Yu Shun Jiang, Zheng Lei, Ladanyi Marc, Asa Sylvia L, Ezzat Shereen
Department of Medicine, Mount Sinai Hospital and University of Toronto, The Freeman Centre for Endocrine Oncology and The Ontario Cancer Institute, Toronto, Ontario, Canada.
Clin Cancer Res. 2004 Oct 1;10(19):6750-8. doi: 10.1158/1078-0432.CCR-04-0223.
Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of differentiating and proliferative actions. FGFR4 is expressed mainly in lung, kidney, pancreas, spleen, and developing muscle. FGFR4 was found to be overexpressed in some human malignancies, where it has been implicated in their pathogenesis. Recently, FGFR4 was found to be overexpressed in pediatric rhabdomyosarcomas, based on cDNA microarray analysis. Using Northern blotting, reverse transcription-polymerase chain reaction, and Western blotting, we classified four human rhabdomyosarcoma-derived cell lines based on their relative expression of FGFR4. We defined a 214 bp (-115/+99) promoter that functioned as a minimal promoter and examined cis-DNA elements implicated in the control of expression of the FGFR4 gene in these cells. Overlapping 40- to 50-bp fragments of the minimal promoter were examined by electrophoretic mobility shift assay using nuclear extracts from cell lines with high (HS729-1015) or low (HS729-1016) FGFR4 expression. Fragment C (-65/-26) formed specific complexes with nuclear extracts from both cell lines. Fragment B (-95/-56), however, formed distinct complexes mainly with the high FGFR4-expressing HS729-1015 cells. Both fragments yielded complexes that were competed by an Sp oligonucleotide and supershifted by Sp1 and by Sp3 antibodies. Transfection of Sp1 but not Sp3 efficiently activated FGFR4 promoter activity, an effect that was significantly more pronounced in the HS729-1015 cell line than in the low FGFR4-expressing HS729-1016 cell line. Deletion of each of the two Sp-binding sites in fragments B and C resulted in loss of promoter activity. In particular, deletion of the 5' Sp-binding site in fragment B was associated with the greatest loss of activity. Sp1 protein expression correlated with FGFR4 expression in cell lines and primary human rhabdomyosarcomas. Furthermore, transfection of Sp1 and methylation inhibition was effective in inducing the endogenous FGFR4 gene in HS729-1015 cells. Our findings point to Sp1 as an important contributor to FGFR4 transcriptional control and elucidate a potential mechanism for the heterogeneous expression of FGFR4 in neoplasms derived from the same cell lineage.
成纤维细胞生长因子受体（FGFRs）与多种分化和增殖作用有关。FGFR4主要在肺、肾、胰腺、脾脏以及发育中的肌肉中表达。研究发现FGFR4在一些人类恶性肿瘤中过度表达，并与这些肿瘤的发病机制有关。最近，基于cDNA微阵列分析发现FGFR4在儿童横纹肌肉瘤中过度表达。我们使用Northern印迹法、逆转录-聚合酶链反应和Western印迹法，根据FGFR4的相对表达对四种人横纹肌肉瘤来源的细胞系进行了分类。我们确定了一个214 bp（-115/+99）的启动子，其作为最小启动子发挥作用，并研究了与这些细胞中FGFR4基因表达调控相关的顺式DNA元件。使用来自FGFR4高表达（HS729-1015）或低表达（HS729-1016）细胞系的核提取物，通过电泳迁移率变动分析检测最小启动子重叠的40至50 bp片段。片段C（-65/-26）与两种细胞系的核提取物形成特异性复合物。然而，片段B（-95/-56）主要与FGFR4高表达的HS729-1015细胞形成不同的复合物。两个片段产生的复合物都能被Sp寡核苷酸竞争，并被Sp1和Sp3抗体超迁移。转染Sp1而非Sp3能有效激活FGFR4启动子活性，这种效应在HS729-1015细胞系中比在FGFR4低表达的HS729-1016细胞系中更明显。片段B和C中两个Sp结合位点的缺失均导致启动子活性丧失。特别是，片段B中5' Sp结合位点的缺失与活性丧失最大相关。Sp1蛋白表达与细胞系和原发性人横纹肌肉瘤中的FGFR4表达相关。此外，转染Sp1和抑制甲基化可有效诱导HS729-1015细胞中的内源性FGFR4基因。我们的研究结果表明Sp1是FGFR4转录调控的重要因素，并阐明了FGFR4在源自同一细胞谱系的肿瘤中异质性表达的潜在机制。
