Ameloblastin upstream region contains structural elements regulating transcriptional activity in a stromal cell line derived from bone marrow

Ameloblastin (AMBN) was first detected in secretory-stage ameloblasts (1, 2) and later in the Hertwig’s epithelial root sheath (HERS) cells and in differentiating odontoblasts (3, 4). AMBN expression has more recently been detected in trauma-induced reparative dentin (5), in bone tissue during early stages of bone formation (6), in cultured mesenchymal hard tissue cells, and in precursor cells from the blood and bone marrow (7). AMBN expression is temporally and spatially restricted during tooth development (8) and diminishes with tooth eruption (9). AMBN expression tends to phase out during the differentiation of bone cells and after completion of bone remodelling (6, 7). The biological function of AMBN remains obscure, and it is now documented that AMBN expression is not restricted to enamel formation (5, 6, 10). In odontogenesis, Ambn is believed to be implicated in enamel biomineralization (11–13) and in interactions between the ameloblasts and the extracellular matrix (14–16). It has been suggested that AMBN could act as a signal molecule in epithelial–mesenchymal interactions (4, 17), and AMBN is presumed to have ‘growth factor’ activity during periodontal ligament formation and regeneration (18). The expression level of enamel proteins is several hundred times lower in odontoblasts than in secretory ameloblasts (16, 19), and the level of AMBN expression during bone formation is also much lower than during amelogenesis (6). This may argue for a possible role of AMBN as a signalling molecule involved in orchestrating cellular events, such as cell proliferation, differentiation, and mineralization, rather than as a structural protein of the extracellular matrix. This assumption is further supported by recent results showing that recombinant AMBN can induce proliferation in stem cells and osteoblasts, osteoclast differentiation, and the expression of genes involved in inflammation and repair in osteoblasts (20). Weak Ambn promoter activity has been demonstrated in mesenchymal cells (gingival and pulp fibroblasts) transfected with a previously characterized promoter (1,616 bp upstream region of the transcriptional start site) (21). The authors found that both positive and negative cis-acting regions are involved in Ambn transcriptional regulation, and demonstrated that bone-related Runt transcription factor 2 (Runx2) interacts with osteoblast-specific element 2 (OSE2) in the region upstream of the Ambn promoter in ameloblast-like cells (22). However, they concluded that the Ambn promoter is inactive in mesenchymal cells. Based on the observations that AMBN is expressed in mesenchymal tissues, we wanted to study the region further upstream of the reported Ambn promoter (21), seeking to identify potential bone-related transcriptional regulatory elements. Here we describe the cloning and activation of the 3,788-bp Ambn promoter in bone marrow-derived stromal cells, and describe potential transcriptional factor-binding sites involved in bone development, maintenance, and repair.