Acute and transient MF activation

Acute and transient MF activation is part of the body’s normal wound healing program, but persistent MFs contribute to fibrosis by excessively producing and contracting collagenous extracellular matrix (ECM) into stiff scar tissue (Hinz et al., 2012). In turn, the stiffness of mature scar promotes mechanical activation of MFs (Hinz, 2010b). In cell therapy, MSCs engrafted into early stages of organ fibrosis were shown to improve healing, but delivery into stiff mature scar further enhanced fibrogenesis in fibrotic lung, kidney, liver, and heart (Breitbach et al., 2007; di Bonzo et al., 2008; Nagaya et al., 2005; Ninichuk et al., 2006; Yan et al., 2007). Substrate mechanics in conjunction with intracellular tension have also been shown to determine the preference of naive MSCs toward specific lineages (Engler et al., 2006; Winer et al., 2009; Yang et al., 2011, 2014), but the functional consequences of MF activation (fibrogenesis) on MSC clonogenicity and lineage differentiation potential have not been systematically investigated.
Results
Discussion MSCs are prone to MF activation by stiff ECM and TGF-β1, but the consequences on their stem cell potential and reversibility have not been systematically assessed. We establish a direct link between α-SMA expression/function, YAP/TAZ activity, and hMSC fate. Different order 740 Y-P isoforms promote specific types of actin organization levels and a shift in the ratios of actin isoforms can reprogram cell differentiation (Lechuga et al., 2014; Tondeleir et al., 2012). α-SMA incorporation into existing stress fibers has been shown to increase actin organization and intracellular tension (Goffin et al., 2006; Hinz et al., 2001). Actin organization as cortical filaments or incorporation into stress fiber bundles has been shown to differentially control YAP/TAZ activation, nuclear localization, and regulation of the Hippo pathway (Gaspar and Tapon, 2014; Halder et al., 2012; Yu et al., 2012a). In epithelia, cell-morphology-dependent actin organization provides positional information to individual cells in a multicellular layer (Aragona et al., 2013; Wada et al., 2011). YAP/TAZ are also central in regulating cell fate decision by interacting with RUNX2 and PPARG (Hong et al., 2005; Hong and Yaffe, 2006; Varelas et al., 2008). Consistently, neo-expression of α-SMA alone was sufficient in our experiments to reduce hMSC differentiation potential, which was rescued by inhibition and downregulation of YAP1. Whether the α-SMA-induced nuclear shift of YAP is responsible for reduced clonogenicity is unclear because YAP1 knockdown did not protect against the reduction of SOX2 and OCT4 transcript levels upon α-SMA-overexpression. Consistently, YAP binds to and is expected to activate gene transcription of SOX2 and OCT4 in embryonic stem (ES) cells (Lian et al., 2010). However, YAP is inactivated in ES cells during differentiation (Lian et al., 2010), whereas it drives specific lineage specification in MSCs (Dupont et al., 2011). Overexpression of OCT4 has been shown to result in increased MSC proliferation, whereas OCT4 knockdown had the opposite effect (Tsai et al., 2012). Similarly, knockdown of SOX2 reduces proliferation in MSC-derived osteoprogenitors that is restored by YAP1 overexpression, which acts downstream of SOX2 in this model (Seo et al., 2013). Hence, the interplay between α-SMA, YAP/TAZ, and self-renewal genes is complex and not entirely understood at present. YAP/TAZ transcription factors provide a direct link between cell mechanosensing and gene regulation (Halder et al., 2012). Inhibition of cell contraction has been shown to decrease nuclear localization of YAP/TAZ and transcription of downstream genes, whereas high intracellular tension drives YAP/TAZ into the nucleus (Calvo et al., 2013; Dupont et al., 2011). Expression of α-SMA is not essential for the localization of YAP/TAZ in the nucleus and ∼10% of the hMSC retained predominantly nuclear YAP even in the presence of the SMA-FP. However, incorporation of α-SMA into existing stress fibers substantially increases contractile force and cell stress (Hinz et al., 2001) and thus accentuates fibrogenic transcription programs. YAP/TAZ have been shown to be involved in regulating α-SMA expression and fibrogenesis using an experimental model of epithelial-to-mesenchymal transition (Speight et al., 2013) and during lung fibroblast-to-MF activation by stiff ECM (Liu et al., 2015). These reports and our works suggest a positive feedback loop between α-SMA and YAP/TAZ signaling that amplifies fibrosis.