Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells

Ponce, Arturo; Larre, Isabel; Castillo, Aida; Flores-Maldonado, Catalina; Verdejo-Torres, Odette; Contreras, Rubén G.; Cereijido, Marcelino

doi:10.1159/000447837

Cited by 20 publications

(23 citation statements)

References 47 publications

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“…As previously mentioned, Na-K-ATPase has been shown to be the receptor that transduces the action of cardiac glycosides in various physiological processes, and we have previously demonstrated that this also happens in the changes that ouabain induces on GJIC and TER of MDCK cells [36][37][38].…”

Section: Involvement Of Na-k-atpase In a The Effect Of Marinobufagenisupporting

confidence: 57%

“…us, we have demonstrated that ouabain influences Tight Junctions (TJ), as reflected by a significant increase in the Transepithelial Electrical Resistance (TER) and to an increased expression of claudins 2 and 4 [36]. It also induces, within minutes, Gap Junctional Intercellular Communication (GJIC) by promoting relocation of connexins 32 and 43 [37,38]. Ouabain 10 nM upregulates Adherens Junctions, enhancing the cellular content of E-cadherin, β-catenin, and c-catenin [39].…”

Section: Introductionmentioning

confidence: 99%

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Influence of Endogenous Cardiac Glycosides, Digoxin, and Marinobufagenin in the Physiology of Epithelial Cells

Toro

Jimenez

Hinojosa

et al. 2019

Cardiology Research and Practice

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Cardiac glycosides are a group of compounds widely known for their action in cardiac tissue, some of which have been found to be endogenously produced (ECG). We have previously studied the effect of ouabain, an endogenous cardiac glycoside, on the physiology of epithelial cells, and we have shown that in concentrations in the nanomolar range, it affects key properties of epithelial cells, such as tight junction, apical basolateral polarization, gap junctional intercellular communication (GJIC), and adherent junctions. In this work, we study the influence of digoxin and marinobufagenin, two other endogenously expressed cardiac glycosides, on GJIC as well as the degree of transepithelial tightness due to tight junction integrity (TJ). We evaluated GJIC by dye transfer assays and tight junction integrity by transepithelial electrical resistance (TER) measurements, as well as immunohistochemistry and western blot assays of expression of claudins 2 and 4. We found that both digoxin and marinobufagenin improve GJIC and significantly enhance the tightness of the tight junctions, as evaluated from TER measurements. Immunofluorescence assays show that both compounds promote enhanced basolateral localization of claudin-4 but not claudin 2, while densitometric analysis of western blot assays indicate a significantly increased expression of claudin 4. These changes, induced by digoxin and marinobufagenin on GJIC and TER, were not observed on MDCK-R, a modified MDCK cell line that has a genetically induced insensitive α1 subunit, indicating that Na-K-ATPase acts as a receptor mediating the actions of both ECG. Plus, the fact that the effect of both cardiac glycosides was suppressed by incubation with PP2, an inhibitor of c-Src kinase, PD98059, an inhibitor of mitogen extracellular kinase-1 and Y-27632, a selective inhibitor of ROCK, and a Rho-associated protein kinase, indicate altogether that the signaling pathways involved include c-Src and ERK1/2, as well as Rho-ROCK. These results widen and strengthen our general hypothesis that a very important physiological role of ECG is the control of the epithelial phenotype and the regulation of cell-cell contacts.

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Section: Involvement Of Na-k-atpase In a The Effect Of Marinobufagenisupporting

confidence: 57%

Section: Introductionmentioning

confidence: 99%

Influence of Endogenous Cardiac Glycosides, Digoxin, and Marinobufagenin in the Physiology of Epithelial Cells

Toro

Jimenez

Hinojosa

et al. 2019

Cardiology Research and Practice

Self Cite

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“…It is well recognized that ouabain promotes cell-to-cell contacts and cell proliferation in cultured cells (8, 9, 43). There is also evidence that ouabain affects embryonic growth and development in mammals, because offspring of mice treated with anti-ouabain antibodies have lower body weight (11) and ouabain supplementation to malnourished pregnant mice prevents adverse development of the kidney in the offspring (64).…”

Section: Discussionmentioning

confidence: 99%

“…S4A). Previous studies by Cereijido et al demonstrated that ouabain, when employed at a subsaturating concentration, plays a major role in regulating the permeability and composition of tight junctions (8,9,43). Furthermore, cell adhesion molecules play a significant role in cancer progression and metastasis, and many of the adhesive molecules phosphorylated in response to ouabain have been suggested as potential therapeutic targets in cancer (44,45).…”

