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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, ?SMA). 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.

SUBMITTER: Li Y

PROVIDER: S-EPMC6816600 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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

Li Yanfen Y Tang Claire B CB Kilian Kristopher A KA

Cellular and molecular bioengineering 20170718 5

<h4>Introduction</h4>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 ...[more]

PMID: 31719870

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Project description:BackgroundFibrosis plays an important role in both normal physiological and pathological phenomena as fibroblasts differentiate to myofibroblasts. The activation of fibroblasts is determined through interactions with the surrounding extracellular matrix (ECM). However, how this fibroblast-to-myofibroblast transition (FMT) is regulated and affected by elastin concentration in a three-dimensional (3D) microenvironment has not been investigated.MethodsWe developed an insoluble elastin-gradient 3D hydrogel system for long-lasting cell culture and studied the molecular mechanisms of the FMT in embedded cells by nanoflow LC-MS/MS analysis along with validation through real-time PCR and immunofluorescence staining.ResultsBy optimizing pH and temperature, four 3D hydrogels containing fibroblasts were successfully fabricated having elastin concentrations of 0, 20, 50, and 80% in collagen. At the low elastin level (20%), fibroblast proliferation was significantly increased compared to others, and in particular, the FMT was clearly observed in this condition. Moreover, through mass spectrometry of the hydrogel environment, it was confirmed that differentiation proceeded in two stages. In the early stage, calcium-dependent proteins including calmodulin and S100A4 were highly associated. On the other hand, in the late stage after several passages of cells, distinct markers of myofibroblasts were presented such as morphological changes, increased production of ECM, and increased α-SMA expression. We also demonstrated that the low level of elastin concentration induced some cancer-associated fibroblast (CAF) markers, including PDGFR-β, and fibrosis-related disease markers, including THY-1.ConclusionUsing our developed 3D elastin-gradient hydrogel system, we evaluated the effect of different elastin concentrations on the FMT. The FMT was induced even at a low concentration of elastin with increasing CAF level via calcium signaling. With this system, we were able to analyze varying protein expressions in the overall FMT process over several cellular passages. Our results suggest that the elastin-gradient system employing nonlinear optics imaging provides a good platform to study activated fibroblasts interacting with the microenvironment, where the ECM plays a pivotal role.

Dataset Information

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

Publications

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

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