Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties

Gekle, Michael; Wünsch, Stefan; Oberleithner, Hans; Silbernagl, Stefan

doi:10.1007/bf00374853

Cited by 180 publications

(191 citation statements)

References 30 publications

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“…Clones were expanded and screened for GFP fluorescence by confocal fluorescence microscopy. Similar experimental results were found in MDCK C7 cells (generous gift of Dr. Hans Oberleithner) (27) stably transfected with GFP-CFTR. Following establishment of cell lines, G418 was reduced to 150 g/ml and was removed 3-4 days prior to experimentation.…”

Section: Transfectionsupporting

confidence: 80%

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Moyer¹,

Loffing²,

Schwiebert³

et al. 1998

Journal of Biological Chemistry

152

170

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The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl ؊ ) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl ؊ secretion by stimulating CFTR Cl ؊ channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patchclamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl ؊ secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl ؊ secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

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Section: Transfectionsupporting

confidence: 80%

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Moyer¹,

Loffing²,

Schwiebert³

et al. 1998

Journal of Biological Chemistry

152

170

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“…Mouse B16F10 melanoma cells and ret transgenic melanoma cells 52,53 were cultured in DMEM or RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 1% L-glutamine. The high-resistance C7 subclone of Madin-Darby canine kidney cells (MDCK-C7) 54 were cultured in supplemented MEM medium (PAA, Pasching, Austria).…”

Section: Cell Culturementioning

confidence: 99%

Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

et al. 2015

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Heparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and glycocalyx thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression. Therefore, we investigated the relevance of HPSE duality in malignant melanoma in vitro as well as in mouse melanoma models basing on the intradermal injection of transfected melanoma cells. In line with its extracellular action, HPSE-deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of VEGF affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-and most prominently MMP-9 upon HPSE knockdown. In vivo, HPSE-deficiency was related to increased lymph node metastasis. While inhibition of its extracellular function heparin was unable to block the gene regulatory impact of HPSE we proposed an intracellular mechanism. Immunostainings revealed a counter-staining of HPSE and NF-B in the nucleus suggesting a close relationship between both proteins. This finding was further supported by the discovery of a direct charge-driven molecular interaction between HPSE and DNA by using atomic force microscopy and a co-precipitation approach. Our findings are novel and point towards a dual identity of HPSE in malignant melanoma with a protumorigenic extracellular activity and a tumor suppressive nuclear action. Identification of molecular strategies to shuttle extracellular HPSE into the nuclei of cancer cells envisions new therapeutic options. AbstractHeparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and glycocalyx thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression.Therefore, we investigated the relevance of HPSE duality in malignant melanoma in vitro as well as in mouse melanoma models basing on the intradermal injection of transfected melanoma cells. In line with its extracellular action, HPSE-deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of VEGF affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-α and most prominently MMP-9 upon HPSE knockdown. In vivo, HPSE-deficiency was related to increased lymph node metastasis.While inhibition of its extracellular function heparin was unable to block the gene regulatory impact of HPSE we proposed an intracellular mechanism. Immunostainings revealed a counter-staining of HPSE and NF-κB in the nucleus suggesting a close relationship between both proteins. This finding was further supported by the discovery of a dir...

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“…The present paper investigated the role of Cl -ions in H + ion transport across plasma membranes of a clone of cultivated MDCK cells, C11 cells, that were obtained by Gekle et al (11) and characterized as a model for renal-collecting duct intercalated cells. Experiments from our laboratory have shown that these cells possess Na + /H + exchangers, H + -ATPase and H + -K + -ATPase in their membranes, the latter transporters being demonstrated during experiments in 0 Na + solutions (12).…”

Section: Discussionmentioning

confidence: 99%

“…A subtype (clone) of MDCK cells denominated C11 (MDCK-C11), obtained from Dr. H. Oberleithner (Department of Physiology, University of Münster, Germany), was used (11). These cells were used from passage 74 to passage 82.…”

Section: Cell Culturementioning

confidence: 99%

Cl- and regulation of pH by MDCK-C11 cells

Tararthuch¹,

Fernández²,

Malnic³

2007

Braz J Med Biol Res

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The interaction between H + extrusion via H + -ATPase and Cl -conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH 4 Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na + Ringer and 0.032 ± 0.003 in the absence of Na + (0 Na + ). With 0 Na + plus the Cl -channel inhibitor NPPB (10 µM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl -channels, increased dpHi/dt in 0 Na + to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 µM Schering 28080. Since it is thought that the Cl -dependence of H + -ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 µM valinomycin at high (100 mM) K + was tested in our cells. In Na + Ringer, dpHi/dt was increased, but no effect was detected in 0 Na + Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl -channels on dpHi/ dt was not due to an adverse electrical gradient. The effect of 100 µM ATP was studied in 0 Na + Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 µM Bapta. We have shown that H + -ATPase present in MDCK C11 cells depends on Clions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H + transport.

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Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties

Cited by 180 publications

References 30 publications

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity

Cl- and regulation of pH by MDCK-C11 cells

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