Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor

We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells.     

Apical cell surface expression of rat dipeptidyl peptidase IV in transfected Madin-Darby canine kidney cells.

Dipeptidyl peptidase IV (DPPIV) is a type II membrane glycoprotein that is predominantly localized to the apical plasma membrane in various epithelial cells. In order to understand in more detail the biogenesis and sorting of DPPIV, the cDNA for rat DPPIV was inserted into a mammalian plasmid expression vector so that DPPIV expression was driven by a control region composed of the SV40 early promoter region fused to the enhancer of the Rous sarcoma virus. Madin-Darby canine kidney cells transfected with this construct were found to express the DPPIV protein. In these transfected cells, the majority of DPPIV was present on the apial cell surface. This observation suggests that the information for apical surface localization is inherent in the DPPIV molecule itself and that this sorting information is decipherable in the epithelial cells of a different species. DPPIV is transported efficiently from the endoplasmic reticulum to the Golgi apparatus as assessed by pulse-chase experiments. Furthermore, evidence is presented which suggests that the majority of DPPIV is sorted intracellularly to the apical cell surface. The same protein has, however, been reported to be sorted by an indirect pathway through transcytosis from the basolateral to the apical cell surface in hepatocytes (Bartles, J.R., Feracci, H., M., Stinger, B., and Hubbard, A.L. (1987) J. Cell Biol. 105, 1241-1251). This study suggests that the same protein can take two different pathways in different cell types for its correct apical cell surface localization.     

Secretion of proteases in serglycin transfected Madin-Darby canine kidney cells

Madin-Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules.     

Direct apical sorting of rat liver dipeptidylpeptidase IV expressed in Madin-Darby canine kidney cells.

In Madin-Darby canine kidney (MDCK) cells, apical and basolateral membrane proteins are segregated from each other in the trans-Golgi network (TGN) and are transported to the appropriate membrane domain via separate vesicle populations. In hepatocytes, however, all plasma membrane proteins are delivered basolaterally. Apical proteins are then selectively retrieved and reach the apical surface by transcytosis. The sorting of apical proteins in different cell types may be the result of differences in the cellular sorting machinery, or alternatively, due to expression of cell-specific sorting signals on the proteins themselves. To test this directly, we have stably expressed cDNA encoding an apical protein from rat liver, dipeptidylpeptidase IV (DPPIV), in MDCK cells. We found that approximately 90% of the exogenous DPPIV is expressed on the apical cell surface at steady state. Furthermore, we demonstrate that this distribution is primarily due to vectorial transport from the TGN to the apical plasma membrane. The small pool of mis-sorted DPPIV that appears basolaterally is slowly endocytosed (t1/2 approximately 60 min) and is subsequently transcytosed. These data are consistent with the notion that both hepatocytes and MDCK cells are capable of correctly sorting rat liver DPPIV, but that this sorting occurs at different sites in the two cell types.     

Endocytosis in filter-grown Madin-Darby canine kidney cells

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three- dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.     

Ion channels in Madin-Darby canine kidney cells.

Ion channels in Madin-Darby canine kidney cells serve transepithelial chloride transport and probably cell volume regulation. Three distinct potassium channels and one anion channel have been revealed by patch clamp studies in Madin-Darby canine kidney cells. The potassium channels are activated by an increase in intracellular calcium activity. A number of hormones activate the potassium channels by an increase in intracellular calcium activity. However, under certain conditions the hormones hyperpolarize the cell membrane without increasing intracellular calcium activity sufficiently to activate the calcium-sensitive potassium channels. Thus, the hormones may activate potassium channels via another, as yet undefined, intracellular mechanism. The anion channel is stimulated by cAMP. Another factor modifying channel activity is cell volume: cell swelling leads probably to subsequent activation of potassium and anion channels. The net result is a variable transient hyperpolarization followed by a sustained depolarization of the cell membrane.     

Lixazinone stimulates mitogenesis of Madin-Darby canine kidney cells

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.     

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120^ctn interacts with E-cadherin, because p120^ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Rho family GTPases; catenin; polarity; sorting; actin     

Immunocytochemical localization of the receptor for asialoglycoprotein in rat liver cells

We used high-resolution immunocytochemistry on ultrathin frozen sections labeled with colloidal gold to study the subcellular distribution of the asialoglycoprotein receptor in rat liver. The receptor was localized along the entire hepatocyte plasma membrane, including the bile capillary membrane, but was scarce intracellularly. Sinusoidal lining (Kupffer) cells and blood cells showed no immunoreactivity. In liver cells of rats injected with 1 to 100 micrograms of asialoorosomucoid (ASOR) 2-15 min before tissue fixation, endocytotic internalization of receptors at the blood front was conspicuous. At all times in this interval, receptor was present in approximately 100-nm vesicles and larger vacuoles adjacent to the sinusoidal plasma membrane. No other significant intracellular receptor was noted during the 15-min exposure to ASOR; in particular, lysosomes and Golgi complex were not labeled. Our observations, in combination with data from the literature which demonstrate that, under these conditions, the ligand is transferred further to the Golgi complex-lysosome region, suggest that the receptor and ligand are dissociated in the vicinity of the plasma membrane, after which the receptor rapidly returns to the cell surface.     

