European Journal of Cell Biology 92 (2013) 363–373 Contents lists available at ScienceDirect European Journal of Cell Biology jo ur nal homepage: www.elsevier.co m/locate/ejcb Effective myofibroblast dedifferentiation by concomitant inhibition of TGF- signaling and perturbation of MAPK signaling Jan Kosla, Marta Dvorakova, Michal Dvorak, Vladimir Cermak ∗ Laboratory of Molecular Virology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, CZ-14220 Prague 4, Czech Republic a r t i c l e i n f o Article history: Received 9 April 2013 Received in revised form 30 October 2013 Accepted 30 October 2013 Keywords: PDGFB Ha-Ras(G12V) EGR4 TGF- Myofibroblast FOXG1 Microarrays a b s t r a c t Fibrotic diseases are a group of pathologies with high incidence and mortality. Despite extensive research efforts, effective therapies are still not available. Understanding the molecular mechanisms driving the onset, progression and possible resolution of fibrosis is a prerequisite to the development of successful therapies. The central role of the TGF- pathway and myofibroblasts in the pathogenesis of fibrosis is now generally accepted. The possible mechanisms of myofibroblast elimination or dedifferentiation, on the other hand, are still almost uncharted territory. Here we show that sustained expression of some com- ponents of MAPK signaling pathway (PDGFB, Ha-Ras(G12V) or the transcription factor EGR4) in primary chicken embryo dermal myofibroblasts results in a loss of autocrine TGF- signaling and suppression of the myofibroblastic phenotype, characterized by the loss of alpha smooth muscle actin fibers and a sub- stantial reduction in the production of extracellular matrix. Detailed analysis of the possible molecular mechanisms employed by EGR4 revealed FOXG1, BAMBI, NAB1, NAB2 and DUSP5 genes forming an EGR4 regulated network counteracting autocrine TGF- signaling. We have also found that a combination of chemical inhibition of TGF- signaling and perturbation of MAPK signaling with phorbol ester mimics the anti-fibrotic effects of PDGFB, Ha-Ras(G12V) and EGR4. © 2013 Elsevier GmbH. All rights reserved. Introduction Fibrosis is a pathological condition characterized by exces- sive and progressive accumulation of extracellular matrix (ECM) material and permanent contraction of the resulting fibrotic tissue (Rosenbloom et al., 2010). Both activities are performed by a specialized cell type – the myofibroblast (Phan, 2008). The myofi- broblastic phenotype is a differentiation status of a mesenchymal cell that can come from various possible sources, including local fibroblasts, circulating fibrocytes, mesenchymal stem cells from the bone marrow, transdifferentiated smooth muscle cells, and even local epithelial or endothelial cells undergoing an epithelial–mesenchymal transition (Hinz et al., 2007). Progressive fibrosis leads to increasing irreversible disruption of the tissue structure of the affected organ, often with fatal consequences. The spectrum of possible sites of fibrosis includes almost all vital organs and the resulting diseases are known as idiopathic pulmonary fibrosis (lungs), liver fibrosis or cirrhosis (liver), systemic fibrosis or scleroderma (skin and internal organs), cardiac fibrosis (heart) and renal fibrosis (kidney), just to name a few examples. Fibrosis is ∗ Corresponding author. Tel.: +420 296443391; fax: +420 241063586. E-mail addresses: kosla@img.cas.cz (J. Kosla), marta@img.cas.cz (M. Dvorakova), mdvorak@img.cas.cz (M. Dvorak), cermak@img.cas.cz (V. Cermak). often a consequence of local chronic inflammation (Lopez-Novoa and Nieto, 2009; Pohlers et al., 2009). The hallmark of all fibrotic lesions is constitutive TGF- signaling, which is responsible for myofibroblastic differentiation and directly stimulates the production of ECM components by myofibroblasts (Flanders, 2004; Gharaee-Kermani et al., 2009; Pohlers et al., 2009). In the early phase of fibrosis, the TGF- cytokine is likely produced by inflammatory cells, chiefly macrophages, with TGF-1 being the major isoform (Demirci et al., 1996). Later, a sustained autocrine loop is established in myofibroblasts, producing TGF-1 and especially TGF-3 (McKaig et al., 1999). In addition to sustained TGF- signaling, the myofibroblastic phenotype also requires the presence of myocardin family transcriptional coactivators that act in concert with serum response factor (SRF) to activate the expression of actin contractility-related genes like alpha smooth muscle actin (ACTA2), transgelin (TAGLN), basic calponin (CNN1) and many others (Gineitis and Treisman, 2001; Long et al., 2008; Miano et al., 2007; Yoshida et al., 2003). It is known that treatment of myofibroblasts with the growth factors FGF2 or PDGF-BB results in suppression of myofibroblastic features and that MAPK signaling plays a crucial role in this effect (Hecker et al., 2011; Ishiguro et al., 2009; Spyrou and Naylor, 2002). EGR4, a member of the EGR family of transcription factors, was first identified in 1991 as a zinc finger protein induced by mitogenic stimulation in resting fibroblasts and T-cells (Muller et al., 1991). 0171-9335/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejcb.2013.10.013