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Ouabain Modulates Ciliogenesis in Epithelial Cells

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Ouabain Modulates Ciliogenesis in Epithelial Cells Ouabain modulates ciliogenesis in epithelial cells Isabel Larrea, Aida Castilloa, Catalina Flores-Maldonadoa, Ruben G. Contrerasa, Ivan Galvanb, Jesus Muñoz-Estradaa, and Marcelino Cereijidoa,1 aDepartment of Physiology, Biophysics and Neurosciences, and bCentral Laboratories, Center for Research and Advanced Studies of the National Polytechnic Institute, Mexico City, DF 07300, Mexico Edited* by Lutz Birnbaumer, National Institute of Environmental Health Sciences, Research Triangle Park, NC, and approved October 27, 2011 (received for review February 24, 2011) The exchange of substances between higher organisms and the Results environment occurs across transporting epithelia whose basic Ouabain Accelerates Ciliogenesis. MDCK cells display procilia features are tight junctions (TJs) that seal the intercellular space, some 12 h after reaching confluence (Fig. 1A). Procilia pro- and polarity, which enables cells to transport substances vecto- gressively lengthen until they become mature cilia. At the third rially. In a previous study, we demonstrated that 10 nM ouabain day almost all cells have a cilium (Fig. 1B). Ouabain increases modulates TJs, and we now show that it controls polarity as well. the length of the cilium (Fig. 1C) but not its thickness (Fig. 1D). We gauge polarity through the development of a cilium at the We also followed ciliogenesis by staining the cells with an anti- apical domain of Madin-Darby canine kidney cells (MDCK, epithe- body against acetylated α-tubulin and counting the number of lial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2- E F + cells at stages without cilium (Fig. 1 ), with procilium (Fig. 1 ), dependent manner. Claudin-2, a molecule responsible for the Na and with a mature cilium (Fig. 1G). Fig. 1H summarizes the and H2O permeability of the TJs, is also present at the cilium, as it progressive increase in the percentage of prociliated and ciliated α colocalizes and coprecipitates with acetylated -tubulin. Ouabain cells that tend to reach 100% (open circles). Ouabain accelerates modulates claudin-2 localization at the cilium through ERK1/2. the kinetics of procilia and cilia formation (Fig. 1H, red filled Comparing wild-type and ouabain-resistant MDCK cells, we show circles), an effect that reaches a 400% increase at 24 h. that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain Role of Homo- and Heterotypic Cell-Cell Contacts. To study the role constitutes a hormone that modulates the transporting epithelial of cell-cell contacts on the effect played by ouabain on cilio- CELL BIOLOGY phenotype, thereby playing a crucial role in metazoan life. genesis, we plated cells at confluency, in mixed populations of 1% MDCK and 99% NRK cells (epithelial rat kidney). Under this E-cadherin | occludin | cell adhesion | cardiotonic steroids condition, most single MDCK cells (Fig. 2A, red) surrounded by NRK cells do not exhibit a cilium (Fig. 2 A and C). Nevertheless, he high affinity and specificity of Na+,K+-ATPase for the ouabain is able to stimulate cilliogenesis even in these MDCK Tplant-derived inhibitor ouabain suggested the possibility that cells totally surrounded by NRK cells (Fig. 2 B and C). there might exist endogenous analogs. Hamlyn et al. (1) dem- In keeping with our previous observations (19, 20) that the TJ onstrated that there is a substance in plasma that cannot yet be is a promiscuous structure that can be established by epithelial distinguished from ouabain, even by electrospray ionization-mass cells from different organs and even from different animal spe- 1 spectrometry, H-NMR, and liquid chromatography (2–4). Fur- cies, we found that monolayers of mixed MDCK (Fig. 2, red) and thermore, the observation that this endogenous ouabain and its NRK cells develop a transepithelial electrical resistance (TER). analogs increase during exercise (5), salty meals (6), and path- This parameter is proportional to the TER in the monolayer of ological conditions [such as arterial hypertension, eclampsia (7), a single-cell type and their proportion in the mixture (Fig. 2D). and myocardial infarction (8)] raised the possibility that it may Mixed cells also express ZO-2 at the TJs (Fig. 2E). Nagafuchi function as a hormone (9). This theory prompted efforts to un- et al. (21) showed that the expression of E-cadherin requires that ravel its physiological role (10). The present work stems from our the two neighboring cells belong to the same animal species previous observations that toxic levels of ouabain invariably af- (homotypic contact). In accordance with this observation, Fig. 2F fect cell-cell and cell-substrate adhesion molecules in Madin- shows that monolayers of pure MDCK (Fig. 2F, red), as well as F Darby canine kidney (MDCK) cells (11), suggesting that low-dose small groups of MDCK cells (Fig. 2 , red), express E-cadherin F G ouabain may be able to modulate cell contacts