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,
,,1
* Geriatric Research Education and Clinical Center, Bedford Veteran’s Affairs Medical Center, and
Neurology,
Pathology, and Psychiatry Departments, Boston University School of Medicine, Boston, Massachusetts, USA;
Department of Neurology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA;
|| Neuroscience Research Program of Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada;
¶ Harvard Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, USA;
Department of Neurology, Weill Medical College of Cornell University and Burke-Cornell Medical Research Institute, White Plains, New York, USA
1Correspondence: The GRECC Unit 182B, VA Medical Center, 200 Springs Rd., Bedford, MA 01730, USA. E-mail: hoonryu{at}bu.edu
SPECIFIC AIMS
Cox-2 is expressed constitutively in the brain and cerebral blood vessels of the newborn, and appears to be developmentally and functionally regulated. The elevated expression of COX-2 in the tissue of neurodegenarative diseases such as Alzheimer’s, amyotrophic lateral sclerosis (ALS), PD, and stroke has implicated that this protein is involved in the pathophysiological process of neurodegenerative diseases. COX-2 is under transcriptional control by NF-
B, CREB, and C/EBP. It reveals that the binding of NF-B is a necessary but not a sufficient step in the induction of COX-2 by hypoxic signaling. Despite reports showing that oxidative stress generated by reactive oxygen species (ROSs) induces COX-2 gene transcription in neuronal cell lines, the transcriptional regulation of COX-2 has not been precisely examined in primary neurons. It is intriguing whether COX-2 expression leads to a prosurvival or a proapoptotic effect in primary neurons under oxidative stress. We recently found that glutathione depletion-induced oxidative stress regulates DNA binding activity of Sp1 and Sp3 in cortical neurons. In primary neurons, Sp1 appears likely to mediate a prosurvival role in response to oxidative stress. We wanted to determine whether the regulation of COX-2 expression is mediated by Sp1 and/or Sp3 activation under oxidative stress. We also examined the role of COX-2 as a downstream pathway in relation to the known prosurvival role of Sp1 in primary neurons subjected to various stress.
PRINCIPAL FINDINGS
1. COX-2 transcripts and protein levels are regulated by oxidative stress in primary neurons
To explore the mechanism by which oxidative stress regulates COX-2 expression and examine the role of COX-2 in neuronal survival and death, we used an established in vitro glutathione depletion-induced oxidative stress model in primary neurons. The level of COX-2 protein was increased dose-dependently (1 and 5 mM) in response to HCA-induced oxidative stress in cortical neurons. RT-polymerase chain reaction (RT-PCR) analysis detected COX-2, COX-1, and acidic ribosomal phosphoprotein (PO) transcripts. We found that the transcripts of COX-2 were also clearly increased by HCA-induced oxidative stress in embryonic cortical neurons after 3 h. HCA (3 mM) treatment did not change the transcripts level of COX-1 or PO. COX-2 protein was induced 4 h after oxidative stress, and its level lasted for 8 h.
2. Two proximal Sp1-like sites are responsible for the basal and oxidative stress-induced COX-2 promoter activity in primary neurons
To investigate the regulation of COX-2 expression by Sp1 and Sp3 and to identify the transcriptional regulatory regions in the human COX-2 promoter, we constructed a nested series of proximal COX-2 promoter fragments containing various lengths of the 5'-flanking region using polymerase chain reaction (PCR). The parental plasmid employed in this study spanned nucleotides –3930 to + 100. Each deletion construct was generated by inserting DNA fragments of various lengths into the luciferase (Luc) gene, then analyzed for promoter activity. Deletion constructs –850/+50, –550/+50, and –350/+50 showed a 3- to 4-fold increase in Luc activity compared with –50/+50, which has no Sp1-like sites. Then we measured the COX-2 promoter activity of deletion constructs in response to oxidative stress induced by HCA. Under oxidative stress, Luc activity of the –850/+50 and –350/+50 was similar and increased by 1.8-fold in comparison with –50 to +50. These results show that the deletion construct (–350/+50) harbors a region necessary not only for basal transcriptional activation but also for oxidative stress-dependent expression of COX-2 through Sp1 sites.
3. Mutation of Sp1 sites abolishes oxidative stress-induced COX-2 promoter activity
To confirm the functional Sp sites in proximal COX-2 promoters, we introduced mutations by site-directed mutagenesis into two GC element sites, –268/–267 and –244/–243, which showed evident Sp1 and Sp3 DNA binding activity by EMSA. As expected, mutations in Sp1 DNA binding sites of the COX-2 promoter abolished the promoter activity. Mutations in GC elements –268/–267 and –244/–243 in the COX-2 promoter decreased basal Luc activity by 2-fold. In addition, double mutations in both –268/–267 and –244/–243 elements abrogated the basal activity of the COX-2 promoter, which is close to the basal expression level of pGL3 vector.
4. Dominant-negative form of Sp1 (Sp1-ZnF) nullifies oxidative stress-induced COX-2 promoter activity
With increasing amounts of expression vectors for the dominant negative (DN) form of Sp1 (pHSV-Flag-Sp1-ZnF), COX-2 promoter activity levels induced by Sp1 and Sp3 were suppressed near to or lower than basal levels. These results indicate that the DN form of Sp1 reduces Sp1 and Sp3 transcriptional activity by replacing the DNA binding activity in both transcription factors. Levels of COX-2 promoter activity induced by oxidative stress were nullified by Sp1-ZnF. Thus, the data provide evidence that COX-2 promoter activity induced by oxidative stress is mediated by a Sp1-dependent mechanism.
