Dibutyryl cyclic AMP resistant MDCK cells in serum free medium have reduced cyclic AMP dependent protein kinase activity and a diminished effect of PGE1 on differentiated function

Prostaglandin E1 (PGE1) has a stimulatory effect both on the growth and the expression of differentiated function of Madin Darby Canine Kidney (MDCK) cells in a hormonally defined medium (Medium K-1). While the stimulatory effect of PGE1 on MDCK cell growth is observed in subconfluent cultures, the effect of PGE1 on differentiated function (i.e., dome formation) is observed at confluency. PGE1 may possibly affect growth and such differentiated functions by separate mechanisms. In order to examine this possibility, dibutyryl cyclic AMP resistant variants of MDCK were selected. All of the variants were partially resistant to the growth inhibitory effects of dibutyryl cyclic AMP and theophylline. The cyclic AMP dependent protein kinase activity of four of the five variant clones studied was significantly reduced as compared with normal MDCK cells. The dependence of the kinase activity of several of the dibutyryl cyclic AMP resistant variants (DBr2 and DBr3) on the cyclic AMP concentration in the reaction mixture was compared with that of normal MDCK cells. At all of the cyclic AMP concentrations tested DBr2 and DBr3 cells had reduced protein kinase activity as compared with normal MDCK cells. This reduced activity could be attributed to a decrease in the Vmax for kinase in the two variants, rather than to a change in the Km of kinase for cyclic AMP. The cyclic AMP phosphodiesterase activity of dibutyryl cyclic AMP resistant variants was also studied. Unlike PGE1 independent clone 1, DBr2 and DBr3 cells did not differ significantly from normal MDCK cells with regard to their ability to degrade cyclic AMP. The growth and functional responsiveness of DBr2 and DBr3 cells to PGE1 was also examined. DBr2 and DBr3 cells were shown to retain a normal growth response to PGE1. However the capacity of DBr2 and DBr3 cells to form domes in response to PGE1 was dramatically reduced as compared with normal MDCK cells. Nevertheless DBr3 cells were shown to still retain the capacity to form domes in response to other inducers. The effect of PGE1 on one of the functional parameters involved in dome formation (the activity of the Na+/K+ATPase) was examined. The rate of ouabain-sensitive Rb+ uptake was observed to be elevated in confluent monolayers of normal MDCK cells maintained in Medium K-1, as compared with monolayers maintained in Medium K-1 minus PGE1.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of the beta1 subunit of the Na,K-ATPase and its glycosylation in cell-cell adhesion

Based on recent data showing that overexpression of the Na,K-ATPase beta(1) subunit increased cell-cell adhesion of nonpolarized cells, we hypothesized that the beta(1) subunit can also be involved in the formation of cell-cell contacts in highly polarized epithelial cells. In support of this hypothesis, in Madin-Darby canine kidney (MDCK) cells, the Na,K-ATPase alpha(1) and beta(1) subunits were detected as precisely co-localized with adherens junctions in all stages of the monolayer formation starting from the initiation of cell-cell contact. The Na,K-ATPase and adherens junction protein, beta-catenin, stayed partially co-localized even after their internalization upon disruption of intercellular contacts by Ca(2+) depletion of the medium. The Na,K-ATPase subunits remained co-localized with the adherens junctions after detergent treatment of the cells. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase beta(1) subunit and the endogenous alpha(1) subunit was easily dissociated from the adherens junctions and cytoskeleton by the detergent extraction. The MDCK cell line in which half of the endogenous beta(1) subunits in the lateral membrane were substituted by unglycosylated beta(1) subunits displayed a decreased ability to form cell-to-cell contacts. Incubation of surface-attached MDCK cells with an antibody against the extracellular domain of the Na,K-ATPase beta(1) subunit specifically inhibited cell-cell contact formation. We conclude that the Na,K-ATPase beta(1) subunit is involved in the process of intercellular adhesion and is necessary for association of the heterodimeric Na,K-ATPase with the adherens junctions. Further, normal glycosylation of the Na,K-ATPase beta(1) subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation.     

Regulation of the Na,K-ATPase activity of Madin-Darby canine kidney cells in defined medium by prostaglandin E1 and 8-bromocyclic AMP.

The role of PGE1 in regulating the activity of the Na+, K(+)-ATPase in Madin Darby Canine Kidney (MDCK) cells has been examined. PGE1 increased the initial rate of ouabain-sensitive Rb+ uptake by MDCK cells, a process that continued to occur over a 5-day period. The increase in the initial rate of ouabain-sensitive Rb+ uptake in MDCK cells treated with PGE1 could be explained by a 1.6-fold increase in the Vmax for ouabain-sensitive Rb+ uptake. The increase in the Vmax for ouabain-sensitive Rb+ uptake observed in MDCK cells under these conditions can be explained either by an increase in the number of active Na+ pumps, or by an increase in the efficiency of the Na+ pumps. Consistent with the former possibility is the observed increase in the number of ouabain binding sites, as well as the increase in Na+, K(+)-ATPase activity in cell lysates obtained from MDCK monolayers treated with PGE1. The involvement of cyclic AMP in mediating these effects of PGE1 on the Na+, K(+)-ATPase in MDCK cells is supported by: (1) the observation of similar effects in 8-bromocyclic AMP treated MDCK monolayers, and (2) a dramatic reduction of the stimulatory effects of PGE1 and 8-bromocyclic AMP on the Vmax for ouabain-sensitive Rb+ uptake, and on the number of ouabain binding sites in dibutyryl cyclic AMP resistant clone 3 (DBr3) (which is defective in cyclic AMP dependent protein kinase activity). PGE1 independent MDCK monolayers exhibit both an increase in the Vmax for ouabain-sensitive Rb+ uptake and an increase in the number of ouabain binding sites in response to 8-bromocyclic AMP. Apparently, the cyclic AMP phosphodiesterase defect in these PGE1 independent cells did not cause cellular cyclic AMP levels to be elevated to a sufficient extent to maximally increase the Na+, K(+)-ATPase activity in these variant cells.     

