Aida M. Castillo

Ouabain modulates epithelial cell tight junction

Epithelial cells treated with high concentrations of ouabain (e.g., 1 μM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell–cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K+ pumping nor disturb the K+ balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (JDEX). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, JDEX, and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell–cell contacts.

Myosin II-actin interaction in MDCK cells: role in cell shape changes in response to Ca2+ variations.

Cultured MDCK cell monolayers respond to a low level of extracellular calcium ([Ca2+]e≤5μm) with a loss of transepithelial electrical resistance and transport function, and changes in position of a circumferential ring of actin filaments tethered to the plasma membrane at the zonula adhaerens. Keeping this cytoskeletal structure in place seems necessary to preserve the architecture of the tight junctions and therefore their sealing capacity. All three effects are reversible upon restituting normal [Ca2+]e. Recent work provided evidence of actin–myosin interactions at the filament ring, thus suggesting a contraction process involved in the alteration of the actin cytoskeleton. We now report that active contraction does occur and causes an extensive morphological transformation of MDCK cells. A marked increase in cell height simultaneous with a decrease in width and area of contact to the substratum was seen within 10min of removal of [Ca2+]e; recovery began immediately after replacing calcium, although it took longer for completion. Conventional and confocal epifluorescence studies showed actin colocalized with myosin II at various planes of resting or contracted cells, in particular at the ring level. Electron-micrographs revealed the circumferential actin ring associated with the plasma membrane in a waist-like constriction when Ca2+ was removed from the cultures. Contraction, as well as relaxation, in response to [Ca2+]e variations were inhibited by cytochalasin-D (an actin-filament disrupting drug), by okadaic acid (an inhibitor of myosin light-chain dephosphorylation), and by 2,3-butanedione monoxime (a blocker of myosin II ATPase activity). Similarly, no response was observed in cells previously depleted of metabolic energy by 2,4-dinitrophenol and 2-deoxy-D-glucose preincubation. The actin–myosin mediated reversible structural transformation of MDCK cells in response to [Ca2+]e poses new questions for the interpretation of in vitro experiments, as well as for the understanding of epithelial function.

Ouabain modulates ciliogenesis in epithelial cells

The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na+ and H2O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life.

Actin Cytoskeleton Participation in the Onset of IL-1β Induction of an Invasive Mesenchymal-like Phenotype in Epithelial MCF-7 Cells

BACKGROUND Interleukin 1 beta (IL-1beta) and other inflammatory cytokines are reported to induce phenotypic changes in epithelial breast cancer tumor cells related to increased invasiveness. Mechanisms involved in the process are not well understood. METHODS The noninvasive breast cancer epithelial cell line MCF-7 was used to investigate the IL-1beta-induced phenotype. Live cells expressing EGFP-actin were monitored for cell morphology changes and actin cytoskeleton dynamics by time-lapse video microscopy in the presence of IL-1beta and specific inhibitors of actin signaling pathways. Chemotaxis, invasion of Matrigel, MMP activity and expression of S100A4 in cells treated with IL-1beta were assessed by migration assays, zymograms and immunoblots. RESULTS Exposure to IL-1beta specifically induced a change in MCF-7 cells from a typical epithelial morphology into elongated cells, showing numerous dynamic actin-rich lamellae and peripheral ruffles characteristic of fibroblasts. These cells could scatter from compact cell colonies and respond to chemoattractants such as the homing-associated chemokine CXCL-12. Pharmacological blockage of actin signaling pathways and negative mutants of RhoGTPases revealed that actin reorganization and enhanced motility are regulated via PI3K/Rac 1 activation. IL-1beta-stimulated cells expressed the metastasis promoter S100A4, increased secretion of active MMP-9 and MMP-2 and invasion of extracellular matrix proteins. CONCLUSIONS IL-1beta induces a PI3K/Rac 1-regulated reorganization of the actin cytoskeleton of MCF-7 cells that is required for cell scattering, elongation and migration. The enhanced motility is accompanied by expression of protein markers correlated with invasive behavior.

Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways

In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

2,3-Butanedione monoxime (BDM), a potent inhibitor of actin-myosin interaction, induces ion and fluid transport in MDCK monolayers

Membrane–cytoskeleton interactions have been shown to be crucial to modulate polarity, cell shape and the paracellular pathway in epithelial MDCK cell monolayers. In particular, actin organization and myosin-dependent contractility play an important role in the regulation of these functions. Participation of myosin in vectorial transport, expressed as formation of domes, was investigated in confluent monolayers of high transepithelial electrical resistance (TER) plated on non-permeable supports. Cells exposed to 2,3-butanedione monoxime, a selective inhibitor of myosin ATPase, showed a remarkable increase in the number of domes. Replacement of extracellular Na+ and Cl− and inhibition of Na+-K+-ATPase blocked the induction of domes. The monoxime also caused a reduction of the TER leading to an increase in the paracellular flux of small molecular weight dextran. However, immunofluorescence microscopy of drug-treated cells showed that the localization and staining pattern of tight junction proteins ZO-1, occludin, and claudin 1, or the actin–myosin ring at the zonula adherens, were not modified. Treatment with the drug produced striking re-arrangements of actin filaments at the microvilli and at the basal level of the cells. Our data show that disruption of actin–myosin interaction at several cellular sites contributed importantly to the increased transport activity and the formation of the domes. These results point to the relevant role for actin–myosin dynamics and actin organization in the regulation of ion and water channel activity in these cells.

Ouabain Increases Gap Junctional Communication in Epithelial Cells

Background/Aims: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range) modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC). Methods: We employed two different approaches: 1) analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys) in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2) measurement of the electrical capacitance. Results: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. Conclusion: Ouabain 10 nM increases GJC in MDCK cells.

The expression of endogenous voltage‐gated potassium channels in HEK293 cells is affected by culture conditions

HEK293 cells are widely used as a host for expression of heterologous proteins; yet, little care has been taken to characterize their endogenous membrane components, including ion channels. In this work, we aimed to describe the biophysical and pharmacological properties of endogenous, voltage‐dependent potassium currents (IKv). We also examined how its expression depends on culture conditions. We used the electrophysiological technique of whole‐cell patch clamp to record ion currents from HEK293 cells. We found that HEK cells express endogenous, voltage‐dependent potassium currents. We also found that diverse culture conditions, such as the passage number, the cell density, the type of serum that complements the culture media and the substratum, affect the magnitude and shape of IKv, resulting from the relative contribution of fast, slow, and noninactivating component currents. Incubation of cells in mature monolayers with trypsin–EDTA, notoriously reduces the magnitude and modifies the shape of voltage‐dependent potassium endogenous currents; nonetheless HEK cells recover IKv′s magnitude and shape within 6 h after replating, with a process that requires synthesis of new mRNA and protein subunits, as evidenced by the fact that actinomycin D and cycloheximide, inhibitors of synthesis of mRNA and protein, respectively, impair the recovery of IKv after trypsinization. In addition to be useful as a model expression system, HEK293 may be useful to understand how cells regulate the density of ion channels on the membrane.

Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells.

Background/Aims: The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. Methods: We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. Results: Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. Conclusion: Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.