Department of Physiology Biophysics and Neurosciences, Center for Research and Advanced Studies, Cinvestav-Ipn, CDMX 07360, Mexico.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Int J Mol Sci. 2019 Apr 29;20(9):2111. doi: 10.3390/ijms20092111.
Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na,K-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine β subunit become adhesive, and those homotypic interactions amongst β subunits of the Na,K-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na,K-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between β subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the β subunit of the Na,K-ATPase (CHO β), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO β cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the β subunit of the Na,K-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO β cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO β cells. Collectively, our findings suggest that the β subunit adhesion is modulated by the expression levels of the Na,K-ATPase at the plasma membrane, which is regulated by ouabain.
黏附是上皮细胞的一个重要特性，它可以形成屏障，使病原体和环境中的有害物质无法进入。上皮细胞通过侧膜上的细胞间连接相互附着，包括紧密连接和黏附连接，以及 Na,K-ATP 酶。我们的研究小组已经表明，转染了犬β亚基的不附着的中国仓鼠卵巢（CHO）细胞变得具有黏附性，并且 Na,K-ATP 酶β亚基的同种型相互作用发生在相邻的上皮细胞之间。哇巴因是一种强心甾体，与 Na,K-ATP 酶的α亚基结合，抑制泵的活性，并在上皮细胞浓度高于 300 nM 时诱导其分离。在非抑制性纳摩尔浓度下，哇巴因通过激活与α亚基相关的信号通路，诱导细胞黏附分子的表达，从而影响上皮细胞的黏附特性。在这项研究中，我们研究了哇巴因是否也会影响β亚基之间的黏附。我们使用稳定表达 Na,K-ATP 酶β亚基的 CHO 成纤维细胞（CHOβ），并研究了哇巴因对细胞黏附的影响。聚集实验表明，哇巴因增加了 CHOβ细胞之间的黏附。免疫荧光和生物素化实验表明，哇巴因（50 nM）增加了细胞膜上 Na,K-ATP 酶β亚基的表达。我们还研究了哇巴因对 CHOβ细胞中信号通路激活的影响，以及它们对细胞黏附的后续影响。我们发现哇巴因激活了 cSrc，因此，它可能调节 CHOβ细胞的黏附特性。总之，我们的研究结果表明，β亚基的黏附受质膜上 Na,K-ATP 酶表达水平的调节，而这一调节受哇巴因的影响。
