Article

FGF-2- and TGF- 1-Induced Downregulation of Lumican and Keratocan in Activated Corneal Keratocytes by JNK Signaling Pathway

Department of Ophthalmology, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Investigative ophthalmology & visual science (Impact Factor: 3.66). 11/2011; 52(12):8957-64. DOI: 10.1167/iovs.11-8078
Source: PubMed

ABSTRACT Downregulation of lumican and keratocan expression is an undesirable phenotypic change that occurs during corneal wound healing. The present study was intended to determine whether the activation of Jun N-terminal kinase (JNK)-signaling pathway is involved in their downregulation in TGF-β1- and FGF-2-activated keratocytes.
Keratocytes, isolated from rabbit corneal stroma, and cultured in a serum-free medium, pretreated or not treated with JNK inhibitor (SP600125), were activated with FGF-2/heparin sulfate (HS) or TGF-β1 in the presence or absence of SP600125. In another set of experiments, keratocytes were transfected with JNK1/2 Dicer-substrate RNA (DsiRNA) and then activated with TGF-β1 or FGF-2/HS. Specific phenotypic changes were analyzed immunocytochemically and correlated with Western blot analyses. The relative levels of specific mRNAs were estimated by quantitative RT-PCR using specific reagents.
The FGF-2/HS- or TGF-β-induced activation of corneal stromal keratocytes to fibroblast- or myofibroblast-phenotype, respectively, resulted in marked decreases in cell surface-associated and secreted keratan sulfate proteoglycans (KSPGs). Both keratocan and lumican proteins and their mRNAs were downregulated in the activated keratocytes. However, JNK inhibition during the activation of keratocytes, pretreated with the JNK inhibitor, suppressed the reduction in the cell-surface associated and secreted KSPGs (lumican and keratocan), and their mRNA transcripts. Downregulation of total KSPGs and their mRNAs was also inhibited by decreasing JNK1 and JNK2 levels via JNK1/2 DsiRNA transfection of keratocytes before their activation.
Extrapolating from the present study, FGF-2- and TGF-β1-activation of JNK signaling pathway may be partly responsible for the downregulation of keratocan and lumican expression in activated corneal keratocytes observed during corneal stromal wound healing.

0 Followers
 · 
117 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation by were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.
    Experimental Eye Research 04/2014; 121. DOI:10.1016/j.exer.2014.02.002 · 3.02 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To assess how wound healing cytokines and the extracellular matrix (ECM) environment regulate the keratocyte mechanical phenotype. Rabbit corneal keratocytes were plated within standard bovine or rat tail type I collagen matrices (2.5 mg/mL), compressed collagen matrices (approximately 100 mg/mL), or on collagen-coated dishes and cultured for up to 7 days in serum-free media, platelet derived growth factor BB (PDGF BB), insulin-like growth factor (IGF), TGFβ1, TGFβ2, or FGF2. F-actin, α-smooth muscle actin (α-SMA) and collagen fibrils were imaged using confocal microscopy. Cell morphology, local matrix reorganization, and global matrix contraction were quantified digitally. IGF and PDGF BB stimulated elongation of keratocytes and extension of dendritic processes within 3-D matrices, without inducing stress fiber formation or collagen reorganization. In contrast, treatment with TGFβ1 and TGFβ2 increased keratocyte contractility, as indicated by stress fiber formation and matrix compaction and alignment. This transformation was enhanced at higher cell densities within standard 3-D matrices, in which α-SMA was incorporated into stress fibers. In contrast, α-SMA was expressed within compressed 3-D matrices even at low cell density. FGF2 did not produce significant cytoskeletal or matrix reorganization in standard 3-D matrices; however, stress fibers were consistently expressed within compressed collagen matrices and on rigid two-dimensional substrates. Inhibiting Rho kinase blocked both TGFβ- and FGF2-induced stress fiber formation. Keratocytes cultured in IGF or PDGF BB maintain a quiescent mechanical phenotype over a range of matrix environments. In contrast, the mechanical phenotypes induced by FGF and TGFβ vary in response to the structural and/or mechanical properties of the ECM.
    Investigative ophthalmology & visual science 03/2012; 53(3):1077-86. DOI:10.1167/iovs.11-8609 · 3.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury.
    Development Growth and Regeneration 04/2012; 54(3):327-40. DOI:10.1111/j.1440-169X.2012.01339.x · 2.18 Impact Factor

Preview

Download
4 Downloads
Available from