Activation of Bone Sialoprotein Gene Transcription by Amelogenin

Objectives: Amelogenins constitute 90% of the extracellular matrix secreted by ameloblasts, and the proteins are cleaved in a regulated process during enamel maturation. Recently, porcine enamel matrix derivative (EMD) have been used clinically for periodontal regenerative treatment, and the activities were attributed to amelogenin and growth factors. While EMD is believed to induce periodontal regeneration, the precise mechanism is not known. Bone sialoprotein (BSP), an early phenotypic marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation. In this study, we examined the ability of amelogenin to regulate BSP gene transcription. Methods: To determine the molecular basis of the transcriptional regulation of BSP expression by amelogenin, we conducted Northern hybridization, luciferase analyses and gel mobility shift assays. Results: Treatment of ROS 17/2.8 cells with recombinant amelogenin (1 μg/ml) for 12h resulted in 2.4-fold increases in the levels of BSP mRNA. In transient transfection analyses, amelogenin (1 μg/ml, 12h) increased luciferase activity of the construct, pLUC3, which encompasses nucleotides -116 to +60 transfected into ROS 17/2.8 cells. Included in the DNA sequence that is unique to pLUC3 (nts -116 to -43) are an inverted CCAAT box (nts -50 to -46), a cAMP response element (CRE; nts -75 to -68), a FGF2 response element (FRE; nts -92 to -85) and a pituitary-specific transcription factor-1 motif (Pit-1; nts -111 to -105). Gel mobility shift assays with radiolabeled FRE ds-oligonucleotides revealed increased binding of nuclear proteins from amelogenin treated ROS 17/2.8 cells. When the inverted CCAAT, CRE and Pit-1 were used as probes, DNA-protein complexes were unaffected following stimulation by amelogenin. Conclusions: These studies have, therefore, identified amelogenin response element in the rat BSP gene promoter is FRE that may mediate the effects of amelogenin on BSP gene transcription.