Ji C, Casinghino S, McCarthy T L, Centrella M
Department of Surgery, Yale University, School of Medicine, New Haven, Connecticut 06520-8041, USA.
J Cell Biochem. 1996 Dec 15;63(4):478-90. doi: 10.1002/(SICI)1097-4644(19961215)63:4%3C478::AID-JCB9%3E3.0.CO;2-Q.
Transforming growth factor (TGF-beta) binds several discrete membrane proteins. Of these, a type 1 receptor appears indispensable for signal transduction. Previous examination of TGF-beta receptor expression has been limited to changes in cell surface protein, and more recently, mRNA abundance. In order to learn more about TGF-beta function and receptor expression during osteogenesis, we have now cloned a 4 kilobase (kb) DNA fragment 5' proximal to the coding region of the rat TGF-beta type I receptor gene. Sequence analysis revealed multiple elements compatible with transcription initiation, including a properly positioned and oriented CCAAT box, six Sp1 binding sites (three defining GC boxes), and two strong AP2 binding sites within a 0.7 kb span directly upstream of the coding region. The 3' terminal 0.3 kb span comprises a GC-enriched (77%) so-called CpG island that, like other similarly organized promoters, lacks a TATA box. Primer extension and RNase protection studies with cRNAs from this area show multiple initiation sites within 220 bp 5' proximal to the initial methionine codon. Transient transfections using nested, deleted, and inverted promoter sequences demonstrated maximal reporter expression by a 1 kb fragment encompassing all of these elements. Truncation of the 1 kb fragment from the 5' and 3' ends indicated the need for several elements for peak promoter activity. These results, and transfections in fetal rat bone and dermal cells, suggest that this promoter contains elements that specify basal and conditional expression of the TGF-beta type I receptor in bone.
转化生长因子（TGF-β）与几种不同的膜蛋白结合。其中，1型受体似乎对信号转导必不可少。先前对TGF-β受体表达的研究仅限于细胞表面蛋白的变化，以及最近的mRNA丰度变化。为了更多地了解成骨过程中TGF-β的功能和受体表达，我们现在克隆了大鼠TGF-β 1型受体基因编码区5'近端的一个4千碱基（kb）DNA片段。序列分析揭示了多个与转录起始兼容的元件，包括一个位置和方向合适的CCAAT框、六个Sp1结合位点（三个定义GC框），以及在编码区上游直接的0.7 kb跨度内的两个强AP2结合位点。3'末端的0.3 kb跨度包含一个富含GC（77%）的所谓CpG岛，与其他类似组织的启动子一样，它缺乏TATA框。对该区域的cRNAs进行引物延伸和RNase保护研究表明，在起始甲硫氨酸密码子5'近端的220 bp内有多个起始位点。使用嵌套、缺失和反向启动子序列进行的瞬时转染表明，包含所有这些元件的1 kb片段能使报告基因表达最大化。从5'和3'末端截断1 kb片段表明，启动子活性峰值需要几个元件。这些结果以及在胎鼠骨和真皮细胞中的转染表明，该启动子包含在骨中指定TGF-β 1型受体基础表达和条件表达的元件。
