Endocrine Abstracts

Particulate guanylyl cyclases are expressed in all endocrine organs and serve as specific receptors of the natriuretic peptides ANP, BNP and CNP. Guanylyl cyclase-B (GC-B/Npr2) receptors specifically mediate the biological effects of CNP in its target tissues. Targeted disruption of either CNP or GC-B results in pituitary growth hormone deficiency and dwarfism as a result of achondroplasia. Despite this, molecular regulation of GC-B expression is poorly elucidated. Therefore, the aim of this study was to characterise the activity of the human GC-B receptor promoter in αT3-1 and LβT2 gonadotrophs and GH3 somatotrophs. A range of GC-B receptor promoter deletion constructs were prepared, and transiently transfected by calcium phosphate and lipofection methods in order to investigate activity in specific regions of the promoter. In αT3-1 and LβT2 gonadotroph cells the highest activity was seen in the −441 base pair (bp) promoter construct, with 5.2±1.5-fold and 4.1±1.1-fold increases in promoter activity over pGL3LUC control (in αT3-1 and LβT2 cells, respectively). In GH3 somatotroph cells the −214 bp promoter construct was most active with 9.3±2.1-fold increase over pGL3LUC. To establish whether the human GC-B promoter was responsive to hormone treatment, similar transfection experiments were performed except transfected cells were treated with 100 nM GnRH (αT3-1 cells) or 100 nM TRH (GH3 cells) for up to 24 h. GnRH treatment significantly stimulated −214GC-B-LUC and −2129GC-B-LUC by 2.8±0.3-fold and 2.5±0.3-fold over the relevant basal. In GH3 cells, TRH caused a 4.7±0.5-fold increase in promoter activity over basal −214GC-B-LUC activity. In silico analysis of the proximal −214 to −441 bp of the GC-B receptor promoter revealed the presence multiple Sp1 and Sp3 transcription factor binding, suggesting a role for these factors in basal and hormone-dependent GC-B gene transcription. These data suggest common and distinct mechanisms control transcription of the human GC-B promoter in gonadotroph and somatotroph lineage cells. Funded by a BBSRC Project Grant (BBD0015601).