without causing (Fig. 2 and , green) at homotypic cell-cell borders. However, irreversible damage (12). In keeping with such possibility, we have this molecule is not observed at heterotypic MDCK/NRK con- G previously shown that ouabain promotes cell-cell contacts as well tacts (Fig. 2 ). Accordingly, a quiescent single MDCK cell (Fig. H as contact-dependent phenomena, such as increases in cell com- 2 ) expresses no E-cadherin at its borders. We conclude that TJ munication, the expression of connexin-32 (13), and the molec- sealing and ouabain stimulation of ciliogenesis occur in cells that ular structure and hermeticity of the tight junction (TJ). This establish homo- and heterotypic borders, even if these express no process occurs by regulating the specific expression and distri- E-cadherin at the plasma membrane. bution of claudin-1, -2, and -4 (12) during differentiation toward Ouabain and Na+,K+-ATPase. Ouabain promotes ciliogenesis when the so-called “epithelial transporting phenotype.” added from the basolateral (Fig. 3 A, B, and J, first and second Pursuing our exploration of the effect of low-dose ouabain on columns) but not from the apical side (Fig. 3C), in accordance cell contacts, we now study whether 10 nM ouabain can modu- late polarity, one of the two basic cell features of the trans- epithelial transporting phenotype (14, 15). We gauge polarity Author contributions: I.L., A.C., C.F.-M., R.G.C., and M.C. designed research; I.L., A.C., C.F.-M., through the expression of a single cilium at the center of the R.G.C., I.G., and J.M. performed research; I.L., A.C., C.F.-M., R.G.C., and M.C. analyzed data; apical domain, a process that relays on the polarized delivery of and I.L., R.G.C., and M.C. wrote the paper. many proteins to this apical compartment (16, 17). At 10 nM, The authors declare no conflict of interest. ouabain causes none of its well-known toxic effects; it does not *This Direct Submission article had a prearranged editor. + + + inhibit the unidirectional transport of K by Na ,K -ATPase 1To whom correspondence should be addressed. E-mail: Cereijido@fisio.cinvestav.mx. + nor distorts the K balance of the cell, and it does not cause cell This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. death (12, 18). 1073/pnas.1102617108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1102617108 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Ouabain, at a concentration of 10 nM, accelerates ciliogenesis. Scanning electron micrographs of a cilium (between arrows) in a monolayer that has been confluent for 12 h (A), or 3 d (B) under control conditions. Cilia length (C) and thickness (D) as measured in scanning electron microscopy micrographs of wild-type MDCK cells, under control (white bars) or 24 h of ouabain treatment (red bars); ***P < 0.001. Monolayers of MDCK cells stained with antiacetylated α-tubulin at zero (E), 24 h (F), or 72 h (G)in confluence. Ciliogenesis as observed in confluent monolayers under control (open circles) and ouabain (red circles) treatment as a function of time (H). with the fact that the Na+,K+-ATPase located at the intercel- lular cell borders is only accessible from the basal side (20, 22– fi 24). This nding suggests that this enzyme may act as receptor Fig. 2. Ciliogenesis in proliferation-arrested MDCK cells does not depend for low concentrations of ouabain acting on cilliogenesis. This on homotypic cell contacts. Stable red color MDCK cells were produced by possibility was further tested using ouabain-resistant R-MDCK transfection of red fluorescent protein. To produce MDCK cells with arrested cells, a stable line produced by Soderberg et al. (25) with very low proliferation, we plated them at confluence in a 1/99 ratio with NRK cells. affinity for ouabain. Procilia and cilia in R-MDCK cells are ru- Forty-two hours later cells were fixed and stained with the indicated anti- fi dimentary (Fig. 3 D and E), and their length (Fig. 3F) and bodies (green) and to detect nucleus with DAPI (blue, NUC). The gure G shows a single MDCK completely surrounded by NRK cells, under control thickness (Fig. 3 ), as well as the number of ciliated and pro- condition (A), and treated with 10 nM ouabain for the last 30 h (B). The ciliated cells (Fig. 3 H, I, and J. third and fourth columns), are cilium is stained with a primary antibody against antiacetylated α-tubulin (Ac not stimulated by ouabain, as observed by scanning electron Tub) and a fluoresceinated secondary one (green). Statistical analysis of microscopy and immunodetection of acetylated α-tubulin. single MDCK cells, either under control (open circles) or ouabain treatment Zampar et al. (26) have clearly demonstrated that acetylated conditions (red circles) (C). Establishment of tight junctions as revealed by fi α-tubulin binds and inhibits Na+,K+-ATPase in cells of rat brain the value of TER (D) in monolayers of pure MDCK cells ( rst column), pure fi ouabain-resistant NRK cells (second column), and a 50/50 mixture of both cell and COS (monkey kidney broblasts), and Menco et al.
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