scholarly journals Expression of Podocalyxin Inhibits Cell–Cell Adhesion and Modifies Junctional Properties in Madin-Darby Canine Kidney Cells

2000 ◽  
Vol 11 (9) ◽  
pp. 3219-3232 ◽  
Author(s):  
Tetsuro Takeda ◽  
William Y. Go ◽  
Robert A. Orlando ◽  
Marilyn Gist Farquhar
Keyword(s):  
Cell Adhesion ◽  
Cho Cells ◽  
Direct Evidence ◽  
Mdck Cells ◽  
Chinese Hamster ◽  
Cell Aggregation ◽  
Madin Darby Canine Kidney ◽  
Adhesion Properties ◽  
Darby Canine Kidney ◽  
Canine Kidney

Podocalyxin is a major membrane protein of the glomerular epithelium and is thought to be involved in maintenance of the architecture of the foot processes and filtration slits characteristic of this unique epithelium by virtue of its high negative charge. However, until now there has been no direct evidence for podocalyxin's function. Podocalyxin is a type 1 transmembrane sialoprotein with an N-terminal mucin-like domain. To assess its function, we cloned rat podocalyxin and examined the effects of its expression on the cell adhesion properties of stably transfected Chinese hamster ovary (CHO)-K1 and Madin-Darby canine kidney (MDCK) cells and inducible ecdysone receptor–expressing (EcR)-CHO cells. In a cell aggregation assay, CHO-K1 cells expressing high levels of podocalyxin showed complete inhibition of cell aggregation, and MDCK transfectants showed greatly reduced aggregation (∼60–80%) compared with parental cells. In EcR-CHO cells, the expression level of podocalyxin induced by increasing levels of ecdysone analogue correlated closely with the antiadhesion effect. The inhibitory effect of podocalyxin was reversed by treatment of the cells with Arthrobacter ureafacienssialidase, indicating that sialic acid is required for inhibition of cell adhesion. Overexpression of podocalyxin also affected transepithelial resistance and the distribution of junctional proteins in MDCK cells by an unknown mechanism that may involve interaction with the actin cytoskeleton. These results provide direct evidence that podocalyxin functions as an antiadhesin that maintains an open filtration pathway between neighboring foot processes in the glomerular epithelium by charge repulsion.

Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin–Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor

2002 ◽  
Vol 361 (3) ◽  
pp. 653-661
Author(s):  
Abdelkarim ABOUSALHAM ◽  
Tom C. HOBMAN ◽  
Jay DEWALD ◽  
Michael GARBUTT ◽  
David N. BRINDLEY
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mdck Cells ◽  
Chinese Hamster ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Madin Darby Canine Kidney ◽  
Adp Ribosylation Factor ◽  
Darby Canine Kidney ◽  
Canine Kidney

Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin—Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-α (PKC-α) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5′-[γ-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3–4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-α binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation.

scholarly journals Par1b Promotes Hepatic-type Lumen Polarity in Madin Darby Canine Kidney Cells via Myosin II- and E-Cadherin–dependent Signaling

2007 ◽  
Vol 18 (6) ◽  
pp. 2203-2215 ◽  
Author(s):  
David Cohen ◽  
Yuan Tian ◽  
Anne Müsch
Keyword(s):  
Cell Adhesion ◽  
Mdck Cells ◽  
Myosin Ii ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Luminal Compartment ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Kidney-derived Madin Darby canine kidney (MDCK) cells form lumina at their apices, and target luminal proteins to an intracellular vacuolar apical compartment (VAC) when prevented from polarizing. Hepatocytes, by contrast, organize their luminal surfaces (the bile canaliculi; BC) between their lateral membranes, and, when nonpolarized, they display an intracellular luminal compartment that is distinct from the VACs of MDCK cells. Overexpression of the serine/threonine kinase Par1b/EMK1/MARK2 induces BC-like lateral lumina and a hepatic-type intracellular luminal compartment in MDCK cells, suggesting a role for Par1b in the branching decision between kidney- and hepatic-type epithelial phenotypes. Here, we report that Par1b promotes lateral lumen polarity in MDCK cells independently of Ca2+-mediated cell–cell adhesion by inhibiting myosin II in a rho kinase-dependent manner. Polarization was inhibited by E-cadherin depletion but promoted by an adhesion-defective E-cadherin mutant. By contrast, apical surface formation in control MDCK cells required Ca2+-dependent cell–cell adhesion, but it occurred in the absence of E-cadherin. We propose that E-cadherin, when in an adhesion-incompetent state at the lateral domain, serves as targeting patch for the establishment of lateral luminal surfaces. E-cadherin depletion also reverted the hepatic-type intracellular luminal compartment in Par1b-MDCK cells to VACs characteristic of control MDCK cells, indicating a novel link between E-cadherin and luminal protein targeting.

