Translocation of calcium-permeable TRPV2 channel to the podosome: Its role in the regulation of podosome assembly
Translocation of calcium-permeable TRPV2 channel to the podosome: Its role in the regulation of podosome assembly
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DOI：
10.1016/j.ceca.2011.12.012
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发表时间：
2012-02-01
期刊：
影响因子：
4
通讯作者：
Kojima, Itaru
中科院分区：
文献类型：
--
作者：
Nagasawa, Masahiro;Kojima, Itaru
本研究旨在探讨TRPV 2通道在小鼠巨噬细胞系TtT/M87中的定位和功能。我们感染了编码TRPV 2的腺病毒载体，该载体在胞外结构域中标记有c-Myc。暴露于细胞表面的c-Myc表位的免疫反应性形成环状结构，该环状结构与podosome的标记物，即β-整联蛋白、桩蛋白和Pyk 2共定位。使用全内反射荧光显微镜在TRPV 2-GFP表达细胞中也观察到环结构。添加甲酰基-Met-Leu-Phe（fMLP）增加了podosome的数量并增加了与podosome相关的TRPV 2信号的强度。质膜下游离钙浓度（[Ca ~（2+）]（pm））的测定显示，在足状体周围[Ca ~（2+）]（pm）升高。fMLP进一步增加该区域的[Ca 2 +]（pm），这被TRPV 2抑制剂钌红消除。在fMLP处理的细胞中检测到磷酸化Pyk 2，并且TRPV 2的敲低降低磷酸化Pyk 2的表达。引入显性负性Pyk 2或敲低TRPV 2增加了podosome的数量。相反，通过添加离子霉素升高[Ca 2 +]（pm）减少了podosome的数量。这些结果表明，TRPV 2大量定位于足状体中，并通过足状体增加[Ca 2 +]（pm）。[Ca 2 +]（pm）的升高对于调节足体的组装是至关重要的。(C)2011爱思唯尔有限公司保留所有权利。
The present study was conducted to investigate localization and function of TRPV2 channel in a mouse macrophage cell line, TtT/M87. We infected an adenovirus vector encoding TRPV2 tagged with c-Myc in the extracellular domain. Immunoreactivity of c-Myc epitope exposed to the cell surface formed a ring structure, which was colocalized with markers of the podosome, namely beta-integrin, paxillin and Pyk2. The ring structure was also observed in TRPV2-GFP-expressing cells using total internal reflection fluorescent microscopy. Addition of formyl-Met-Leu-Phe (fMLP) increased the number of podosome and increased the intensity of the TRPV2 signal associated with the podosome. Measurement of subplasmalenmal free calcium concentration ([Ca2+](pm)) revealed that [Ca2+](pm) was elevated around the podosome. fMLP further increased [Ca2+](pm) in this region, which was abolished by a TRPV2 inhibitor ruthenium red. Phosphorylated Pyk2 was detected in fMLP-treated cells, and knockdown of TRPV2 reduced the expression of phospho-Pyk2. Introduction of dominant-negative Pyk2 or knockdown of TRPV2 increased the number of podosome. Conversely, elevation of [Ca2+](pm) by the addition of ionomycin reduced the number of podosome. These results indicate that TRPV2 is localized abundantly in the podosome and increases [Ca2+](pm) by the podosome. The elevation of [Ca2+](pm) is critical to regulate assembly of the podosome. (C) 2011 Elsevier Ltd. All rights reserved.