"Functional mapping of the promoter region of the GNB2L1 human gene coding for RACK1 scaffold protein"
"Functional mapping of the promoter region of the GNB2L1 human gene coding for RACK1 scaffold protein"
复制标题
DOI：
10.1016/j.gene.2008.10.005
复制
发表时间：
2009-02-01
期刊：
影响因子：
3.5
通讯作者：
Racchi, Marco
中科院分区：
文献类型：
--
作者：
Del Vecchio, Igor;Zuccotti, Annalisa;Racchi, Marco
RACK1（激活 C 激酶 1 受体）是不同激酶和膜受体的支架蛋白。之前，我们描述了 RACK1 蛋白表达随年龄的下降，可以用 DHEA（脱氢表雄酮）来抵消 [Corsini, E., et al. 2017]。 2002. 体内脱氢表雄酮可恢复与年龄相关的蛋白激酶 C 信号转导途径缺陷和相关功能反应。 J.免疫学。 168，1753-1758。和 Corsini，E. 等人。 2005.人类白细胞中RACK-1表达与年龄相关的下降与血浆脱氢表雄酮水平相关。 J.洛科克。生物。 77、247-256。]。假设 DHEA 直接控制 RACK1 表达，我们研究了其编码基因 GNB2L1 的尚未表征的人类启动子区域。 FLOE（荧光标记寡核苷酸延伸）用于绘制转录起始位点和新型 Gateway 荧光素酶载体（GW luc basic；Del Vecchio. I.、Zuccotti, A.、Canneva, F.、Lenzken, S.C.、Racchi, M., 2007。开发第一个 Gateway 萤火虫荧光素酶载体并在 FLOE（荧光标记）中使用逆转录酶寡核苷酸延伸)反应。质粒58, 269-274.)以获得启动子区域突变体。使用人SH-SY5Y、THP1和类淋巴母细胞进行瞬时转染，并用脂多糖（LPS）、肉豆蔻酸佛波酯（PMA）、DHEA和皮质醇（前两种分子不同地激活NF-kB，一种能够调节小鼠Gnb2l1基因表达的转录复合物，而DHEA和皮质醇，因为它们已知在衰老过程中不平衡并对免疫功能具有抵消作用）进行治疗。引物延伸证明存在两个替代转录起始位点，分别位于 GNB2L1 Genbank mRNA 条目的 5' 约 230 和 300 nt。此外，作为荧光素酶研究的结果，我们能够证明大约 300 nt 的小区域保留了足够的报告基因表达元件。我们还报道了 GNB2L1 内源表达的 DHEA 调节不能用荧光素酶测定来重现。事实上，启动子通过 LPS 和 PMA 处理而受到显着调节，但不使用 DHEA。不同的是，皮质醇的使用使我们证明，只有在糖皮质激素核受体的结合位点存在的情况下，荧光素酶活性才会在生物学上显着降低。有趣的是，在计算机上鉴定了转录因子的其他结合位点：不同的 c-Rel (NF-kB) 和一些心肌炎特异性顺式作用元件。所有这些数据表明，DHEA 介导的 GNB2L1 调节是由远程元件（增强子/沉默子）调节的，而 LPS、PMA 和皮质醇效应可以直接作用于映射的 GNB2L1 启动子。总之，我们假设衰老过程中 DHEA 和皮质醇之间的不平衡对于先前证明的 RACK1 表达的恢复可能很重要。 (C) 2008 Elsevier B.V. 保留所有权利。
RACK1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. Previously, we characterized an age-dependent decline of RACK1 protein expression which could be counteracted with DHEA (dehydroepiandrosterone) [Corsini, E., et al. 2002. In vivo dehydroepiandrosterone restores age-associated defects in the protein kinase C signal transduction pathway and related functional responses. J. Immunol. 168,1753-1758. and Corsini, E., et al. 2005. Age-related decline in RACK-1 expression in human leukocytes is correlated to plasma levels of dehydroepiandrosterone. J. Leukoc. Biol. 77, 247-256.]. Hypothesizing a direct control of RACK1 expression by DHEA we studied the not yet characterized human promoter region of its coding gene GNB2L1. The FLOE (Fluorescently Labeled Oligonucleotide Extension) was used to map the transcription start site and a novel Gateway luciferase vector (GW luc basic; Del Vecchio. I., Zuccotti, A., Canneva, F., Lenzken, S.C., Racchi, M., 2007. Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions. Plasmid 58, 269-274.) to obtain promoter region mutants. Human SH-SY5Y, THP1 and lymphoblastoid cells were used for transient transfections and treatments with lipopolysaccharide (LPS), phorbol myristate acetate (PMA), DHEA and cortisol (the first two molecules to differently activate NF-kB, a transcription complex able to regulate the murine Gnb2l1 gene expression, whereas DHEA and cortisol since they are known to be imbalanced during the aging and possess counteracting actions on the immune function). The primer extension demonstrated the existence of two alternative start sites of transcription respectively located at about 230 and 300 nt 5' of the Genbank mRNA entry for GNB2L1. Moreover, as a result of the luciferase study we were able to demonstrate that a little region of approximately 300 nt conserved sufficient elements for reporter expression. We also reported that the DHEA modulation of GNB2L1 endogenous expression could not be recapitulated with the luciferase assays. Indeed, the promoter was significantly modulated by means of LPS and PMA treatments but not using DHEA. Differently the use of cortisol led us to demonstrate a biologically significant decrease of luciferase activity only in the presence of a binding site for nuclear receptors of glucocorticoids. Interestingly, other binding sites for transcriptional factors were identified in silico: different c-Rel (NF-kB) and some cardiomyocitic specific cis-acting elements. All this data suggest that the DHEA mediated GNB2L1 regulation is modulated by distant elements (enhancers/silencers), whereas LPS, PMA and cortisol effect can act directly on the mapped GNB2L1 promoter. In conclusion we hypothesize that the imbalance between DHEA and cortisol during aging could be important in the previously demonstrated recovery of the RACK1 expression. (C) 2008 Elsevier B.V. All rights reserved.