Regulation of myofibroblast differentiation by cardiac glycosides

Abstract

Myofibroblast differentiation is a key process in pathogenesis of fibrotic diseases. Cardiac glycosides (ouabain, digoxin) inhibit Na(+)-K(+)-ATPase, resulting in increased intracellular [Na(+)]-to-[K(+)] ratio in cells. Microarray analysis suggested that increased intracellular [Na(+)]/[K(+)] 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(+)/Ca(2+) exchanger, KB-R4943. Ouabain inhibited transforming growth factor-β (TGF-β)-induced Rho activation, stress fiber formation, serum response factor activation, and the expression of smooth muscle α-actin, collagen-1, and fibronectin. These effects were recapitulated by an increase in intracellular [Na(+)]/[K(+)] ratio through the treatment of cells with K(+)-free medium or with digoxin. Although inhibition of COX-2 or of the Na(+)/Ca(2+) exchanger blocked ouabain-induced PKA activation, this failed to reverse the inhibition of TGF-β-induced Rho activation or myofibroblast differentiation by ouabain. Together, these data demonstrate that ouabain, through the increase in intracellular [Na(+)]/[K(+)] 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-β. However, COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-β by ouabain, suggesting that additional mechanisms must exist.

Keywords: cyclooxygenase-2; digoxin; intracellular sodium-to-potassium ion ratio; ouabain.

Fig. 1.

Ouabain-induced cyclooxygenase (COX)-2 expression and protein kinase A (PKA) activation in human lung fibroblasts (HLF). Serum-starved HLFs were treated with 100 nM ouabain with or without 1 μM NS-398 for the times indicated. Cells were analyzed by real-time qPCR for COX-2 mRNA levels (A) or by Western blotting for COX-2 expression and vasodilator-stimulated phosphoprotein (VASP) shift (A–C). C: HLFs were transfected with a scramble or COX-2 small-interfering RNAs (siRNAs), serum-starved for 24 h, and treated with 100 nM ouabain for 24 h. Shown are the Western blots for COX-2 and VASP.

Fig. 2.

Effect of ouabain, nicardipine, and KB-R7943 on ⁴⁵Ca²⁺ influx and on COX-2 expression and VASP shift. A: HLFs were preincubated for 24 h in the presence of 100 nM ouabain, 1 mM nicardipine, or 3 mM KB-R7943, and the rate of ⁴⁵Ca²⁺ influx was measured as described in experimental procedures. Means ± SE from experiments performed in triplicate are shown. B: HLFs were treated with 100 nM ouabain, 1 mM nicardipine, or 3 mM KB-R7943 for 24 h, and cell extracts were examined by Western blotting for COX-2 expression and VASP shift.

Fig. 3.

Ouabain distorts actin stress fibers without affecting HLF viability. A: serum-starved HLFs were treated with or without 100 nM ouabain for times indicated. Shown are the phase-contrast microscopy images of the cells. B: effect of 100 nM ouabain or 1 mM H₂O₂ on HLF viability as assessed by lactate dehydrogenase (LDH) release. C: phalloidin staining of HLFs treated with or without 100 nM ouabain for 48 h. Percentile intensity of phalloidin is indicated.

Fig. 4.

Ouabain and digoxin inhibit myofibroblast differentiation. Serum-starved HLFs were treated with or without 1 ng/ml transforming growth factor-β (TGF-β) in the presence or absence of 30 or 100 nM ouabain (A) or 100 nM digoxin (B) for 48 h. Cell extracts were assessed by Western blotting for COX-2 expression, VASP shift, and myofibroblast differentiation markers as indicated.

Fig. 5.

Ouabain inhibits TGF-β-induced stress fiber formation and serum response factor (SRF) activation. A: serum-starved HLF cells were treated with 1 ng/ml TGF-β in the presence or absence of ouabain for 48 h. Cell extracts were either lysed (total) or were processed for isolation of stress fibers, followed by Western blotting with desired antibodies. B: effect of increasing concentrations of ouabain (100 nM, 48 h) on SRF-luciferase reporter activity.

Fig. 6.

Effect of K⁺-free medium on intracellular Na⁺ content and on COX-2 expression, VASP phosphorylation, and myofibroblast differentiation. HLFs were treated with a control medium ([K⁺]_o = 5 mM), K⁺-free medium, or with 100 nM ouabain in a control medium for 48 h, with or without 1 ng/ml TGF-β for 48 h as indicated. A: intracellular Na⁺ concentration was measured as described in experimental procedures. B: cell extracts were analyzed by Western blotting with desired antibodies as indicated.

Fig. 7.

Dose-dependent actions of ouabain on intracellular Na⁺ and K⁺ content, COX-2 expression, and expression of myofibroblast differentiation markers. A: HLFs were treated with ouabain for 48 h and assessed for intracellular Na⁺ and K⁺ content. Means ± SE from experiments performed in triplicate are shown. B: HLFs were treated with 1 ng/ml TGF-β in the presence of increasing doses of ouabain for 48 h. Cell extracts were examined by Western blotting for COX-2 expression and myofibroblast differentiation markers. Shown are representative images from three independent experiments. C: densitometry of ECL for collagen-1, SM α-actin, and fibronectin Western blots (mean ± SD from 3 independent experiments). *P < 0.05 and **P < 0.01.

Fig. 8.

Inhibition of COX-2 or of Na⁺/Ca²⁺ exchanger does not abolish the inhibitory action of ouabain on TGF-β-induced myofibroblast differentiation. HLFs were treated with 1 μM NS-398 (A), pretransfected with a control or COX-2 siRNA (B), or treated with 3 μM KB-R7943 (C) followed by incubation with 100 nM ouabain or 1 ng/ml TGF-β for 48 h as indicated. Cells were lysed, and the cell extracts were analyzed by Western blotting with the desired antibodies as indicated.