Cyclooxygenase-2 is a neuronal target gene of NF-κB
© Kaltschmidt et al; licensee BioMed Central Ltd. 2002
Received: 2 September 2002
Accepted: 4 December 2002
Published: 4 December 2002
NF-κB is implicated in gene regulation involved in neuronal survival, inflammmatory response and cancer. There are relatively few neuronal target genes of NF-κB characterized.
We have identified the neuronal cyclooxygenase-2 (COX-2) as a NF-κB target gene. In organotypic hippocampal slice cultures constitutive NF-κB activity was detected, which was correlated with high anti-COX-2 immunoreactivity. Aspirin a frequently used painkiller inhibits neuronal NF-κB activity in organotypic cultures resulting in a strong inhibition of the NF-κB target gene COX-2. Based on these findings, the transcriptional regulation of COX-2 by NF-κB was investigated. Transient transfections showed a significant increase of COX-2 promoter activity upon stimulation with PMA, an effect which could be obtained also by cotransfection of the NF-κB subunits p65 and p50. In the murine neuroblastoma cell line NB-4, which is characterized by constitutive NF-κB activity, COX-2 promoter activity could not be further increased with PMA or TNF. Constitutive promoter activity could be repressed upon cotransfection of the inhibitory subunit IκB-α. EMSA and mutational analysis conferred the regulatory NF-κB activity to the promoter distal κB-site in the human COX-2 promoter.
NF-κB regulates neuronal COX-2 gene expression, and acts as an upstream target of Aspirin. This extends Aspirin's mode of action from a covalent modification of COX-2 to the upstream regulation of COX-2 gene expression in neurons.
NF-κB a transcription factor with inducible activity, present in most cell types. This factor is crucially involved in regulation of genes relevant in neuronal survival, inflammmatory response, cancer and innate immunity [1, 2]. The activation of NF-κB is mainly controlled at the posttranscriptional level by complex formation with the inhibitory subunit IκB in the cytoplasm . Phosphorylation of IκB prior to degradation is catalyzed by the activation of a complex consisting of two kinases (IKK-α and IKK-β)  together with a modifying subunit called NEMO  or IKK-γ . Binding of NEMO is important to mediate the cytokine response in a aktivation of the kinases . Recently it was shown that mutations of NEMO/ IKK-γ were linked to human genetic diseases (for review see ).
NF-κB is also frequently found in different cells of the nervous system (for review see . Many neurons of the central nervous system contain NF-κB as a heterodimer of the DNA-binding subunits p50 and p65, complexed with IκB . Constitutive activity of NF-κB is present in fields of the hippocampus and in the cerebral cortex . These data suggest an endogenous, physiological stimulus, which controls the activity of NF-κB. One candidate is the neurotransmitter glutamate, which can activate NF-κB in cerebellar granule cells and hippocampal neurons [11–15]. Furthermore the presence of inducible NF-κB in synaptosomes [16, 17] and the transport of GFP-tagged p65 from neurites to the nucleus  suggest that NF-κB could be involved in connecting synaptic activity with gene expression. This notion is also supported by the ultrastructural localization of activated NF-κB in dendrites . A gene induced by synaptic activity is the inducible cyclooxygenase or prostaglandin H (PGH) synthase-2 (COX-2). In contrast to peripheral tissues the cyclooxygenase-2 activity and expression is high in normal brain, where it is restricted to neurons [19, 20]. We investigated wether COX-2 is regulated by NF-κB. COX-2 and activated NF-κB immunoreactivity colocalized in hippocampal and cortical neurons. Aspirin, a described inhibitor of NF-κB  inhibited neuronal NF-κB, leading to a robust inhibition of COX-2 protein expression. These data were further corroborated by an analysis of the COX-2 promoter. A promoter distal κB element was identified as the only functional κB-site in NB-4 neuroblastoma cells. In addition this element is also responsible for the constitutive promoter activity. Thus the previously described constitutive COX-2 activity in neurons  is dependent on constitutive NF-κB activity.
