-signalling counterbalanced by Abcg2.     

Activation of the canonical Wnt pathway by the antipsychotics haloperidol and clozapine involves dishevelled-3

Protein kinase B (Akt), glycogen synthase kinase-3 (GSK-3) and members of the Wnt signal transduction pathway were recently found to be altered in schizophrenia and targeted by antipsychotic drugs. In the current study, selected Wnt signalling proteins were investigated to determine if they are altered by the antipsychotics clozapine or haloperidol in the rat prefrontal cortex. Pheochromocytoma (PC12) and neuroblastoma (SH-SY5Y) cells were also used to elucidate how antipsychotics generated the pattern of changes observed in vivo . Western blotting (WB) revealed that treatment with haloperidol or clozapine caused an up-regulation of Wnt-5a, dishevelled-3, Axin, total and phosphorylated GSK-3 and β-catenin protein levels. Treatment of PC12 and SH-SY5Y cells with a variety of pharmacological agents as well as the over-expression of several Wnt related proteins failed to mimic the pattern observed in vivo following antipsychotic treatment. However, the over-expression of dishevelled-3 nearly perfectly duplicated the changes observed in vivo . Immunoprecipitations (IP) conducted using protein isolated from the rat prefrontal cortex indicated that dishevelled-3 is associated with the D₂ dopamine receptor thereby suggesting that antipsychotics may act on dishevelled-3 via D₂ dopamine receptors to initiate a cascade of downstream changes involving Axin, GSK-3 and β-catenin that may help to alleviate psychosis in schizophrenic patients.     

Modulation of p-glycoprotein function by caveolin-1 phosphorylation

p-glycoprotein (p-gp) is an ATP-binding cassette transporter and its overexpression is responsible for the acquisition of the multidrug resistance phenotype in human tumors. p-gp is localized at the blood-brain barrier and is involved in brain cytoprotection. Our previous work used immunoprecipitation to show that caveolin-1 can interact with p-gp. In this study, we provide evidence that caveolin-1 regulates p-gp transport activity in a rat brain endothelial cell line (RBE4). Down-regulation of caveolin-1 by siRNA reduced the interaction between p-gp and caveolin-1, followed by a decrease in [3H]-Taxol and [3H]-Vinblastine accumulation in RBE4 cells. The latter result showed that down-regulation of caveolin-1 enhanced p-gp transport activity. RBE4 cells were also transfected with Sarcoma in order to modulate caveolin-1 phosphorylation. Overexpression of Sarcoma, a protein tyrosine kinase, stimulated caveolin-1 phosphorylation and increased both [3H]-Taxol and [3H]-Vinblastine accumulation as well as Hoechst 33342 accumulation. Transfection of caveolin-1 inhibits p-gp transport activity. Conversely, transfection of the mutant cavY14F decreased the p-gp/caveolin-1 interaction and reduced accumulation of the two p-gp substrates. Thus, our data show that caveolin-1 regulates p-gp function through the phosphorylation state of caveolin-1 in endothelial cells from the blood-brain barrier.     

Concomitant degradation of beta-catenin and GSK-3 beta potently contributes to glutamate-induced neurotoxicity in rat hippocampal slice cultures

Increasingly, published evidence links glutamate with the pathogenesis of Alzheimer's disease. We investigated the molecular mechanism underlying glutamate-induced neurotoxicity in hippocampus, which is primarily linked to cognitive dysfunction in Alzheimer's disease. Acute exposure of rat hippocampal slices to glutamate significantly induced cell death, as determined by media lactate dehydrogenase levels and PI staining. Moreover, this was accompanied by Ca²⁺ influx and calpain-1 activation, as confirmed by the proteolytic pattern of spectrin. Notably, glutamate-induced calpain-1 activation decreased the level of β-catenin, and this process appeared to be independent of glycogen synthase kinase 3beta (GSK-3β), since glutamate also led to loss of GSK-3β. Calpeptin, a calpain inhibitor, attenuated the glutamate-mediated degradations of spectrin, synaptophysin, and β-catenin except GSK-3β and modestly increased cell survival. In contrast, the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) effectively reduced all glutamate-evoked responses, i.e., the breakdowns of spectrin, synaptophysin, β-catenin and GSK-3β, and cell death. Pharmacological studies and in vitro calpain-1 proteolysis confirmed that in the glutamate-treated hippocampus, calpain-1-mediated decrease of β-catenin could occur independently of GSK-3β and of proteasome, and that GSK-3β degradation is independent of calpain-1. These findings together provide the first direct evidence that glutamate promotes the down-regulations of β-catenin and GSK-3β, which potently contribute to neurotoxicity in hippocampus during excitotoxic cell death, and a molecular basis for the protection afforded by calpeptin and APV from the neurotoxic effect of glutamate.     

