Dexamethasone-Associated Cross-Linked Actin Network Formation in Human Trabecular Meshwork Cells Involves β3 Integrin Signaling
Dexamethasone-Associated Cross-Linked Actin Network Formation in Human Trabecular Meshwork Cells Involves β3 Integrin Signaling
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DOI：
10.1167/iovs.10-6618
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发表时间：
2011-05-01
影响因子：
4.4
通讯作者：
Peters, Donna M.
中科院分区：
文献类型：
--
作者：
Filla, Mark S.;Schwinn, Marie K.;Peters, Donna M.
目的.为了确定地塞米松（DEX）处理的人小梁网（HTM）细胞中形成的交联肌动蛋白网络（CLANs）是否与β 3整合素激活后形成的那些在结构上相似并且涉及α v β 3整合素信号传导。使用两种HTM细胞株和α v β 3整联蛋白过表达永生化TM细胞系。将DEX-或乙醇-处理的HTM细胞铺在具有或不具有β 3整联蛋白活化mAb AP-5的纤连蛋白上。免疫荧光显微术用于鉴定鬼笔环肽标记的CLAN，并确定其中α-辅肌动蛋白、PIP 2和多配体蛋白聚糖-4的存在。使用PI 3-激酶（LY 294002）或Rac 1（NSC 23766）抑制剂测定β 3整联蛋白信号传导参与。α v β 3整联蛋白表达水平和β 3整联蛋白活化状态通过荧光激活细胞分选仪分析和免疫荧光显微镜测定。与DEX治疗或β 3整联蛋白活化相关的CLAN含有多配体蛋白聚糖-4、PIP 2和α-辅肌动蛋白。在不存在mAb AP-5的情况下，LY 294002不影响DEX相关的CLAN形成，而NSC 23766使CLAN阳性细胞的百分比降低80%。在mAb AP-5的存在下，两种抑制剂都降低了DEX相关的CLAN形成。与对照细胞相比，DEX预处理使β 3整合素诱导的CLAN形成增加近6倍，α v β 3整合素表达和活化水平增加3倍。在DEX处理的细胞中，活化的β 3整合素阳性粘附增加了近5倍。α v β 3整合素在TM-1细胞中的过度表达使CLAN形成增加两倍。DEX相关的CLAN在结构上与mAb AP-5诱导的CLAN相似，并且涉及α v β 3整联蛋白的表达和活化增加。因此，糖皮质激素诱导的CLAN形成可能涉及HTM细胞中增强的β 3整合素信号传导，可能是通过由内而外的信号传导机制。（Invest Ophthalmol维斯科学。2011;52：2952-2959）DOI：10.1167/iovs.10-6618
PURPOSE. To determine whether cross-linked actin networks (CLANs) formed in dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells are structurally similar to those formed after beta 3 integrin activation and involve alpha v beta 3 integrin signaling.METHODS. Two HTM cell strains and an alpha v beta 3 integrin-overexpressing immortalized TM cell line were used. DEX- or ethanol-retreated HTM cells were plated on fibronectin with or without beta 3 integrin-activating mAb AP-5. Immunofluorescence microscopy was used to identify phalloidin-labeled CLANs and to ascertain the presence of alpha-actinin, PIP 2, and syndecan-4 within them. beta 3 Integrin signaling involvement was determined using a PI3-kinase (LY294002) or Rac1 (NSC23766) inhibitor. alpha v beta 3 Integrin expression levels and the beta 3 integrin activation state were determined by fluorescence-activated cell sorter analysis and immunofluorescence microscopy.RESULTS. CLANs associated with either DEX treatment or beta 3 integrin activation contained syndecan-4, PIP 2, and alpha-actinin. In the absence of mAb AP-5, LY294002 did not affect DEX-associated CLAN formation, whereas NSC23766 decreased the percentage of CLAN-positive cells by 80%. In the presence of mAb AP-5, both inhibitors decreased DEX-associated CLAN formation. DEX pretreatment increased beta 3 integrin-induced CLAN formation nearly sixfold and the level of alpha v beta 3 integrin expression and activation threefold compared with control cells. Activated beta 3 integrin-positive adhesions increased nearly fivefold in DEX-treated cells. alpha v beta 3 Integrin overexpression in TM-1 cells increased CLAN formation twofold.CONCLUSIONS. DEX-associated CLANs were structurally similar to those induced by mAb AP-5 and involved both increased expression and activation of alpha v beta 3 integrins. Thus, glucocorticoid-induced CLAN formation may involve enhanced beta 3 integrin signaling in HTM cells, possibly by an inside-out signaling mechanism. (Invest Ophthalmol Vis Sci. 2011;52:2952-2959) DOI:10.1167/iovs.10-6618