NF-κB and cyclooxygenase-2 colocalize in subsets of cortical and hippocampal neurons
COX-2 was identified as a gene induced after seizures . Basal expression of this enzyme is high in brain, in comparison to other organs were COX-1 is the major isoenzyme. COX-2 expression in brain is dependent on normal neuronal activity, as demonstrated with intra-ocular tetrodotoxin injection which blocks COX-2 expression in the visual cortex. Moreover COX-2 expression in the CNS is obligate neuronal . Here we tested if COX-2, as a marker of neuronal activity, is present in the same neurons, that show activated NF-κB. Double labeling immunofluorescence was used to correlate the activation of NF-κB with COX-2 protein amount at single cell level. Previously we developed a monoclonal antibody specific for the activated form of p65 . This antibody is directed against an epitope of the nuclear localization signal (NLS) of p65. In the non-activated cytoplasmic form of NF-κB the NLS is predominantly covered by the inhibitory subunit IκB, making binding of the antibody impossible. Upon stimulation active NF-κB is generated after IκB degradation. This active NF-κB can be visualized with the activity specific antibody.
In addition to the abundant colocalization of both stainings, cells with distinct staining for both COX-2 and p65 could be detected in the cortex and hippocampus. The specificity of both antibodies was analyzed by incubation without primary antibody, which showed no significant staining (data not shown).
Inhibition of NF-κB leads to down regulation of COX-2 expression
Thus the inhibition by aspirin suggests that COX-2 is a neuronal NF-κB target gene. To further corroborate this notion we performed a promoter analysis of the COX-2 gene.
Two conserved κB-binding sites are present in the human cyclooxygenase-2 promoter
The cyclooxygenase-2 promoter is strongly induced by NF-κB activating stimuli
Constitutive NF-κB activity in NB-4 neuroblastoma cells is essential for cyclooxygenase-2 promoter activity
The activity of the cyclooxygenase-2 promoter is only dependent on the promoter-distal NF-κB binding-site
Here the regulation of the human COX-2 promoter was analyzed. Immunocytochemistry was used to colocalize COX-2 immunoreactivity and activated NF-κB in neurons in vivo. This was investigated in the rat hippocampus and cortex cerebri, using an antibody specific for the activated form of NF-κB. In cultured hippocampal slices the specific NF-κB inhibitor aspirin, inhibited both, NF-κB activation and COX-2 expression. The promoter region of the human COX-2 gene contains, in contrast to the mouse promoter region, two putative NF-κB binding sites. It was found, that only the conserved NF-κB binding site, present in the mouse and human COX-2 promoter region (see Fig. 4A), is of functional relevance in neuronal cells.
Evidence for COX-2 as a neuronal NF-κB target gene
Non-neuronal cells were used to characterize the mechanisms of promoter-induction, since neuronal cells support already a full blown constitutive COX-2 promoter activity (see below). In HeLa cells the human COX-2 promoter is fully inducible with PMA, but only to a low amount with TNF, whereas a synthetic NF-κB-dependent promoter is readily activated with TNF. This difference might be exaggerated through the lower COX-2 promoter activity, which is the result of only one functional NF-κB binding site. During the analysis of NF-κB-subunits mediating an induction of the COX-2 promoter, we found that the NF-κB subunits p50 and p65 were active. In contrast to p65, which contains a transactivating domain, the p50 subunit does not contain its own transactivation domain. This effect might be mediated by interaction of p50 with Bcl-3, which can provide the transactivating function. This transactivating effect of the NF-κB p50 subunit is not a characteristic of the COX-2 promoter but is now frequently observed also in other promoters .
In accordance with the inter-species conservation of the promoter-distal κB1 element (Fig. 4A), DNA-binding of NF-κB proteins to this element was detected. The promoter proximal κB2 element is different in one nucleotide from the NF-κB consensus binding site and could not be bound by NF-κB subunits. In a recent approach recombinant NF-κB subunits were used to select target sequences bound by the DNA-binding domains (target detection assay). In accordance with our data the κB2 element was not selected as a binding site .
