Characterization of the promoter region of the porcine opn (osteopontin, secreted phosphoprotein 1) gene

Osteopontin (secreted phosphoprotein‐1, Opn) is a phosphorylated glycoprotein expressed by transformed cells, macrophages, activated T‐lymphocytes, specialized epithelial cells and bone cells that is characteristically enriched in milk and in the mineralized matrix of bone. The synthesis of Opn by bone cells is regulated by glucocorticoids and growth factors, which promote bone formation, and by the osteotropic hormone calcitriol (1,25‐dihydroxycholecalciferol) and retinoic acid, which mediate bone resorption, indicating a bifunctional role for this protein in bone remodelling. To study the transcriptional regulation of the opn gene, two genomic clones (10 and 15 kb) encoding the opn gene were isolated from a porcine liver genomic library cloned into λ phage. From the 15‐kb clone a 4‐kb Eco RI fragment containing the first two exons and 2.6 kb of the 5′ flanking region of the opn gene was sequenced, and the transcriptional start site determined by primer extension analysis and S1 nuclease mapping. To identify the opn promoter, chimeric chloramphenicol acetyltransferase constructs were prepared using fragments from the first intron and the 5′ flanking region of the opn gene. Transient transfection of porcine bone cells with these constructs showed strong promoter activity located within 74bp upstream from the transcription initiation site. Within this region a TATA sequence, TTTAAA, was identified at positions −26 to −31. However, the highest transcription rate was observed in a construct extending 180 bp upstream that included a CCGCCC Sp1 binding sequence (−63 to −68), and an AP1 site (−74 to −80). Further upstream in the 5′ flanking region and within the first intron of the opn , a number of consensus sequensus could be identified. Chimeric constructs containing a GGGTCAtatGGTTCA direct repeat consensus sequence for a vitamin D 3 response element located at nucleotides –2245 to –2259 responded to the addition of 0.1μM calcitriol by a 2.5‐fold stimulation of transcription, although a > 2‐fold increase was also observed in shorter constructs – 180 to – 905 lacking such a consensus. Promoter activity was also exhibited by a region containing a TTTAAA sequence in the first intron that corresponded to the putative promoter site reported for mouse opn in macrophages (Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S. & Yamamoto, S. (1990) J. Biol. Chem. 265 , 14432–14438). However, Primer extension and hybridization analysis of both porcine and monocyte/macrophage and bone mRNA failed to reveal an Opn mRNA transcribed from the alternative promoter, indicating that the same promter regulates transcription of the opn gene in monocytes and macrophages as well as in bone.