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

ABSTRACT 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
28 Reads
  • 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
  • Source
    • "TGFb has been shown to have a critical role in the generation and maintenance of corneal stromal myofibroblasts (Funderburgh et al., 2001; Bourlier et al., 2012; Andrianifahanana et al., 2013; Weber et al., 2013). Studies by Jester et al. (2002) showed that TGFb induces keratocyte proliferation and myofibroblast differentiation through activation of a PDGF autocrine loop. PDGF blocked in the stroma confirmed a role for PDGF in myofibroblast generation and suggested that in rabbit cells PDGF acts at a specific point of transition from VþAÀDÀ to VþAþDÀ myofibroblasts (Kaur et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Myofibroblasts, the primary cells associated with corneal stromal haze (opacity), can be derived from both cornea-derived and bone marrow-derived precursor cells. In the present study, the role of TGFβ or PDGF blockage on bone marrow-derived myofibroblast development was investigated using a green fluorescent protein (GFP) chimeric bone marrow mouse model and plasmid vectors that blocked TGFβ or PDGF signaling. At the peak of corneal haze one month after irregular phototherapeutic keratectomy the central stroma had significantly less alpha-smooth muscle actin (α-SMA)-positive cells derived from GFP+ bone marrow-derived cells or GFP- keratocyte/corneal fibroblast-derived cells when corneas were treated with the TGFβ blocking vector pGFPC1.TGFRBKDEL or the PDGF blocking vector pCMV.PDGFRB.23KDEL compared with the corresponding empty vector treated or untreated control groups. In individual animals, 30 to 60% of myofibroblasts were derived from bone marrow-derived precursor cells and 40 to 70% of myofibroblasts were derived from keratocyte-derived precursor cells. TGFβ and PDGF regulate corneal myofibroblast development from bone marrow-derived precursor cells and keratocyte/corneal fibroblast-derived precursor cells.
    Experimental Eye Research 04/2014; 121. DOI:10.1016/j.exer.2014.02.013 · 2.71 Impact Factor
Show more