FGF-2- and TGF-β1-induced downregulation of lumican and keratocan in activated corneal keratocytes by JNK signaling pathway.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Ophthalmology, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Jian
J
Chen
Julie
J
Wong-Chong
Nirmala
N
SundarRaj
D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics / Q000378:metabolism*; D060527:Corneal Keratocytes / Q000166:cytology / Q000502:physiology*; D003319:Corneal Stroma / Q000166:cytology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D007632:Keratan Sulfate / Q000235:genetics / Q000378:metabolism*; D000071234:Lumican; D048055:Mitogen-Activated Protein Kinase 8 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014945:Wound Healing / Q000502:physiology
D000873:Anthracenes; D011508:Chondroitin Sulfate Proteoglycans; D005136:Eye Proteins; D000071234:Lumican; D011509:Proteoglycans; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2; C432165:pyrazolanthrone; D007632:Keratan Sulfate; D048056:Mitogen-Activated Protein Kinase 9; D048055:Mitogen-Activated Protein Kinase 8