[show abstract][hide abstract] ABSTRACT: The neuropeptide calcitonin gene-related peptide (CGRP) plays a key role in migraine. CGRP gene expression involves an enhancer that is active in neurons, yet inactive in glia. In this report, we analyze epigenetic modifications that allow enhancer activation in glia.
DNA methylation and histone acetylation states were measured in rat and human- model cell lines and primary cultures of rat trigeminal ganglia glia. The functional consequence of altering the chromatin state was determined by quantitative measurements of both calcitonin (CT) and CGRP mRNAs.
A hypermethylated CpG island flanking the enhancer was identified in glia and non-expressing cell lines. In addition, the chromatin was hypoacetylated. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced CT mRNA ~30-fold in glial cultures. Treatment with a histone deacetylase inhibitor alone had little effect; however, the combination of inhibitors yielded a synergistic ~80-fold increase in CT and ~threefold increase in CGRP mRNA. Treated glia contained CT precursor (pro-CT) immunoreactivity.
Epigenetic modulation is sufficient to induce the CGRP gene in glia. Because the CGRP gene is systemically activated by inflammatory conditions, this suggests that glial pro-CT may be an unexplored biomarker during migraine.
[show abstract][hide abstract] ABSTRACT: The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. However, the transcription factors controlling CGRP expression in the migraine-relevant trigeminal ganglion neurons are unknown. Previous in vitro studies demonstrated that upstream stimulatory factor (USF) 1 and USF2 bind to the CGRP neuroendocrine-specific 18-bp enhancer, yet discrepant overexpression results in cell lines, and the ubiquitous nature of the USF cast doubts about its role. To test the functional role of USF, we first demonstrated that small interfering RNAs directed against USF1 and USF2 reduced endogenous CGRP RNA and preferentially targeted the USF binding site at the 18-bp enhancer in the neuronal-like CA77 cell line. In cultured rat trigeminal ganglion neurons, knockdown of either USF1 or USF2 reduced CGRP promoter activity. Conversely, overexpression of USF1 or USF2 increased promoter activity. The activation was even greater upon cotransfection with an upstream activator of mitogen-activated protein kinases and was synergistic in a heterologous cell line. To begin to address the paradox of how ubiquitous USF proteins might direct neuronal-specific activity, we examined USF expression and used a series of adenoviral reporters in the cultured ganglia. Unexpectedly, there was more intense USF immunostaining in neurons than nonneuronal cells. Importantly, the 18-bp USF enhancer driving a minimal promoter was sufficient for neuronal specificity, although it was not the only site that directed neuronal expression. These results demonstrate that USF1 and USF2 are important contributors to neuronal-specific and mitogen-activated protein kinase regulation of the CGRP gene in trigeminal ganglion neurons.
Journal of Biological Chemistry 03/2008; 283(9):5441-51. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The calcitonin gene-related peptide (CGRP) gene is specifically expressed and regulated in a subset of cell types. Abnormal
regulation of CGRP gene expression and/or sensitivity to CGRP actions may contribute to certain pathologies. In this chapter,
we will summarize the current state of knowledge on the CGRP promoter and the use of a genetic strategy to sensitize mice
to CGRP actions. The CGRP promoter is regulated by two distinct elements: a proximal cyclic AMP response element and a distal,
regulated cell-specific enhancer. Both of these elements are activated by mitogen-activated protein kinase (MAPK) signaling
pathways. MAPK can stimulate the CGRP promoter in response to proinflammatory cytokines. In addition, CGRP can directly activate
these pathways to boost its own expression and potentially increase CGRP actions in a feedback loop. These pathways may contribute
to the elevated CGRP levels in migraine and sepsis. Genetic enhancement of the CGRP receptor subunit, receptor activity-modifying
protein-1 (RAMP1), increases CGRP actions in vascular smooth muscle and neurons. Potential implications of CGRP elevation
and sensitization in migraine will be discussed.