Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line:: effects upon signal transduction and cellular proliferation
Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line:: effects upon signal transduction and cellular proliferation
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DOI：
10.1016/j.yjmcc.2005.11.014
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发表时间：
2006-05-01
影响因子：
5
通讯作者：
Re, Richard N.
中科院分区：
文献类型：
--
作者：
Cook, Julia L.;Mills, Sarah J.;Re, Richard N.
本研究的目的是确定血管细胞内与细胞外血管紧张素II-AT（1）受体相互作用的功能结果。用编码大鼠血管紧张素II（pECFP/AII，编码融合在增强的青色荧光蛋白下游的AII）和大鼠AT 1 α受体（pAT（1）R/EYFP，编码融合在增强的黄色荧光蛋白上游的大鼠AT（1 α）受体）的荧光融合蛋白的质粒，单独和同时转染大鼠血管平滑肌细胞系A10。全荧光融合蛋白不具有分泌信号肽，并且建立了主要在细胞核中维持在这些细胞内的去卷积显微镜。当AT（1）R/EYFP单独表达或在未处理的细胞中表达时，细胞核中不存在AT（1）R/EYFP，但在外源性All处理后或与ECFP/AII共表达时，AT（1）R/EYFP在细胞核中积累。此外，ECFP/AII的表达促进A10血管平滑肌细胞（VSMCs）增殖1.6倍（P < 0.05）。转染对照pECFP/AII（C）（其编码与ECFP融合的乱序A11肽）没有生长效应。鉴于ECFP/AII的细胞内生长效应，我们试图阐明潜在的信号通路。我们发现，细胞外的所有治疗A10细胞激活cAMP反应元件结合蛋白（CREB）确定的单杂交试验和免疫印迹。细胞内ECFP/AII的表达类似地激活CREB。然而，细胞内和细胞外的所有激活CREB通过不同的磷酸化途径。通过Western印迹分析和单杂交测定，A10细胞的外源性All处理激活了p38 MAPK和ERK 1/2磷酸化。p38 MAPK抑制剂SB 203580和ERK激酶抑制剂PD 98059各自部分抑制外源性A11赋予的CREB活化，证实p38 MAPK和ERK 1/2在该系统中介导CREB磷酸化。相反，在A10 VSMCs中ECFP/AII（细胞内A11）的表达激活p38 MAPK，但不激活ERK 1/2; SB 203580抑制p38 MAPK抑制细胞内A11诱导的CREB磷酸化。总之，细胞外AII刺激细胞内AII共有的至少一种途径。在外源性ALL的情况下，这一共同途径可能反映了受体配体复合物内化后的细胞内信号传导。细胞外ALL还刺激独特的途径，显然反映了与质膜相关ATIR的相互作用。(c)2006爱思唯尔有限公司保留所有权利。
The objective of the study was to identify the functional outcome of intracellular versus extracellular angiotensin II-AT(1) receptor interactions in vascular cells. Rat vascular smooth muscle cell line A10 was transfected, independently and concurrently, with plasmids encoding fluorescent fusion proteins of rat angiotensin II (pECFP/AII, encodes AII fused downstream of enhanced cyan fluorescent protein) and the rat AT,, receptor (pAT(1)R/EYFP, encodes the rat AT(1a) receptor fused upstream of enhanced yellow fluorescent protein). The All fluorescent fusion protein possesses no secretory signal peptide and deconvolution microscopy established that is maintained within these cells predominantly in the nucleus. AT(1)R/EYFP was absent from the nucleus when expressed exclusively or in untreated cells but accumulated in the nucleus following exogenous All treatment or when co-expressed with ECFP/AII. Furthermore, expression of ECFP/AII Stimulated proliferation of A10 vascular smooth muscle cells (VSMCs) 1.6-fold (P < 0.05). Transfection of a control, pECFP/AII(C) (which encodes a scrambled All peptide fused to ECFP) had no growth effect. In light of the intracellular growth effects of ECFP/AII, we sought to elucidate the underlying signaling pathways. We found that extracellular All treatment of A10 cells activated cAMP response element-binding protein (CREB) as determined by one-hybrid assays and immunoblots. Expression of intracellular ECFP/AII similarly activated CREB. However, intracellular and extracellular All activated CREB through different phosphorylation pathways. Exogenous All treatment of A10 cells activated p38MAPK and ERK1/2 phosphorylation as determined by Western blot analyses and one-hybrid assays. The p38MAPK inhibitor, SB203580, and the ERK kinase inhibitor, PD98059 each partially inhibited exogenous All-conferred CREB activation confirming that p38MAPK and ERK1/2 mediate CREB phosphorylation in this system. In contrast, expression of ECFP/AII (intracellular All) in A 10 VSMCs activated p38MAPK but not ERK1/2; inhibition of p38MAPK by SB203580 inhibited intracellular All-induced CREB phosphorylation. In summary, extracellular All stimulates at least one pathway common to intracellular AII. This common pathway, in the case of exogenous All, likely reflects intracellular signaling following internalization ofreceptorligand complex. Extracellular All also stimulates a unique pathway, apparently reflecting interaction with plasma membrane-associated ATIR. (c) 2006 Elsevier Ltd. All rights reserved.