Ouabain Suppresses IL-6/STAT3 Signaling and Promotes Cytokine Secretion in Cultured Skeletal Muscle Cells

The cardiotonic steroids (CTS), such as ouabain and marinobufagenin, are thought to be adrenocortical hormones secreted during exercise and the stress response. The catalytic alpha-subunit of Na,K-ATPase (NKA) is a CTS receptor, whose largest pool is located in skeletal muscles, indicating that muscles are a major target for CTS. Skeletal muscles contribute to adaptations to exercise by secreting interleukin-6 (IL-6) and plethora of other cytokines, which exert paracrine and endocrine effects in muscles and non-muscle tissues. Here, we determined whether ouabain, a prototypical CTS, modulates IL-6 signaling and secretion in the cultured human skeletal muscle cells. Ouabain (2.5-50 nM) suppressed the abundance of STAT3, a key transcription factor downstream of the IL-6 receptor, as well as its basal and IL-6-stimulated phosphorylation. Conversely, ouabain (50 nM) increased the phosphorylation of ERK1/2, Akt, p70S6K, and S6 ribosomal protein, indicating activation of the ERK1/2 and the Akt-mTOR pathways. Proteasome inhibitor MG-132 blocked the ouabain-induced suppression of the total STAT3, but did not prevent the dephosphorylation of STAT3. Ouabain (50 nM) suppressed hypoxia-inducible factor-1 alpha (HIF-1 alpha), a modulator of STAT3 signaling, but gene silencing of HIF-1 alpha and/or its partner protein HIF-1 beta did not mimic effects of ouabain on the phosphorylation of STAT3. Ouabain (50 nM) failed to suppress the phosphorylation of STAT3 and HIF-1 alpha in rat L6 skeletal muscle cells, which express the ouabain-resistant alpha 1-subunit of NKA. We also found that ouabain (100 nM) promoted the secretion of IL-6, IL-8, GM-CSF, and TNF-alpha from the skeletal muscle cells of healthy subjects, and the secretion of GM-CSF from cells of subjects with the type 2 diabetes. Marinobufagenin (10 nM), another important CTS, did not alter the secretion of these cytokines. In conclusion, our study shows that ouabain suppresses the IL-6 signaling via STAT3, but promotes the secretion of IL-6 and other cytokines, which might represent a negative feedback in the IL-6/STAT3 pathway. Collectively, our results implicate a role for CTS and NKA in regulation of the IL-6 signaling and secretion in skeletal muscle.