Structural analysis and characterization of tissue and hormonal responsive expression of the avian bone sialoprotein...

Bone sialoprotein (BSP) is an extracellular matrix protein that has a highly restricted expression to mineralized skeletal tissues. The chicken bone sialoprotein-encoding gene (bsp) was isolated and shown to contain two less exons than similar mammalian genes, with the absence of an untranslated 5' exon and the fusion of the first two exons that encode the signal peptide and amino terminal end of the mature BSP peptide. Primer extension analysis showed one strong transcriptional start point (tsp) in mRNA prepared from embryonic bone. Comparison of the avian bsp promoter sequence to those of other genes expressed in vertebrate skeletal tissues, identified the presence of homeobox protein binding sequence motifs for engrailed (en-1) and Msx 2 (Hox 8.1), and two collagen type II gene silencer elements. Two TATA sequences one at -21 bp and the second at -172 bp to the tsp were identified. For the first TATA element no CCAAT sequence was observed at an appropriate cis position however two Sp1 sequences (GGGCGG) were identified at -66 and -85 bp. A CCAAT element was seen in an appropriate cis position in relationship to the second upstream TATA, but transient expression analysis in embryonic chicken calvaria osteoblasts using two separate promoter/reporter constructs (+24 to -1244 bp or -121 to -1244 bp), confirmed that only the proximal TATA and Sp1 elements were functional. The +24 to -1244 bp promoter sequence demonstrated 33.6, 13.2, and 3.2 fold activity above base line respectively, within cells prepared from embryonic chicken calvaria bone, cephalic sterna, a cartilage that undergoes mineralization and caudal sterna, a cartilage that does not mineralize during embryogenesis. Only base line activity was observed within cells prepared from embryonic dermal fibroblasts a non-skeletal tissue, which does not express BSP. These same cells demonstrated comparable steady state mRNA levels, corroborating that this segment of promoter DNA had tissue specific activity. A series of nested deletions from the 5' end of the -1244 construct demonstrated that a portion of the tissue specific regulation was controlled by the presence of a silencer element(s) between -1244 and -620 bp since deletion of this segment of DNA resulted in a 6 fold increase in the promoter activity in dermal skin fibroblasts. The -1244-(+)24 nt promoter construct was shown to be stimulated by dexamethasome approximately 1.5 fold over control, inhibited by 1,25(OH)2D3 approximately 60% of control and was strongly stimulated approximately 5.0 fold by parathyroid hormone (PTH) in embryonic calvaria osteoblasts. These data define the proximal promoter of the avian bsp gene and identify several potential regulatory elements that have been observed in the promoters of other genes expressed in skeletal tissues. These elements imparted both tissue and hormone specific promoter activity to bsp expression within skeletal cells.