Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork.
Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork.
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DOI：
10.1167/iovs.16-19610
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发表时间：
2016-11-01
影响因子：
4.4
通讯作者：
Zode GS
中科院分区：
文献类型：
--
作者：
Kasetti RB;Phan TN;Millar JC;Zode GS
小梁网 (TM) 中细胞外基质 (ECM) 的异常积累与 POAG 中房水流出设施减少和眼压升高有关。此前，我们通过表达人类突变体肌纤蛋白（MYOC）（一种已知的 POAG 遗传原因）开发了 POAG 转基因小鼠模型（Tg-MYOCY437H）。本研究的目的是检查突变型肌纤蛋白的表达是否会导致 Tg-MYOCY437H 小鼠和培养的人 TM 细胞的流出能力减少和异常 ECM 积累。使用回弹眼压计测量不同年龄的 Tg-MYOCY437H 小鼠的清醒眼压。在 10 个月大的 Tg-MYOCY437H 小鼠中测量了流出能力。通过实时 PCR、蛋白质印迹和免疫染色，在稳定表达突变肌纤蛋白的人 TM-3 细胞和原代人 TM 细胞 (n = 4) 以及 Tg-MYOCY437H 小鼠的 TM 中检查选定的 ECM 蛋白。此外，用 5 mM 4-苯基丁酸钠 (PBA) 处理表达 WT 或突变型肌纤蛋白的 TM 细胞，并通过蛋白质印迹和免疫染色检查 ECM 蛋白。从 3 个月大开始，Tg-MYOCY437H 小鼠与野生型 (WT) 同窝小鼠相比表现出显着的 IOP 升高。 Tg-MYOCY437H 小鼠的流出能力显着降低（Tg-MYOCY437H 小鼠为 0.0195 μl/min/mm Hg，而 WT 同窝小鼠为 0.0332 μl/min/mm Hg）。与 WT 同窝小鼠相比，在 Tg-MYOCY437H 小鼠的 TM 中观察到纤连蛋白、弹性蛋白以及 IV 型和 I 型胶原蛋白的积累增加。此外，ECM 蛋白的增加还与 Tg-MYOCY437H 小鼠 TM 中内质网 (ER) 应激标记物 GRP78 和 CHOP 的诱导有关。与表达WT MYOC的TM细胞相比，稳定表达DsRed标记的Y437H突变体MYOC的人TM-3细胞表现出肌纤蛋白分泌及其细胞内积累的抑制。 TM-3 细胞或人原代 TM 细胞中突变 MYOC 的表达诱导 ER 应激，并增加纤连蛋白、弹性蛋白、层粘连蛋白以及胶原 IV 和 I 的细胞内蛋白水平。此外，与表达 WT 肌纤蛋白的 TM-3 细胞相比，表达突变肌纤蛋白的 TM-3 细胞在条件培养基中表现出基质金属蛋白酶 (MMP)-2 和 MMP-9 活性形式的减少。有趣的是，细胞内积累的纤连蛋白和 I 型胶原蛋白均与突变型肌纤蛋白以及 ER 标记 KDEL 共定位，进一步表明这些蛋白质在 TM 细胞的 ER 中进行细胞内积累。此外，通过 PBA 减少 ER 应激会减少原代 TM 细胞中选定的 ECM 蛋白。这些研究表明，突变型 myocilin 会诱导 TM 细胞 ER 中 ECM 异常积累，这可能是 myocilin 相关青光眼中流出设施减少和 IOP 升高的原因。
Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 μl/min/mm Hg in Tg-MYOCY437H vs. 0.0332 μl/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.
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影响因子：
4.4
作者：
Millar, J. Cameron;Phan, Tien N.;Clark, Abbot F.
通讯作者：
Clark, Abbot F.
DOI：
10.1007/978-1-61737-998-7_4
发表时间：
2011-01-01
期刊：
PLANT IMMUNITY: METHODS AND PROTOCOLS
影响因子：
--
作者：
Elmore, James M.;Coaker, Gitta
通讯作者：
Coaker, Gitta
影响因子：
4.4
作者：
Gobeil, S;Rodrigue, MA;Raymond, V
通讯作者：
Raymond, V
影响因子：
4.4
作者：
Aroca-Aguilar, Jose-Daniel;Sanchez-Sanchez, Francisco;Escribano, Julio
通讯作者：
Escribano, Julio
影响因子：
3.4
作者：
Fuchshofer, R;Welge-Lussen, U;Lütjen-Drecoll, E
通讯作者：
Lütjen-Drecoll, E