SFE2004 Oral Communications Young Endocrinologist Session (7 abstracts)
C-type natriuretic peptide (CNP) is encoded by the Nppcgene and is abundant in the hypothalamus and anterior pituitary gonadotrophs. Nppc deletion leads to skeletal deformity, dwarfism, early death and female infertility. To investigate regulation of Nppcexpression and action in the hypothalamic-pituitary-gonadal axis, we investigated transcriptional regulation of the Nppc promoter in alphaT3-1 gonadotroph cells by transient transfection of reporter gene constructs, localised CNP immunoreactivity in mouse gonadotrophs by electronmicroscopy and determined the effect of CNP on GnRH and PACAP-dependent gene transcription and intracellular signalling. A 5kb, 5' upstream region of the mouse Nppcgene was specifically activated in gonadotroph alphaT3-1 cells, compared to GH3 (somatotrophs) and AtT-20 (corticotrophs) cells. Treatment with GnRH (100nM, 24h) resulted in a 3.3plus/minus0.3-fold increase in the activity of a truncated -50bp Nppc-LUC construct, an effect that was completely blocked by pre-treatment with a PKC inhibitor (GF109203X, 1uM) or a calcium channel antagonist (nifedipine, 1uM). The GC-rich region of the proximal -50bp promoter specifically bound Sp1 and Sp3 transcription factors, as homo and heterodimers, as determined by gel-shift analysis. Treatment of alphaT3-1 cells with GnRH or the PKC activator, PMA (both 100nM, 24h) led to a 2.2plus/minus0.2- and 2.7plus/minus0.2-fold increase in total (intra and extracellular) CNP content and electronmicroscopy revealed specific immunogold labelling over secretory granules in mouse gonadotrophs, using a CNP-specific antibody. Finally, CNP potently inhibited PACAP-stimulated transcription of the human alphaGSU gene (to 59plus/minus6.1%), but significantly enhanced extracellular calcium entry in PACAP stimulated alphaT3-1 cells (by 40plus/minus8%), without altering the effect of PACAP on cAMP production. These data demonstrate a regulatory role for GnRH, Sp1 and Sp3 in Nppcgene transcription in gonadotrophs, and reveal contrasting effects of CNP on PACAP-stimulated intracellular signalling pathways. Supported in part by a Needham-Cooper Fellowship (CAM) and the Wellcome Trust (JMB).