Epidermal growth factor receptor activation differentially regulates claudin expression and enhances transepithelial resistance in Madin-Darby canine kidney cells

Tight junctions (TJs) are the most apical cell-cell junctions, and claudins, the recently identified TJ proteins, are critical for maintaining cell-cell adhesion in epithelial cell sheets. Based on their in vivo distribution and the results of overexpression studies, certain claudins, including claudin-1 and -4, are postulated to increase, whereas other claudins, especially claudin-2, are postulated to decrease the overall transcellular resistance. The overall ratio among claudins expressed in a cell/tissue has been hypothesized to define the complexity of TJs. Disruption of the TJs contributes to various human diseases, and a correlation between reduction of TJ function and tumor dedifferentiation has been postulated. The epidermal growth factor (EGF) receptor (EGFR) is overexpressed in a wide spectrum of epithelial cancers, and its expression correlates with a more metastatic cancer phenotype. However, normal functioning of EGFR is essential for normal epithelial cell proliferation and differentiation. The role of EGFR-dependent signaling in the development and maintenance of epithelial TJ integrity has not been studied in detail. This study demonstrates that, in polarized Madin-Darby canine kidney II cells, EGF-induced EGFR activation significantly inhibited claudin-2 expression while simultaneously inducing cellular redistribution and increased expression of claudin-1, -3, and -4. Accompanying these EGF-induced changes in claudin expression was a 3-fold increase in transepithelial resistance, a functional measure of TJs. In contrast, there were no alterations in protein expression and/or intracellular localization of other TJ-related proteins (ZO-1 and occludin) or adherens junction-associated proteins (E-cadherin and beta-catenin), suggesting that EGF regulates TJ function through selective and differential regulation of claudins.     

The action of epinephrine on Madin-Darby canine kidney cells

We have used cultured monolayers of Madin-Darby canine kidney (MDCK) cells, which form epithelial layers of high transepithelial resistance, grown on Millipore filters, for transport studies. In the absence of hormones net ion transport is of small magnitude and is consistent with a net absorptive flow (apical to basal) of Na+. Epinephrine, effective only from the basolateral cell surface, stimulates a net secretion (basal to apical) of Cl-. A substantial portion of net Cl- secretion is inhibited by loop diuretics such as furosemide applied to the basolateral cell aspects. The participation of a diuretic-sensitive cotransport system for Na+, K+, and Cl-, similar to that found in other cells, in transepithelial Cl- flux is postulated. The action of catecholamines on MDCK cell adenylate cyclase and on a Ca2+-activated K+ conductance is described.     

Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells

The polymeric immunoglobulin receptor (pIg-R) is responsible for the receptor-mediated transcytosis of polymeric immunoglobulins (IgA and IgM) across various epithelia. We have expressed the cDNA for the pIg-R in Madin-Darby canine kidney (MDCK) cells and found that this system mimics that found in vivo (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). We have now investigated the postendocytotic pathway of the ligand for the pIg-R. After a 5-min internalization at the basolateral surface, approximately 45% of internalized ligand recycles to the basolateral medium and 30% is transcytosed to the apical medium. We have also examined why transcytosis of ligand is unidirectional, going only from basolateral to apical, but not from apical to basolateral. Several factors could explain this, such as proteolytic cleavage of the pIg-R at the apical surface, decreased apical endocytosis of ligand, or an intracellular sorting event. In this report, we show that the protease inhibitor, leupeptin, inhibits the cleavage of the pIg-R but does not alter the unidirectionality of transcytosis. In addition, we demonstrate that there is a significant amount of apical endocytosis of ligand (70% of that observed basolaterally). Finally, we demonstrate that apically endocytosed ligand can return only to the apical surface. Thus, once ligand reaches the apical surface, it is "trapped" and cannot return to the basolateral surface. We propose that the unidirectionality of transcytosis is the result of intracellular sorting, and that this results from a signal(s) present on the pIg-R.     

Isolation and expression of cDNA clones for a rat liver asialoglycoprotein receptor

cDNA clones for the major rat liver asialoglycoprotein (ASGP) receptor were isolated from a phage gtl 1 library using synthetic oligonucleotide probes corresponding to two regions of the protein sequence. The longest clone obtained encoded all but the first 11 codons of the receptor. The cDNA was completed with synthetic oligonucleotides and was used to direct the synthesis of mRNA for the receptorin vitro. Subsequent translation in a wheat germ lysate produced authentic ASGP receptor which assembled correctly into microsomal membranes.     

