Cloning and characterization of the murine β ₃ integrin gene promoter: Identification of an interleukin-4 responsive element and regulation by STAT-6

Expression of the α _vβ ₃ integrin by murine bone marrow macrophages is regulated by cytokines such as IL-4 and GM-CSF through transcriptional activation of the β ₃ subunit gene. To characterize the molecular mechanisms by which such regulation occurs, we isolated the murine β ₃ integrin promoter. To this end, we first cloned a full length β ₃ cDNA and used the 5′UTR and leader peptide coding sequence to identify genomic clones containing the β ₃ promoter region. The transcriptional start site, identified by primer extension and S1 nuclease assay, is 34 nt upstream of the translation initiation codon. A 1.1 kb fragment of the promoter region drives IL-4 responsive transcription in transiently transfected murine bone marrow macrophages. Deletion analysis of the β ₃ promoter indicates the IL-4 responsive element lies between -465 to -678 nt relative to the transcriptional start site. This promoter fragment contains two overlapping STAT consensus recognition sites and nuclear extracts from BMMs contain an IL-4-inducible DNA binding factor, identified by super shift analysis, as STAT-6. Furthermore, an oligonucleotide which includes the two STAT recognition sites residing in the IL-4 responsive region of the β ₃ promoter, competes for STAT-6 binding. Confirming IL-4 induction of the integrin subunit is specifically mediated by STAT-6, β ₃ mRNA is not enhanced in BMMs derived from STAT-6 deleted mice, which however, retain their capacity to respond to GM-CSF.