CLONING, CHARACTERIZATION, AND EXPRESSION OF THE TRANSFORMING GROWTH-FACTOR-BETA TYPE-I RECEPTOR PROMOTER IN FETAL-RAT BONE-CELLS

Transforming growth factor (TGF-beta) binds several discrete membrane proteins. Of these, a type I receptor appears indispensable for signal transduction. Previous examination of TGF-beta receptor expression ha s been limited to changes in cell surface protein, and more recently, mRNA abundance. In order to learn more about TGF-beta function and rec eptor 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 co mpatible with transcription initiation, including a properly positione d and oriented CCAAT box, six Spl binding sites (three defining CC box es), and two strong AP2 binding sites within a 0.7 kb span directly up stream of the coding region. The 3' terminal 0.3 kb span comprises a G C-enriched (77%) so-called CpG island that, like other similarly organ ized promoters, lacks a TATA box. Primer extension and RNase protectio n studies with cRNAs from this area show multiple initiation sites wit hin 220 bp 5' proximal to the initial methionine codon. Transient tran sfections using nested, deleted, and inverted promoter sequences demon strated maximal reporter expression by a 1 kb fragment encompassing al l of these elements. Truncation of the 1 kb fragment from the 5' and 3 ' ends indicated the need for several elements for peak promoter activ ity. These results, and transfections in fetal rat bone and dermal cel ls, suggest that this promoter contains elements that specify basal an d conditional expression of the TGF-beta type I receptor in bone. (C) 1996 Wiley-Liss, Inc.