Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch
Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch
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DOI：
10.1167/iovs.03-0213
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发表时间：
2003-12-01
影响因子：
4.4
通讯作者：
Acott, TS
中科院分区：
文献类型：
--
作者：
Bradley, JMB;Kelley, MJ;Acott, TS
目的.小梁网（TM）基质金属蛋白酶（MMP）和组织抑制剂（TIMP）对机械拉伸的反应变化似乎是眼内压（IOP）稳态的中心。本研究旨在确定TM细胞在机械拉伸作用下MMP-2和MMP-14表达增加的信号转导途径。对猪TM细胞进行机械拉伸，并通过明胶酶谱法和Western免疫印迹分析测定MMP-2和-14水平的变化。分析信号转导通路抑制剂对MMP水平的影响。磷酸特异性抗体用于鉴定选择途径中间体中的磷酸化变化。对MMP-2和-14 mRNA的5'非翻译区（UTR）进行计算机二级结构分析。持续机械拉伸TM细胞24小时后MMP-2和-14的增加被雷帕霉素阻断。Wortmannin阻断MMP-2的增加，但不阻断MMP-14的增加。拉伸可显著增加S473和T308细胞上的蛋白激酶B（PKB）磷酸化。在p70/p85 S6激酶中T389的雷帕霉素敏感性磷酸化也增加。S209上的翻译起始因子eIF-4 E及其抑制性结合蛋白4 E-BP 1在T70上的磷酸化均被拉伸增加。MMP-2和MMP-14的mRNA的5'UTR的二级结构的计算自由能为负且相对较大。MMP-2在其UTR的5'端也有嘧啶区。TM MMP-2和-14蛋白水平响应于机械拉伸的增加似乎至少部分由mTOR（雷帕霉素的哺乳动物靶标（mTOR））转导。渥曼青霉素敏感性暗示磷酸肌醇3-激酶作为MMP-2的调节剂，而不是MMP-14的增加。整合素连接激酶（ILK）、磷酸肌醇依赖性激酶（PDK-1）和PKB与MMP-2的增加有关。涉及eIF-4 E及其抑制性结合蛋白4 E-BP 1的翻译起始似乎参与MMP-2和MMP-14随拉伸的增加，并且通常受mTOR调节。这些转录物的5'UTR中的高度二级结构通常是对通过该途径的调节特别敏感的基因的指示。P70/p85 S6激酶可能在mTOR和PKB的下游参与调节MMP-2的翻译，MMP-2在其5' UTR具有嘧啶片段。这些转导途径的操纵可能提供新的治疗性IOP调节的方法。
PURPOSE. Trabecular meshwork (TM) matrix metalloproteinase (MMP), and tissue inhibitor (TIMP) changes in response to mechanical stretching appear to be central to intraocular pressure (IOP) homeostasis. Studies were conducted to define the signal transduction pathway responsible for the increases in MMP-2 and -14 that occur in response to mechanical stretching of TM cells.METHODS. Porcine TM cells were subjected to mechanical stretching, and changes in MMP-2 and -14 levels were determined by gelatin zymography and Western immunoblot analysis. Effects of signal transduction pathway inhibitors on MMP levels were analyzed. Phosphospecific antibodies were used to identify phosphorylation changes in select pathway intermediates. In silico secondary structure analysis was conducted on the 5' untranslated regions (UTRs) of MMP-2 and -14 mRNAs.RESULTS. The increases in MMP-2 and -14 that occur 24 hours after sustained mechanical stretching of TM cells were blocked by rapamycin. Wortmannin blocked the MMP-2 but not the MMP-14 increase. Protein kinase B (PKB) phosphorylation on S473 and T308 was increased significantly by stretching. Rapamycin-sensitive phosphorylation of T389 in p70/p85 S6 kinase was also increased. The phosphorylations of the translation initiation factor eIF-4E on S209 and of its inhibitory binding protein 4E-BP1 on T70 were both increased by stretch. The calculated free energies of secondary structures of the 5' UTRs of the mRNAs for MMP-2 and -14 were negative and relatively large. MMP-2 also had pyrimidine tracts in the extreme 5' region of its UTR.CONCLUSIONS. The increases in TM MMP-2 and -14 protein levels in response to mechanical stretching appear to be transduced at least in part by mTOR, the mammalian target of rapamycin (mTOR). The wortmannin sensitivity implicates phosphoinositide 3-kinase as a modulator of the MMP-2 but not the MMP-14 increase. Integrin-linked kinase (ILK), phosphoinositide-dependent kinase (PDK-1), and PKB are implicated in the MMP-2 increase. Translational initiation involving eIF-4E and its inhibitory binding protein 4E-BP1 appear to be involved in both the MMP-2 and -14 increases with stretching and are normally regulated by mTOR. The high degree of secondary structure in the 5' UTRs of these transcripts is typically an indicator of genes specifically sensitive to regulation through this pathway. P70/p85 S6 kinase is probably involved downstream from mTOR and PKB in regulating translation of MMP-2, which has pyrimidine tracts in its 5' UTR. Manipulation of these transduction pathways may provide new approaches to therapeutic IOP regulation.