cgs-12066b and Thyroid-Neoplasms

We have investigated the mechanisms underlying regulation of the calcitonin gene-related peptide (CGRP) cell-specific enhancer. Recently, we reported that this enhancer is inhibited by serotonin type-1 (5-HT1) agonists, similar to currently used antimigraine drugs. We have now tested whether this repression involves a mitogen-activated protein (MAP) kinase pathway. We first demonstrate that the CGRP enhancer is strongly (10-fold) activated by a constitutively active MAP kinase kinase (MEK1), yielding reporter activities 100-fold above the enhancerless control. The involvement of a MAP kinase pathway was confirmed by down-regulation of reporter activity upon cotransfection of a dominant negative Ras. Activation of the enhancer by MEK1 was blocked in a dose-dependent manner by the 5-HT1 receptor agonist CGS 12066A (CGS). Since it is not known whether the CGRP enhancer factors are immediate targets of MAP kinases, we then used EIk-1- and c-Jun-dependent reporter genes that are directly activated by the ERK (extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinase) MAP kinases. CGS treatment repressed the activation of both of these reporters, suggesting that at least two MAP kinases are the immediate targets of CGS-mediated repression. We further demonstrate that 5-HT1 agonists inactivate ERK by dephosphorylation, even in the presence of constitutively activated MEK1. This inactivation appears to be due to a marked increase in the level of MAP kinase phosphatase-1. These results have defined a novel and general mechanism by which 5-HT1 receptor agonists can repress MAP kinase activation of target genes, such as CGRP.

Topics: Animals; Calcitonin Gene-Related Peptide; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Medullary; Cell Cycle Proteins; DNA-Binding Proteins; Dual Specificity Phosphatase 1; Enhancer Elements, Genetic; ets-Domain Protein Elk-1; Genes, jun; Immediate-Early Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Phosphatase 1; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Quinoxalines; Rats; Serotonin; Serotonin Receptor Agonists; Thyroid Neoplasms; Transcription Factors; Transfection; Tumor Cells, Cultured

We have investigated the control of calcitonin gene-related peptide (CGRP) expression by a serotonergic agonist that is related pharmacologically to currently used antimigraine drugs. During migraines, CGRP levels are elevated but then returned to normal by a 5-HT1 receptor agonist, sumatriptan. However, neither the molecular nor cellular targets of this drug are known. Trigeminal neurons are the major source of cerebrovascular CGRP, and thus we have used trigeminal primary cultures and the neuronal-like CA77 thyroid C-cell line as a model. We first demonstrate that sumatriptan and another 5-HT1 agonist, CGS 12066A (CGS), cause a robust and prolonged increase with oscillations in intracellular calcium in CA77 cells. CGS caused a similar increase in trigeminal cultures. We then show that CGS treatment leads to a decrease in CGRP mRNA levels in the CA77 cells. This decrease is attributable to the repression of promoter activity through two discrete elements: (1) the cAMP-responsive region, via a cAMP-independent mechanism; and (2) the cell-specific enhancer, which binds the upstream stimulatory factor helix-loop-helix protein and a cell-specific activator. These results demonstrate that activation of the endogenous 5-HT1 receptor is coupled to calcium signaling pathways and leads to inhibition of CGRP gene transcription.

Topics: Animals; Calcitonin Gene-Related Peptide; Calcium; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Enhancer Elements, Genetic; Gene Expression Regulation; Genes, Reporter; Neurons; Promoter Regions, Genetic; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; RNA, Messenger; Serotonin Receptor Agonists; Sumatriptan; Thyroid Neoplasms; Time Factors; Transcription, Genetic; Trigeminal Ganglion; Tumor Cells, Cultured