Myofibroblasts Exhibit Enhanced Fibronectin Assembly That Is Intrinsic to Their Contractile Phenotype

Elizabeth E Torr,Caitlyn R Ngam,Ksenija Bernau,Bianca Tomasini-Johansson,Benjamin Acton,Nathan Sandbo

doi:10.1074/jbc.m114.606186

Elizabeth E Torr, Caitlyn R Ngam + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m114.606186

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Abstract

Myofibroblasts have increased expression of contractile proteins and display augmented contractility. It is not known if the augmented contractile gene expression characterizing the myofibroblast phenotype impacts its intrinsic ability to assemble fibronectin (FN) and extracellular matrix. In this study we investigated whether myofibroblasts displayed increased rates of FN fibril assembly when compared with their undifferentiated counterparts. Freshly plated myofibroblasts assemble exogenous FN (488-FN) into a fibrillar matrix more rapidly than fibroblasts that have not undergone myofibroblast differentiation. The augmented rate of FN matrix formation by myofibroblasts was dependent on intact Rho/Rho kinase (ROCK) and myosin signals inasmuch as treatment with Y27632 or blebbistatin attenuated 488-FN assembly. Inhibiting contractile gene expression by pharmacologic disruption of the transcription factors megakaryoblastic leukemia-1 (MKL1)/serum response factor (SRF) during myofibroblast differentiation resulted in decreased contractile force generation and attenuated 488-FN incorporation although not FN expression. Furthermore, disruption of the MKL1/SRF target gene, smooth muscle α-actin (α-SMA) via siRNA knockdown resulted in attenuation of 488-FN assembly. In conclusion, this study demonstrates a linkage between increased contractile gene expression, most importantly α-SMA, and the intrinsic capacity of myofibroblasts to assemble exogenous FN into fibrillar extracellular matrix.

Highlights

Myofibroblasts have heightened expression of contractile genes and drive extracellular matrix formation during pulmonary fibrosis
Collagen Matrix Formation by Myofibroblasts Is Dependent on the Assembly of a FN Matrix—Human lung fibroblasts (HLF) undergoing differentiation to myofibroblasts by treatment with TGF-␤ demonstrate a linear increase in the expression of DOC-soluble FN and collagen over time (Fig. 1A, left panel), which serves as a critical source of matrix proteins for subsequent incorporation into the extracellular matrix (ECM)
This demonstrates the dependence of collagen matrix formation on assembly of fibrillar FN matrix by HLF (Fig. 1C), consistent with previous reports establishing the requirement of FN matrix assembly for collagen deposition [4]

Summary

Background

Myofibroblasts have heightened expression of contractile genes and drive extracellular matrix formation during pulmonary fibrosis. Conclusion: This study demonstrates a linkage between contractile gene expression and increased assembly of fibronectin fibrils by myofibroblasts. This study demonstrates a linkage between increased contractile gene expression, most importantly ␣-SMA, and the intrinsic capacity of myofibroblasts to assemble exogenous FN into fibrillar extracellular matrix. In granulation tissue and fibrotic lung from patients with idiopathic pulmonary fibrosis, myofibroblasts are found in association with a dense fibronectin matrix [12, 13] Despite their association with abundant ECM in wound healing and fibrotic disorders, until now it has not been known if myofibroblasts exhibit an intrinsic capacity to more rapidly assemble a fibrillar FN matrix. We further observed that the increased contractile gene expression characterizing myofibroblasts is required for this effect These findings establish a relationship between heightened contractile gene expression in myofibroblasts and ECM deposition and remodeling in fibrotic disorders of the lung

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION