Cardiotonic steroids such as ouabain, digoxin, and marinobufagenin are known ligands for the plasma membrane receptor Na/K-ATPase (NKA). These ligands are able to stimulate interaction of the NKA with other membrane and cytosolic proteins leading to cellular events such as activation of kinase cascades and gene transcription. Endogenous cardiotonic steroids have been detected in human circulation and interestingly their levels were elevated in human patients with hypertension, congestive heart failure and diabetes, all of which were associated with chronic systemic inflammation. However, the role of cardiotonic steroids in systemic inflammation and immunity has not been well studied. We previously discovered that ouabain stimulated macrophages to produce pro-inflammatory cytokines, many of which are known targets of the transcription factor, NF-κB. Therefore, we hypothesized that ouabain activates NF-κB pathway leading to pro-inflammatory cytokine production in macrophages. Using Western blot and densitometry analysis, we showed that physiological concentrations of ouabain promoted IκBα degradation (as low as 5 nM ouabain decreased IκBα level by 66.8%±7.4%, n=4). This was accompanied by NF-κB translocation from cytoplasm to the nuclei as shown by immunocytochemistry (% of nuclei NF-κB signals increased from 30.5%±2.3% in control to 62.2%±2.6% in ouabain-treated macrophages, n>25). Moreover, via quantitative RT-PCR (n=3), we found that ouabain increased mRNA levels of pro-inflammatory cytokines such as MCP-1 (3.2±1.1 fold), TNF-α (59.7±35.6 fold), and CXCL-10 (2.8±1.6 fold), all of which are known NF-κB targets. Consistent with the increase in mRNA level, we found that MCP-1 protein levels were elevated in both cell lysates (1.8±0.3 fold) and culture media (1.4±0.1 fold; n=4). Addition of an NF-κB inhibitor blocked MCP-1 production induced by ouabain (n=4). Mechanistically, ouabain stimulated interaction between NKA and TLR4 as shown by Co-Immunoprecipitation (n=3). Blockade of TLR4 signaling using a specific peptide inhibitor, CLI-095, abolished the ouabain effect on NF-κB activation (n=3). We conclude that ouabain activates NF-κB through NKA/TLR4 complex leading to pro-inflammatory cytokine production by macrophages.
COVID-19 Pathology on Various Organs and Regenerative Medicine and Stem Cell-Based Interventions
