Parathyroid hormone (PTH) binds to its receptor on osteoblasts to regulate gene transcription primarily through the elevation of the second messenger cAMP. A number of genes regulated by PTH in osteoblasts contain GC-rich and Sp-binding sites. Osterix (Osx, Sp7) is a transcription factor required for the differentiation of osteoblasts that can bind to Sp-binding sites on gene promoters and regulate their expression. Here, we report the effect of PTH (1–34) on Osx expression in osteoblastic UMR-106-01 cells and murine calvaria. PTH (1–34) and PTH (1–31) inhibited Osx mRNA and protein expression, and this effect could be mimicked by forskolin, 8-bromo-cAMP, or expression of constitutively active Gsα (caGsα). Treatment of the cells with PTH (3–34) or the EPAC-selective agonist 8CPT-2Me-cAMP had no effect on Osx mRNA, whereas PTH (7–34) or expression of caGqα-stimulated Osx mRNA levels. PTH (1–34) treatment did not require new protein synthesis and did not involve changes in Osx mRNA stability. Osx promoter fragments coupled to a luciferase reporter were inhibited by PTH (1–34) treatment in a similar manner to the inhibition of Osx mRNA and protein. Deletion analysis localized PTH inhibition to two regions flanking the Osx1 start site; −304/−119 and −71/+91. These results demonstrate that prolonged exposure to PTH inhibits Osx expression in osteoblasts through sites on its proximal promoter and this suppression occurs through PTH stimulation of cellular cAMP.
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Shara H H Hong, Xianghuai Lu, Mark S Nanes, and Jane Mitchell
D S Kim, J H Yoon, S K Ahn, K E Kim, R H Seong, S H Hong, K Kim, K Ryu, and S D Park
Our previous studies demonstrated that at least two DNA regions with upstream limits between positions −223 to −190 and positions −151 to −135 of the human TSH gene are important for transcriptional regulation by TRH in GH3 rat pituitary cells. The proximal region (−151 to −135 bp) including the cAMP-responsive element (CRE) was required for the induction of the TSH gene by TRH, while the distal region (−223 to −190 bp) containing an element similar to the binding site for the pituitary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear protein, in addition to the CRE-binding protein, is involved in the molecular mechanism of TRH regulation, a gel retardation assay and Southwestern blot analysis were performed on the distal region with GH3 cell nuclear extracts. GH3 extracts generated a distinct DNA—protein complex that was effectively eliminated in the presence of excess unlabelled DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat prolactin gene inhibited formation of the complex, but mutation of the Pit-1 consensus sequence in the distal region did not eliminate the complex. In addition, Southwestern experiments showed that a 33 kDa nuclear protein present in GH3 cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maximal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also resulted in a loss of binding affinity. Taken together, these data indicate that the interaction of a pituitary-specific nuclear protein, identical or closely related to Pit-1, with the distal region may be involved in the TRH stimulation of human TSH gene expression.