通过不同的β1和β3整合素途径汇聚对人小梁网（HTM）细胞中交联肌动蛋白网络（CLAN）形成的调节
Regulation of cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells by convergence of distinct beta1 and beta3 integrin pathways.
作者信息
Filla Mark S, Schwinn Marie K, Sheibani Nader, Kaufman Paul L, Peters Donna M
机构信息
Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
出版信息
Invest Ophthalmol Vis Sci. 2009 Dec;50(12):5723-31. doi: 10.1167/iovs.08-3215. Epub 2009 Jul 30.
PURPOSE
To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM.
METHODS
Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2.
RESULTS
HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it.
CONCLUSIONS
Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
目的
确定β1/β3整合素介导的调节人小梁网（HTM）细胞中交联肌动蛋白网络（CLAN）形成的信号通路。CLAN在青光眼和类固醇处理的小梁网细胞中形成，这可能导致小梁网房水流出易度降低。
方法
通过流式细胞术评估CD47（αvβ3整合素共受体/血小板反应蛋白-1受体）以及整合素αvβ3和β1的表达。在不存在或存在β3整合素激活单克隆抗体AP-5的情况下，将细胞接种于纤连蛋白（一种β1整合素配体）上诱导CLAN形成，并用鬼笔环肽标记进行鉴定。通过用抑制剂PP2和EPA（Src激酶）、LY294002（PI-3激酶）或NSC23766（Rac1）处理细胞来确定Src激酶、PI-3激酶（PI-3K）、Rac1和CD47的作用。使用Tiam1和Trio小干扰RNA以及显性负性Tiam1来确定涉及哪种Rac1特异性鸟嘌呤核苷酸交换因子。使用血小板反应蛋白-1衍生的激动剂肽4N1K和CD47功能阻断抗体B6H12.2来确定CD47的作用。
结果
HTM细胞表达CD47以及整合素αvβ3和β1。β3整合素或CD47激活显著增加CLAN形成，使其超过β1整合素诱导的水平，而抗CD47单克隆抗体B6H12.2抑制这种增加。PP2、NSC23766和Trio小干扰RNA分别使β3诱导的CLAN形成减少72%、45%和67%，而LY294002和显性负性Tiam1没有作用。LY294002使β1整合素介导的CLAN形成减少42%，而PP2完全阻断它。
结论
不同的β1和αvβ3整合素信号通路汇聚以增强CLAN形成。β1介导的CLAN形成依赖于PI-3K，而β3介导的CLAN形成依赖于CD47和Rac1/Trio，可能受血小板反应蛋白-1调节。两条整合素信号通路均依赖于Src。
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