TGFβ/Smad4-Dependent and -Independent Regulation of Human Lens Epithelial Cells
TGFβ/Smad4-Dependent and -Independent Regulation of Human Lens Epithelial Cells
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DOI：
10.1167/iovs.08-3223
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发表时间：
2009-11-01
影响因子：
4.4
通讯作者：
Wormstone, Ian Michael
中科院分区：
文献类型：
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作者：
Dawes, Lucy Jean;Sleeman, Matthew Alexander;Wormstone, Ian Michael
目的。转化生长因子 (TGF)-β 可以通过 Smad4 依赖性和非依赖性信号通路介导纤维化反应。 TGFβ诱导的晶状体上皮细胞向肌成纤维细胞的转分化和基质收缩有助于白内障术后后囊膜混浊（PCO）。本研究的主要目的是确定 Smad4 在 TGF beta 2 诱导的人晶状体上皮细胞转分化、基质收缩和 Smad 信号转导中的重要性。方法。使用人晶状体细胞系 FHL 124。 Smad4 敲低是通过使用 siRNA 技术实现的，并分别通过实时 PCR 和蛋白质印迹在信息和蛋白质水平上测试效率。通过免疫细胞化学测定 TGF β 2 刺激 (10 ng/mL) 后的 Smad4 和 Smad2/3 核分布。在存在和不存在 TGF beta 2 (10ng/mL) 的情况下，通过实时 PCR 测定转分化标记物、α-平滑肌肌动蛋白和纤连蛋白以及抑制性 Smad、Smad7 的基因表达。通过贴片收缩测定评估TGFβ2诱导的收缩。使用悬浮珠阵列技术来测定 2 小时内对 TGF beta 2 反应的 pERK、pP38 和 pJNK 水平。 结果。 siRNA 靶向 Smad4，在初始转染期和去除转染条件后成功降低了 Smad4 信息和蛋白的表达。这些结果通过 Smad4 的免疫细胞化学得到了证实，结果显示，在存在和不存在 10 ng/mL TGF beta 2 的情况下，相对于对照 siRNA，siSmad4 处理后核 Smad4 显着减少。Smad4 敲低细胞表现出 TGF beta 2 诱导的 Smad2/3 核转位，与表达 Smad4 的细胞没有显着差异。实时 PCR 分析显示，在表达 Smad4 的细胞系（对照 siRNA 组）中，α SMA 和纤连蛋白转录物显着诱导。这些诱导在 Smad4 敲除细胞中受到抑制。 FHL 124 细胞中 TGF β 诱导的 mSmad7 表达不受 Smad4 敲低的影响。在存在或不存在 10 ng/mL TGF beta 2 的情况下，表达 Smad4 的细胞在培养 24 小时后并未表现出显着的收缩反应。然而，在 TGF beta 2 处理后，Smad4 敲低细胞表现出斑块面积显着减少（即收缩）。珠阵列分析表明，相对于未刺激的对照，TGF beta 2 显着增加了 pERK 和 pP38 水平。 pJNK 没有观察到显着变化。结论。 Smad4 对于转分化标记物、α SMA 和纤连蛋白的表达至关重要；相反，Smad4 信号传导对于 TGF β 诱导的 Smad7 表达并不是必需的。 Smad4 似乎不是 TGF β 诱导的基质收缩所必需的。 TGF beta 确实激活人晶状体上皮细胞中不依赖于 Smad 的通路。因此，TGF β 2 对转分化和基质收缩的调节似乎是由不同的 TGF β 信号通路介导的。 （投资眼科可见科学。2009；50：5318-5327）DOI：10.1167/iovs.08-3223
PURPOSE. Transforming growth factor (TGF)-beta can mediate fibrotic responses via Smad4-dependent and-independent signaling pathways. TGF beta-induced transdifferentiation of lens epithelial cells to myofibroblasts and matrix contraction contribute to posterior capsule opacification (PCO) after cataract surgery. The primary objective of the study was to determine the importance of Smad4 in TGF beta 2-induced transdifferentiation, matrix contraction, and Smad signaling by human lens epithelial cells.METHODS. The human lens cell line FHL 124 was used. Smad4 knockdown was achieved by using siRNA technology and efficiency tested at the message and protein level by real-time PCR and Western blots, respectively. Smad4 and Smad2/3 nuclear distribution after TGF beta 2 stimulation (10 ng/mL) was determined by immunocytochemistry. Gene expression of transdifferentiation markers, alpha-smooth muscle actin and fibronectin and the inhibitory Smad, Smad7, in the presence and absence of TGF beta 2 (10ng/mL) was determined with real-time PCR. TGF beta 2-induced contraction was assessed with a patch contraction assay. Suspended bead array technology was used to determine pERK, pP38, and pJNK levels in response to TGF beta 2 over a 2-hour time course.RESULTS. SiRNA targeted against Smad4, successfully reduced expression of Smad4 message and protein after both the initial transfection period and removal of transfection conditions. These findings were confirmed by immunocytochemistry for Smad4, which showed a significant reduction in nuclear Smad4 after siSmad4 treatment relative to control siRNA in both the presence and absence of 10 ng/mL TGF beta 2. Smad4 knockdown cells exhibited TGF beta 2-induced Smad2/3 nuclear translocation which did not differ significantly from Smad4-expressing cells. Real-time PCR analysis showed significant induction of alpha SMA and fibronectin transcript in the Smad4-expressing cell line (control siRNA group). These inductions were suppressed in Smad4-knockdown cells. TGF beta-induced mSmad7 expression in FHL 124 cells was unaffected by Smad4 knockdown. Smad4-expressing cells did not exhibit a significant contractile response after 24 hours of culture in the presence or absence of 10 ng/mL TGF beta 2. However, Smad4-knockdown cells demonstrated a significant reduction in patch area (i.e., contraction), after TGF beta 2 treatment. Bead array analysis showed that TGF beta 2 significantly increased pERK and pP38 levels relative to unstimulated control. No significant change was observed with pJNK.CONCLUSIONS. Smad4 is critical for the expression of transdifferentiation markers, alpha SMA, and fibronectin; in contrast, Smad4 signaling is not essential for TGF beta-induced Smad7 expression. Smad4 does not appear to be necessary for TGF beta-induced matrix contraction. TGF beta does activate Smad-independent pathways in human lens epithelial cells. Therefore, TGF beta 2 regulation of transdifferentiation and matrix contraction appears to be mediated by distinct TGF beta signaling pathways. (Invest Ophthalmol Vis Sci. 2009;50:5318-5327) DOI:10.1167/iovs.08-3223