FGF-2- and TGF-β1-induced downregulation of lumican and keratocan in activated corneal keratocytes by JNK signaling pathway.
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.
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ABSTRACT: In mammals, tissue damage is usually repaired by activation of a fibrotic response which saves the life of the organism, but which can never restore function to the damaged organ. In addition, fibrotic responses form the basis for diverse pathologies, including many that occur in the eye. It is intriguing, therefore, to observe the occasional circumstances in which repair in mammals appears to take on a regenerative character, such as during fetal wound healing or in certain types of corneal wounds. The thesis of this chapter is that the choice between regeneration or fibrosis lies in the control of fibroblast phenotype. The cornea of the eye has several features which make it a particularly useful model for the study of fibroblast phenotype. Studies discussed herein, identify failure to activate the transcription factor NF-kappaB as a control mechanism for inhibiting fibroblast activation in the cornea. Evidence is further presented for the view that transition in fibroblast phenotype in repair tissue is not simply a matter of differential gene expression, but is a developmental event which reflects changes in the hard wiring of signalling pathways by which the cell responds to environmental input.Progress in Retinal and Eye Research 08/1999; 18(4):529-51. · 9.45 Impact Factor
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ABSTRACT: Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera-/- cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera-/- cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera-/- mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.Journal of Biological Chemistry 07/2003; 278(24):21672-7. · 4.77 Impact Factor
International Ophthalmology Clinics 02/2002; 42(3):13-22.