It was reported earlier that many neurons of the cortex cerebri and the hippocampus contain constitutive NF-κB activity . Using an antibody specific for activated p65 , here a colocalization of constitutive NF-κB activity and basal level COX-2 expression was detected. In accordance to the constitutive activity detected in vivo we also found that NB-4 cells contain constitutive activity based on the following criteria: 1.) A high-level basic activity of a promoter containing 6 κB elements could not be further augmented after treatment with PMA or TNF. 2.) Constitutive promoter activity could be repressed with cotransfection of IκB. The COX-2 promoter in NB-4 cells showed the same level of constitutive activity that could not be augmented strongly with PMA or TNF, but is repressed after cotransfection of IκB. Therefore we conclude that NB-4 neuroblastoma cells are a suitable model for the constitutive NF-κB activity found in vivo in neurons of the cortex and hippocampus. Here we show that a mutation of the COX-2 promoter κB1 element entirely abolishes the constitutive activity of this promoter in NB-4 cells.
Recent studies have shown, that NF-κB can be activated in cerebellar granule cells via stimulation of glutamate receptors [11–13, 34]. In addition it was shown that the transcription of NF-κB subunits p50 and p65 was increased during seizure activity . Taken together NF-κB is one of the transcription factors regulated via neuronal activity (see ). One of the important physiological consequences of NF-κB activation might be the induction of a compensatory neuroprotective gene expression program [14, 35–38].
COX-2 mediated inflammatory pathways may play important roles in pathogenesis of neurodegenrative diseases such as Alzheimer disease . In Alzheimer disease patients early plaque stages are surrounded by neurons with activated NF-κB [37, 39]. Moreover high levels of NF-κB binding activity could be strongly correlated to high levels of COX-2 transcription in Alzheimer disease and age matched control brains . There is evidence reviewed by  that induction of high level of COX-2 epression might be responsible for patho-physiological changes which are also seen in a COX-2 overexpressing mouse model . But the activation of COX-2 might also serve protective functions as shown in seizure paradigms. Seizures can activate NF-κB , which in turn may lead to prostaglandin production after COX-2 gene induction. These newly produced prostaglandins might exert a protective effect against new seizure attacks .
Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin are the major therapy for inflammatory pain. In addition to its action as an inhibitor of prostaglandin synthesis aspirin also acts as an inhibitor of NF-κB. A recent study has shown that inflammatory pain is induced via COX-2, expressed in neurons within the CNS. Here we have shown that neuronal COX-2 expression is essentially dependent on NF-κB activity. In addition the NSAID aspirin inhibits neuronal NF-κB, which results in strongly reduced COX-2 activity. Similarly an enantiomer of the NSAID flurbiprofen which could not repress COX-2 enzyme activity is still acting anti-phlogistically as an inhibitor of NF-κB .
We found that COX-2 is a neuronal target gene of NF-κB. Aspirin inhibited both, NF-κB activation and COX-2 expression in neurons. Thus preventing COX-2 gene transcription via NF-κB inhibition might provide novel means of normalizing pain sensitivity.
Human genomic DNA was from Promega, Heidelberg, Germany; Taq-Polymerase and PCR-reagents from Stratagene, Heidelberg, Germany. Sequencing was performed using reagents and equipment from Applied Biosystems (Weiterstadt, Germany). D-Luciferin, PMA and TNF were obtained from Sigma, Deisenhofen, Germany; Lipofectin from Gibco, Karlsruhe, Germany.
The human COX-2 promoter was cloned by PCR using the following primers: tgcagctcttgactcatcgg and cccaagcttgacaattggtcgctaaccga according to a published sequence . The obtained sequences were cloned in front of the luciferase gene into the promoter-less luciferases reporter vector pGL-2 (Promega, Heidelberg, Germany) and verfied by sequencing. For mutational analysis an EcoRI site was introduced in the κB1 element (κB1mut:gagagaattctccctgcgc) with a PCR-mediated strategy.
Organotypic hippocampal slice cultures
Hippocampal slice cultures (N= 30) were prepared from slices 350 μm thick taken from five-day-old pubs of Wistar rats. After 6 days in vitro [47, 48] all cultures were treated with GABA antagonists to mimick the glutamatergic input present in vivo. All used drugs were from Sigma, Deisenhofen, Germany. Treatment with the GABA antagonists bicuculline (200 μM) and picrotoxine (1000 μM) of all slice cultures was done as described  for 3 days. Cultures were co-treated with aspirin (5 mM for 15 min as described  and assayed for immunoreactivity after 6 h post treatment. Cultures were fixed with 4% formaldehyd (pH 7.2) for 12–24 h at room temperature, kryoprotected in 30% sucrose solution (overnight at 4°C) and sectioned with a kryostat (20 μm).
