TGFβ Induced Myofibroblast Differentiation of Rabbit Keratocytes Requires Synergistic TGFβ, PDGF and Integrin Signaling

Department of Ophthalmology, University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75390-9057, USA.
Experimental Eye Research (Impact Factor: 2.71). 01/2003; 75(6):645-57. DOI: 10.1006/exer.2002.2066
Source: PubMed


There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFbeta). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFbeta. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFbeta1 stimulated cell cycle entry of serum-free cultured rabbit corneal keratocytes. AntiPDGF treatment also markedly reduced the TGFbeta1-induced intracellular actin filament re-organization, fibronectin fibril assembly, and focal contact formation as well as reducing by 80% the expression of alpha-smooth muscle (alpha-SM) specific isoform of actin characteristic of myofibroblast differentiation. Although PDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFbeta1 as did quiescent keratocytes. Furthermore, blocking TGFbeta1 induction of myofibroblast differentiation with the Arg-Gly-Asp containing peptide, GRGDdSP, for 3 days followed by allowing progression of myofibroblast differentiation by removing GRGDdSP did not change the temporal response or tyrosine phosphorylation cascade (2-72 hr) leading to myofibroblast differentiation. Nor did PDGF treatment of keratocytes reverse the RGD blockade of TGFbeta1 induced myofibroblast differentiation. Overall these cumulative findings indicate that myofibroblast differentiation in the rabbit corneal keratocyte requires synergistic growth factor/integrin signaling involving TGFbeta, PDGF, and the fibronectin receptor. Additionally, the similar TGFbeta1 temporal response of PDGF-stimulated compared to nai;ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.

Download full-text


Available from: Walter Matthew Petroll, Dec 01, 2014
36 Reads
  • Source
    • "A similar increase in stress fiber formation and myofibroblast transformation of corneal keratocytes is observed within compressed 3-D collagen matrices; further demonstrating that increased substrate stiffness enhances TGFb-induced myofibroblast transformation of corneal keratocytes. Both connective tissue growth factor (CTGF) and PDGF have been shown to participate in TGFb-induced myofibroblast differentiation through an autocrine feedback loop, which would be amplified at higher cell density (Garrett et al., 2004; Jester et al., 2002). While stress fibers were observed in all cells within compressed ECM irrespective of cell density, the percentage of cells with a-SM-actin incorporated into stress fibers was greater at higher cell density (~60% versus ~20%), suggesting that both mechanical stiffness and autocrine signaling promote myofibroblast transformation in vitro (Lakshman and Petroll, 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The generation of cellular forces and the application of these physical forces to the ECM play a central role in mediating matrix patterning and remodeling during fundamental processes such as developmental morphogenesis and wound healing. In addition to growth factors and other biochemical factors that can modulate the keratocyte mechanical phenotype, another key player in the regulation of cell-induced ECM patterning is the mechanical state of the ECM itself. In this review we provide an overview of the biochemical and biophysical factors regulating the mechanical interactions between corneal keratocytes and the stromal ECM at the cellular level. We first provide an overview of how Rho GTPases regulate the sub-cellular pattern of force generation by corneal keratocytes, and the impact these forces have on the surrounding ECM. We next review how feedback from local matrix structural and mechanical properties can modulate keratocyte phenotype and mechanical activity. Throughout this review, we provide examples of how these biophysical interactions may contribute to clinical outcomes, with a focus on corneal wound healing. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Experimental Eye Research 04/2015; 133. DOI:10.1016/j.exer.2014.09.003 · 2.71 Impact Factor
  • Source
    • "TGFβ signaling also plays multiple roles in the anterior segment of the eye: TGFβ1 plays a predominant role in the differentiation of keratocytes to myofibroblasts [12], [73], TGFβ2 is immunosuppressive in normal human and rabbit aqueous humor [74], [75] and is a key cytokine that can influence corneal wound healing [76]. In this study, no alteration to TGFß2 was observed with separate knockdown of either YAP or TAZ. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ), two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFβ). In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFβ2 and CTGF, were investigated. The results demonstrate (a) that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b) that CTGF is predominantly regulated by YAP and not TAZ, and (c) that TGFβ2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and β-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression.
    PLoS ONE 10/2014; 9(10):e109811. DOI:10.1371/journal.pone.0109811 · 3.23 Impact Factor
  • Source
    • "The TGFβ1 and 2 ligands are present in their inactive form in the corneal epithelium under homeostatic conditions, while in their active cleaved forms they are present in the epithelium and to a lesser extent in the stroma during injury and infections [52]–[55]. Earlier studies have shown that keratocytes respond to TGF β1 stimulus, however these were conducted in the context of myofibroblastic changes, ECM production and fibrosis [56]–[59]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Keratoconus (KC) is a complex thinning disease of the cornea that often requires transplantation. The underlying pathogenic molecular changes in this disease are poorly understood. Earlier studies reported oxidative stress, metabolic dysfunctions and accelerated death of stromal keratocytes in keratoconus (KC) patients. Utilizing mass spectrometry we found reduced stromal extracellular matrix (ECM) proteins in KC, suggesting ECM-regulatory changes that may be due to altered TGFβ signals. Here we investigated properties of stromal cells from donor (DN) and KC corneas grown as fibroblasts in serum containing DMEM: F12 or in serum-free medium containing insulin, transferrin, selenium (ITS). Phosphorylation of SMAD2/3 of the canonical TGFβ pathway, was high in serum-starved DN and KC fibroblast protein extracts, but pSMAD1/5/8 low at base line, was induced within 30 minutes of TGFβ1 stimulation, more so in KC than DN, suggesting a novel TGFβ1-SMAD1/5/8 axis in the cornea, that may be altered in KC. The serine/threonine kinases AKT, known to regulate proliferation, survival and biosynthetic activities of cells, were poorly activated in KC fibroblasts in high glucose media. Concordantly, alcohol dehydrogenase 1 (ADH1), an indicator of increased glucose uptake and metabolism, was reduced in KC compared to DN fibroblasts. By contrast, in low glucose (5.5 mM, normoglycemic) serum-free DMEM and ITS, cell survival and pAKT levels were comparable in KC and DN cells. Therefore, high glucose combined with serum-deprivation presents some cellular stress difficult to overcome by the KC stromal cells. Our study provides molecular insights into AKT and TGFβ signal changes in KC, and a mechanism for functional studies of stromal cells from KC corneas.
    PLoS ONE 09/2014; 9(9):e106556. DOI:10.1371/journal.pone.0106556 · 3.23 Impact Factor
Show more