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.

Chronic Ouabain Targets Pore-Forming Claudin-2 and Ameliorates Radiation-Induced Damage to the Rat Intestinal Tissue Barrier

“…The ROCK (Rho-associated coiled-coil forming kinase) signaling pathway is considered a key regulator of the cytoskeleton components [ 90 ]. In cultured epithelial (MDCK) cells, ouabain (10 nM) has been shown to induce transcript changes and activation of ROCK signaling [ 91 ], and this finding opens a new field of further research on claudin-2 regulation. Whether occludin, which is associated with the actin cytoskeleton and regulated by the ROCK signaling pathway [ 92 ], may be involved in the regulation of claudin-2 expression triggered by ouabain remains to be determined.…”

Sensational site: the sodium pump ouabain-binding site and its ligands

“…The ouabain-promoted gap junction formation is apparently mediated by Src and ERK1/2 because junction formation is blocked by selective blockers of these PKs ( 518 ). Recently, the Cereijido and Ponce group reported that ouabain-promoted formation of tight junctions also is mediated by the small GTPase, RhoA, and Rho-associated PK (ROCK) signaling, and is prevented by inhibitors of RhoA activation and ROCK ( 519 ). Whether RhoA/ROCK lie downstream of the Src/ERK1/2 signaling pathway, or are in an independent pathway, is unknown.…”