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

Abstract

Upregulation of CCR2 chemokine receptor expression by dorsal root ganglion (DRG) neurons is an important process in the development and maintenance of neuropathic pain. CCR2 is not expressed by DRG neurons under normal conditions but is upregulated in several animal models of neuropathic pain where its signaling is excitatory. However, the molecular mechanisms underlying neuronal upregulation of CCR2 have not been investigated. We examined the promoter region of the CCR2 gene and found that a binding site for the nuclear factor of activated T-cells (NFAT) was conserved among species. The NFAT element was functional since the CCR2 promoter was activated by a constitutively active form of calcineurin A, whereas a point mutation in the NFAT binding site abrogated it. Activation of the NFAT pathway in the DRG neuronal cell line F11 increased CCR2 promoter activity and induced CCR2 transcription. Moreover, depolarization of cultured DRG neurons induced de novo synthesis of CCR2 mRNA, which was blocked by the calcineurin inhibitors cyclosporin A and FK506. These data indicate that CCR2 is a target of the NFAT pathway and suggest that tonic excitation of DRG neurons in association with chronic pain may lead to neuronal CCR2 upregulation via activation of the NFAT pathway.

Figure 1. The genomic organization of the mouse CCR2 gene

A) Reported mRNA species and their coverage on genomic DNA sequences (numbered boxes: exons; lines between exons: introns; a black box: protein coding region). B) Primer extension experiment. The location of the primer is denoted in the diagram on the left (W: WEHI265.1 monocytic cell line). C) RT-PCR experiments. Relative location of primers is denoted on the upper diagram (arrow heads: primers). Two sets of primers were used: E1up/E3dn and E2up/E3dn (left two and right two lanes, respectively). The structure of the PCR product is denoted in the left and right diagrams (W: WEHI265.1 monocytic cell line; F: F11 DRG neuronal cell line). D) The sequence of the exon 1 (E1). Exonic sequence is in upper cases and non-exonic sequence in lower cases. An additional intronic sequence within the exon 1 is underlined. AG and GT consensus splice dinucleotides are in bold letters. An mRNA with this sequence spliced out corresponds to the shorter band in the leftmost lane in C.

Figure 2. Cloning of a functional CCR2 promoter

A) Cell type-specific CCR2 transcriptional activity. CCR2 was expressed by the WEHI265.1 (W) monocytic cell line but not by the F11 DRG neuronal cell line (F) under basal condition (left panels). The half-life of CCR2 mRNA was examined in WEHI265.1 (right panels). After the addition of a transcriptional inhibitor actinomycin D (ActD: 1 µg/ml), the CCR2 mRNA level rapidly decreased. 28S rRNA was used as an internal control. B) Cell type specific CCR2 promoter activity. 1.0 kb and 0.5 kb fragments upstream of exon 1 (E1) were cloned into a luciferase reporter vector (Luc). Promoter activity was high in WEHI265.1 cells but very low in F11 cells (**p<0.01 and *p<0.05 between WEHI265.1 and F11, unpaired t-test).

Figure 3. An NFAT element in the promoter

A) The NFAT binding element near the transcription initiation site is conserved (*) between mouse and human (E1: exon 1). The core binding sequences are in bold. B) A specific NFAT binding activity in the nuclear extract of WEHI265.1 cells (Co: cold competitors; S: self-competitors; M: GGAAA → GGTTT mutated competitors; NE: nuclear extract from WEH265.1 cells; a filled arrowhead: specific NFAT binding activity; an empty arrowhead: binding activity that is not specific to the core NFAT sequence; Ab: antibody; IgG: an isotype-matched control antibody; αNFATc: a pan-specific antibody to NFATc). C) The NFAT binding element in the CCR2 promoter is functional. The CCR2 promoter (WT) or the promoter with a point mutation in the NFAT binding site (Mut: GGAAA → GGTTT) was co-transfected with a constitutively active form of calcineurin A (caCnA) or an empty vector (***p<0.001; N.S.: not significant, a post hoc Bonferroni’s multiple comparison test following one-way ANOVA).

Figure 4. The components of the NFAT pathway in DRG neurons and F11 cells

A) Calcineurin A (CnA) and NFATc1-4 were expressed by cultured DRG neurons and F11 DRG neurons (RTase: reverse transcriptase). RTase-negative control reaction was performed for each group. B) CCR2 promoter is activated by calcium ionophore ionomycin (IM: 1 µM) in F11 DRG neurons in a calcineurin inhibitor cyclosporin A (CsA: 1 µM)-sensitive way (**p<0.01, a post hoc Bonferroni’s multiple comparison test following one-way ANOVA).

Figure 5. NFAT-dependent transcription of CCR2 in F11 and WEHI265.1 cells

A) NFAT-dependent de novo transcription of CCR2 in F11 DRG neurons. Ionomycin (IM: 1 µM) treatment induced CCR2 mRNA which was blocked by a transcriptional inhibitor actinomycin D (ActD: 1 µg/ml). The induction was significantly inhibited by a calcineurin inhibitor cyclosporin A (CsA: 1 µM). 28S rRNA was used as an internal control. B) NFAT-dependent transcription contributes to cell type-specific CCR2 expression. Calcineurin inhibitors CsA or FK506 (0.2 µM each) decreased baseline levels of CCR2 in WEHI265.1 monocytic cell line. GAPDH was used as an internal control.

Figure 6. Depolarization-induced transcription of CCR2 in DRG neurons

A) NFAT-dependent transcription of CCR2 is induced in DRG neurons by depolarization. Acutely isolated DRG neurons were depolarized by high external potassium (43K: 43 mM) for 24 hr with or without calcineurin inhibitors (CsA/FK506: 0.2 µM each), which were added 30 min prior to depolarization. Control culture medium contained 3 mM of potassium (3K). Osmorality of different media was identical. Total RNA was isolated from neurons (see supplementary figure 1) and subjected to RT-PCR. B) Real-time quantitative RT-PCR was used to quantify mRNA levels of different chemokine receptors in conditions described in A (n=3–5 independent DRG cultures) (**p<0.01, *p<0.05 vs. 3K and ††p<0.01, †p<0.05 vs. 43K, a post hoc Bonferroni’s multiple comparison test following one-way ANOVA).