Cellular and regional specific changes in multidrug efflux transporter expression during recovery of vasogenic edema in the rat hippocampus and piriform cortex

In the present study, we investigated the characteristics of drug efflux transporter expressions following status epilepticus (SE). In the hippocampus and piriform cortex (PC), vasogenic edema peaked 3-4 days after SE. The expression of breast cancer resistance protein (BCRP), multidrug resistance protein-4 (MRP4), and p-glycoprotein (p-GP) were decreased 4 days after SE when vasogenic edema was peaked, but subsequently increased 4 weeks after SE. Multidrug resistance protein-1 (MRP1) expression gradually decreased in endothelial cells until 4 weeks after SE. These findings indicate that SE-induced vasogenic edema formation transiently reduced drug efflux pump expressions in endothelial cells. Subsequently, during recovery of vasogenic edema drug efflux pump expressions were differentially upregulated in astrocytes, neuropils, and endothelial cells. Therefore, we suggest that vasogenic edema formation may be a risk factor in pharmacoresistent epilepsy. [BMB Reports 2015; 48(6): 348-353]     

Aspirin induces apoptosis in mesenchymal stem cells requiring Wnt/β-catenin pathway

Background and Objectives:  Mesenchymal stem cells (MSC) are multipotent progenitor cells that are have found use in regenerative medicine. We have previously observed that aspirin, a widely used anti-inflammatory drug, inhibits MSC proliferation. Here we have aimed to elucidate whether aspirin induces MSC apoptosis and whether this is modulated through the Wnt/β-catenin pathway.
Materials and methods:  Apoptosis of MSCs was assessed using Hoechst 33342 dye and an Annexin V–FITC/PI Apoptosis Kit. Expression of protein and protein phosphorylation were investigated using Western blot analysis. Caspase-3 activity was detected by applying a caspase-3/CPP32 Colorimetric Assay Kit.
Results:  In these MSCs, aspirin induced morphological changes characteristic of apoptosis, cytochrome  c release from mitochondria, and caspase-3 activation. Stimulating the Wnt/β-catenin pathway by both Wnt 3a and GSK-3β inhibitors (LiCl and SB 216763), blocked aspirin-induced apoptosis and protected mitochondrial function, as demonstrated by decreased cytochrome  c release and caspase-3 activity. Aspirin initially caused a time-dependent decrease in COX-2 expression but subsequently, and unexpectedly, elevated the latter. Stimulation of COX-2 expression by aspirin was further enhanced following stimulation of the Wnt/β-catenin pathway. Application of the COX-2 inhibitor NS-398 suppressed elevated COX-2 expression and promoted aspirin-induced apoptosis.
Conclusion:  These results demonstrate that the Wnt/β-catenin pathway is a key modulator of aspirin-induced apoptosis in MSCs by regulation of mitochrondrial/caspase-3 function. More importantly, our findings suggest that aspirin may influence MSC survival under certain conditions; therefore, it should be used with caution when considering regenerative MSC transplantation in patients with concomitant chronic inflammatory diseases such as arthritis.     

