Downregulation of TGF-β Receptor-2 Expression and Signaling through Inhibition of Na/K-ATPase

Transforming growth factor-beta (TGF-β) is a multi-functional cytokine implicated in the control of cell growth and differentiation. TGF-β signals through a complex of TGF-β receptors 1 and 2 (TGFβR1 and TGFβR2) that phosphorylate and activate Smad2/3 transcription factors driving transcription of the Smad-target genes. The Na⁺/K⁺-ATPase is an integral plasma membrane protein critical for maintaining the electro-chemical gradient of Na⁺ and K⁺ in the cell. We found that inhibition of the Na⁺/K⁺ ATPase by ouabain results in a dramatic decrease in the expression of TGFβR2 in human lung fibrobalsts (HLF) at the mRNA and protein levels. This was accompanied by inhibition of TGF-β-induced Smad phosphorylation and the expression of TGF-β target genes, such as fibronectin and smooth muscle alpha-actin. Inhibition of Na⁺/K⁺ ATPase by an alternative approach (removal of extracellular potassium) had a similar effect in HLF. Finally, treatment of lung alveolar epithelial cells (A549) with ouabain also resulted in the downregulation of TGFβR2, the inhibition of TGF-β-induced Smad phosphorylation and of the expression of mesenchymal markers, vimentin and fibronectin. Together, these data demonstrate a critical role of Na⁺/K⁺-ATPase in the control of TGFβR2 expression, TGF-β signaling and cell responses to TGF-β.