Characterization of an element within the rat parathyroid hormone/parathyroid hormone-related peptide receptor gene promoter that enhances expression in osteoblastic osteosarcoma 17/2.8 cells.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) mediate their actions via a common G-protein-coupled receptor. High levels of PTH/PTHrP receptor expression have been detected in many tissues including bone and kidney. This study has demonstrated specific PTH/PTHrP receptor expression from the U3 promoter in the osteoblastic osteosarcoma ROS 17/2.8 cell line, which expresses the endogenous PTH/PTHrP receptor, compared to rat 2 fibroblasts which do not express the endogenous PTH/PTHrP receptor gene. Transient transfection studies revealed cell-specific expression of a construct containing 4391 bp of DNA upstream of exon U3 of the PTH/PTHrP receptor gene fused to a luciferase reporter gene. Deletion mapping of the 5' region of U3 revealed that a construct containing 206 bp upstream of U3 confers cell-specific expression. These data suggest that cell-specific expression in ROS 17/2.8 involves cell-specific elements within the PTH/PTHrP receptor promoter.     

Analysis of the promoter region of the human FcRn gene

The 5'-flanking region of the human FcRn alpha-chain gene was analyzed for its ability to directly express the chloramphenicol acetyltransferase (CAT) reporter gene in NIH3T3 and Lu106 cells. Transient transfection of the CAT constructs revealed that there was promoter activity in the region -660 to +300 of the 5'-flanking sequence. Electrophoretic mobility-shift assays showed that there are functional binding sites for Sp1 or Sp1-like factors, AP1 or a related factor, and additional unidentified proteins in the promoter region.     

Recent studies on the biological action of parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor in cartilage and bone

Mice with a targeted deletion of parathyroid hormone (PTH)-related peptide (PTHrP) develop a form of dyschondroplasia resulting from diminished proliferation and premature maturation of chondrocytes. Abnormal, heterogeneous populations of chondrocytes at different stages of differentiation were seen in the hypertrophic zone of the mutant growth plate. Although the homozygous null animals die within several hours of birth, mice heterozygous for PTHrP gene deletion reach adulthood, at which time they show evidence of osteopenia. Therefore, PTHrP appears to modulate cell proliferation and differentiation in both the pre and post natal period. PTH/PTHrP receptor expression in the mouse is controlled by two promoters. We recently found that, while the downstream promoter controls PTH/PTHrP receptor gene expression in bone and cartilage, it is differentially regulated in the two tissues. 1alpha,25-dihydroxyvitamin D3 downregulated the activity of the downstream promoter in osteoblasts, but not in chondrocytes, both in vivo and in vitro. Most of the biological activity of PTHrP is thought to be mediated by binding of its amino terminus to the PTH/PTHrP receptor. However, recent evidence suggests that amino acids 87-107, outside of the amino terminal binding domain, act as a nucleolar targeting signal. Chondrocytic cell line, CFK2, transfected with wild-type PTHrP cDNA showed PTHrP in the nucleoli as well as in the secretory pathway. Therefore, PTHrP appears to act as a bifunctional modulator of both chondrocyte proliferation and differentiation, through signal transduction linked to the PTH/PTHrP receptor and by its direct action in the nucleolus.     

Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells. Roles of the oct-1 element in the 5'-flanking region and the AP-2 element in the untranslated exon 1

The human hematopoietic prostaglandin D synthase (H-PGDS) gene is highly expressed in human megakaryoblastic cells, in which phorbol ester induces its expression. We characterized the promoter activity of the 5'-flanking region and the untranslated exon 1 (-1044 to +290) of the human H-PGDS gene in human megakaryoblastic Dami cells. Transient expression analysis using the luciferase reporter gene revealed that the 5'-flanking region and the untranslated exon 1 were sufficient for efficient expression of the H-PGDS gene in Dami cells, but not in monocytic U937 cells. Deletion and site-directed mutagenesis of the Oct-1 element in the 5'-flanking region decreased the promoter activity by approximately 30% compared with that of the entire region from -1044 to +290. An electrophoretic mobility shift assay demonstrated that Oct-1 specifically bound to the promoter region. Interestingly, even only untranslated exon 1 (+1 to +290) showed approximately 60% of the promoter activity of the entire region from -1044 to +290. Site-directed mutagenesis of the AP-2 element within the untranslated exon 1 abolished the basal promoter activity as well as its phorbol ester-mediated up-regulation. In AP-2-deficient HepG2 cells, the H-PGDS promoter activity was enhanced by coexpression with AP-2alpha. These findings indicate that the Oct-1 element in the 5'-flanking region acts as a positive cis-acting element and that the AP-2 element in the untranslated exon 1 is crucial for both basal and phorbol ester-mediated up-regulation of human H-PGDS gene expression in megakaryoblastic Dami cells.     

Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.