Use of the H,K-ATPase beta subunit to identify multiple sorting pathways for plasma membrane delivery in polarized cells

A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase beta subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the beta subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase beta subunits associate with the endogenous Na,K-ATPase alpha(1) subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase beta subunit with the Na,K-ATPase alpha(1) subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the beta subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase beta subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the beta subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric beta subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase beta subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis.     

Inverse correlation between the extent of N-glycan branching and intercellular adhesion in epithelia. Contribution of the Na,K-ATPase beta1 subunit

The majority of cell adhesion molecules are N-glycosylated, but the role of N-glycans in intercellular adhesion in epithelia remains ill-defined. Reducing N-glycan branching of cellular glycoproteins by swainsonine, the inhibitor of N-glycan processing, tightens and stabilizes cell-cell junctions as detected by a 3-fold decrease in the paracellular permeability and a 2-3-fold increase in the resistance of the adherens junction proteins to extraction by non-ionic detergent. In addition, exposure of cells to swainsonine inhibits motility of MDCK cells. Mutagenic removal of N-glycosylation sites from the Na,K-ATPase beta(1) subunit impairs cell-cell adhesion and decreases the effect of swainsonine on the paracellular permeability of the cell monolayer and also on detergent resistance of adherens junction proteins, indicating that the extent of N-glycan branching of this subunit is important for intercellular adhesion. The N-glycans of the Na,K-ATPase beta(1) subunit and E-cadherin are less complex in tight renal epithelia than in the leakier intestinal epithelium. The complexity of the N-glycans linked to these proteins gradually decreases upon the formation of a tight monolayer from dispersed MDCK cells. This correlates with a cell-cell adhesion-induced increase in expression of GnT-III (stops N-glycan branching) and a decrease in expression of GnTs IVC and V (promote N-glycan branching) as detected by real-time quantitative PCR. Consistent with these results, partial silencing of the gene encoding GnT-III increases branching of N-glycans linked to the Na,K-ATPase beta(1) subunit and other glycoproteins and results in a 2-fold increase in the paracellular permeability of MDCK cell monolayers. These results suggest epithelial cells can regulate tightness of cell junctions via remodeling of N-glycans, including those linked to the Na,K-ATPase beta(1)-subunit.     

Identification and characterization of the cis-elements regulating the rat AMOG (adhesion molecule on glia)/Na,K-ATPase beta 2 subunit gene.

We have identified a positive regulatory cis-acting element of the adhesion molecule on glia (AMOG)/Na,K-ATPase beta 2 subunit gene as GAGGCGGGG at position -87 to -79 by transient transfection assay using B103 cells (rat neuroblastoma cell line). The newly identified AMOG regulatory element (AMRE) enhanced the promoter activity in a mutually compensating manner with the Sp1 element at position -147 to -142. AMRE acts as a positive regulatory element not only in B103 cells but also in 3Y1 (rat embryo cell line) cells to roughly the same extent and in MDCK (canine kidney cell line) cells to a lesser extent. AMRE also enhances other gene promoters, such as myelin basic protein (MBP) and herpes simplex virus (HSV) thymidine kinase (TK) gene promoters. The element is not a typical enhancer element because when it is introduced downstream of the HSV TK promoter, it has no enhancing activity.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.     

Recombinant addition of N-glycosylation sites to the basolateral Na,K-ATPase beta1 subunit results in its clustering in caveolae and apical sorting in HGT-1 cells

In most polarized cells, the Na,K-ATPase is localized on the basolateral plasma membrane. However, an unusual location of the Na,K-ATPase was detected in polarized HGT-1 cells (a human gastric adenocarcinoma cell line). The Na,K-ATPase alpha1 subunit was detected along with the beta2 subunit predominantly on the apical membrane, whereas the Na,K-ATPase beta1 subunit was not found in HGT-1 cells. However, when expressed in the same cell line, a yellow fluorescent protein-linked Na,K-ATPase beta1 subunit was localized exclusively to the basolateral surface and resulted in partial redistribution of the endogenous alpha1 subunit to the basolateral membrane. The human beta2 subunit has eight N-glycosylation sites, whereas the beta1 isoform has only three. Accordingly, up to five additional N-glycosylation sites homologous to the ones present in the beta2 subunit were successively introduced in the beta1 subunit by site-directed mutagenesis. The mutated beta1 subunits were detected on both apical and basolateral membranes. The fraction of a mutant beta1 subunit present on the apical membrane increased in proportion to the number of glycosylation sites inserted and reached 80% of the total surface amount for the beta1 mutant with five additional sites. Clustered distribution and co-localization with caveolin-1 was detected by confocal microscopy for the endogenous beta2 subunit and the beta1 mutant with additional glycosylation sites but not for the wild type beta1 subunit. Hence, the N-glycans linked to the beta2 subunit of the Na,K-ATPase contain apical sorting information, and the high abundance of the beta2 subunit isoform, which is rich in N-glycans, along with the absence of the beta1 subunit, is responsible for the unusual apical location of the Na,K-ATPase in HGT-1 cells.     

Cloning of the H,K-ATPase beta subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase beta subunits

We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase beta subunit. A bovine abomasum lambda gt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase beta subunit. A single positive phage clone containing an approximately 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase beta subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase beta subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length beta subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase beta subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase beta 2 subunit and shares a number of structural similarities with Na,K-ATPase beta subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase beta subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase beta subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.