Control of the Calcitonin Gene-related Peptide Enhancer by Upstream Stimulatory Factor in Trigeminal Ganglion Neurons

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.

Epigenetic regulation of the calcitonin gene–related peptide gene in trigeminal glia

“…Rat2 cell line (ATCC, Manassas, VA, USA) was maintained in Dulbecco’s Modified Eagle’s Medium (high glucose), 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 μg/ml) at 37°C in 5% CO 2 . Rat trigeminal ganglia cells were isolated and dispersed as described (17). Rat trigeminal glial cultures were prepared as described (14), with minor modifications.…”

“…Primers were: CT/CGRP (GenBank M11597.1) exon 2 sense 5′-CCCTTTCCTGGTTGTCAGCATCTT-3′, exon 3 antisense 5′-CATGCCTGGGCTGC TTTCTAAGGTT-3′, CT (GenBank V01228.1) exon 4 antisense 5′-AGCATGCAGGTACTCAGATTCCCA-3′. CGRP (GenBank M11597) exon 3 sense and exon 5 antisense have been described (17). For NCI-H460 cells: 730 ng of RNA was reversed transcribed.…”

“…The Q-PCR protocol was 50°C, 2 minutes, 95°C, 3 minutes, then 40 cycles at 95°C, 15 seconds, 60.7°C, 30 seconds, 72°C, 1 minute. Each sample was measured in triplicate with delta-delta Ct normalization to 18S rRNA Ct values as described (17). Controls included samples with no RT enzyme added to the RT reaction and samples with H 2 O added in place of cDNA.…”

“…This enhancer is sufficient for cell-specific CALCA gene expression in neuronal-like cell lines and trigeminal ganglia neurons (15–17). The enhancer is activated by the bHLH-Zip upstream stimulatory factor (USF) (17–19). USF can also act with the forkhead protein FoxA2 to synergistically activate the enhancer, although FoxA2 is not present in trigeminal ganglia (19).…”

Reactive Oxygen Species Induce Procalcitonin Expression in Trigeminal Ganglia Glia

“…Much has been discovered about the regulation of the Calca gene in recent years. Transcription factors USF1 and USF2 are activators of the gene 23 , but their ubiquitous expression pattern led to questions about how tissue specificity is maintained. We previously reported that the gene was kept silent in glia through an epigenetic mechanism 24 .…”