Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells
Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells
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DOI：
10.1016/j.exer.2009.01.008
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发表时间：
2009-06-01
影响因子：
3.4
通讯作者：
Fuchshofer, Rudolf
中科院分区：
文献类型：
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作者：
Junglas, Benjamin;Yu, Alice H. L.;Fuchshofer, Rudolf
患有原发性开角型青光眼 (POAG) 的人眼小梁网 (TM) 的主要结构变化是 TM 近小管区域细胞外基质 (ECM) 的增加。有证据表明，TGF-β2 治疗可诱导培养的人 TM 细胞中 ECM 沉积，并且 TGF-β2 与 POAG 中 JCT ECM 增加有关。在本研究中，我们研究了结缔组织生长因子 (CTGF) 对培养的人类 TM 细胞生物学的影响。 CTGF 是 TGF-β2 信号传导的下游介质，在人 TM 中原位大量表达。用含有人CTGF编码序列的真核表达质粒转染HEK293细胞。通过色谱法分离和纯化分泌的CTGF。原代人TM细胞与浓度为2.5-100 ng/ml的CTGF一起孵育24小时。用 CTGF 处理后，通过实时 RT-PCR 和蛋白质印迹分析研究了 JCT 中表达的各种 ECM 成分、基质金属蛋白酶 (MMP) 和整合素的表达。此外，通过明胶酶谱法研究了 MMP 的活性。通过在 TGF-β 2 处理前用 CTGF 特异性小干扰 (si) RNA 转染永生化 HTM 细胞，研究 CTGF 沉默对 TGF-β 2 诱导的基因表达的影响。 CTGF处理的人TM细胞显示纤连蛋白、I型、III型、IV型和VI型胶原蛋白以及整合素亚基aV和β1的表达增加。较低浓度的CTGF引起CTGF表达的自诱导。未观察到对 MMP-2、MMP-9 和纤溶酶原激活剂抑制剂-1 (PAI-1) 的表达和活性的影响。用 CTGF 特异性 siRNA 转染可抑制 TGF-β 2 诱导的 CTGF 和纤连蛋白上调。我们的结果表明，用重组 CTGF 处理人 TM 细胞会引起基因表达的明显变化，并且 CTGF 是 TGF-β 2 对人 TM 细胞 ECM 合成影响的关键介质。本研究数据支持的一个有趣的方面是，CTGF 的药理调节可能是开发新的治疗策略的有用方法，以预防或逆转 POAG 眼 TM 中发生的结构变化。 (C) 2008 Elsevier Ltd. 保留所有权利。
The major structural change in the human trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG) is an increase in extracellular matrix (ECM) in the juxtacanalicular region of the TM. There is evidence that treatment with TGF-beta 2 causes an induction of ECM deposition in cultured human TM cells and that TGF-beta 2 is causatively involved in the JCT ECM increase in POAG. In the present study, we investigated the effects of connective tissue growth factor (CTGF) on the biology of cultured human TM cells. CTGF is a downstream mediator of TGF-beta 2-signaling, which is expressed at high amounts in the human TM in situ. HEK293 cells were transfected with an eukaryotic expression plasmid containing the coding sequences of human CTGF. Secreted CTGF was isolated and purified by chromatography. Primary human TM cells were incubated for 24 h with CTGF at concentrations of 2.5-100 ng/ml. Following treatment with CTGF, the expression of various ECM components that are expressed in the JCT, matrix metal loproteinases (MMPs) and integrins was investigated by real-time RT-PCR and western blot analyses. In addition, the activity of MMPs was investigated by gelatine zymography. The effect of CTGF silencing on TGF-beta 2-induced gene expression was investigated by transfection of immortalized HTM cells with CTGF-specific small interfering (si)RNA before TGF-beta 2 treatment. CTGF-treated human TM cells showed an increase in the expression of fibronectin, collagen types I, III, IV and VI, as well as in the integrin subunits aV and beta 1. Lower concentrations of CTGF caused an autoinduction of CTGF expression. No effects were observed on the expression and activity of MMP-2, MMP-9 and plasminogen activator inhibitor-1 (PAI-1). Transfection with CTGF-specific siRNA inhibited the TGF-beta 2-induced upregulation of CTGF and fibronectin. Our results indicate that treatment of human TM cells with recombinant CTGF causes distinct changes in gene expression and that CTGF is a critical mediator of the effects of TGF-beta 2 on ECM synthesis in human TM cells. An intriguing aspect supported by the data of the present work is that the pharmacologic modulation of CTGF might be a useful approach to develop novel therapeutic strategies to prevent or to reverse the structural changes that occur in the TM of eyes with POAG. (C) 2008 Elsevier Ltd. All rights reserved.