Regulation of myofibroblast differentiation by cardiac glycosides

Abstract

] ratio may promote the expression of cyclooxygenase-2 (COX-2), a critical enzyme in the synthesis of prostaglandins. Given antifibrotic effects of prostaglandins through activation of protein kinase A (PKA), we examined if cardiac glycosides stimulate COX-2 expression in human lung fibroblasts and how they affect myofibroblast differentiation. Ouabain stimulated a profound COX-2 expression and a sustained PKA activation, which was blocked by COX-2 inhibitor or by COX-2 knockdown. Ouabain-induced COX-2 expression and PKA activation were abolished by the inhibitor of the Na ϩ /Ca 2ϩ exchanger, KB-R4943. Ouabain inhibited transforming growth factor-␤ (TGF-␤)-induced Rho activation, stress fiber formation, serum response factor activation, and the expression of smooth muscle ␣-actin, collagen-1, and fibronectin. These effects were recapitulated by an increase in intracellular [Na ϩ ]/[K ϩ ] ratio through the treatment of cells with K ϩ -free medium or with digoxin. Although inhibition of COX-2 or of the Na ϩ /Ca 2ϩ exchanger blocked ouabain-induced PKA activation, this failed to reverse the inhibition of TGF-␤-induced Rho activation or myofibroblast differentiation by ouabain. Together, these data demonstrate that ouabain, through the increase in intracellular [Na ϩ ]/[K ϩ ] ratio, drives the induction of COX-2 expression and PKA activation, which is accompanied by a decreased Rho activation and myofibroblast differentiation in response to TGF-␤. However, COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-␤ by ouabain, suggesting that additional mechanisms must exist. ouabain; digoxin; intracellular sodium-to-potassium ion ratio; cyclooxygenase-2 IDIOPATHIC PULMONARY FIBROSIS (IPF) is a progressive fatal disease characterized by parenchymal fibrosis and structural distortion of the lungs. Age-adjusted mortality due to pulmonary fibrosis is increasing, and it poses a vexing clinical challenge given the lack of efficacious therapy. IPF is thought to be a disorder of abnormal wound healing (54), in which the initial trigger to the fibrotic response is injury to the alveolar epithelial cell, followed by an exuberant nonresolving woundhealing response (50). Injury of alveolar epithelial cells is thought to result in the elaboration of a fibrinous provisional matrix and activation of several proinflammatory, procoagulant, and profibrotic mediators, of which transforming growth factor-␤1 (TGF-␤1) is the most established (47). Fibroblasts, under stimulation of TGF-␤1, respond by altering their gene expression profile with de novo expression of cytoskeletal and contractile proteins normally found within smooth muscle (SM) cells, modified focal adhesion complexes, and components of the extracellular matrix (18,27). These SM-like fibroblasts are called myofibroblasts and display a phenotype that is in an intermediate state between fibroblasts and SM cells (10,19). Several cytoskeletal and SM proteins are expressed in myofibroblasts, including SM ␣-actin, the most established m...

Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation

“…To our knowledge, there are no reports specifically demonstrating the ability of cardiac glycosides to prevent CAF differentiation. Interestingly, recently published data indicate an anti-fibrotic effect of ouabain through upregulation of COX-2 in lung fibroblasts, likely sharing a mechanism with our findings at the level of SMAD activity [55]. Given the abundance of published reports demonstrating that CAFs are able to facilitate the invasiveness of otherwise quiescent tumor cells, work toward the development of CAF-targeting therapeutic agents is warranted [8].…”

Cardiotonic Steroids and the Sodium Trade Balance: New Insights into Trade-Off Mechanisms Mediated by the Na+/K+-ATPase

“…A number of important studies have shown the role of NKA inhibition and elevation of the [Na + ]i/[K + ]i ratio in the activation of Src-EGFR-MAPK- and Akt-mediated signaling pathways triggered by CTS, and readers are referred to a recent review which more thoroughly addresses this topic [ 83 ]. The importance of this concept is highlighted by recent data showing that NKA inhibition by CTS triggers TGFβ-induced fibrosis in cultured human lung fibroblasts via [Na + ]i/[K + ]i-mediated signaling which results in augmented expression of COX-2 [ 84 ] and downregulation of TGFβ2R [ 85 ]. Cumulatively, these studies have broadened the understanding of the role of CTS signaling through the NKA in human disease.…”

Na+i,K+i-Dependent and -Independent Signaling Triggered by Cardiotonic Steroids: Facts and Artifacts

“…Elevation of [Ca 2+ ] i , in turn, leads to its interaction with calmodulin and other Ca 2+ i sensors and activation of Ca 2+ response elements detected within promoters in hundreds of genes (for a comprehensive review, see [ 135 , 136 , 137 ]). Thus, for example, sharp elevation of cyclooxygenase 2 (COX-2) mRNA content seen in ouabain-treated human lung fibroblasts was completely abolished by Na + /Ca 2+ exchanger inhibitor KB-R4943 [ 138 ].…”

“…Importantly, the action of ouabain was absent in p85-KO mice, thus suggesting that side-by-side with the positive inotropic effect, the treatment with Digitalis prevents pathological cardiac hypertrophy and heart failure through activation of PI3KIA [ 167 ]. Our recent study demonstrated that anti-fibrotic action of cardiac glycosides documented by the suppression of myofibroblast differentiation in TGF-β-treated human lung fibroblasts is mediated by dissipation of transmembrane gradients of monovalent cations [ 138 , 168 ]. To the best of our knowledge, the role of Na + ,K + -ATPase inhibition and the elevated [Na + ] i /K + ] i ratio in PI3K/Akt-mediated signaling triggered by CTS has not been explored yet.…”