scholarly journals Recombinant human endothelin-converting enzyme ECE-1b is located in an intracellular compartment when expressed in polarized Madin–Darby canine kidney cells

1998 ◽  
Vol 333 (2) ◽  
pp. 439-448 ◽  
Author(s):  
Arezou AZARANI ◽  
Guy BOILEAU ◽  
Philippe CRINE
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Mdck Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Converting Enzyme ◽  
Madin Darby Canine Kidney ◽  
Endothelin Converting Enzyme ◽  
Darby Canine Kidney ◽  
Canine Kidney

Endothelin-converting enzyme (ECE) is a phosphoramidon-sensitive membrane-bound metalloprotease responsible for the conversion of big-endothelins into endothelins [Yanagisawa, Kurihara, Kimura, Tomobe, Kobayashi, Mitsui, Yazaki, Goto and Masaki (1988) Nature (London) 332, 411–415]. Several distinct isoforms of ECE have been cloned and identified. ECE-1a, b and c have the same ectodomain and differ only by their cytosolic tails [Schweizer, Valdenaire, Nelböck, Deuschle, Edwards, Stumpf and Löffler (1997) Biochem. J. 328, 871–877]. The ectodomain common to ECE-1 a, b and c shares extensive sequence similarities with neprilysin, a major kidney brush border metallopeptidase. To study the sorting of ECE in polarized cells, ECE-1b cDNA was expressed by transfection in polarized Madin–Darby canine kidney (MDCK) cells. Cell-surface biotinylation and immunofluorescence studies showed that ECE-1b is not expressed on the cell-surface but was rather located in intracellular compartments that could also be labelled with anti-Rab-5 and Rab-7 antibodies and was thus tentatively identified as early and late endosomes. Similar results were also obtained when ECE-1b was expressed in non-polarized Chinese hamster ovary cells for comparison purposes. When MDCK or Chinese hamster ovary transfected cells were pre-treated with the ECE inhibitor phosphoramidon, a 3-fold increase in the level of ECE-1b was observed both by Western blotting and by enzymic activity. However, no change in the level of neprilysin or the β-chain of meprin, two apical membrane metallopeptidases, was observed in MDCK cells transfected under similar conditions. Northern blotting showed that the increase in the level of ECE-1b was not owing to changes in the ECE mRNA transcription rate or stability. Rather, pulse-chase experiments followed by immunoprecipitation showed a decrease in the rate of degradation of ECE-1b in phosphoramidon-treated cells. Half-lives were determined to be 2.8 and 7.5 h for non-treated and phosphoramidon-treated cells, respectively. Confocal microscopy showed accumulation of ECE-1b immunoreactive material in the lysosomes of phosphoramidon-treated cells. Taken together, these results suggest that ECE-1b turns over very rapidly between endosomal and lysosomal compartments and that lysosomal degradation of the enzyme is slowed down by phosphoramidon.

scholarly journals PAR1b Promotes Cell–Cell Adhesion and Inhibits Dishevelled-mediated Transformation of Madin-Darby Canine Kidney Cells

2006 ◽  
Vol 17 (8) ◽  
pp. 3345-3355 ◽  
Author(s):  
Maya Elbert ◽  
David Cohen ◽  
Anne Müsch
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Wnt Signaling ◽  
Mdck Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Mammalian Par1 is a family of serine/threonine kinases comprised of four homologous isoforms that have been associated with tumor suppression and differentiation of epithelial and neuronal cells, yet little is known about their cellular functions. In polarizing kidney epithelial (Madin-Darby canine kidney [MDCK]) cells, the Par1 isoform Par1b/MARK2/EMK1 promotes the E-cadherin–dependent compaction, columnarization, and cytoskeletal organization characteristic of differentiated columnar epithelia. Here, we identify two functions of Par1b that likely contribute to its role as a tumor suppressor in epithelial cells. 1) The kinase promotes cell–cell adhesion and resistance of E-cadherin to extraction by nonionic detergents, a measure for the association of the E-cadherin cytoplasmic domain with the actin cytoskeleton, which is critical for E-cadherin function. 2) Par1b attenuates the effect of Dishevelled (Dvl) expression, an inducer of wnt signaling that causes transformation of epithelial cells. Although Dvl is a known Par1 substrate in vitro, we determined, after mapping the PAR1b-phosphorylation sites in Dvl, that PAR1b did not antagonize Dvl signaling by phosphorylating the wnt-signaling molecule. Instead, our data suggest that both proteins function antagonistically to regulate the assembly of functional E-cadherin–dependent adhesion complexes.