Claudin-8 expression in Madin-Darby canine kidney cells augments the paracellular barrier to cation permeation

Claudins are a family of integral membrane proteins of the tight junction that are thought to participate in the permeation of solutes across epithelia via the paracellular pathway. Claudin-8 is expressed in the distal renal tubule, which has a characteristically low passive permeability to monovalent cations. To test the hypothesis that claudin-8 plays a role in forming a tight paracellular barrier to cations, stably transfected Madin-Darby canine kidney II cell lines with inducible expression of claudin-8 were generated. Induction of claudin-8 expression was associated with down-regulation of endogenous claudin-2 protein. Other tight junction proteins were expressed and targeted normally, and the number of junctional strands was minimally altered. By Ussing chamber and radiotracer flux studies, claudin-8 expression was found to reduce paracellular permeability to monovalent inorganic and organic cations and to divalent cations but not to anions or neutral solutes. The size selectivity, charge dependence, and activation energy of paracellular cation permeation were all unchanged. These observations are consistent with a model in which claudin-2 encodes a highly cation-permeable channel, whereas claudin-8 acts primarily as a cation barrier. When exogenous claudin-8 is expressed, it replaces endogenous claudin-2, inserting in its place into existing tight junction strands, thereby reducing the apparent number of functional cation pores. Our findings suggest that claudin-8 plays an important role in the paracellular cation barrier of the distal renal tubule.     

Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.     

Effect of trifluoperazine on renal epithelioid Madin-Darby canine kidney cells

Following exposure to a number of hormones, the cell membrane in Madin-Darby Canine Kidney (MDCK) cells is hyperpolarized by increase of intracellular calcium activity. The present study has been performed to elucidate the possible role of calmodulin in the regulation of intracellular calcium activity and cell membrane potential. To this end trifluoperazine has been added during continuous recording of cell membrane potential or intracellular calcium. Trifluoperazine leads to a transient increase of intracellular calcium as well as a sustained hyperpolarization of the cell membrane by activation of calcium sensitive K+ channels. Half-maximal effects are observed between 1 and 10 mumol/L trifluoperazine. A further calmodulin antagonist, chlorpromazine, (50 mumol/L), similarly hyperpolarizes the cell membrane. The effects of trifluoperazine are virtually abolished in the absence of extracellular calcium. Pretreatment of the cells with either pertussis toxin or phorbol-ester TPA does not interfere with the hyperpolarizing effect of trifluoperazine. In conclusion, calmodulin is apparently involved in the regulation of calcium transfer across the cell membrane but not in the stimulation of K+ channels by intracellular calcium.     

Subcellular trafficking of exogenously expressed interferon-beta in Madin-Darby canine kidney cells

We have recently demonstrated that when IFN-beta was exogenously expressed in epithelial cells, transiently expressed IFN-beta was predominantly secreted from the cell side to which the transfection was performed, while stably expressed one was almost equally secreted to the apical and basolateral sides. In the present study, we analyzed the subcellular transport of IFN-beta using confocal imaging with green fluorescent protein (GFP)-tagged IFN-beta in Madin-Darby canine kidney (MDCK) cells. Stably expressed and transiently expressed human IFN-beta (HuIFN-beta)-GFPs were seen in upper regions of the nucleus. In stable HuIFN beta-GFP-producing transformants, transiently expressed mouse IFN-beta (MuIFN-beta) was apparently co-localized with the bulk of the constitutive HuIFN beta-GFP proteins at TGN, and a significant quantity of them then appeared to pass into distinct post-TGN vesicles, accepting either type of IFN. Meanwhile, when cells were co-transfected with both expression vectors, transiently expressed both IFNs tended to co-localize not only at TGN but in post-TGN vesicles. These results suggest that stably and transiently expressed IFN-betas, albeit co-localized at TGN, were transported through apparently discriminated post-TGN routes.     

Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells

We have characterized the muscarinic AChreceptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK)strain II epithelial cells. Binding studies with themembrane-impermeable antagonist N-[³H]methylscopolaminedemonstrated that mAChRs are ~2.5 times more abundant on thebasolateral than on the apical surface. Apical, but not basolateral,mAChRs inhibited forskolin-stimulated adenylyl cyclase activity inresponse to the agonist carbachol. Neither apical nor basolateralmAChRs exhibited detectable carbachol-stimulated phospholipase Cactivity. Carbachol application to the apical or the basolateralmembrane resulted in a threefold increase in intracellularCa²⁺ concentration, which wascompletely inhibited by pertussis toxin on the apical side andpartially inhibited on the basolateral side. RT-PCR analysis showedthat MDCK cells express the M₄ and M₅ receptor mRNAs. These datasuggest that M₄ receptors reside on the apical and basolateral membranes of polarized MDCK strain IIcells and that the M₅ receptor mayreside in the basolateral membrane of a subset of cells.

     

RhoB-dependent modulation of postendocytic traffic in polarized Madin-Darby canine kidney cells

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein-tagged wild-type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin-Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant-negative RhoBN19 or dominant-active RhoBV14 altered postendocytic traffic of ligand-receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral-to-apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system.