Section: Ouabain Regulates Proteins Responsible For the Oscillatory Cmentioning

confidence: 99%

Ouabain‐regulated phosphoproteome reveals molecular mechanisms for Na ⁺ , K ⁺ ‐ATPase control of cell adhesion, proliferation, and survival

et al. 2019

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Ouabain is a cardiotonic steroid with a history of medical uses. It acts as a ligand to the ubiquitous ion pump Na+,K+‐ATPase which it inhibits at high concentrations. At lower concentrations it triggers calcium oscillations and activation of MAP kinases, stimulates cell proliferation as well as adhesion and acts anti‐apoptotic. In light of this, it has been proposed that the pump may have a dual function as a signaling transducer. To investigate this, we have analyzed the phosphoproteome of COS‐7 kidney cells after treatment with sub‐saturating levels (100 nM) of ouabain, which were high enough to induce Ca2+ oscillations but below those needed to modulate the ion pumping function of Na+,K+‐ATPase. Gene ontology analysis was performed to find distinct cellular processes regulated by ouabain after 10 and 20 min of treatment in the phopshoproteomic analysis. The analysis revealed 2580 ouabain‐regulated phosphorylation sites, many of which were associated with cell adhesion and proliferation. Two of the regulated phospho‐proteins were the inositol triphosphoate receptor (InsP3) and stromal interaction molecule (STIM), both of which are essential for ouabain‐induced calcium oscillations. We used siRNA and Western blotting for target confirmation. One confirmed target, calcium/calmodulin‐dependent protein kinase II gamma (CAMK2γ), were shown to be involved in the anti‐apoptotic effect of ouabain; siRNA silencing of CaMK2γ in primary renal cells eliminates the protective effect of ouabain against apoptosis induced by either serum starvation or glucose. In conclusion, our findings support a role for Na+,K+‐ATPase as a signal transducer that serves to protect cell and tissue integrity. Support or Funding Information This study was supported by the Swedish Research Council and Erling‐Persson Family Foundation. D.S. is supported by a Novo Nordisk postdoctoral fellowship run in partnership with Karolinska Institutet. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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“…Previous studies demonstrated that binding of cardiotonic steroids such as ouabain to the sodium pump stimulates multiple kinase cascades [57]. Therefore, the effect of ouabain on the β 1 -β 1 interaction could be explained by the potential activation of some components from the signaling machinery that is known to modulate cell junctions such as TJs, AJs and GAP junctions [29,58]. We first screened for signaling proteins of the family of tyrosine kinases that were differentially activated compering between CHOβ 1 and CHO wild type cells.…”

Section: The β 1 -β 1 Adhesion Induced By Ouabain Depends On the Actimentioning

confidence: 99%

Ouabain enhances cell-cell adhesion mediated by β₁-subunits of the Na⁺,K⁺-ATPase in CHO fibroblasts

Vilchis-Nestor

Roldán

Padilla‐Benavides

et al. 2019

Preprint

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Adhesion is an important characteristic of epithelial cells to provide a crucial barrier to pathogens and substances. In polarized epithelial cells, cell-adhesion depends on tight junctions, adherent junctions and the Na + ,K + -ATPase. All these are located in the basolateral membrane of the cells. The hormone ouabain, a cardiotonic steroid, binds to the α subunit of the Na + ,K + -ATPase, and inhibits the pump activity when used at above μM concentrations. At physiological nM concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through activation of signaling pathways associated to the α subunit. Our group showed that non-adherent CHO cells transfected with the canine β 1 subunit become adhesive, and that homotypic interactions between β 1 subunits of the Na + ,K + -ATPase occur between neighboring epithelial cells. Therefore, in this study we investigated whether the adhesion between β 1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the β 1 subunit of the Na + ,K + -ATPase (CHO-β 1 ) and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO-β 1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the β1 subunit of the Na + ,K + -ATPase at the cell membrane. We also screened the effect of ouabain on activation of signaling pathways in CHO-β 1 cells, and their effect on cell adhesion. We found that c-Src, is activated by ouabain and is therefore likely to regulate the adhesive properties of CHO-β 1 cells. Collectively, our findings suggest that the β 1 subunits adhesion is modulated by the levels of expression and activation of the Na + ,K + -ATPase at the plasma membrane, which is regulated by ouabain.

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Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells

Cited by 20 publications

References 47 publications

Influence of Endogenous Cardiac Glycosides, Digoxin, and Marinobufagenin in the Physiology of Epithelial Cells

Influence of Endogenous Cardiac Glycosides, Digoxin, and Marinobufagenin in the Physiology of Epithelial Cells

Ouabain‐regulated phosphoproteome reveals molecular mechanisms for Na ⁺ , K ⁺ ‐ATPase control of cell adhesion, proliferation, and survival

Ouabain enhances cell-cell adhesion mediated by β₁-subunits of the Na⁺,K⁺-ATPase in CHO fibroblasts

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Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells

Cited by 20 publications

References 47 publications

Influence of Endogenous Cardiac Glycosides, Digoxin, and Marinobufagenin in the Physiology of Epithelial Cells

Influence of Endogenous Cardiac Glycosides, Digoxin, and Marinobufagenin in the Physiology of Epithelial Cells

Ouabain‐regulated phosphoproteome reveals molecular mechanisms for Na + , K + ‐ATPase control of cell adhesion, proliferation, and survival

Ouabain enhances cell-cell adhesion mediated by β1-subunits of the Na+,K+-ATPase in CHO fibroblasts

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Product

Resources

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Ouabain‐regulated phosphoproteome reveals molecular mechanisms for Na ⁺ , K ⁺ ‐ATPase control of cell adhesion, proliferation, and survival

Ouabain enhances cell-cell adhesion mediated by β₁-subunits of the Na⁺,K⁺-ATPase in CHO fibroblasts