Armoni Michal, Quon Michael J, Maor Gila, Avigad Smadar, Shapiro David N, Harel Chava, Esposito Diana, Goshen Yaacov, Yaniv Isaac, Karnieli Eddy
Institute of Endocrinology, Diabetes and Metabolism, Rambam Medical Center, Technion-Israel Institute of Technology, Haifa 31096, Israel.
J Clin Endocrinol Metab. 2002 Nov;87(11):5312-24. doi: 10.1210/jc.2002-020318.
Increased levels of glucose uptake and increased expression of the glucose transporter (GLUT) genes are characteristic features of tumors. In the muscle-derived tumor alveolar rhabdomyosarcoma (ARMS), a chromosomal translocation t(2:13) generates the PAX3/forkhead homolog in rhabdomyosarcoma (FKHR) oncoprotein. In muscle tissues, glucose transport is primarily mediated by GLUT4. However, the mechanisms that regulate GLUT4 gene expression in tumor tissues are largely unknown. Therefore, we evaluated the role of PAX3/FKHR in the regulation of GLUT4 gene expression in muscle tumorigenesis. GLUT4 mRNA and protein were detected in ARMS-derived human biopsies and in ARMS-derived RH30 myoblasts, which both express the PAX3/FKHR chimeric protein, but not in either C2C12 or embryonal rhabdomyosarcoma-derived myoblasts. GLUT4 was functionally active in RH30 cells, because insulin induced a 1.4-fold stimulation of basal 2-deoxyglucose uptake rates. Coexpression of PAX3/FKHR increased basal transcriptional activity from a GLUT4 promoter reporter (GLUT4-P) in C2C12, SaOS-2, and Chinese hamster ovary-K1 cells in a dose-dependent and tissue-specific manner. PAX3/FKHR mutants with deletions in either the homeodomain (DeltaHD) or the FKHR-derived activation domain (DeltaFKHR), or in which the PAX3-derived paired domain (PD) was point-mutated (PD-R56L), were unable to activate GLUT4-P. Progressive 5'-deletion analysis of GLUT4-P further identified a specific region of the promoter, -66/+163 bp, which retained about 65% of the full transactivation effect. EMSA studies established that the PAX3/FKHR protein directly and specifically binds to this region and to a shorter fragment, -4/+36 bp, that contains potential binding sites for HD and PD, but not to a -4/+36-bp fragment whose HD and PD sites have been mutated. Thus, the functional interaction of PAX3/FKHR with GLUT4-P appears to require all of the functional domains of PAX3/FKHR, as well as a -4/+36-bp region within the GLUT4 promoter. Taken together, the data suggest that the GLUT4 gene is a downstream target of PAX3/FKHR and that GLUT4 is aberrantly transactivated by this oncoprotein both in vivo and in vitro.
葡萄糖摄取水平升高以及葡萄糖转运蛋白（GLUT）基因表达增加是肿瘤的特征性表现。在源自肌肉的肿瘤肺泡横纹肌肉瘤（ARMS）中，染色体易位t(2:13)产生了横纹肌肉瘤中的PAX3/叉头同源物（FKHR）致癌蛋白。在肌肉组织中，葡萄糖转运主要由GLUT4介导。然而，调节肿瘤组织中GLUT4基因表达的机制在很大程度上尚不清楚。因此，我们评估了PAX3/FKHR在肌肉肿瘤发生过程中对GLUT4基因表达调控的作用。在源自ARMS的人类活检组织和源自ARMS的RH30成肌细胞中检测到了GLUT4 mRNA和蛋白，这两种细胞均表达PAX3/FKHR嵌合蛋白，但在C2C12或源自胚胎性横纹肌肉瘤的成肌细胞中均未检测到。GLUT4在RH30细胞中具有功能活性，因为胰岛素诱导基础2-脱氧葡萄糖摄取率增加了1.4倍。PAX3/FKHR的共表达以剂量依赖性和组织特异性方式增加了C2C12、SaOS-2和中国仓鼠卵巢-K1细胞中GLUT4启动子报告基因（GLUT4-P）的基础转录活性。在同源结构域（DeltaHD）或FKHR衍生的激活结构域（DeltaFKHR）中存在缺失，或者PAX3衍生的配对结构域（PD）发生点突变（PD-R56L）的PAX3/FKHR突变体无法激活GLUT4-P。对GLUT4-P进行逐步的5'-缺失分析进一步确定了启动子的一个特定区域，即-66/+163 bp区域，该区域保留了约65%的完全反式激活效应。电泳迁移率变动分析（EMSA）研究表明，PAX3/FKHR蛋白直接且特异性地结合到该区域以及一个较短的片段，即-4/+36 bp片段，该片段包含同源结构域（HD）和配对结构域（PD）的潜在结合位点，但不结合HD和PD位点已发生突变的-4/+36 bp片段。因此，PAX3/FKHR与GLUT4-P的功能相互作用似乎需要PAX3/FKHR的所有功能结构域以及GLUT4启动子内的-4/+36 bp区域。综上所述，这些数据表明GLUT4基因是PAX3/FKHR的下游靶点，并且GLUT4在体内和体外均被这种致癌蛋白异常反式激活。