Accumulation of β-catenin by lithium chloride in porcine myoblast cultures accelerates cell differentiation

The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation to determine cell fate during embryogenesis. Lithium chloride (LiCl) is known to activate canonical Wnt signaling by inhibiting glycogen synthetase kinase-3β and consequently stabilizing free cytosolic β-catenin. To understand the role of the Wnt/β-catenin pathway in the regulation of porcine myoblast differentiation, we studied the effects of LiCl on cultured porcine myoblasts and β-catenin expression. A supplementation of 25 mM LiCl induced myoblast differentiation into myotubes over 3 days of culture. By semi-quantitative RT-PCR analyses, levels of mRNA encoding MyoD, Myogenin, Myf5 and several Wnt-responsive genes in the cultured myoblast cells were significantly increased after LiCl treatment. Using Western blotting and immunofluorescence analysis, we found that the protein levels of β-catenin were consistently increased by LiCl. Meanwhile, phosphorylated GSK-3β at Ser9 levels were also increased as an indicator of GSK-3β inactivation. Additionally, the nuclear staining of endogenous β-catenin was also significantly increased in porcine myoblasts 48 h after LiCl treatment. These results provided additional evidence that Wnt/β-catenin is a significant pathway that regulates myogenic differentiation. An enhanced level of β-catenin plays a positive role in porcine myoblast differentiation.     

The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

Barrier characteristics of brain endothelial cells forming the blood–brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.     

Calcineurin dephosphorylates glycogen synthase kinase-3 beta at serine-9 in neuroblast-derived cells

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3β (GSK-3β). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3β at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1–401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3β, and an increase in the phosphorylation of tau. K_m of calcineurin for a GSK-3β phosphopeptide was 469.3 μM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3β were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3β. We conclude that in vitro, calcineurin can dephosphorylate GSK-3β at Ser-9 and form a stable complex with GSK-3β, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3β in vivo .     

Cover Image

The human central nervous system (CNS) vasculature expresses a distinctive barrier phenotype, the blood–brain barrier (BBB). As the BBB contributes to low efficiency in CNS pharmacotherapy by restricting drug transport, the development of an in vitro human BBB model has been in demand. Here, we present a microfluidic model of CNS angiogenesis having three-dimensional (3D) lumenized vasculature in concert with perivascular cells. We confirmed the necessity of the angiogenic tri-culture system (brain endothelium in direct interaction with pericytes and astrocytes) to attain essential phenotypes of BBB vasculature, such as minimized vessel diameter and maximized junction expression. In addition, lower vascular permeability is achieved in the tri-culture condition compared to the monoculture condition. Notably, we focussed on reconstituting the functional efflux transporter system, including p-glycoprotein (p-gp), which is highly responsible for restrictive drug transport. By conducting the calcein-AM efflux assay on our 3D perfusable vasculature after treatment of efflux transporter inhibitors, we confirmed the higher efflux property and prominent effect of inhibitors in the tri-culture model. Taken together, we designed a 3D human BBB model with functional barrier properties based on a developmentally inspired CNS angiogenesis protocol. We expect the model to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.     

SLFN5 suppresses cancer cell migration and invasion by inhibiting MT1-MMP expression via AKT/GSK-3β/β-catenin pathway

Human SLFN5 inhibits invasions of IFNα-sensitive renal clear-cell carcinoma and melanoma cells. However, whether this inhibition is confined to these IFNα-sensitive cancers is unclear. Here we show that SLFN5 expressions on both mRNA and protein levels are significantly higher in non/low-invasive cancer cell lines (breast cancer cell line MCF7, colorectal cancer cell line HCT116 and lung cancer cell line A549) than in highly-invasive cancer cell lines (fibrosarcoma cell line HT1080 and renal clear cell cancer cell line 786-0). SLFN5 knockdown in non/low-invasive cancer cell lines enhanced MT1-MMP expression and increased migration and invasion in vitro, and in vivo. Furthermore, SLFN5 overexpression in HT1080 and 786-0 inhibited MT1-MMP expression and repressed migration and invasion. MT1-MMP is instrumental in SLFN5-controlled inhibition of cancer cell migration and invasion, as shown by MT1-MMP-knockdown and -overexpression analyses. SLFN5 knockdown activated AKT/GSK-3β/β-catenin pathway by promotion AKT phosphorylation and subsequent GSK-3β phosphorylation, further β-catenin translocation into nucleus as un-phosphorylated protein at Ser33, 37 and 45 and Thr41 sites. This is the first study to report that SLFN5 inhibits cancer migration and invasiveness in several common cancer cell lines by repressing MT1-MMP expression via the AKT/GSK-3β/β-catenin signalling pathway, suggesting that SLFN5 plays wide inhibitory roles in various cancers.     