Immunocytochemistry was done as detailed below.
Culture of cell lines
Cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). HeLa and 293 cells were grown in DMEM (Gibco) containing 10% fetal calf serum. NB-4 cells were cultivated in Ham's F10 medium (Sigma, Deisenhofen, Germany) with addition of 15% horse serum, 2.5% fetal calf serum, antibiotics and glutamine. For luciferase assays, cells were plated in six-well plates (25.000 cells per well with 2 ml of culture medium).
Transfection of cells and luciferase reporter assays
The Rc/CMV derived expression vectors for p50 and p65 were described earlier . The tk(NF-κB)6 luciferase reporter construct contains 6 reiterated copies of the HIV-1 κB-site in front of the truncated Herpes simplex thymidine kinase (tk) promoter spanning position -105 to +51 . HeLa and 293 cells were transfected according to a modified calcium phosphate protocol . NB-4 cultures were transfected using lipofectin according to the instructions of the manufacturer (Gibco, Karlsruhe, Germany). Twenty hours after transfection, cells were lysed and assayed for luciferase activity . At least three independent transfection experiments were performed in triplicates using different cell and DNA preparations. All experiments gave the same qualitative result. One representative experiment is shown. In several experiments cells were stimulated for 6 h with either 50 ng/ml PMA (Sigma, Deisenhofen, Germany) or 2 ng/ml (200 U/ml) human TNF-α (Roche, Mannheim, Germany).
Electrophoretic mobility shift assay (EMSA)
Nuclear proteins were prepared as previously described . Briefly, cells were lysed in buffer A containing 20 mM HEPES, 0.35 M NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM MgCl2, 0.5 M EDTA, 0.1 mM EGTA, 5 mM dithiothreitol, phenylmethylsulfonyl fluoride and aprotinin. Nuclei were extracted with buffer C and stored at -80°C for EMSA. EMSAs were performed using 3.5 μg nuclear proteins to bind to 32P-labeled oligonucleotides, encompassing the κB-site from the murine κ light chain enhancer (Promega, Heidelberg, Germany), κB1: gagaggggattccctgcg and κB2:agtgggactaccccctc from the human COX-2 promoter.
Brains were dissected from adult Wistar rats and embedded in OTC-compound (Miles-Bayer, Leverkusen, Germany). 8 μm cryo-sections were cut from snap frozen material with a Leica cryostat (Leica Instruments, Heidelberg, Germany). The sections were collected on gelatine coated slides and dried. After fixation in methanol at -20°C for 5 min, the sections were blocked in 5% goat serum. For double-label immunofluorescence the sections were incubated with the two primary antibodies (diluted 1:50): a monoclonal antibody against p65 (Roche, Germany, see  and a rabbit polyclonal antibody against murine COX-2 (cyclooxygenase-2, Cayman Chemical Company, Ann Arbor, USA). Bound antibodies were detected with an anti-mouse IgG antibody coupled with Cy3 (1:1000, Dianova, Hamburg, Germany) and an anti-rabbit IgG coupled with DTAF (1:100, Dianova, Hamburg). Nuclei were stained with DAPI (4',6-Diamidine-2'phenylindole dihydrochloride, Roche, Germany). Microphotographs were taken with a Zeiss Axioskop equipped with epifluorescence. Mounting of colour plates was done on an Apple PowerPC with Adobe Photoshop.
List of abbreviations
CCAAT/enhancer binding protein
electrophoretic mobility shift assay
Inhibitor of kappaB-alpha
NF-kappaB Essential MOdulator
nuclear factor kappa B
tumor necrosis factor alpha.
This work was supported by grants to C. K. from the Deutsche Forschungsgemeinschaft and the Volkswagen-Stiftung. We thank Prof. Frotscher for continuos support.
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