A role for CCN3 (NOV) in calcium signalling
A role for CCN3 (NOV) in calcium signalling
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DOI：
10.1136/mp.55.4.250
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发表时间：
2002-08-01
期刊：
影响因子：
--
通讯作者：
Perbal, B
中科院分区：
文献类型：
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作者：
Li, CL;Martinez, V;Perbal, B
目的：在动物和人类中，在钙是关键调节剂的组织中检测到CCN 3（NOV）的表达增加，所述组织例如肾上腺、中枢神经系统、骨和软骨、心肌和肾脏。由于CCN蛋白的多模块结构强烈表明，这些细胞生长调节剂是金属蛋白，本研究探讨了CCN 3在离子通量和运输过程中可能发挥的作用，在发展中，控制细胞增殖，分化，和pathobiology.Methods：CCN 3的合作伙伴的分离进行了通过双杂交系统。酵母共转染HL 60 cDNA文库融合到GAL 4转录因子的反式激活结构域，并与表达CCN 3融合到GAL 4的DNA结合结构域的质粒。用β-半乳糖苷酶试验筛选基于亮氨酸、组氨酸和色氨酸原养型选择的重组克隆。在CCN 3与其分子相互作用后，用GST（谷胱甘肽S-转移酶）pull down试验检测推定的配偶体，通过克隆鉴定阳性克隆。为了确定CCN 3蛋白是否影响钙离子通量，使用动态成像显微镜系统，其允许荧光测量细胞内钙浓度。在测定中使用的蛋白质是GST与CCN 3或CCN 2（CTGF）融合的蛋白质，并且单独的GST作为对照。双杂交系统鉴定了S100A4（mts 1）钙结合蛋白作为CCN 3的伴侣，并且GST融合蛋白的使用表明，将CCN 3和CCN 2添加到G59胶质母细胞瘤和SK-N-胶质母细胞瘤中。SH神经母细胞瘤细胞引起细胞内钙的显著但短暂的增加，源于细胞外钙的进入和细胞内储存的动员。CCN 3与S100 A4的相互作用可能部分解释了CCN 3与癌发生及其在正常条件下的表达模式的相关性。CCN 3和CCN 2引起的细胞内钙离子浓度升高涉及不同的过程，其中电压非依赖性钙通道可能在调节与细胞生长控制、运动和铺展相关的钙流中具有相当重要的作用。这些观察结果首次赋予CCN 3蛋白生物学功能，并指出CCN蛋白在钙离子信号传导中具有更广泛的作用。
Aims: In animals and humans increased expression of CCN3 (NOV) is detected in tissues where calcium is a key regulator, such as the adrenal gland, central nervous system, bone and cartilage, heart muscle, and kidney. Because the multimodular structure of the CCN proteins strongly suggests that these cell growth regulators are metalloproteins, this study investigated the possible role of CCN3 in ion flux and transport during development, control of cell proliferation, differentiation, and pathobiology.Methods: The isolation of CCN3 partners was performed by means of the two hybrid system. Yeasts were cotransfected with an HL60 cDNA library fused to the transactivation domain of the GAL4 transcription factor, and with a plasmid expressing CCN3 fused to the DNA binding domain of GAL4. Screening of the recombinant clones selected on the basis of leucine, histidine, and tryptophan prototrophy was performed with a beta-galactosidase assay. After the interaction between CCN3 and its . putative partners was checked with a GST (glutathione S-transferase) pull down assay, the positive clones were identified by cloning. To establish whether the CCN3 protein affected calcium ion flux, a dynamic imaging microscopy system was used, which allowed the fluorometric measurement of the, intracellular calcium concentration. The proteins used in the assays were GST fused with either CCN3 or CCN2 (CTGF) and GST alone as a control.Results: The two hybrid system identified the S100A4 (mts 1 ) calcium binding protein as a partner of CCN3 and the use of the GST fusion proteins showed that the addition of CCN3 and CCN2 to G59 glioblastoma and SK-N-SH neuroblastoma cells caused a pronounced but transient increase of intracellular calcium, originating from both the entry of extracellular calcium and the mobilisation of intracellular stores.Conclusions: The interaction of CCN3 with S100A4 may account, in part, for the association of CCN3 with carcinogenesis and its pattern of expression in normal conditions. The increased intracellular calcium concentrations induced by CCN3 and CCN2 both involve different processes, among which voltage independent calcium channels might be of considerable importance in regulating the calcium flux associated with cell growth control, motility, and spreading. These observations assign for the first time a biological function to the CCN3 protein and point out a broader role for the CCN proteins in calcium ion signalling.