Kawai S, Sugiura T
Laboratory for Bone Research, Discovery Research Laboratories, Hoechst Marion Roussel, Ltd., Kawagoe, Saitama, Japan.
Bone. 2001 Jul;29(1):54-61. doi: 10.1016/s8756-3282(01)00470-7.
Among the bone morphogenetic protein (BMP) family, which plays a crucial role not only in bone formation but also in development, BMP-2, -4, and -7 participate predominantly in various aspects. To undertake complex tasks, their expression is strictly controlled. In this study we isolated and analyzed the 5'-flanking regions of the human BMP-4 and -7 genes to elucidate the mechanism of their temporally and spatially specific expression. As for BMP-4 expression, a reverse transcription-polymerase chain reaction (RT-PCR) assay with specially designed sets of primers demonstrated that osteoblastic SaOS-2 and Hos cells expressed two types of transcripts comprising one of the 5'-untranslated first exons, whereas MG63 cells displayed only the transcript with the BMP-4 proximal first exon. Likewise, RT-PCR revealed that Hos and MG63 cells expressed BMP-7. Subsequent 5'-RACE confirmed an alternative usage of the BMP-4 first exons with clustered multiple transcription start sites in the distal exon and the sole start site in the proximal exon. The transcription start site of the BMP-7 gene was found to be far upstream (764 bp) of the initiation ATG codon. We constructed a series of deletion mutants of fusions between these BMP promoters and the luciferase gene and examined their activity by transient transfection into osteoblastic Hos and renal COS-7 cells. The degree of distal and proximal BMP-4 promoter activity was in accordance with the expression level of the corresponding transcripts. Both distal and proximal BMP-4 promoters possessed suppressor elements that are operative only in Hos cells. The positive and negative elements identified in the BMP-7 promoter were more remarkably effective in Hos cells. The activities of the respective BMP-4 promoters and BMP-7 promoter were all stimulated upon the cotransfection of a potential sonic hedgehog (SHH) mediator, Gli1 or Gli3 into COS-7 cells, providing direct evidence that the Gli proteins are capable of inducing the BMP expression. Our systems are helpful for assessment of the complicated interactions of molecules involved in the skeletogenesis and developmental processes.
在不仅在骨形成而且在发育中起关键作用的骨形态发生蛋白（BMP）家族中，BMP-2、-4和-7主要参与各个方面。为了承担复杂的任务，它们的表达受到严格控制。在本研究中，我们分离并分析了人BMP-4和-7基因的5'侧翼区域，以阐明它们在时间和空间上特异性表达的机制。关于BMP-4表达，用专门设计的引物组进行的逆转录-聚合酶链反应（RT-PCR）分析表明，成骨细胞SaOS-2和Hos细胞表达两种类型的转录本，其中包含一个5'非翻译的第一个外显子，而MG63细胞仅显示带有BMP-4近端第一个外显子的转录本。同样，RT-PCR显示Hos和MG63细胞表达BMP-7。随后的5'-RACE证实了BMP-4第一个外显子的替代使用，在远端外显子中有多个成簇的转录起始位点，而在近端外显子中有唯一的起始位点。发现BMP-7基因的转录起始位点在起始ATG密码子上游很远（764 bp）。我们构建了一系列这些BMP启动子与荧光素酶基因之间融合的缺失突变体，并通过瞬时转染成骨细胞Hos和肾COS-7细胞来检测它们的活性。远端和近端BMP-4启动子活性的程度与相应转录本的表达水平一致。远端和近端BMP-4启动子都具有仅在Hos细胞中起作用的抑制元件。在BMP-7启动子中鉴定出的正负元件在Hos细胞中更显著有效。当将潜在的音猬因子（SHH）介质Gli1或Gli3共转染到COS-7细胞中时，各个BMP-4启动子和BMP-7启动子的活性均受到刺激，提供了Gli蛋白能够诱导BMP表达的直接证据。我们的系统有助于评估参与骨骼发生和发育过程的分子之间复杂的相互作用。
