The Ski–Zeb2–Meox2 pathway provides a novel mechanism for regulation of the cardiac myofibroblast phenotype

Ryan H. Cunnington 1 2 Josette M. Northcott 0 1 Saeid Ghavami 1 2 Krista L. Filomeno 1 2 Fahmida Jahan 0 1 Morvarid S. Kavosh 1 2 Jared J. L. Davies 1 2 Jeffrey T. Wigle 0 1 Ian M. C. Dixon () 1 2 0 Department of Biochemistry and Medical Genetics, University of Manitoba , Winnipeg, MB R3E 0J9 , Canada 1 Institute of Cardiovascular Sciences, University of Manitoba , Winnipeg, MB R3E 0J9 , Canada 2 Department of Physiology, University of Manitoba , Winnipeg, MB R3E 0J9 , Canada Cardiac fibrosis is linked to fibroblast-to-myofibroblast phenoconversion and proliferation but the mechanisms underlying this are poorly understood. Ski is a negative regulator of TGF-b-Smad signaling in myofibroblasts, and might redirect the myofibroblast phenotype back to fibroblasts. Meox2 could alter TGF-b-mediated cellular processes and is repressed by Zeb2. Here, we investigated whether Ski diminishes the myofibroblast phenotype by de-repressing Meox2 expression and function through repression of Zeb2 expression. We show that expression of Meox1 and Meox2 mRNA and Meox2 protein is reduced during phenoconversion of fibroblasts to myofibroblasts. Overexpression of Meox2 shifts the myofibroblasts into fibroblasts, whereas the Meox2 DNA-binding mutant has no effect on myofibroblast phenotype. Overexpression of Ski partially restores Meox2 mRNA expression levels to those in cardiac fibroblasts. Expression of Zeb2 increased during phenoconversion and Ski overexpression reduces Zeb2 expression in first-passage myofibroblasts. Furthermore, expression of Meox2 is decreased in scar following myocardial infarction, whereas Zeb2 protein expression increases in the infarct scar. Thus Ski modulates the cardiac myofibroblast phenotype and function through suppression of Zeb2 by upregulating the expression of Meox2. This cascade might regulate cardiac myofibroblast phenotype and presents therapeutic options for treatment of cardiac fibrosis. - INTRODUCTION Fibroblast-to-myofibroblast phenoconversion in the heart is a crucial event in the onset of many cardiovascular diseases. Cardiac fibroblasts are an understudied cell, despite the fact that they constitute the majority of cells in the heart (Weber and Brilla, 1991). These relatively quiescent cells reside in the cardiac interstitium and synthesize cardiac fibrillar collagen types (e.g. types I and III), as well as the majority of other extracellular matrix proteins. Cardiac stress and injury both induce differentiation of fibroblasts into hypersecretory and contractile myofibroblasts, which can persist in the myocardium for months (and even years) after the initial pathological insult (Willems et al., 1994). The myofibroblast is associated with fibrillar collagen deposition, leading to reduced cardiac efficiency and, ultimately, decompensated heart failure. Initially, increased collagen deposition is beneficial, but when left to persist for years, it can transform healthy myocardium with normal extracellular matrix complement into overtly fibrosed tissue with abnormal expansion of the interstitium (Freed et al., 2005). Fibroblast-to-myofibroblast phenoconversion is marked by increased expression of a-smooth muscle actin (a-SMA) (Darby et al., 1990), extra domain A (ED-A) fibronectin (Serini et al., 1998), and non-muscle myosin heavy chain b (SMemb) (Frangogiannis et al., 2000). Although not well understood, this process is typically thought of as being a one-way pathway. However, Hinz and colleagues have provided data that both forward and reverse phenoconversion are possible in these cells, depending upon the compressibility of the underlying substrate (Hinz et al., 2001). Still, the molecular mechanisms for fibroblastmyofibroblast inter-conversion have yet to be fully elucidated. The canonical TGF-b signaling cascade has been implicated as an important inducer of phenoconversion. High levels of this cytokine are associated with cardiac fibrosis (Brooks and Conrad, 2000). The diverse functions of TGF-b necessitate the tight control of membrane-to-nucleus signal transduction; in particular, levels of the endogenous inhibitors Smad7 and Ski. The protooncoprotein Ski has a complex set of functions and is linked to skeletal muscle hypertrophy (Sutrave et al., 1990a; Sutrave et al., 1990b), as well as developmental defects in the neural tube and cranial mesenchyme (Berk et al., 1997). Ski binds to R-Smad proteins (Sun et al., 1999; Suzuki et al., 2004; Ueki and Hayman, 2003; Xu et al., 2000) and might inhibit TGF-b-mediated effects through both nuclear and cytosolic mechanisms (Akiyoshi et al., 1999; Ferrand et al., 2010; Nagata et al., 2006; Suzuki et al., 2004). We have previously demonstrated the anti-fibrotic and anti-contractile effects of overexpression of Ski in TGF-b1stimulated primary cardiac myofibroblasts, and have shown these inhibitory effects to be orchestrated at the nuclear level (Cunnington et al., 2011). Mesenchyme homeobox 2 (Meox2, also known as growth arrest specific homeobox protein, Gax) and Meox1 comprise the Meox family of homeodomain proteins. Meox1 and Meox2 proteins share 95% sequence identity within the homeodomain region, but are otherwise highly divergent (Douville et al., 2011). Both Meox1 and Meox2 are required for proper bone and muscle formation in developing mouse embryos (Mankoo et al., 1999; Mankoo et al., 2003). Meox2 expression is decreased by mitogen stimulation of vascular smooth muscle cells (Gorski et al., 1993) and in mechanically damaged arteries (Weir et al., 1995), indicating that Meox2 expression is sensitive to a range of stimuli. Meox2 could enhance TGF-b-mediated inhibition of cell proliferation (Valcourt et al., 2007) or, conversely, might block TGF-b-induced epithelial-to-mesenchymal transition (EMT) (Valcourt et al., 2007). Meox2 expression is repressed by the Zeb2 protein [also called Smad-interacting protein 1 (Sip1 or Zfhx1b)], a zinc-finger E-box binding protein (Chen et al., 2010). Zeb2 expression itself has been shown to be positively regulated by TGF-b signaling during EMT (Comijn et al., 2001). In this study, we provide data to support a novel mechanism through which Ski regulates the cardiac fibroblast phenotype. We show for the first time that both Meox1 and Meox2 are downregulated during fibroblast-to-myofibroblast phenoconversion, and that overexpression of Ski can rescue Meox2 expression. Moreover, our data establish a link between increased Meox2 expression and a reduction of the myofibroblast phenotype. Finally, we identify a putative signaling cascade that might modulate fibroblast-to-myofibroblast phenoconversion through differential expression of Ski, Zeb2 and Meox2 proteins. RESULTS Meox1 and Meox2 are downregulated during phenoconversion Marker proteins frequently used to monitor fibroblast-tomyofibroblast phenoconversion include an increase in expression of a-SMA (Darby et al., 1990), ED-A fibronectin (Serini et al., 1998) and SMemb (Frangogiannis et al., 2000). Myofi (...truncated)