ECE2006 Oral Communications Signal transduction OC1 Novartis Oncology Young Investigator Award (8 abstracts)
The ubiquitous transcription factor, CREB, is responsible for mediating gene transcription in many endocrine tissues. Recent studies reveal that the activation of several signalling pathways can lead to the phosphorylation of CREB at Ser133, including PKC, MAPKs, CaMKs, and PI3K. In the pituitary gonadotroph, there are consensus cAMP response elements (CREs) in several of the gonadotrophin subunit promoters as well as in the GnRH receptor promoter. We have previously shown that activators of the cAMP pathway can phosphorylate CREB on Ser133 in αT3-1 cells, and others have shown GnRH to activate CREB in rat pituitaries. To establish which signalling pathways are involved in GnRH-stimulated CREB activation, we have used Western blotting, reporter gene assays and electrophoretic mobility shift assays (EMSA) to examine CREB phosphorylation, transcriptional activity and DNA binding. αT3-1 and LβT2 cells treated with 100 nM GnRH for 15 min showed enhanced phosphorylation of CREB (at Ser133) as well as ATF-2. We transiently transfected αT3-1 and LβT2 cells with a TKCRE-LUC reporter construct and stimulated with GnRH, PMA, PACAP (100 nM) or Forskolin (10 μM) for 24 h. GnRH (6.4±0.2-fold, 3.9±0.5-fold) and PMA (3.3±0.9-fold, 6.9±0.1-fold) strongly activated TKCRE activity in αT3-1 and LβT2 cells respectively (P<0.01). Pre-treatment of LβT2 cells for 30 min with nifedipine (Ca2+ channel antagonist) or GF109203X (PKC inhibitor) (both 1 μM), but not H-89 (PKA inhibitor), significantly inhibited GnRH-stimulated TKCRE activity (P<0.05). EMSA analysis of nuclear protein extracted from αT3-1 and LβT2 cells treated with GnRH for 15 min revealed enhanced binding of CREB, ATF-2 and other CREB-related proteins to the consensus CRE response element from the glycoprotein hormone α-subunit promoter. These data suggest GnRH enhances CREB phosphorylation at Ser133, increases CRE-mediated transcription by calcium influx and PKC activity, and enhances CREB binding in pituitary gonadotrophs; effects which are apparently cAMP-independent.