Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes
Following injury and refractive surgery, corneal wound healing can initiate a protracted fibrotic response that interferes with ocular function. This fibrosis is related, in part, to the myofibroblast differentiation of corneal keratocytes in response to transforming growth factor beta 1 (TGF-β1). P...
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Frontiers Media S.A.
2022-05-01
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author | Daniel P. Maruri Krithika S. Iyer David W. Schmidtke David W. Schmidtke W. Matthew Petroll Victor D. Varner Victor D. Varner |
author_facet | Daniel P. Maruri Krithika S. Iyer David W. Schmidtke David W. Schmidtke W. Matthew Petroll Victor D. Varner Victor D. Varner |
author_sort | Daniel P. Maruri |
collection | DOAJ |
description | Following injury and refractive surgery, corneal wound healing can initiate a protracted fibrotic response that interferes with ocular function. This fibrosis is related, in part, to the myofibroblast differentiation of corneal keratocytes in response to transforming growth factor beta 1 (TGF-β1). Previous studies have shown that changes in the mechanical properties of the extracellular matrix (ECM) can regulate this process, but the mechanotransductive pathways that govern stiffness-dependent changes in keratocyte differentiation remain unclear. Here, we used a polyacrylamide (PA) gel system to investigate how mechanosensing via focal adhesions (FAs) regulates the stiffness-dependent myofibroblast differentiation of primary corneal keratocytes treated with TGF-β1. Soft (1 kPa) and stiff (10 kPa) PA substrata were fabricated on glass coverslips, plated with corneal keratocytes, and cultured in defined serum free media with or without exogenous TGF-β1. In some experiments, an inhibitor of focal adhesion kinase (FAK) activation was also added to the media. Cells were fixed and stained for F-actin, as well as markers for myofibroblast differentiation (α-SMA), actomyosin contractility phosphorylated myosin light chain (pMLC), focal adhesions (vinculin), or Smad activity (pSmad3). We also used traction force microscopy (TFM) to quantify cellular traction stresses. Treatment with TGF-β1 elicited stiffness-dependent differences in the number, size, and subcellular distribution of FAs, but not in the nuclear localization of pSmad3. On stiff substrata, cells exhibited large FAs distributed throughout the entire cell body, while on soft gels, the FAs were smaller, fewer in number, and localized primarily to the distal tips of thin cellular extensions. Larger and increased numbers of FAs correlated with elevated traction stresses, increased levels of α-SMA immunofluorescence, and more prominent and broadly distributed pMLC staining. Inhibition of FAK disrupted stiffness-dependent differences in keratocyte contractility, FA patterning, and myofibroblast differentiation in the presence of TGF-β1. Taken together, these data suggest that signaling downstream of FAs has important implications for the stiffness-dependent myofibroblast differentiation of corneal keratocytes. |
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spelling | doaj.art-b54775678df54c3da907717c08ab648a2022-12-22T02:23:17ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2022-05-011010.3389/fcell.2022.886759886759Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal KeratocytesDaniel P. Maruri0Krithika S. Iyer1David W. Schmidtke2David W. Schmidtke3W. Matthew Petroll4Victor D. Varner5Victor D. Varner6Department of Bioengineering, University of Texas at Dallas, Richardson, TX, United StatesDepartment of Bioengineering, University of Texas at Dallas, Richardson, TX, United StatesDepartment of Bioengineering, University of Texas at Dallas, Richardson, TX, United StatesDepartment of Surgery, UT Southwestern Medical Center, Dallas, TX, United StatesDepartment of Ophthalmology, UT Southwestern Medical Center, Dallas, TX, United StatesDepartment of Bioengineering, University of Texas at Dallas, Richardson, TX, United StatesDepartment of Surgery, UT Southwestern Medical Center, Dallas, TX, United StatesFollowing injury and refractive surgery, corneal wound healing can initiate a protracted fibrotic response that interferes with ocular function. This fibrosis is related, in part, to the myofibroblast differentiation of corneal keratocytes in response to transforming growth factor beta 1 (TGF-β1). Previous studies have shown that changes in the mechanical properties of the extracellular matrix (ECM) can regulate this process, but the mechanotransductive pathways that govern stiffness-dependent changes in keratocyte differentiation remain unclear. Here, we used a polyacrylamide (PA) gel system to investigate how mechanosensing via focal adhesions (FAs) regulates the stiffness-dependent myofibroblast differentiation of primary corneal keratocytes treated with TGF-β1. Soft (1 kPa) and stiff (10 kPa) PA substrata were fabricated on glass coverslips, plated with corneal keratocytes, and cultured in defined serum free media with or without exogenous TGF-β1. In some experiments, an inhibitor of focal adhesion kinase (FAK) activation was also added to the media. Cells were fixed and stained for F-actin, as well as markers for myofibroblast differentiation (α-SMA), actomyosin contractility phosphorylated myosin light chain (pMLC), focal adhesions (vinculin), or Smad activity (pSmad3). We also used traction force microscopy (TFM) to quantify cellular traction stresses. Treatment with TGF-β1 elicited stiffness-dependent differences in the number, size, and subcellular distribution of FAs, but not in the nuclear localization of pSmad3. On stiff substrata, cells exhibited large FAs distributed throughout the entire cell body, while on soft gels, the FAs were smaller, fewer in number, and localized primarily to the distal tips of thin cellular extensions. Larger and increased numbers of FAs correlated with elevated traction stresses, increased levels of α-SMA immunofluorescence, and more prominent and broadly distributed pMLC staining. Inhibition of FAK disrupted stiffness-dependent differences in keratocyte contractility, FA patterning, and myofibroblast differentiation in the presence of TGF-β1. Taken together, these data suggest that signaling downstream of FAs has important implications for the stiffness-dependent myofibroblast differentiation of corneal keratocytes.https://www.frontiersin.org/articles/10.3389/fcell.2022.886759/fullTGF-β1extracellular matrixtraction force microscopyFAKmechanobiology |
spellingShingle | Daniel P. Maruri Krithika S. Iyer David W. Schmidtke David W. Schmidtke W. Matthew Petroll Victor D. Varner Victor D. Varner Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes Frontiers in Cell and Developmental Biology TGF-β1 extracellular matrix traction force microscopy FAK mechanobiology |
title | Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes |
title_full | Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes |
title_fullStr | Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes |
title_full_unstemmed | Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes |
title_short | Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes |
title_sort | signaling downstream of focal adhesions regulates stiffness dependent differences in the tgf β1 mediated myofibroblast differentiation of corneal keratocytes |
topic | TGF-β1 extracellular matrix traction force microscopy FAK mechanobiology |
url | https://www.frontiersin.org/articles/10.3389/fcell.2022.886759/full |
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