Long-term Ouabain treatment promotes ATP1A1-dependent IL-6 expression in a fetal human lens epithelial cell line (FHL124)

The cardiac glycoside ouabain (OUA) selectively inhibits Na + /K + ‐ATPase (NKA) governing the steady state maintenance of the electro‐chemical gradient of Na + and K + across the cell membranes. OUA, following binding to the NAK, activates several growth‐related signaling pathways: the EGFR/Src–Ras–ERK pathway and the PI3K1A–PDK–Akt pathway (Xie and Askari, Eur J Biochem 269(10):2434–2439, 2002). OUA also influences pro‐ and anti‐inflammation (Cavalcante‐Silva et al., Frontiers in Physiology, 8 :895, 2017) and cell proliferation depending on OUA concentration and incubation time, and cell type (Kulikov et al., Biochim et Biophys Acta 1768: 1691–1702, 2007 and Nguyen et al. JASN 18 (1): 46–57, 2007). Our group proposed and showed the direct interaction between NKA and BcL2 family proteins, which promote or inhibit apoptosis in human fetal lens epithelial cell line (FHL124) (Lauf et al., Cell Physiol Biochem 31:257–276, 2013; Lauf et al., Am J Physiol. Cell Physiol. 308, C51‐60, 2015). Here, we aimed to study the time dependence of OUA action at a concentration of 10 − 7 M on cell viability and transcriptional gene regulation of various pro‐apoptotic and pro‐inflammatory related genes in FHL124 cells. Cells were incubated with either 1–100 μM OUA in 7 μM DMSO or 7 μM DMSO alone (solvent control) relative to an untreated control for 1, 24 and 48 h. The OUA‐treated cells cultured for 24 h revealed a slightly decreased cell viability, however, 48 h incubation dramatically decreased the number of cells by 75–80% while DMSO alone reduced cell viability by 40–50% compared to untreated cells. Like all epithelial cells, FHL124 cells possess α1 ( atp1a1 ) isoform of NAK. The 5′‐untranslated region (5′UTR) of the atp1a1 gene contains two p53 and three NF‐kB (transcription factors nuclear factor‐kappa‐B) response elements. To test for the functional expression of the p53 and NFKB target genes, we selected mdm2, which negatively regulates the p53 gene and IL‐6 (interleukin‐6), which is induced by NF‐KB. Quantitative polymerase chain reaction (qPCR) method, using TaqMan probes, was employed to measure comparative gene expression of the atp1a1, mdm2, and IL‐6 genes . C ells OUA‐treated for 48 h promoted an increase of the IL‐6 expression by ~eight fold and the atp1a1 expression by two fold, but not the mdm2 expression. These findings suggest that OUA, in addition to utilizing existing signaling pathways mentioned above, may act either in complex with cytosolically internalized NKA (see Stricker et al., this FASEB meeting 2018) or derived therefrom through to be determined gene regulation factors proximal to the NF‐kB response elements. Further studies are aimed to confirm NKA‐dependent IL‐6 expression after long‐treatment with OUA. Support or Funding Information Supported by the P&T Graduate program and WSU Foundation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .