Activation of the nuclear factor of activated T-cells (NFAT) mediates upregulation of CCR2 chemokine receptors in dorsal root ganglion (DRG) neurons: A possible mechanism for activity-dependent transcription in DRG neurons in association with neuropathic pain
Introduction
Long-term changes in the profile of gene transcription in dorsal root ganglion (DRG) cells and in the central nervous system (CNS) are believed to underlie the heightened pain sensitivity observed in neuropathic pain states. Many genes are differentially regulated by DRG neurons and glia in different animal models of chronic pain, suggesting multiple signaling pathways may contribute to this process (Basbaum and Woolf, 1999, Ji and Strichartz, 2004, Mogil et al., 2000). However, the mechanism by which such changes occur is poorly understood. Expression of the CCR2 chemokine receptor has been found to be upregulated by DRG neurons in association with several animal models of neuropathic pain (Bhangoo et al., 2006, White et al., 2005b). Under normal circumstances neither CCR2 nor its ligand MCP-1/CCL2 are expressed by DRG neurons, whereas their expression is upregulated under pathological conditions (Bhangoo et al., 2006, Sun et al., 2006, White et al., 2005b). Importantly, DRG neurons from animals exhibiting neuropathic pain are strongly excited by the application of MCP-1, whereas neurons from naïve animals are not (Sun et al., 2006, White et al., 2005b). Moreover, CCR2 knockout mice do not develop neuropathic pain following spared nerve injury (Abbadie et al., 2003), suggesting that upregulation of CCR2 is a crucial step in the generation and/or maintenance of neuropathic pain. However, the transcriptional regulation of CCR2 receptors in DRG neurons has not been examined. Consequently, we wished to define the signaling pathway responsible for the upregulation of these receptors in DRG neurons.
In addition to its diverse roles in the development and function of the immune system, it has recently been shown that the nuclear factor of activated T-cells (NFAT) family of transcription factors can play an important role in mediating long-term changes in neuronal excitability (Graef et al., 1999). NFAT cannot normally enter the nucleus until it is dephosphorylated, but can be activated by a Ca2+-dependent phosphatase calcineurin (CN). Thus, an increase in [Ca2+]i turns on the transcription of NFAT-dependent genes and this is believed to link transient neuronal excitation to a long-lasting transcriptional activation (Graef et al., 1999, Zanzouri et al., 2006).
Here we report that CCR2 is a target gene of the NFAT pathway in DRG neurons. We demonstrate that there is a functional and conserved NFAT binding element in the promoter region of the CCR2 gene. Activation of the NFAT pathway in DRG neurons and the DRG neuronal cell line F11 increased the CCR2 promoter activity and initiated CCR2 transcription. These data suggest that repetitive excitation of DRG neurons under pathological circumstances might turn on activity-dependent transcription of CCR2 via NFAT signaling. Hence, antagonism of the NFAT pathway may be a novel point for therapeutic intervention in treating neuropathic pain.
Section snippets
The structure of the mouse CCR2 gene
We examined the genomic organization of the mouse CCR2 gene. It is composed of three exons with the entire protein coding region residing in the third exon (Fig. 1A). The length of the 5′-untranslated region (5′-UTR) varies among the reported mRNA sequences (Fig. 1A). The expected length of the first exon deduced from the longest reported sequence (AK046579) was 258 nt (Fig. 1A). In order to see if this represents the transcription initiation site, we performed a primer extension experiment
Discussion
Sustained pain hypersensitivity and neuronal hyperexcitability associated with neuropathic pain is believed to result from long-term changes in gene expression in DRG and central dorsal horn neurons. Many genes have been identified whose expression changes in association with the development of neuropathic pain using several animal models, although the molecular mechanisms responsible for such changes are largely unknown (Mogil et al., 2000). In particular, the CCR2 chemokine receptor appears
Plasmid constructions and materials
The fragments of 5′-flanking region of CCR2 was amplified from mouse genomic DNA (from a CD-1 mouse) by polymerase chain reaction (PCR) and cloned into pGL3-Basic (Promega). A point mutation in the NFAT binding site was introduced using QuickChange site-directed mutagenesis kit (Stratagene). The constitutively active calcineurin A construct (caCnA) was a kind gift from Dr. Neil A. Clipstone (Loyola University Chicago, Maywood, IL) (Clipstone et al., 1994). All PCR products were cloned into
Acknowledgments
This work was supported by the National Institutes of Health Grants DA013141, NS043095, and MH040165 to RJM. The authors would like to thank Dr. Fletcher A. White (Loyola University Chicago, Maywood, IL) for his advice when carrying out these studies and with preparation of the manuscript.
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