Differential sensitivity of Madin-Darby canine kidney (MDCK) cells to epinephrine

2015 ◽  
Vol 20 (5) ◽  
pp. 486-493 ◽  
Author(s):  
P. Muthuraman ◽  
P. C. Nagajyothi ◽  
M. Chandrasekaran ◽  
G. Enkhtaivan ◽  
B. Venkitasamy ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Differential Sensitivity ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Accumulation of natural and synthetic betaines by a mammalian renal cell line

1996 ◽  
Vol 74 (2) ◽  
pp. 283-287 ◽  
Author(s):  
K. Randall ◽  
M. Lever ◽  
B. A. Peddie ◽  
S. T. Chambers
Keyword(s):  
Osmotic Stress ◽  
Glycine Betaine ◽  
Mdck Cells ◽  
Intracellular Accumulation ◽  
Madin Darby Canine Kidney ◽  
Renal Cell Line ◽  
High Concentrations ◽  
Inhibition Studies ◽  
Darby Canine Kidney ◽  
Canine Kidney

Intracellular accumulation of different betaines was compared in osmotically stressed Madin Darby canine kidney (MDCK) cells to model the betaine accumulation specificity of the mammalian inner medulla and to show how this accumulation differed from that of bacteria. All betaines accumulated less than glycine betaine. Arsenobetaine (the arsenic analogue of glycine betaine) accumulated to 12% of the glycine betaine levels and the sulphur analogue dimethylthetin accumulated to >80%. Most substituted glycine betaine analogues accumulated to 2–5% of intracellular glycine betaine concentrations, however, serine betaine accumulated to <0.5% of glycine betaine levels. Inhibition studies to distinguish the betaine ports were performed by the addition of proline. Butyrobetaine and carnitine accumulation was not proline sensitive, whereas that of omer betaines was. As with glycine betaine, the accumulation of propionobetaine and dimethylthetin was proline sensitive and osmoregulated. Pyridinium betaine was accumulated by both proline-sensitive and -insensitive systems, with a small increase under osmotic stress. High concentrations (10 times that of glycine betaine) of the dietary betaines proline betaine and trigonelline inhibited total betaine accumulation. Because α-substituted betaines are accumulated by bacteria and not by MDCK cells, these betaines may be the basis for design of antimicrobial agents.Key words: MDCK cells, betaine accumulation, osmolytes, betaine analogues.

Damaging effects of high energy shock waves on cultured Madin Darby Canine Kidney (MDCK) cells

1990 ◽  
Vol 18 (4) ◽  
pp. 255-258 ◽  
Author(s):  
W. L. Strohmaier ◽  
K. -H. Bichler ◽  
P. Deetjen ◽  
S. Kleinknecht ◽  
M. Pedro ◽  
...  
Keyword(s):  
Shock Waves ◽  
Mdck Cells ◽  
High Energy ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Damaging Effects

scholarly journals TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

1994 ◽  
Vol 5 (10) ◽  
pp. 1093-1103 ◽  
Author(s):  
A K Rajasekaran ◽  
J S Humphrey ◽  
M Wagner ◽  
G Miesenböck ◽  
A Le Bivic ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Protein Localization ◽  
Evolutionary Relationship ◽  
Cytoplasmic Domain ◽  
Chimeric Proteins ◽  
Madin Darby Canine Kidney ◽  
Surface Domains ◽  
Darby Canine Kidney ◽  
Canine Kidney

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.

Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures

1987 ◽  
Vol 7 (4) ◽  
pp. 1326-1337
Author(s):  
S L Warren ◽  
W J Nelson
Keyword(s):  
Tyrosine Kinases ◽  
Mdck Cells ◽  
Growth Potential ◽  
Junctional Complex ◽  
Madin Darby Canine Kidney ◽  
Epithelial Monolayers ◽  
Zonula Occludens ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) cells form polarized, multicellular epithelial structures in vitro. Low-level expression of pp60v-src in MDCK cells elicits plasticity in these multicellular structures. Plasticity was revealed by the displacement of cells from mechanically stressed regions of the epithelial monolayers; however, the two-dimensional relationship between the cells in the remainder of the monolayer was maintained. Electron microscopy of multicellular structures revealed abnormal separation of the lateral membranes of adjacent cells and selective uncoupling of the junctional complex; the zonula adherens was disrupted, but the zonula occludens and desmosomes were retained. Significantly, this result was not accompanied by transformation of the cells, as judged by the absence of anchorage-independent growth potential. These results demonstrate a nonmitogenic biological activity of pp60v-src which is experimentally dissociable from transformation. This morphoregulatory action on higher-order epithelial structures may reflect a function of related cellular tyrosine kinases.

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