Ouabain modulates ciliogenesis in epithelial cells

Abstract

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 H(2)O 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.

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).

Fig. 2.

Ciliogenesis in proliferation-arrested MDCK cells does not depend on homotypic cell contacts. Stable red color MDCK cells were produced by transfection of red fluorescent protein. To produce MDCK cells with arrested proliferation, we plated them at confluence in a 1/99 ratio with NRK cells. Forty-two hours later cells were fixed and stained with the indicated antibodies (green) and to detect nucleus with DAPI (blue, NUC). The figure shows a single MDCK completely surrounded by NRK cells, under control condition (A), and treated with 10 nM ouabain for the last 30 h (B). The cilium is stained with a primary antibody against antiacetylated α-tubulin (Ac Tub) and a fluoresceinated secondary one (green). Statistical analysis of single MDCK cells, either under control (open circles) or ouabain treatment conditions (red circles) (C). Establishment of tight junctions as revealed by the value of TER (D) in monolayers of pure MDCK cells (first column), pure ouabain-resistant NRK cells (second column), and a 50/50 mixture of both cell types (third column) cultured for 48 h; ***P < 0.001. In mixed monolayers of MDCK (red) and NRK cells, the cytoplasmic protein ZO-2 is detected by immunofluorescence (green) (E). Immunodetection of E-cadherin (green) in a monolayer of pure MDCK cells (3 d old) (F), or in mixed monolayers (48 h old) showing a small group (G) or a single (H) MDCK cell surrounded by NRK cells. (Scale bars, 10 μm.)

Fig. 3.

Role of Na⁺,K⁺-ATPase in the effect of ouabain. Monolayers of wild MDCK cells, under control (A) and 48-h treatment with ouabain (B and C), added from the basolateral (A and B) or the apical side (C). Ouabain-resistant MDCK cells (MDCK-R) display very short cilia (D) that do not become larger (E and F) nor thicker (E and G), nor increase the number of ciliated cells (H vs. I and J) in response to 2 d of incubation with ouabain. Cilia were stained with an antibody against acetylated α-tubulin. Statistical analysis of ciliogenesis in wild-type MDCK cells under control (J, first column) and ouabain (J, second column). Third and fourth columns indicate that ouabain-resistant MDCK cells do not increase the number of ciliated cells when treated with ouabain for 48 h (J); ***P < 0.001. In a 4-d old monolayer of pure MDCK cells (the last 3 d at confluence), Na⁺,K⁺-ATPase (green) is localized at all cell borders, and it was never observed at the cilium (red) (K). In 1.5-d mixed monolayers of MDCK (red) with NRK (unstained), the enzyme is only observed at homotypic MDCK-MDCK borders (L, arrow). In keeping with this observation, a single MDCK cell does not express the protein because all its borders contact NRK cells (M). (Scale bars, 20 μm, except for D and E, which are 1 μm.)

Fig. 4.

Ouabain induces ciliogenesis through ERK1/2. Cilia stained with an antibody against acetylated α-tubulin in a control (A) and a monolayer treated with ouabain for 48 h (B). Results corresponding to experiments similar to A and B, but in the presence of PD (25 μM), an inhibitor of ERK1/2 (C and D). Statistical analysis of the percentage of ciliated cells under control, ouabain-stimulated, control with PD, and ouabain treatment conditions in the presence of PD (columns 1–4) (E); ***P < 0.001.

Fig. 5.

Claudin-2, besides of being a typical TJ protein, is also a ciliary protein that is affected by ouabain via ERK1/2 signaling. Immunofluorescence pictures of monolayers incubated with ouabain for 48 h to stain claudin-2 (A, D, and G, green) and -1 (J, green) and acetylated α-tubulin (B, E, H, and K, red). C, F, I, and L correspond to the superposition of the corresponding images. D, E, and F are lateral views of monolayer showing claudin-2 at the cilium. (G, H, and I) x/y projections showing that claudin-2 and acetylated α-tubulin colocalize along the whole cilium at the apical domain. Statistical analysis of the percentage of cells with ciliar claudin-2 observed in images of immunofluorescent cells costained with antibodies against this protein and acetylated α-tubulin, under control, ouabain-stimulated, control with PD, and ouabain treatment in the presence of PD (columns 1–4) (M); ***P < 0.001. Total cell content of acetylated α-tubulin (N, open squares) and claudin-2 (N, open circles). The correspondent effect of PD is shown in gray circles for claudin-2 and gray squares for acetylated α-tubulin. To gain information on the association between acetylated α-tubulin and claudin-2, we coprecipitated the first and blotted for the second (O). Lane 1 corresponds to total homogenate (Total Ext), and the lanes 2–5 depict immunoprecipitation with an antibody against acetylated α-tubulin (IP: AcTub), blotted with an antibody against the same acetylated α-tubulin (WB: AcTub) or claudin-2 (WB: claudin-2). Ouabain effect is shown in the lane 3 (ouabain). Lane 4 shows the effect of PD. Interestingly, this inhibitor increases by itself the association. The effect of ouabain does not depend on ERK1/2 as PD does not block the effect (lane 5). Claudin-2 (green) in a MDCK clone that does not express this protein in the cilia, identified as usual with fluorescent red label, in a x/y (P) and z/x image (Q). Ciliary claudin-2 is not required for the development of the cilium. (Magnification: A–F and J –L, 400×; G and H, 1,200×; P and Q, 600×.) (Scale bar in J, 10μm.)