Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA.
Invest Ophthalmol Vis Sci. 2012 Jul 26;53(8):4952-62. doi: 10.1167/iovs.12-9681.
Connective tissue growth factor (CTGF) is a matricellular protein presumed to be involved in the pathobiology of various fibrotic diseases, including glaucoma. We investigated the effects of Rho GTPase-dependent actin cytoskeletal integrity on CTGF expression and CTGF-induced changes in gene expression profile in human trabecular meshwork (HTM) cells.
CTGF levels were quantified by immunoblotting and ELISA. CTGF-induced changes in gene expression, actin cytoskeleton, myosin light chain (MLC) phosphorylation, and extracellular matrix (ECM) proteins were evaluated in trabecular meshwork (TM) cells by cDNA microarray, q-PCR, fluorescence microscopy, and immunoblot analyses. The effects of neuromedin U (NMU) on aqueous humor (AH) outflow were determined in enucleated porcine eyes.
Expression of a constitutively active form of RhoA (RhoAV14), activation of Rho GTPase by bacterial toxin, or inhibition of Rho kinase by Y-27632 in HTM cells led to significant but contrasting changes in CTGF protein levels that were detectable in cell lysates and cell culture medium. Stimulation of HTM cells with CTGF for 24 hours induced actin stress fiber formation, and increased MLC phosphorylation, fibronectin, and laminin levels, and NMU expression. NMU independently induced actin stress fibers and MLC phosphorylation in TM cells, and decreased AH outflow facility in perfused porcine eyes.
These data revealed that CTGF influences ECM synthesis, actin cytoskeletal dynamics, and contractile properties in TM cells, and that the expression of CTGF is regulated closely by Rho GTPase. Moreover, NMU, whose expression is induced in response to CTGF, partially mimics the effects of CTGF on actomyosin organization in TM cells, and decreases AH outflow facility, revealing a potentially important role for this neuropeptide in the homeostasis of AH drainage.
结缔组织生长因子（CTGF）是一种基质细胞蛋白，据推测其参与多种纤维化疾病的病理生物学过程，包括青光眼。我们研究了 Rho GTPase 依赖性细胞骨架完整性对 CTGF 表达的影响，以及 CTGF 在人眼小梁细胞（HTM）中诱导的基因表达谱变化。
通过免疫印迹和 ELISA 定量检测 CTGF 水平。通过 cDNA 微阵列、q-PCR、荧光显微镜和免疫印迹分析评估 CTGF 诱导的基因表达、细胞骨架、肌球蛋白轻链（MLC）磷酸化和细胞外基质（ECM）蛋白变化在 TM 细胞中的变化。在去眼猪眼中测定神经调节素 U（NMU）对房水（AH）流出的影响。
在 HTM 细胞中表达组成型激活型 RhoA（RhoAV14）、细菌毒素激活 Rho GTPase 或 Rho 激酶抑制剂 Y-27632 导致 CTGF 蛋白水平的显著但相反的变化，这些变化可在细胞裂解物和细胞培养基中检测到。CTGF 刺激 HTM 细胞 24 小时诱导肌动蛋白应力纤维形成，并增加 MLC 磷酸化、纤连蛋白和层粘连蛋白水平以及 NMU 表达。NMU 独立诱导 TM 细胞肌动蛋白应力纤维和 MLC 磷酸化，并降低灌注猪眼中的 AH 流出率。
这些数据表明，CTGF 影响 TM 细胞的 ECM 合成、细胞骨架动力学和收缩特性，并且 CTGF 的表达受到 Rho GTPase 的紧密调节。此外，NMU 的表达响应 CTGF 而被诱导，其部分模拟 CTGF 对 TM 细胞中肌动球蛋白组织的作用，并降低 AH 流出率，表明这种神经肽在 AH 引流的动态平衡中具有重要作用。
