FGF-2 和 TGF-β1 通过 JNK 信号通路诱导激活的角膜基质细胞中 lumican 和 keratocan 的下调。
FGF-2- and TGF-β1-induced downregulation of lumican and keratocan in activated corneal keratocytes by JNK signaling pathway.
机构信息
Department of Ophthalmology, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
出版信息
Invest Ophthalmol Vis Sci. 2011 Nov 21;52(12):8957-64. doi: 10.1167/iovs.11-8078.
PURPOSE
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.
目的
层粘连蛋白和角膜蛋白聚糖表达的下调是角膜创伤愈合过程中发生的一种不良表型改变。本研究旨在确定 TGF-β1 和 FGF-2 激活的角膜基质细胞中，Jun N-末端激酶（JNK）信号通路的激活是否参与了它们的下调。
方法
从兔角膜基质中分离出角膜基质细胞，在无血清培养基中培养，用或不用 JNK 抑制剂（SP600125）预处理，然后用 FGF-2/肝素硫酸盐（HS）或 TGF-β1 激活，同时存在或不存在 SP600125。在另一组实验中，用 JNK1/2 Dicer 底物 RNA（DsiRNA）转染角膜基质细胞，然后用 TGF-β1 或 FGF-2/HS 激活。通过免疫细胞化学分析和 Western blot 分析来分析特定的表型变化，并将其与 Western blot 分析相关联。使用特定的试剂通过定量 RT-PCR 估计特定 mRNA 的相对水平。
结果
FGF-2/HS 或 TGF-β1 诱导的角膜基质角膜基质细胞向成纤维细胞或肌成纤维细胞表型的激活，导致细胞表面相关和分泌的角膜蛋白聚糖（KSPGs）显著减少。激活的角膜基质细胞中，角膜蛋白聚糖和层粘连蛋白蛋白及其 mRNA 均下调。然而，在用 JNK 抑制剂预处理的角膜基质细胞激活过程中抑制 JNK，可抑制细胞表面相关和分泌的 KSPGs（层粘连蛋白和角膜蛋白聚糖）及其 mRNA 转录物的减少。通过在激活前用 JNK1/2 DsiRNA 转染角膜基质细胞降低 JNK1 和 JNK2 水平，也抑制了总 KSPGs 及其 mRNA 的下调。
结论
从本研究推断，FGF-2 和 TGF-β1 激活的 JNK 信号通路可能部分负责在角膜基质创伤愈合过程中观察到的激活的角膜基质细胞中角膜蛋白聚糖和层粘连蛋白表达的下调。
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