In the present report, we provide the first demonstration that COMT gene expression is downregulated by TNFα in primary rat astrocytes at both protein and mRNA levels. As the P2-COMT promoter controls expression of MB-COMT, the main COMT transcript in brain, this promoter was cloned from human genomic DNA and transfected in H4 astroglioma cells. The activity of the cloned promoter was substantially suppressed by TNFα in a time-dependent manner.
A number of putative regulatory elements have been described in P1
- and P2-COMT
promoters, including estrogen response (ER) elements [50
] that likely mediate estradiol-dependent downregulation of COMT expression in cell culture [55
also contains abundant methylation sites associated with cancer development [56
], schizophrenia, and bipolar disorder [57
]. We identified a novel putative regulatory site – a κB consensus sequence that is a potential target for TNFα-dependent NF-κB activation.
Next, we demonstrated that TNFα-dependent COMT downregulation was indeed mediated by the NF-κB pathway. Transient expression of p65, the essential component of NF-κB complexes, or IKKβ, the major positive regulator of NF-κB activition, significantly decreased P2-COMT reporter expression. In addition, H4 IκBα-SR cells lost the ability to regulate P2-COMT promoter expression in response to TNFα treatment. The TNFα-mediated suppression of endogenous COMT expression was also abrogated in H4 IκBα-SR cells. Moreover, we confirmed that TNFα activated NF-κB in H4 astroglioma cells through the canonical IκB degradation pathway to trigger p65 nuclear translocation and DNA binding.
Our data strongly suggest that the putative κB binding site 5'-GGGGACGCCC-3' at position -109 of the P2-COMT promoter region is a functional site for NF-κB-mediated regulation of COMT expression as deletion of the P2-COMT region containing this site abrogated TNFα-dependent inhibition of P2-COMT activity in H4 astroglioma cells. Furthermore, competition experiments performed with the wild type or mutant site-specific oligonucleotides showed that TNFα indeed induced recruitment of p65 to this κB consensus binding site of the promoter.
Although NF-κB-mediated activation of transcription is well known, the mechanisms of NF-κB-mediated repression are poorly established. Probably, the best studied example of transcriptional repression by NF-κB complex is described for Dorsal transcription factor, a Drosophila
Rel family member that can either activate or repress gene expression through the recruitment of coactivators such as CBP or corepressors such as Groucho [58
]. Furthermore, a number of examples have been reported in mammals. NF-κB can repress transcription by competing with steroid receptors for a common promoter cis
-DNA element [59
N-myc recruitment to the glutamate transporter gene promoter [53
] and through inhibiting histone H4 acetylation at the cytochrome P-450 1A1 promoter [60
]. Thus, further experiments should be conducted to address the specific mechanism underlying NF-κB-dependent inhibition of COMT
gene expression. Interestingly, consequent deletions of 5'fragments of the P2-COMT
promoter led to a significant increase in overall basal promoter activity. This result would suggest the presence of a number of putative negatively regulating elements along the P2-COMT
promoter other than ER- and κB-response elements. Although, to date, no studies have systematically searched for regulators of COMT expression, this finding clearly warrants further research.
Our results demonstrating that COMT expression is downregulated in astrocytes under inflammatory conditions are in line with those of other studies showing a positive correlation between astrocyte activation and exaggerated pain responses [24
]. Intrathecal injection of gp120 (human immunodeficiency virus-1 envelope glycoprotein) induces mechanical allodynia via
the release of proinflammatory cytokines and NF-κB activation in spinal cord astrocytes, but not in microglial cells or neurons [62
]. Selective inactivation of astroglial NF-κB in transgenic mice expressing a dominant negative form of the inhibitor IκBα leads to a dramatic improvement in functional recovery after contusive spinal cord injury (SCI) [46
] and decreases formalin-induced pain [47
]. Additionally, several recent studies report cell type-specific NF-κB activation by cytokines. For example, in rat brain cultures IL-1 induces NF-κB activation in astrocytes, but not in neurons [63
]. Taken together, these studies unequivocally link NF-κB activation in astrocytes to pain states.
Although activation of the NF-κB pathway has been deemed critical for the development of pain [40
], there are few reports studying NF-kB-dependent pro-nociceptive signaling. Historically, these studies have focused on NF-kB-dependent up-regulation of pro-inflammatory cytokines [25
], cyclooxygenase-2 (COX-2) [39
], inducible and neuronal nitric oxide synthases (iNOS and nNOS) [38
], c-src [48
], and c-fos [38
]. However, recent studies from our group demonstrated that genetic variants of COMT
coding for low enzymatic activity are associated with heightened experimental pain sensitivity and the onset of a myofacial pain condition in humans [5
]. Additionally, pharmacologic inhibition of COMT in a rat model of inflammation resulted in elevated pain sensitivity [8
]. Together, these data suggest that an NF-κB-mediated decrease in COMT expression is likely to contribute to heightened pain sensitivity under inflammatory conditions. A series of in vivo
experiments further addressing this hypothesis are currently being conducted in our laboratory.