Matrix Rigidity Mediates TGFβ1-induced Epithelial-Myofibroblast Transition by Controlling Cytoskeletal Organization and MRTF-A Localization

Abstract

Myofibroblasts mediate normal wound healing and upon chronic activation can contribute to the development of pathological conditions including organ fibrosis and cancer. Myofibroblasts can develop from epithelial cells through an epithelial-mesenchymal transition (EMT) during which epithelial cells exhibit drastic morphological changes and upregulate cytoskeletal associated proteins that enable exertion of large contractile forces and remodeling of the surrounding microenvironment. Increased matrix rigidity is a hallmark of fibrosis and tumor progression and mechanical tension has been identified as a regulator of EMT; however, the mechanisms governing the mechanical regulation of EMT are not completely understood. Here, we find that matrix rigidity regulates transforming growth factor (TGF)-β1-induced EMT, with rigid substrata enabling increased myofibroblast marker expression, cell morphology changes, and cytoskeletal reorganization while soft matrices block these changes. Furthermore, we find that matrix rigidity controls the subcellular localization of myocardin related transcription factor (MRTF)-A, a regulator of cytoskeletal protein expression that contributes to the acquisition of myogenic features during EMT. Results from these studies provide insight into how biophysical cues contribute to myofibroblast development from epithelial cells and may suggest ways to enhance wound healing or to engineer therapeutic solutions for fibrosis and cancer.

Substrate stiffness-dependent regulation of the SRF−Mkl1 co-activator complex requires the inner nuclear membrane protein Emerin

“…S1A), but the maximum level of Mkl1 nuclear accumulation 5 min after serum stimulation was reduced in Emd WT MEFs on soft substrates (≤1 kPa) relative to the stiffest substrate (50 kPa) (Fig. 1C,D), consistent with previous reports demonstrating that Mkl1 nuclear accumulation is substrate stiffnessdependent (McGee et al, 2011;O'Connor et al, 2015;Varney et al, 2016). Emerin was required for maximal Mkl1 nuclear C ratio in cells plated on collagen-coated polyacrylamide hydrogels of the indicated stiffness before or 5 min after serum stimulation (mean±s.e.m., n=3).…”

“…In response to stimuli that initiate actin polymerization, Mkl1 accumulates in the nucleus and, together with SRF, induces the expression of genes important for myofibroblast differentiation (Cen et al, 2003;Crider et al, 2011;Esnault et al, 2014;Miralles et al, 2003). Mkl1 nuclear localization after stimulation is significantly increased in cells on stiff substrates when compared to cells plated on more compliant substrates (McGee et al, 2011;O'Connor et al, 2015;Varney et al, 2016). We tested whether the nuclear lamina is important for mechanosensing in wild-type (Emd WT ) vs Emerin-knockout (Emd KO ) mouse embryonic fibroblasts (MEFs) cultured on glass coverslips and collagencoated polyacrylamide hydrogels with elastic moduli between 0.2−50 kPa, which spans the range observed in vertebrate tissues in vivo (Butcher et al, 2009).…”

“…The co-activator complex comprising serum response factor (SRF) and megakaryoblastic leukemia 1 protein (Mkl1; also known as MRTF-A, MAL) (SRF−Mkl1) is critical for myofibroblast differentiation (Crider et al, 2011;Small et al, 2010). Though chemical signals including TGFβ1 induce SRF−Mkl1-dependent gene activation during wound healing, tension in the actin cytoskeleton developed through cell-ECM interactions is required for this response (Gomez et al, 2010;Huang et al, 2012;McGee et al, 2011;O'Connor et al, 2015;Varney et al, 2016). Moreover, a feedforward loop that couples tissue stiffness and SRF−Mkl1-dependent transcription is thought to underlie persistent myofibroblast activation (Zhou et al, 2013).…”

RhoA/ROCK Signaling Regulates TGFβ-Induced Epithelial-Mesenchymal Transition of Lens Epithelial Cells through MRTF-A

“…These transcription factors have been implicated in EMT (33)(34)(35)(36)(37)(38); however, little is known about their role in the lens (39). The two isoforms, MRTF-A and -B, are widely expressed in numerous tissues, where they are normally bound to monomeric actin (G-actin).…”

“…Nuclear MRTF forms a complex with serum response factor (SRF) activating transcription of cytoskeletal genes associated with EMT, including αSMA (34,(40)(41)(42). Indeed, TGFβ has been shown to stimulate nuclear accumulation of MRTF-A in several organ fibrosis models (38,43,44), including in recent work from our group showing TGFβ-induced nuclear localization of MRTF-A in lens epithelial cells (39). However, whether TGFβ-induced changes in MTRF-A in the lens are ROCK-dependent is not known.…”