肿瘤坏死因子与白细胞介素-1在小梁网中诱导基质金属蛋白酶-3的协同作用。

Synergism of TNF and IL-1 in the induction of matrix metalloproteinase-3 in trabecular meshwork.

作者信息

Kelley Mary J, Rose Anastasia Y, Song Kaili, Chen Yanwen, Bradley John M, Rookhuizen Derek, Acott Ted S

机构信息

Casey Eye Institute, Oregon Health and Science University, Portland, Oregon 97239-4197, USA.

出版信息

Invest Ophthalmol Vis Sci. 2007 Jun;48(6):2634-43. doi: 10.1167/iovs.06-1445.

DOI:10.1167/iovs.06-1445

PMID:17525194

Abstract

PURPOSE

TNF and IL-1 increase matrix metalloproteinase-3 (MMP-3) expression in the trabecular meshwork (TM). TNF-alpha, in combination with IL-1alpha or IL-1beta, produces highly synergistic MMP-3 increases. Possible mechanisms for this synergism in TM cells were investigated.

METHODS

Porcine and human TM cells were treated with TNF-alpha, IL-1alpha, IL-1beta and their combinations. Western immunoblots were used to evaluate MMP-3, MMP-9, MMP-12, TNF-alpha, IL-1alpha, IL-1beta, IL-6, TNF receptor I (RI), IL-1 RI, and IL-1 RII levels and the phosphorylation of Erk, JNK, and p38 MAP kinases. Dose-response effects for TNF-alpha, IL-1alpha and IL-1beta on MMP-3 were evaluated. Microarray and quantitative RT-PCR were used to determine mRNA levels. MMP-3 transcription rate was assessed by transfecting TM cells with an MMP-3 promoter/reporter construct. Combined cytokine effects on outflow facility were appraised in perfused anterior segment organ culture.

RESULTS

TNF-alpha, IL-1alpha, and IL-1beta each individually increased MMP-3 levels, whereas TNF-alpha in combination with IL-1alpha or IL-1beta produced highly synergistic increases. MMP-9 and MMP-12 levels were also elevated, but only MMP-12 showed synergism. IL-1alpha, IL-1beta, and IL-6, but not TNF-alpha mRNA or protein level, were elevated by these cytokines. Maximum MMP-3 production for individual cytokines, even at high doses, was far less than with dual cytokine doses. Erk 1 and 2, JNK 1 and 2, and p38 alpha and beta phosphorylation increased, but not synergistically. However, phosphorylation of novel isoforms of JNK and p38 delta and gamma did show synergism. MMP-3 mRNA levels and transcription rates also demonstrated synergism. TNF-alpha significantly increased IL-1 RI levels. Synergism in outflow facility was observed with TNF-alpha and IL-1alpha.

CONCLUSIONS

TNF-alpha, in combination with IL-1alpha or IL-1beta, produced intense synergistic increases in MMP-3 and MMP-12 but not in MMP-9. Induction of IL-1 RI by TNF-alpha partially explains the synergism. Responses of novel JNK and p38 MAP kinase delta and gamma isoforms also partially account for the synergism. Understanding this strong synergistic effect may provide useful insight into optimizing therapeutic regulation of intraocular pressure in glaucoma.

摘要

目的

肿瘤坏死因子（TNF）和白细胞介素-1（IL-1）可增加小梁网（TM）中基质金属蛋白酶-3（MMP-3）的表达。TNF-α与IL-1α或IL-1β联合使用时，会产生高度协同的MMP-3增加效应。本研究探讨了TM细胞中这种协同作用的可能机制。

方法

用TNF-α、IL-1α、IL-1β及其组合处理猪和人的TM细胞。采用蛋白质免疫印迹法评估MMP-3、MMP-9、MMP-12、TNF-α、IL-1α、IL-1β、IL-6、TNF受体I（RI）、IL-1 RI和IL-1 RII的水平以及细胞外信号调节激酶（Erk）、应激活化蛋白激酶（JNK）和p38丝裂原活化蛋白激酶（MAPK）的磷酸化情况。评估TNF-α、IL-1α和IL-1β对MMP-3的剂量反应效应。利用基因芯片和定量逆转录聚合酶链反应（RT-PCR）测定mRNA水平。通过用MMP-3启动子/报告基因构建体转染TM细胞来评估MMP-3转录率。在灌注的眼前节器官培养中评估细胞因子联合作用对房水流出易度的影响。

结果

TNF-α、IL-1α和IL-1β各自单独作用时均可增加MMP-3水平，而TNF-α与IL-1α或IL-1β联合使用时则产生高度协同的增加效应。MMP-9和MMP-12水平也升高，但只有MMP-12表现出协同作用。这些细胞因子可升高IL-1α、IL-1β和IL-6水平，但不影响TNF-α的mRNA或蛋白质水平。即使高剂量的单一细胞因子诱导产生的MMP-3产量也远低于双细胞因子剂量诱导产生的产量。Erk 1和2、JNK 1和2以及p38α和β的磷酸化增加，但无协同作用。然而，JNK和p38δ和γ新亚型的磷酸化确实表现出协同作用。MMP-3的mRNA水平和转录率也表现出协同作用。TNF-α可显著增加IL-1 RI水平。在TNF-α和IL-1α联合作用下观察到房水流出易度的协同作用。

结论

TNF-α与IL-1α或IL-1β联合使用时，可使MMP-3和MMP-12产生强烈的协同增加，但对MMP-9无此作用。TNF-α诱导IL-1 RI可部分解释这种协同作用。新型JNK和p38 MAPKδ和γ亚型的反应也部分说明了这种协同作用。了解这种强烈的协同效应可能为优化青光眼眼内压的治疗调控提供有用的见解。