Cloning of the murine beta(5) integrin subunit promoter - Identification of a novel sequence mediating granulocyte-macrophage colony-stimulating factor-dependent repression of beta(5) integrin gene transcription

We previously noted that the initial receptor by which murine osteoclast pr ecursors bind matrix is the integrin alpha(v)beta(5) and that granulocyte-m acrophage colony-stimulating factor (GM-CSF) decreases expression of this h eterodimer by suppressing transcription of the beta(5) gene. We herein repo rt cloning of the beta(5) integrin gene promoter and identification of a GM -CSF-responsive sequence. A 13-kilobase (kb) genomic fragment containing pa rt of the beta(5) gene was isolated by screening a mouse genomic library wi th a probe derived from the most 5'-end of a murine beta(5) cDNA. A combina tion of primer extension and S1 nuclease studies identifies two transcripti onal start sites, with the major one designated +1. A 1-kb subclone contain ing sequence -875 to + 110 is transcriptionally active in a murine myeloid cell line. This 1-kb fragment contains consensus binding sequences for basa l (Sp1), lineage-specific (PU.1), and regulatable (signal transducer and ac tivator of transcription) transcription factors. Reflecting our earlier fin dings, promoter activity is repressed in transfected myeloid cells treated with GM-CSF. Using deletion mutants, we localized a 109-base pair (bp) prom oter region responsible for GM-CSF-inhibited beta(5) transcription. We furt her identified a 19-bp sequence within the 109-bp region that binds GM-CSF- induced nuclear proteins by gel shift/competition assays. Mutation of the 1 9-bp sequence not only ablates its capacity to bind nuclear proteins from G M-CSF-treated cells, in vitro, but the same mutation, when introduced in th e 1-kb promoter, abolishes its ability to respond to GM-CSF treatment. Nort hern analysis demonstrates that cycloheximide treatment abrogates the capac ity of GM-CSF to decrease beta(5) mRNA levels. In summary, we have identifi ed a 19-bp cis-element mediating GM-CSF-induced down-regulation of beta(5) by a mechanism requiring protein synthesis.