Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

Demeuse, Philippe; Frágner, P; Leroy-Noury, C.; Mercier, Claire; Payen, Léa; Fardel, Olivier; Couraud, Pierre Olivier; Roux, F.

doi:10.1046/j.1471-4159.2003.02071.x

Cited by 29 publications

(13 citation statements)

References 38 publications

(61 reference statements)

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“…There are two isoforms of the mdr1 gene in rodents: mdr1a and mdr1b. Both forms are present in primary cultured rodent brain microvessel endothelial cells and immortalized brain endothelial cells [32, 33]. Our recent studies by microarray and real-time PCR have shown that mdr1b is highly expressed and shows stronger upregulation by TNF in both mouse cerebral microvessels and cultured RBE4 cells.…”

Section: Discussionmentioning

confidence: 99%

Neuroinflammation Activates Mdr1b Efflux Transport Through NFκB: Promoter Analysis in BBB Endothelia

Argyropoulos

Zhang

et al. 2008

Cell Physiol Biochem

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Background/aims: Although it is known that drug delivery across the blood-brain barrier (BBB) may be hampered by efflux transport activity of the multidrug resistance (mdr) gene product P-glycoprotein, it is not clear how inflammation regulates efflux transporters. In rat brain endothelial (RBE4) cells of BBB origin, the proinflammatory cytokine TNF mainly induced transcriptional upregulation of mdr1b, and to a lesser extent mdr1a, resulting in greater efflux of the substrates. This study further determines the mechanisms by which TNF activates mdr1b promoter activity. Methods/Results: Luciferase reporter assays and DNA binding studies show that (1) maximal basal promoter activity was conferred by a 476 bp sequence upstream to the mdr1b transcriptional initiation site; (2) TNF induced upregulation of promoter activity by NFκB nuclear translocation; and (3) the NFκB binding site of the mdr1b promoter was solely responsible for basal and TNF-activated gene transcription, whereas the p53 binding site was not involved. Binding of the p65 subunit of NFκB to nuclear DNA from RBE4 cells was shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. Conclusion: NFκB mediates TNF-induced upregulation of mdr1b promoter activity, illustrating how inflammation activates BBB efflux transport.

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Section: Discussionmentioning

confidence: 99%

Neuroinflammation Activates Mdr1b Efflux Transport Through NFκB: Promoter Analysis in BBB Endothelia

Argyropoulos

Zhang

et al. 2008

Cell Physiol Biochem

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“…Elevation of intra-endothelial cyclic AMP and stimulation of protein kinase C were shown to increase Pgp activity in GP8 cells (Deli et al, 2001). Also, chronic treatment of GP8 and GPNT cells with the selective agent puromycin was found to increase Pgp expression and activity with a selective increase in mdr1a mRNA expression (Demeuse et al, 2004). (1) Gomes and Soares-da-Silva, 1999;…”

Section: Use Of Immortalized Rbe Cell Lines For the Study Of Blood-brmentioning

confidence: 98%

Rat Brain Endothelial Cell Lines for the Study of Blood–Brain Barrier Permeability and Transport Functions

2005

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(1) In vitro models of the BBB have been developed from cocultures between bovine, porcine, rodent or human brain capillary endothelial cells with rodent or human astrocytes. Since most in vivo BBB studies have been performed with small laboratory animals, especially rats, it is important to establish a rat brain endothelial (RBE) cell culture system that will allow correlations between in vitro and in vivo results. The present review will constitute a brief description of the best characterized RBE cell lines (RBE4, GP8/3.9, GPNT, RBEC1, TR-BBBs and rBCEC4 cell lines) and will summarize their recent and important contribution to our current knowledge of the BBB transport functions and permeability to blood-borne solutes, drugs, and cells. (2) In most cases, primary cultures of RBE cells were transduced with an immortalizing gene (SV40 or polyoma virus large T-antigen or adenovirus E1A), either by transfection of plasmid DNA or by infection using retroviral vectors. In one case however, the conditionally immortalized TR-BBB cell line was derived from primary cultures of brain endothelial cells of SV40-T-expressing transgenic rats. (3) All cell lines appear to have an endothelial morphology. The absence of foci formation would mean that the cells are not transformed. The endothelial origin is shown by the expression of Factor VIII-related antigen. Immortalized RBE cells express all the enzymes and transporters that are considered as specific for the blood-brain barrier endothelium, with similar characteristics to those expected from in vivo analyses, but at a significantly lower level. Some RBE cell lines are responsive to astroglial factors, such as RBE4 cells, rBEC4, and TR-BBB cells. None of the immortalized RBE cell lines appear to generate the necessary restrictive paracellular barrier properties that would allow to use them in transendothelial permeability screening. (4) RBE cell lines have been used to demonstrate that transporters such as organic cation transporter/carnitine transporter, serotonin transporter, and the ATA2 system A isoform are expressed in rat brain endothelium. When the transporter is shown to be expressed with the same properties in the immortalized RBE cells as in vivo, regulation studies may be initiated even if the transporter is down-regulated. Pharmacological applications have been proposed with well-characterized transporters such as monocarboxylic acid transporter-1, large neutral amino acid tansporter-1, nucleoside carrier systems, and P-glycoprotein. RBE cell monolayers have also been used to investigate the mechanism of the transendothelial transport of large molecules, such as immunoliposomes or nanoparticles, potentially useful as drug delivery vectors to the brain. (5) RBE4 and GP8 cell lines have been extensively used to demonstrate that intercellular adhesion molecule-1 (ICAM-1) engagement in brain endothelial cells triggers multiple signal transduction pathways. Using functional assays, it was established that ICAM-1 signaling is intimately and actively involved in facili...

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“…Capillary fragments were directly seeded onto Falcon inserts (3-m pore; Fisher Scientific) coated with collagen type IV/ fibronectin (10 g/ml; Sigma-Aldrich) in DMEM with 10% FBS (Fisher Scientific), 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 50 g/ml gentamicin (Sigma-Aldrich). Isolated capillaries were cultured for 2 days in puromycin-supplemented medium (4 g/ml; SigmaAldrich) to eliminate contaminating perivascular smooth muscle cells, followed by culture in medium supplemented with basic fibroblast growth factor (2 ng/ml; Roche) (40,41). After reaching confluence, cells were cultured in serum-free DMEM/Ham's F12 (Sigma-Aldrich) containing 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, and 50 g/ml gentamicin (Sigma-Aldrich) supplemented with hydrocortisone (550 nM; SigmaAldrich) (32).…”

Section: Brain Capillary Endothelial Cells (Bcec)mentioning

confidence: 99%

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases

Zozulya

Reinke

Baiu

et al. 2007

The Journal of Immunology

107

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Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1α increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1α-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.

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Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

Cited by 29 publications

References 38 publications

Neuroinflammation Activates Mdr1b Efflux Transport Through NFκB: Promoter Analysis in BBB Endothelia

Neuroinflammation Activates Mdr1b Efflux Transport Through NFκB: Promoter Analysis in BBB Endothelia

Rat Brain Endothelial Cell Lines for the Study of Blood–Brain Barrier Permeability and Transport Functions

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases

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