Cloning of the Murine β5 Integrin Subunit Promoter

Abstract

We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin alphav beta5 and that granulocyte-macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the beta5 gene. We herein report cloning of the beta5 integrin gene promoter and identification of a GM-CSF-responsive sequence. A 13-kilobase (kb) genomic fragment containing part of the beta5 gene was isolated by screening a mouse genomic library with a probe derived from the most 5'-end of a murine beta5 cDNA. A combination of primer extension and S1 nuclease studies identifies two transcriptional start sites, with the major one designated +1. A 1-kb subclone containing sequence -875 to + 110 is transcriptionally active in a murine myeloid cell line. This 1-kb fragment contains consensus binding sequences for basal (Sp1), lineage-specific (PU.1), and regulatable (signal transducer and activator of transcription) transcription factors. Reflecting our earlier findings, promoter activity is repressed in transfected myeloid cells treated with GM-CSF. Using deletion mutants, we localized a 109-base pair (bp) promoter region responsible for GM-CSF-inhibited beta5 transcription. We further 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 19-bp sequence not only ablates its capacity to bind nuclear proteins from GM-CSF-treated cells, in vitro, but the same mutation, when introduced in the 1-kb promoter, abolishes its ability to respond to GM-CSF treatment. Northern analysis demonstrates that cycloheximide treatment abrogates the capacity of GM-CSF to decrease beta5 mRNA levels. In summary, we have identified a 19-bp cis-element mediating GM-CSF-induced down-regulation of beta5 by a mechanism requiring protein synthesis.