Cloning of the Murine β5 Integrin Subunit Promoter: IDENTIFICATION OF A NOVEL SEQUENCE MEDIATING GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR-DEPENDENT REPRESSION OF β5 INTEGRIN GENE TRANSCRIPTION

Xu Feng,Steven L Teitelbaum,Marisol E Quiroz,Dwight A Towler,F Patrick Ross

doi:10.1074/jbc.274.3.1366

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.

Highlights

We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin ␣v␤5 and that granulocyte-macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the ␤5 gene
The reciprocal changes in the two functional integrins are replicated by treating osteoclast precursors with GM-CSF,1 a cytokine that is important for osteoclastogenesis [13,14,15]
Expression of the integrin ␣v␤5 falls in parallel with appearance of ␣v␤3, events mimicked by treatment of osteoclast precursors with GM-CSF [12]

Summary

Introduction

We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin ␣v␤5 and that granulocyte-macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the ␤5 gene. We have demonstrated that murine osteoclast precursors initially express and utilize the integrin ␣v␤5 and not ␣v␤3 for attachment to matrix [12] In this circumstance, ␣v␤3 and ␣v␤5 levels rise and fall, respectively, during formation of boneresorbing polykaryons. The reciprocal changes in the two functional integrins are replicated by treating osteoclast precursors with GM-CSF, a cytokine that is important for osteoclastogenesis [13,14,15] Reflecting their surface heterodimers, ␤5 and ␤3 mRNAs fall and rise, respectively, in response to GM-CSF, whereas those of ␣v are unaltered. We identify a 19-bp sequence within the ␤5 promoter that mediates the GM-CSF responsiveness of this gene This sequence does not contain consensus sequences for any transcription factor known to be regulated by GM-CSF and, as such, represents a novel GMCSF response element

Methods

Results

Conclusion