Characterization of human bone morphogenetic protein (BMP)-4 and-7 gene promoters: Activation of BMP promoters by Gli, a sonic hedgehog mediator

Among the bone morphogenetic protein (BMP) family, which plays a crucial ro le not only in bone formation but also in development, BMP-2, -4, and -7 pa rticipate predominantly in various aspects. To undertake complex tasks, the ir expression is strictly controlled. In this study we isolated and analyze d 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) a ssay 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 tran script with the BMP-4 proximal first exon. Likewise, RT-PCR revealed that H os and MG63 cells expressed BMP-7. Subsequent 5'-RACE confirmed an alternat ive usage of the BMP-4 first exons with clustered multiple transcription st art 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 an d renal COS-7 cells. The degree of distal and proximal BMP-4 promoter activ ity was in accordance with the expression level of the corresponding transc ripts. Both distal and proximal BMP-4 promoters possessed suppressor elemen ts that are operative only in Hos cells. The positive and negative elements identified in the BMP-7 promoter were more remarkably effective in Hos cel ls. The activities of the respective BMP-4 promoters and BMP-7 promoter wer e all stimulated upon the cotransfection of a potential sonic hedgehog (SHH ) mediator, Glil or Gli3 into COS-7 cells, providing direct evidence that t he Gli proteins are capable of inducing the BMP expression. Our systems are helpful for assessment of the complicated interactions of molecules involv ed in the skeletogenesis and developmental processes. (C) 2001 by Elsevier Science Inc. All rights reserved.