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Matrix Mechanics Influence Fibroblast–Myofibroblast Transition by Directing the Localization of Histone Deacetylase 4

Abstract

Introduction

The propagation of mechanochemical signals from the extracellular matrix to the cell nucleus has emerged as a central feature in regulating cellular differentiation and de-differentiation. This process of outside-in signaling and the associated mechanotransduction pathways have been well described in numerous developmental and pathological contexts. However, it remains less clear how mechanotransduction influences the activity of chromatin modifying enzymes that direct gene expression programs.

Objectives

The primary objective of this study was to explore how matrix mechanics and geometric confinement influence histone deacetylase (HDAC) activity in fibroblast culture.

Methods

Polyacrylamide hydrogels were formed and functionalized with fibronectin patterns using soft lithography. Primary mouse embryonic fibroblasts (MEFs) were cultured on the islands until confluent, fixed, and immunolabeled for microscopy.

Results

After 24 h MEFs cultured on soft hydrogels (0.5 kPa) show increased expression of class I HDACs relative to MEFs cultured on stiff hydrogels (100 kPa). A member of the class II family, HDAC4 shows a similar trend; however, there is a pronounced cytoplasmic localization on soft hydrogels suggesting a role in outside-in cytoplasmic signaling. Pharmacological inhibitor studies suggest that the opposing activities of extracellular related kinase 1/2 (ERK) and protein phosphatase 2a (PP2a) influence the localization of HDAC4. MEFs cultured on the soft hydrogels show enhanced expression of markers associated with a fibroblast–myofibroblast transition (Paxillin, αSMA).

Conclusions

We show that the phosphorylation state and cellular localization of HDAC4 is influenced by matrix mechanics, with evidence for a role in mechanotransduction and the regulation of gene expression associated with fibroblast–myofibroblast transitions. This work establishes a link between outside-in signaling and epigenetic regulation, which will assist efforts aimed at controlling gene regulation in engineered extracellular matrices.

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Acknowledgments

The authors would like to thank Dr. Quanxi Li in the Department of Comparative Biosciences at the University of Illinois for the generous isolation and donation of MEFs and Dr. Karin Jensen in the Department of Bioengineering at the University of Illinois for guidance with western blots.

Conflict of interest

Yanfen Li, Claire B. Tang, and Kristopher A. Kilian declare that they have no conflicts of interest.

Funding

Funding was provided by the National Science Foundation Grant No. 1454616 CAR. Y.L. was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE – 1144245.

Research Involved in Human and Animal Rights

No humans or animals were employed in this research by the authors.

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Correspondence to Kristopher A. Kilian.

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Associate Editor Alyssa Panitch oversaw the review of this article.

Kristopher A. Kilian

received B.S. and M.S. degrees in Chemistry from the University of Washington in 1999 and 2003 respectively. He worked for Merck Research Labs in the Methods Development group from 2000 to 2004 before travelling to Sydney, Australia to do his PhD with Justin Gooding at the University of New South Wales. In 2007, he joined the laboratory of Milan Mrksich at the University of Chicago as a postdoctoral fellow to investigate new methods for directing the differentiation of stem cells. Kris joined the faculty of the University of Illinois at Urbana-Champaign in 2011 with appointments in the Departments of Materials Science and Engineering, and Bioengineering. Kris is a 2008 recipient of the NIH Ruth L. Kirchstein National Research Service Award, a 2014 Kavli Fellow of the 19th German-American Frontiers of Science, and a 2015 recipient of the National Science Foundation’s CAREER award. His research interests include the design and development of model extracellular matrices for cell engineering and fundamental studies in cell biology.

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Li, Y., Tang, C.B. & Kilian, K.A. Matrix Mechanics Influence Fibroblast–Myofibroblast Transition by Directing the Localization of Histone Deacetylase 4. Cel. Mol. Bioeng. 10, 405–415 (2017). https://doi.org/10.1007/s12195-017-0493-8

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Keywords

  • Myofibroblast differentiation
  • Soft lithography
  • Substrate stiffness
  • Histone deacetylase
  • Mechanotransduction