Ouabain Induces Transcript Changes and Activation of RhoA/ROCK Signaling in Cultured Epithelial Cells (MDCK)

Abstract

Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na⁺/K⁺-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.

Keywords: MDCK; MYO9A; Na+/K+-ATPase; cell–cell contacts; epithelia; gene expression; ouabain.

Figure 1

Differentially regulated transcripts by the effect of ouabain: (A) MA plot; (B) volcano plot; (C,D) heatmap indicating up and downregulated genes grouped according to their expression levels. Duplicate samples are shown. Transcripts in ouabain-treated MDCK cells are shown in left blue; control, untreated cells are indicated with red. Down-regulation is indicated with violet color, while up-regulation is shown in yellow according to color key present in (D).

Figure 2

Validation of ouabain-induced differential expression of mRNA by RT-qPCR. The four pairs of bars show the average and standard error of the relative expression of mRNA in RT-qPCR assays of four different genes, which, according to the seq mRNA assay, had shown more variation. The grey circles denote the individual values obtained from 5 independent trials. The (*) symbol above the slash denotes a statistically significant difference (p < 0.05). Welch’s t-test, equal variances not assumed.

Figure 3

Functional protein association network (interactome) diagram of Myo9a. The interaction analysis of myo9a (green arrow) shows that there is evidence of interaction with recognized members of the Rho/ROCK signaling pathway, among which RhoA (pink arrow) stands out, and that it also includes upstream components such as Arhgef11 (blue arrow), a guanosine exchange factor. Lines of different colors indicate the type of evidence of the interaction. The green and red colors of the components indicate two levels of clustering. The permalink to access the diagram is: https://version-11-5.string-db.org/cgi/network?networkId=b8BzUXMHHAIQ, accessed on 11 May 2023.

Figure 4

Ouabain induces a transient reduction in the amount of MYOA’s mRNA. Circles represent the averages (±S.E.) of the values estimated by RT-qPCR assays of the (standardized) amount of mRNA of MYO9A in the different times tested and are the result of 5 independent trials. The red circles correspond to the values obtained from ouabain treated cells, and the white circles are from untreated cells (control). The asterisks denote a statistically significant difference (*: p < 0.05; ***: p < 0.001). Student, t-test, equal variances assumed.

Figure 5

Ouabain promotes activation of RhoA. Bars are proportional to the average value (±S.E.) of the amount of GTP-loaded RhoA obtained by luminometry of 9 independent tests in which cells in mature monolayers were either treated with ouabain 10 nmol/L for 1 h (red bar) or not (white bar). (*) denotes a statistically significant difference (p < 0.05). The grey circles denote the individual values obtained in each case. Student, t-test, equal variances assumed.

Figure 6

Inhibition of RhoA and ROCK activation abolishes the enhancement of TJS induced by ouabain. (A) Comparison of the time course of TER as a function of treatment time with none (white circles) ouabain (red circles), Y27 (purple circles), C3 (blue circles), Y27 + ouabain (orange circles), or C3 + ouabain (purple circles). (B) Comparison and statistical significance of abolishment of the enhancing effect on TJS induced by ouabain caused by C3 and Y27 after 72 h of treatment. TER values are the average (±S.E.) of 15 replications, 5 from each of three independent trials. The grey circles denote the individual values obtained on each trial. (**) denotes a statistically significant difference with p < 0.005. Welch’s t-test, equal variances not assumed.

Figure 7

Inhibition of RhoA and ROCK activation abolishes the enhancement of GJIC induced by ouabain. (A) Phase contrast images of representative dye transfer trials of each of the different treatments to probe the effect of C3 and Y27 with or without ouabain. The green color is from staining with lucifer yellow. (B) Box plot shows the abolishment of the enhancing effect of GJIC induced by ouabain, caused by Rho and ROCK inhibitors C3 and Y27. Continuous and dotted lines inside boxes denote mean and median of number of cells stained. The number above each bar indicates the number of trials. The combination treatments are indicated in the below boxes. (***) denotes a statistically significant difference (p < 0.0001). Multiple comparisons versus control group (Holm–Sidak method).

Figure 8

Schematic representation of the main findings of this work. Short-term treatment with ouabain 10 nmol/L causes a decreased content of MYO9A mRNA. The fact that Y27 and C3 inhibited ouabain-induced enhancement of GJIC and TJS indicates participation of Rho-A and ROCK in the signaling cascade activated.