Abstract 578: Na/K-ATPase Forms a Signaling Complex with CD36 and Toll-like Receptor 4 to Mediate NF-kappaB Activation in Macrophages

Abstract

Cardiotonic steroids such as ouabain, digoxin, and marinobufagenin are known ligands for the plasma membrane receptor, Na/K-ATPase (NKA). These ligands stimulate complex formation of NKA with other membrane and cytosolic proteins leading to cellular events such as activation of kinase cascades and gene transcription independent of the ion transporting function. Endogenous cardiotonic steroids have been detected within human circulation and their levels are elevated in patients with diseases associated with chronic systemic inflammation. However, the role of cardiotonic steroids and NKA-mediated signaling in inflammation has not been well studied. We now report that ouabain activates the NF-κB pathway in human monocyte-derived macrophages, leading to pro-inflammatory responses. Using Western blot and densitometry analysis, we found that physiological concentrations of ouabain stimulated IκBα degradation (n=3). Using quantitative RT-PCR (n=4), we found that ouabain increased mRNA levels of pro-inflammatory genes MCP-1 (3.2±1.1 fold), TNF-α (59.7±35.6 fold), CXCL-10 (2.8±1.6 fold), ICAM1 (2.9±0.5 fold), and PTGS2 (3.1±0.1 fold). Consistent with the increase in mRNA level, MCP-1 protein levels in post-culture media assessed by ELISA were elevated by 1.4±0.1 fold (n=4). Mechanistically, when macrophages were incubated with ouabain for 20 min and then subjected to IP, we found that antibodies to NKA co-precipitated more TLR4 (2.2±0.8 fold) compared to unstimulated cells. Additionally, antibodies to CD36 co-precipitated more NKA (1.8±0.6 fold) and more TLR4 (2.2±0.5 fold) (n=3). Blockade of TLR4 signaling using a specific inhibitor, CLI-095, abolished ouabain-induced MCP-1 production as assessed by ELISA (n=4). Next, we used siRNA to specifically knock down either NKA α1, CD36, TLR4 or MyD88 (the adaptor protein downstream of TLR4) in THP-1 cells stably transfected with a NF-κB reporter construct. After siRNA transfection for 48h, cells were treated with ouabain for additional 24h. Knockdown of any one of these proteins fully suppressed ouabain-induced NF-κB activation by NF-κB reporter assay (n=4). In conclusion, ouabain activates NF-κB through an NKA/CD36/TLR4 complex leading to pro-inflammatory responses in human macrophages.