Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression.
The physiological actions of nitric oxide (NO) as a signaling molecule in endothelial and brain cells and as a toxic molecule used by activated immune cells have been the focus of a wide range of studies. Nevertheless, the downstream effector molecules of this important neuromodulator are not well understood. We have previously demonstrated that expression of the gene for the reproductive neuropeptide, GnRH, is repressed by the glutamate/NO/cyclic GMP (cGMP) signal transduction pathway through cGMP-dependent protein kinase in the hypothalamic GnRH-secreting neuronal cell line GT1-7. This repression localized within a previously characterized 300-bp neuron-specific enhancer. Here, we find that mutation of either of two adjacent elements within the enhancer eliminates repression by this pathway. An AT-rich sequence located at -1695 has homology to the octamer motif known to bind POU-homeodomain proteins, while the adjacent element at -1676 has homology to the C/EBP (CCAAT/enhancer-binding protein) protein family consensus sequence. Antibody supershift assays reveal that one of the proteins bound at the -1695 sequence is Oct-1, and one of the proteins bound to the element at -1676 is C/EBPbeta. These two proteins can bind simultaneously to the adjacent -1695 and -1676 binding sites in vitro. In nuclear extracts of GT1-7 cells treated with an NO donor, the intensity of the Oct-1 complex is increased. However, although Western blot analysis indicates that neither Oct-1 nor C/EBPbeta protein levels are increased, the relative binding affinity of Oct-1 is increased. Dephosphorylation of the nuclear extracts decreases binding of the Oct-1 complex to the -1695 site only in NO donor-treated extracts. Thus, we conclude that Oct-1 and C/EBPbeta are both downstream transcriptional regulators involved in the repression of GnRH gene expression by the glutamate/NO/ cGMP signal transduction pathway.
Belsham
D D
DD
Department of Reproductive Medicine, The Center for Molecular Genetics, University of Califonia, La Jolla 92093, USA. d.belsham@utoronto.ca
Mellon
P L
PL
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line / Q000187:drug effects; D006152:Cyclic GMP / Q000378:metabolism* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D018698:Glutamic Acid / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D051863:Host Cell Factor C1; D007031:Hypothalamus / Q000166:cytology; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D050811:Octamer Transcription Factor-1; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C497693:Hcfc1 protein, mouse; D051863:Host Cell Factor C1; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse; C494168:Pou2f1 protein, rat; D012097:Repressor Proteins; D014157:Transcription Factors; D009599:Nitroprusside; D009569:Nitric Oxide; D007987:Gonadotropin-Releasing Hormone; D018698:Glutamic Acid; D016202:N-Methylaspartate; D006152:Cyclic GMP