Contacts and cooperation between cells depend on the hormone ouabain

Abstract

Cell adhesion is a crucial step in proliferation, differentiation, migration, apoptosis, and metastasis. In previous works we have shown that cell adhesion is modulated by ouabain, a highly specific inhibitor of Na+,K+-ATPase, recently found to be a hormone. In the present work we pursue the investigation of the effect of ouabain on a special type of cell-cell interaction: the rescue of ouabain-sensitive MDCK cells (W) by ouabain-resistant cells (R). In cultured monolayers of pure W cells, ouabain triggers the "P-->A mechanism" (from pump/adhesion) consisting of a cascade of phosphorylations that retrieves adhesion-associated molecules occludin and beta-catenin and results in detachment of the cell. When W cells are instead cocultured with R cells, the P-->A reaction is blocked, and W cells are rescued. Furthermore, in these R/W cocultures ouabain promotes cell-cell communication by means of gap junctions by specifically enhancing the expression of connexin 32 and addressing this molecule to the plasma membrane. Ouabain also promotes the internalization of the beta-subunit of the Na+,K+-ATPase. These observations open the possibility that the crucial processes mentioned at the beginning would be under the control of the hormone ouabain.

Fig. 1.

R cells may not protect W cells through humoral factors. (A) Effect of 1.0 μM ouabain for 24 h on MDCK adhesion assayed as the amount of protein of W cells remaining attached to the multiwell plates. Open bars correspond to control conditions, and gray bars correspond to ouabain-treated monolayers. The first pair of bars shows detachment produced by added ouabain. The second pair of bars shows the effect of ouabain in W cells incubated in media that had been bathing W monolayers for 24 h (COND. MEDIUM W). The third pair of bars shows detachment of W cells bathed in media that had been conditioned in R cells for 24 h. (B) Cells (small cylinders) were cultured in Transwell permeable supports. The filter compartment always contained W cells, and the well part contained either W or R cells as indicated. Ouabain was always added to the well compartment. (C) W cells in the filter are not protected from ouabain; regardless, the well compartment contains a monolayer of W cells (first pair of bars) or R cells (second pair of bars). (D and E) Pictures correspond to W cells cultured on the filter in the absence and presence of ouabain, respectively. (F and G) Cocultures in the well, where R cells are stained in red in the absence and presence of ouabain, respectively. The plasma membrane was stained with an antibody against the β-subunit of Na⁺,K⁺-ATPase (green). Despite detaching W cells cultured on the filter (E), ouabain does not detach them in the R/W coculture in the same monolayer (G).

Fig. 2.

Gap junctions and their sensitivity to ouabain. (A–E) Cocultures of R and W cells. (A) Lucifer yellow can be observed directly under the same microscope used to impale the cells. (B) Neurobiotin does not fluoresce under this optics; therefore, to gauge the injection this probe was coinjected with fluorescent (FITC)-dextran of 20 kDa (green) that does not diffuse to neighboring cells. Monolayers were then fixed and stained with TRITC-conjugated streptavidin (red). (C) Confocal micrograph of a cell injected with dextran (∗) plus neurobiotin that diffuses to several nearest neighbors. (D) In ouabain-treated monolayers neurobiotin propagates to secondarily connected cells, and the area of stained cells becomes considerably larger. This picture was taken at one-half of the magnification of the other six to show the whole area of stained cells. (E) The first pair of bars corresponds to cells injected with Lucifer yellow, and the second pair of bars corresponds to cells injected with neurobiotin both in the absence (open bars) and presence (gray bars) of ouabain. Neurobiotin does not diffuse to neighboring cells in confluent monolayers of pure cell types. (F) An R cell (of 10) showing that the probe remains within the injected cell. (G) Another R cell (of 10) treated with ouabain for 24 h. (H) A W cell (of 10) cultured under control conditions. This test cannot be performed in W cells treated with ouabain because they detach and abandon the monolayer.

Fig. 3.

Ouabain specifically up-regulates the expression of connexin 32. (A) Western blot of connexins 43, 32, and 26 in R/W monolayers with or without 1.0 μM ouabain for 24 h. (B) By using the band of actin to normalize the density of connexin bands, ouabain was found to enhance the expression of connexin 32 without affecting the expression of Cx43 and Cx26. Immunofluorescence images of mixed monolayers show that each connexin distributes according to a particular pattern (C, E, and G under control condition), which is disarranged by 1.0 μM ouabain (D, F, and H). (I) Expression of connexin 32 in monolayers of R cells.

Fig. 4.

Effect of ouabain on the P→A mechanism. (A, D, and G) Immunofluorescence images of the β-subunit of the Na⁺,K⁺-ATPase, occludin, and β-catenin in W cells under control conditions. (B, E, and H) The same markers as affected by 1.0 μM ouabain for 12 h (B and E) or 8 h (H). Nuclei in H were stained in red with propidium iodine. (C, F, and I) The same markers in monolayers of mixed W and R cells (red) treated with ouabain for 24 h. Note that in B, E, and H we used a shorter incubation time to observe the distribution of markers in W cells because this cell type would not have withstood this concentration of ouabain for 24 h. (J) Western blots of occludin and β-catenin in pure monolayers of W or R cells or in cocultures of W/R cells under control and 24 h of ouabain treatment. (K) TER under the same experimental conditions.

Fig. 5.

Role of Na⁺,K⁺-ATPase and its modulation by ouabain. (A) The enzyme consists of a catalytic α-subunit and a β-subunit that has a long extracellular domain with adhesive properties (23). (B) Because of β/β adhesiveness the enzyme stabilizes at the intercellular space, which is sealed at the outermost end by the TJ and opens toward the interstitial side. Because of the firmness of the α/β union, the whole enzyme resides at the borders of the intercellular space. (C) Monolayers of MDCK in transverse optical sections show the Na⁺,K⁺-ATPase (green) at the cell–cell borders but not at the apical or basal domains. Nuclei were stained with propidium iodine (red). (D) Pumps located in the lateral cell border facing the intercellular space lower the Na⁺ concentration in the cytoplasm, and the gradient of this ion across the apical border acts as a driving force for cotransporters and countertransporters (brown and gray circles) translocating H⁺, K⁺, Ca²⁺, sugars, amino acids, etc. (E) Ouabain (pink circles) causes endocytosis of pumps as in Fig. 4B, thereby diminishing the driving force for translocating mechanisms and decreasing the flux of nutrients. (F) When one of the cells is a ouabain-resistant one (R), cell–cell attachment molecules are not retrieved from its membrane or from the membrane of its neighbor W, ouabain-binding pumps of the W cell are not endocytosed (Fig. 4C), and cotransporters remain in operation because the Na⁺ gradient across the apical membrane does not dissipate. Ionic balance of the W cells is maintained by the ouabain-resistant pumps of R cells (14) as well as the presence of gap junctions, which, because of the enhancement of connexin 32 (Fig. 3), increases cell–cell communication (Fig. 2).