Hepatocyte growth factor inhibits TGF-β1-induced myofibroblast differentiation in tendon fibroblasts: role of AMPK signaling pathway

Abstract

The transforming growth factor-β1 (TGF-β1)-induced myofibroblastic differentiation in tendon fibroblasts was thought to be one of the most important features of scar fibrosis formation, which is associated with occurrence of re-rupture. Previously, we reported that hepatocyte growth factor (HGF) inhibited TGF-β1-induced myofibroblast differentiation and extracellular matrix deposition in the Achilles tendon of rats. Here, we investigated the potential molecular mechanisms underlying the inhibitory effect of HGF on TGF-β1-induced myofibroblast differentiation. We found that treatment with HGF (10, 20, and 40 ng/ml) increased phosphorylation of adenosine monophosphate kinase (AMPK) and acetyl-CoA carboxylase (ACC) in tendon fibroblasts. Pharmacological inhibition of the AMPK signaling pathway using compound C, a specific blocker of AMPK signaling, remarkably attenuated the inhibitory effect of HGF on TGF-β1-induced myofibroblastic differentiation in tendon fibroblasts. Moreover, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 subunit decreased the inhibitory effect of HGF on TGF-β1-induced myofibroblastic differentiation in tendon fibroblasts. Finally, overexpression of constitutively active AMPKα1, which led to constitutive activation of the AMPK signaling pathway in tendon fibroblasts, mimicked the inhibitory effect of HGF on the TGF-β1-induced myofibroblastic differentiation. Our study therefore suggests that HGF inhibits TGF-β1-induced myofibroblastic differentiation via an AMPK signaling pathway-dependent manner in tendon fibroblasts.

Fig. 1

Treatment with HGF activated AMPK signaling pathway in tendon fibroblasts. a Primary tendon fibroblasts were treated with three different concentrations of HGF (10, 20, and 40 ng/ml) and then the intracellular AMPK phosphorylation (p-AMPK to AMPK ratio) was determined by Western blotting analysis. Actin was used as a loading control. N = 6. *P < 0.05 versus CTRL (control). b The phosphorylation of ACC (p-ACC to ACC ratio), a critical downstream factor of AMPK, in HGF-treated tendon fibroblasts was also determined. N = 6. *P < 0.05 versus CTRL

Fig. 2

AMPK inhibitor compound C attenuated the inhibitory effect of HGF on the TGF-β1-induced myofibroblastic differentiation in tendon fibroblasts. a Typical cell morphology of tendon fibroblasts under stimulation by TGF-β1 and HGF. b–e Tendon fibroblasts were treated with TGF-β1 (10 ng/ml), TGF-β1 (10 ng/ml) + HGF (20 ng/ml), or TGF-β1 (10 ng/ml) + HGF (20 ng/ml) + compound C (20 μM) for 48 and 72 h. Then, the mRNA levels of α-SMA (b), Col I α1 (c), Col III (d), and fibronectin (e), four markers of myofibroblastic differentiation, were measure by real-time quantitative PCR analysis; β-actin was used as a housekeeping gene for reference. All data were normalized to β-actin expression (2^−ΔΔCt methods). N = 8. *P < 0.05

Fig. 3

Knocking down of AMPKα1 disrupted the inhibitory effect of HGF on the TGF-β1-induced myofibroblastic differentiation in tendon fibroblasts. Wild-type tendon fibroblasts, scramble-siRNA-transfected tendon fibroblasts, and AMPKα1-targeting siRNA-transfected tendon fibroblasts were treated with TGF-β1 (10 ng/ml) or TGF-β1 (10 ng/ml) + HGF (20 ng/ml) for 72 h. a Typical cell morphology of control, siRNA-scramble transfected, and siRNA-AMPKα1 transfected tendon fibroblasts under stimulation by TGF-β1 and HGF. b–e The mRNA levels of α-SMA (b), Col I α1 (b), Col III (d), and fibronectin (e), four markers of myofibroblastic differentiation, were measure by real-time quantitative PCR analysis; β-actin was used as a housekeeping gene for reference. All data were normalized to β-actin expression (2^−ΔΔCt methods). N = 8. *P < 0.05

Fig. 4

Overexpression of constitutively active AMPKα1 plasmid (caAMPKα1). a Schematic diagram of the wild-type and constitutive active AMPKα1 protein. b Tendon fibroblasts cells were transfected with either caAMPKα1 (2 or 4 μg/ml) or control vector. Whole cell lysates were examined for AMPK, caAMPKα1, and phosphorylation of caAMPKα1 by immunoblotting. Anti-AMPK antibody having an N-terminal epitope detected exogenous caAMPKα1 (approximately 35 kDa) and endogenous wild-type AMPK (approximately 62 kDa). Actin was used as an internal loading control

Fig. 5

Overexpression of constitutively active AMPKα1 plasmid (caAMPKα1) mimics the inhibitory effect of HGF on the TGF-β1-induced myofibroblastic differentiation in tendon fibroblasts. a Typical cell morphology of control, vector transfected, and ca-AMPKα1 transfected tendon fibroblasts under stimulation by TGF-β1. b–e The mRNA levels of α-SMA (b), Col I α1 (c), Col III (d), and fibronectin (e) were measure by real-time quantitative PCR analysis; β-actin was used as a housekeeping gene for reference. All data were normalized to β-actin expression (2^−ΔΔCt methods). N = 8. *P < 0.05