Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Department of Ophthalmology, Tri-Service General Hospital; and School of Medicine, National Defense Medical Center, Number 325, Section 2, Chang-gong Rd, Nei-Hu District, 114, Taipei, Taiwan, Republic of China.
J Biomed Sci. 2019 Oct 9;26(1):72. doi: 10.1186/s12929-019-0566-1.
Transforming growth factor (TGF) family members play important roles in the regulation of corneal integrity, and the pathogenesis of corneal fibrosis. Currently, there are no effective agents targeting TGF-β signaling to diminish corneal fibrosis. Glucosamine (GlcN), which is widely used in the treatment of osteoarthritis, abrogates the morphologic effects of TGF-β2 on retinal pigmented epithelial cells in a mouse disease model. Here, we sought to determine whether GlcN would exert beneficial effects against TGF-β1-induced corneal fibrosis.
In human corneal fibroblasts (HCFs) treated with GlcN, the expression of Krüppel-like factor 4 (KLF4) and its downstream signaling effects were determined in the presence and absence of TGF-β1 using immunoblot analysis. We further explored GlcN inhibition of fibroblast-to-myofibroblast differentiation via KLF4 siRNA. The effect of cycloheximide on KLF4 protein levels with or without GlcN administration was assessed to determine whether GlcN affects the stability of the KLF4 protein.
In HCFs, GlcN induced the expression of KLF4, which regulated the maturation and maintenance of the ocular surface. GlcN partially suppressed the TGF-β1-induced expression of alpha-smooth muscle actin (α-SMA) and reduced the collagen contraction capacity in HCFs, suggesting a decrease in fibroblast-to-myofibroblast differentiation. This effect appeared to be mediated through suppression of Smad2 phosphorylation and ERK-dependent signaling. The levels of KLF4 mRNA were increased by GlcN and decreased by TGF-β1 and the TGF-β1-induced α-SMA mRNA expression was upregulated when the KLF4 gene was silenced. GlcN also appeared to stabilize the KLF4 protein, reducing its turnover in corneal fibroblasts.
These findings shed light on a novel mechanism by which GlcN suppresses TGF-β1-induced fibroblast-to-myofibroblast differentiation through the upregulation of KLF4 expression. Current strategies for treating corneal fibrosis were not effective. Elevating KLF4 levels through the use of GlcN might provide an effective alternative to alleviate the development and progression of corneal fibrosis.
转化生长因子 (TGF) 家族成员在调节角膜完整性和角膜纤维化发病机制方面发挥着重要作用。目前，尚无针对 TGF-β 信号通路的有效药物来减少角膜纤维化。葡糖胺 (GlcN) 广泛用于治疗骨关节炎，可在小鼠疾病模型中消除 TGF-β2 对视网膜色素上皮细胞的形态效应。在这里，我们试图确定 GlcN 是否会对 TGF-β1 诱导的角膜纤维化产生有益影响。
在接受 GlcN 处理的人角膜成纤维细胞 (HCF) 中，使用免疫印迹分析在存在和不存在 TGF-β1 的情况下确定 Krüppel 样因子 4 (KLF4) 的表达及其下游信号作用。我们进一步通过 KLF4 siRNA 探索 GlcN 抑制成纤维细胞向肌成纤维细胞分化。评估有无 GlcN 给药时细胞松弛素 D 对 KLF4 蛋白水平的影响，以确定 GlcN 是否影响 KLF4 蛋白的稳定性。
在 HCF 中，GlcN 诱导 KLF4 的表达，KLF4 调节眼表的成熟和维持。GlcN 部分抑制 TGF-β1 诱导的 HCF 中α-平滑肌肌动蛋白 (α-SMA) 的表达，并降低 HCF 的胶原收缩能力，表明成纤维细胞向肌成纤维细胞分化减少。这种作用似乎是通过抑制 Smad2 磷酸化和 ERK 依赖性信号传导来介导的。GlcN 增加 KLF4 mRNA 的水平，TGF-β1 降低 KLF4 mRNA 的水平，沉默 KLF4 基因时 TGF-β1 诱导的 α-SMA mRNA 表达上调。GlcN 似乎还稳定了 KLF4 蛋白，减少了角膜成纤维细胞中 KLF4 蛋白的周转。
这些发现揭示了 GlcN 通过上调 KLF4 表达抑制 TGF-β1 诱导的成纤维细胞向肌成纤维细胞分化的新机制。目前治疗角膜纤维化的策略并不有效。通过使用 GlcN 提高 KLF4 水平可能为减轻角膜纤维化的发展和进展提供一种有效的替代方法。
