AMP-activated protein kinase inhibits transforming growth factor-β-induced Smad3-dependent transcription and myofibroblast transdifferentiation

In wound healing, myofibroblast transdifferentiation (MFT) is a metaplastic change in phenotype producing profibrotic effector cells that secrete and remodel the extracellular matrix. Unlike pathways that induce MFT, the molecular mechanisms that negatively regulate MFT are poorly understood. Here, we report that AMP-activated protein kinase (AMPK) blocks MFT in response to transforming growth factor-beta (TGF beta). Pharmacological activation of AMPK inhibited TGF beta-induced secretion of extracellular matrix proteins collagen types I and IV and fibronectin. AMPK activation also prevented induction of the myofibroblast phenotype markers alpha-smooth muscle actin and the ED-A fibronectin splice variant. AMPK activators did not prevent MFT in cells transduced with an adenovirus expressing dominant negative, kinase-dead AMPK alpha 2. Moreover, AMPK activators did not inhibit MFT induction in AMPK(alpha 1,2)(-/-) fibroblasts, demonstrating a requirement for AMPK alpha expression. Adenoviral transduction of constitutively active AMPK(alpha 2) was sufficient to prevent TGF beta-induced collagen I, alpha-smooth muscle actin, and ED-A fibronectin. AMPK did not reduce TGF beta-stimulated Smad3 COOH-terminal phosphorylation and nuclear translocation, which are necessary for MFT. However, AMPK activation inhibited TGF beta-induced transcription driven by Smad3-binding cis-elements. Consistent with a role for AMPK in transcriptional regulation, nuclear translocation of AMPK alpha 2 correlated with the appearance of active AMPK alpha in the nucleus. Collectively, these results demonstrate that AMPK inhibits TGF beta-induced transcription downstream of Smad3 COOH-terminal phosphorylation and nuclear translocation. Furthermore, activation of AMPK is sufficient to negatively regulate MFT in vitro.