ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-β1

Maruri, Daniel P.; Miron-Mendoza, Miguel; Kivanany, Pouriska; Hack, Joshua M.; Schmidtke, David W.; Petroll, Walter M; Varner, Victor D.

doi:10.1016/j.bpj.2020.08.040

Cited by 23 publications

(62 citation statements)

References 52 publications

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“…The signaling pathways downstream of transforming growth factor-beta 1 (TGF- β 1) are key to this process ( Jester et al, 1996 ; Jester and Ho-Chang, 2003 ; Torricelli et al, 2013 ), and, in some cases, can lead to a protracted myofibroblast response that causes corneal hazing and impaired ocular function ( Boote et al, 2012 ). Corneal wound healing is also associated with changes in tissue stiffness ( Raghunathan et al, 2017 ), and recent work has shown that the mechanical properties of the ECM can regulate the TGF- β 1-mediated myofibroblast differentiation of corneal keratocytes ( Dreier et al, 2013 ; Petroll and Miron-Mendoza, 2015 ; Maruri et al, 2020 ). But it is still unclear how keratocytes sense changes in ECM stiffness and how mechanotransductive signaling mediates stiffness-dependent changes in myofibroblast differentiation.…”

Section: Introductionmentioning

confidence: 99%

Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes

Maruri

Iyer

Schmidtke

et al. 2022

Front. Cell Dev. Biol.

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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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Section: Introductionmentioning

confidence: 99%

Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes

Maruri

Iyer

Schmidtke

et al. 2022

Front. Cell Dev. Biol.

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“…Interestingly, αvβ6 is also expressed by corneal epithelial cells (Stepp, 2006) and they were also found to secrete latent-TGFβ-binding protein through extracellular vesicles (Han et al, 2017). Moreover, a recent study observed an increase in TGFβ expression in corneal keratocytes upon an increase in substrate stiffness (Maruri et al, 2020). A similar mechanism might be at play in the corneal epithelium for stiffness induced differentiation mediated by SMAD2/3.…”

Section: Discussionmentioning

confidence: 94%

Biomechanical property of limbal niche maintains stemness through YAP

Bhattacharya

Mukherjee

Pisano

et al. 2021

Preprint

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Stem cells (SCs) decision to self-renew or differentiate largely depends on the extracellular environment and elasticity of their niche. A well-described mediator of the mechanotransduction pathway is the co-transcriptional activator Yes-associated protein (YAP), known to shuttle into the nucleus of cells grown on stiff matrices. YAP is also known to be essential for stemness, but confusingly, SCs often reside in soft niches. Furthermore, the role of matrix rigidity in niche formation and SC function in vivo is poorly understood. Here we report that the post-natal development of the murine corneal epithelium involves matrix stiffening and loss of YAP activity that is associated with the formation of differentiation compartment. Importantly, manipulating the matrix crosslinking enzyme, Lox, perturbed SC mark expression and resulted in loss of corneal transparency. In agreement, we found that YAP and mechanotransduction pathways are essential for stemness in the soft niche compartment, wound healing response, and dedifferentiation of committed cells into SCs following SC depletion. In vitro experiments revealed that stiffer substrates induced cytoplasmic YAP localization through activation of LATS1/2, facilitating SMAD2/3-mediated cell differentiation. Taken together, we propose that the soft environment of the corneal SC niche maintains YAP activity to support SC regulation during morphogenesis, adult homeostasis and regeneration by the niche.

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“…1, the expressions of α-SMA and collagen I were up-regulated in a time-dependent manner when rabbit keratocytes were treated with 2 ng/ml of TGF-β1, suggesting TGF-β1-induced keratocyte activation. In addition to the increase of α-SMA and collagen I expressions, it has been reported that corneal keratocytes treated with TGF-β1 also showed other fibrosis-related changes including ECM stiffness [14].…”

Section: Tgf-β1 Induced Myofibroblast Transformation Of Rabbit Keratocytesmentioning

confidence: 99%

MicroRNA-21a-5p regulates TGF-?1-induced myofibroblast transformation of rabbit keratocytes by targeting Smad7

Bai¹,

Zhang²,

Ding³

et al. 2022

ScienceAsia

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The transforming growth factor β1 (TGF-β1)-induced transformation of keratocytes to myofibroblasts is a very common process during corneal wound healing, but a dysregulated TGF-β1 activity may induce unfavorable fibrosis and scar formation in the cornea. MicroRNA (miR)-21a-5p and Smad7 have been reported to play an important role in the fibrosis process. However, how miR-21a-5p involves in the TGF-β1-induced myofibroblasts transformation of keratocytes remains unclear. In the present study, we found an increase in the expressions of α-SMA and collagen I at protein level as well as Smad7 and miR-21a-5p at mRNA level when keratocytes were treated with TGF-β1Here; but the Smad7 at protein level kept unchanged. The overexpression of miR-21a-5p attenuated Smad7 protein expression, and miR-21a-5p inhibition promoted Smad7 protein expression without affecting its mRNA expression. Furthermore, inhibition of miR-21a-5p could decrease TGF-β1-induced α-SMA expression, whereas the addition of Smad7 knockdown would rescue the expression of α-SMA. These results suggested that miR-21a-5p had a promoting effect on TGF-β1induced myofibroblast transformation of keratocytes by targeting Smad7 at its translation stage.

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ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-β1

Cited by 23 publications

References 52 publications

Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes

Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-β1-Mediated Myofibroblast Differentiation of Corneal Keratocytes

Biomechanical property of limbal niche maintains stemness through YAP

MicroRNA-21a-5p regulates TGF-?1-induced myofibroblast transformation of rabbit keratocytes by targeting Smad7

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