Conservation of intracellular Wnt signaling components in dorsal-ventral axis formation in zebrafish

 The mechanism of early dorso-ventral axis specification in zebrafish embryos is not well understood. While β-catenin has been clearly implicated as a determinant of the axis, the factors upstream and downstream of β-catenin in this system are not defined. Unlike in Xenopus, where a sperm-induced cortical rotation is used to localize β-catenin on the future dorsal side of the embryo, zebrafish do not have an obviously similar morphogenetic movement. Recently, a GSK-3 (Glycogen Synthase Kinase-3) binding protein (GBP) was identified as a novel member of the Wnt pathway required for maternal dorsal axis formation in Xenopus. GBP stabilizes β-catenin levels by inhibiting GSK-3 and potentially provides a link between cortical rotation and β-catenin regulation. Since zebrafish may use a different mechanism for regulating β-catenin, we asked whether zebrafish also express a maternal GBP. We report the isolation of the zebrafish GBP gene and show that it is maternally expressed and is present as mRNA ubiquitously throughout early embryonic development. Over-expression of zebrafish GBP in frogs and fish leads to hyper-dorsalized phenotypes, similar to the effects resulting from over-expression of β-catenin, indicating that components upstream of β-catenin are conserved between amphibians and teleosts. We also examined whether Tcf (T cell factor) functions in zebrafish embryos. As in frogs, ectopic expression of a dominant negative form of XTcf-3 ventralizes zebrafish embryos. In addition, ectopic β-catenin expression activates the promoter of the Tcf-dependent gene siamois, indicating that the step immediately downstream of β-catenin is also conserved between fish and frogs. Received: 23 July 1998 / Accepted: 2 September 1998     

Differential Cellular Phosphorylation of Neurofilament Heavy Side-Arms by Glycogen Synthase Kinase-3 and Cyclin-Dependent Kinase-5

Abstract: To investigate the cellular mechanisms regulating neurofilament-heavy subunit (NF-H) side-arm phosphorylation, we studied the ability of three putative neurofilament kinases, glycogen synthase kinase-3 (GSK-3)α, GSK-3β, and cyclin-dependent kinase-5 (cdk-5), to phosphorylate NF-H in transfected cells. We analysed NF-H phosphorylation by using a panel of phosphorylation-dependent antibodies and also by monitoring the electrophoretic mobility of the transfected NF-H on sodium dodecyl sulphate-polyacrylamide gel electrophoresis because this is known to be affected by side-arm phosphorylation. Our results demonstrate that whereas GSK-3α, GSK-3β, and cdk-5 will all phosphorylate NF-H, they generate different antibody reactivity profiles. GSK-3α and GSK-3β induce a partial retardation of a proportion of the transfected NF-H, but only cdk-5 alters the rate of electrophoretic migration to that of NF-H from brain. We conclude that cdk-5 and GSK-3 phosphorylate different residues or sets of residues within NF-H sidearms in cells. We further show that cdk-5 is active in both the CNS and the PNS but that this activity is not dependent on expression of its activator, p35. This suggests that there are other activators of cdk-5.     

Localization and Developmental Changes of τ Protein Kinase I/Glycogen Synthase Kinase-3β in Rat Brain

Abstract: τ protein kinase I (TPKI) purified from bovine brain extract has been shown to phosphorylate τ and to form paired helical filament (PHF) epitopes and was found recently to be identical to glycogen synthase kinase-3β (GSK-3β). Before elucidating a role of TPKI/GSK-3β in PHF formation, it is necessary to investigate the normal function of the enzyme. To study the distribution and developmental changes of the enzyme, specific polyclonal antibodies were prepared against TPKI and GSK-3α. Immunoblot analysis demonstrated that TPKI was nearly specifically localized in the brain of adult rats. The level of TPKI in the rat brain was high at gestational day 18, peaked on postnatal day 8, and then decreased rapidly to a low level, which was sustained up to 2 years. Immunohistochemistry indicated primarily neuronal localization of TPKI. Growing axons were stained most intensely in the developing cerebellum, but the immunoreactivity became restricted to the gray matter in the mature tissue. Parallel fibers had a high level of TPKI and also stained intensely for τ. These findings indicate that τ is one of the physiological substrates of TPKI and suggest that the enzyme plays an important role in the growth of axons during development of the brain.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.