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. 2009 Mar 16;5:13.
doi: 10.1186/1744-8069-5-13.

Characterization of NF-kB-mediated Inhibition of catechol-O-methyltransferase

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Free PMC article

Characterization of NF-kB-mediated Inhibition of catechol-O-methyltransferase

Inna E Tchivileva et al. Mol Pain. .
Free PMC article

Abstract

Background: Catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, has recently been implicated in the modulation of pain. Specifically, low COMT activity is associated with heightened pain perception and development of musculoskeletal pain in humans as well as increased experimental pain sensitivity in rodents.

Results: We report that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) downregulates COMT mRNA and protein in astrocytes. Examination of the distal COMT promoter (P2-COMT) reveals a putative binding site for nuclear factor kappaB (NF-kappaB), the pivotal regulator of inflammation and the target of TNFalpha. Cell culture assays and functional deletion analyses of the cloned P2-COMT promoter demonstrate that TNFalpha inhibits P2-COMT activity in astrocytes by inducing NF-kappaB complex recruitment to the specific kappaB binding site.

Conclusion: Collectively, our findings provide the first evidence for NF-kappaB-mediated inhibition of COMT expression in the central nervous system, suggesting that COMT contributes to the pathogenesis of inflammatory pain states.

Figures

Figure 1
Figure 1
COMT expression is downregulated by TNFα in primary astrocytes. Administration of TNFα (20 ng/ml) decreases COMT protein expression in primary astrocytes as indicated by (A) a representative Western blot and (B) quantitative analysis of Western blots from experiments performed in triplicate. (C) Administration of TNFα (20 ng/ml) also decreases MB-COMT mRNA expression in primary astrocytes. Data are Means ± SEM. *P < 0.05 and **P < 0.01 different from untreated control.
Figure 2
Figure 2
Activity of P2-COMT promoter is downregulated by TNFα in H4 astroglial cells. (A) Schematic diagram of human COMT gene. The exon-intron organization is not to scale. The positions of translation start codons for MB-COMT (MB-ATG) and S-COMT (S-ATG) polypeptides, translation stop codon (TGA), putative polyadenylation signal (AATTAA), promoter regions, and primers (U1 and D1) are shown. (B) Administration of TNFα (20 ng/ml) inhibits activity of transfected P2-COMT/Luc reporter in H4 but not in H4 IκBα-SR cells. Data are Means ± SEM. *P < 0.05, ***P < 0.001 H4 different from H4 IκBα-SR cells at 16 and 24 h, respectively. (C) TNFα-mediated inhibition is specific to P2-COMT promoter. The activity of 3x-κB/Luc reporter (κB consensus sites from the MHC class I promoter) was tested after treatment with TNFα (20 ng/ml) for 24 h. *P < 0.05 different from untreated control.
Figure 3
Figure 3
TNFα-mediated repression of COMT is attenuated in H4 IκBα-SR astroglial cells. Administration of TNFα (100 ng/ml) does not reduce COMT expression in H4 IκBα-SR cells as indicated by (A) a representative Western blot, (B) quantitative analysis of Western blots from experiments performed in triplicate, and (C) quantitative real-time RT-PCR analysis of MB-COMT mRNA. P > 0.05 different from untreated control.
Figure 4
Figure 4
NF-κB is required for COMT downregulation. (A) Cotransfection of pCMV-SPORT-M-p65 or pCMV-SPORT-M-IKKβ inhibits P2-COMT/Luc reporter activity in H4 cells. Data are Means ± SEM. **P < 0.01 different from P2-COMT/Luc alone. Western blot analysis was performed with protein extracts from H4 cells treated with TNFα (20 ng/ml) for the indicated timepoints. Expression of IκBα was evaluated in cytoplasmic fraction of H4 cells (B), whereas p65 and p50 protein levels were tested in nuclear extracts (C).
Figure 5
Figure 5
Identification of TNFα-responsive region in the P2-COMT promoter. (A) Schematic diagram of the P2-COMT/Luc construct and its serial deletions Del.1 and Del.2. The effect of TNFα (20 ng/ml, 8 h of treatment) on the relative activity of the P2-COMT/Luc, Del.1 and Del.2 constructs transfected into H4 (B) and H4 IκBα-SR (C) cells. Data are Means ± SEM. *P < 0.05 different from untreated control. (D) Binding of p65 to a plate-immobilized oligonucleotide containing a κB binding site was measured by TransAM NF-κB assay in the nuclear extracts from H4 cells 30 min post TNFα treatment. This activation was prevented by competition with a wild-type binding control (WT BC) κB consensus oligonucleotide or a wild-type P2-COMT κB consensus oligonucleotide (WT P2). Oligonucleotides with mutated κB binding sites – a mutant binding control (MT BC) and a mutant P2-COMT (MT P2-COMT) – had little or no effect. Data are Means ± SEM. ###P < 0.001 TNFα-treated different from untreated control, ***P < 0.001 TNFα-treated different from TNFα+WT BC and WT P2 oligos, *P < 0.05 TNFα-treated different from TNFα+MT BC oligo.

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