Parathyroid hormone regulation of bone sialoprotein (BSP) gene transcription is mediated through a pituitary-specific transcription factor-1 (Pit-1) motif in the rat BSP gene promoter.

Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. Parathyroid hormone (PTH), which regulates serum calcium through its actions on bone cells, increases the expression of BSP in the rat osteosarcoma cell line (ROS 17/2.8). At 10(-8) M PTH (human 1-34 PTH), stimulation of BSP mRNA was first evident at 3 h ( approximately 3.8-fold), reached maximal levels at 6 h ( approximately 4.7-fold), and declined slowly thereafter. The effects of PTH, which were abrogated by cycloheximide (28 microg/ml), did not alter the stability of the BSP mRNA. The increased transcription was mimicked by both forskolin (10(-6) M) and isoproterenol (10(-7) M), and was also increased by 3-isobutyl-1-methylxanthine (IBMX; 10(-5) M), while the transcriptional activity induced by PTH was inhibited by the protein kinase A inhibitor, H89 (5x10(-6) M). From transient transfection assays using various BSP promoter-luciferase constructs, a pituitary-specific transcription factor-1 (Pit-1) regulatory element (nts -111 to -105) was identified as the target of transcriptional activation by PTH. Thus, transcriptional activity of constructs including the Pit-1 was enhanced approximately 4.7-fold by 10(-8) M PTH while 5'-ligation of the Pit-1 element conferred PTH regulation in an SV40 promoter construct. Binding of a nuclear protein, recognized by anti-Pit-1 antibodies, to a radiolabelled Pit-1-BSP probe was decreased in nuclear extracts prepared from PTH, forskolin and isoproterenol-stimulated ROS 17/2.8 cells. Moreover, co-transfection of ROS cells with a double-stranded Pit-1 oligonucleotide also increased luciferase activity. Collectively, these results indicate that PTH acts through a protein kinase A pathway involving cAMP to stimulate BSP transcription by blocking the action of a Pit-1-related nuclear protein that suppresses BSP transcription by binding a cognate element in the BSP promoter. Thus, we have identified a novel Pit-1 suppressor element in the rat BSP gene promoter that is the target of PTH-stimulated transcription of the BSP gene.