5. Enforced expression of Sp1 and Sp3 up-regulates COX-2 mRNA and protein levels in primary cortical neurons
To assess the COX-2 protein level in response to Sp1 and Sp3 overexpression using HSV-recombinant vectors, primary neurons were infected with a range of MOI. Enforced expression of Sp1 and Sp3 for 16 to 18 h increased COX-2 mRNA without a change in transcripts level of COX-1 or PO protein. HCA-induced COX-2 promoter activity was abolished by the DN form of Sp1 (pHSV-Flag-Sp1-ZnF). The overexpression of wild-type (WT) Sp1 and Sp3 increased the level of COX-2 protein. A low MOI (1 MOI) of coinfection with Sp1 and Sp3 showed a synergistic elevation of COX-2 protein levels in primary cortical neurons.
6. COX-2–/– cortical neurons are more susceptible to DNA damage
To confirm whether COX-2 is implicated in the pathway of DNA damage-induced neuronal death, we testedthe susceptibility of COX-2 knockout (COX-2–/–) cortical neurons to camptothecin. COX-2–/– cells were significantly more susceptible to CPT-induced DNA damage than WT (COX-2+/+) and heterogygous (COX-2±) cells (Fig. 1
). These data indicate that neurons are more vulnerable to genotoxic stress in the absence of functional COX-2.
7. COX-2 expression protects ischemia-induced neuronal DNA damage in vivo
The in vivo effect of the transient expression of COX-2 on the ischemic injury-induced neuronal DNA damage was determined by transduction of Ad-COX-2 into the brain by microinjection. Ad-COX-2 infected cells were resistant to DNA damage induced by global ischemia but Ad-Track infected cells were not. Surprisingly, a comparison of TUNEL-positive cells in the region of adenovirus transduction showed that neurons with COX-2 overexpression were 3-fold more resistant to DNA damage than ischemic-induced neurons with Ad-Track transduction.
CONCLUSIONS AND SIGNIFICANCE
In the present study we found that the role of Sp1 and Sp3 in the neuronal induction of COX-2 is a sufficient requirement and mediates a high basal level of transcription. On the other hand, both Sp1 and Sp3 activity are required for efficient COX-2 reporter activation, COX-2 mRNA transcription, and COX-2 protein levels in HCA-treated primary neurons. Our results demonstrate that both Sp1 and Sp3 bind in several GC-rich regions of human COX-2 promoter and induce transcriptional activation of COX-2 in cortical neurons. Sp3 synergistically increases Sp1-induced COX-2 expression. One of the most striking results is that a putative recognition motif for Sp1 located in the –280/–261 region of the human COX-2 promoter sequence increased transcription in response to oxidative stress in primary neurons. This suggests that this putative Sp1 motif represents a bona fide oxidative stress response element that apparently plays an important role as a transcription activator on oxidative stress stimuli in primary neurons (Fig. 2
). Thus, our finding that Sp1 and Sp3 sites determine COX-2 expression in basal conditions and in response to oxidative stress provides a novel insight into molecular mechanisms underlying the regulation of the COX-2 gene in primary neurons.
We recently found that enhanced neuronal protection by histone deacetylase inhibitors in response to oxidative stress is mediated by Sp1 acetylation in primary neurons and in an animal model of Huntington’s disease. The fact that acute oxidative stress induces Sp1 acetylation represents the protective role of Sp1 as the frustrated attempt of neurons to protect themselves from oxidative stress-induced cell death. These studies strongly imply that Sp1 promotes neuronal survival in response to oxidative stress. However, the mechanism of neuronal protection and the downstream target of Sp1 in response to oxidative stress remain to be examined. In the present study, we demonstrated the role of the COX-2 gene as a downstream target of Sp1 that mediates the protective role of Sp1 in primary neurons under oxidative stress (Fig. 2)
. The up-regulation of COX-2 is both protective and deleterious, depending on stimuli and many other factors that determine the pathophysiological fate of COX-2. For example, overexpression of COX-2 in endothelial cells up-regulates the vasoactive prostaglandin I2 (PGI2). PGI2 suppresses monocyte activation and its adhesion to endothelial surface. These data show that the increased level of COX-2 in endothelial cells is linked to its protective role in vascular injury. Moreover, PGs produced by COX-2 are important for the early steps of liver regeneration after partial hepatectomy. Other lines of data also suggest that COX-2 can reduce cell death in cardiac tissue and that long-term inhibition of COX-2 can enhance ishemic injury in hearts.
Associations between COX-2, ischemic injury, and oxidative stress-induced cell death suggest that COX-2 plays a role in the onset and progress of disease, but the mechanisms of the pathogenic function of COX-2 have not been clearly defined. Our current data showing that COX-2 expression diminishes TUNEL-positive cells in the cerebral region of ischemic brain suggest that COX-2 directly mediates neuronal cell survival by preventing DNA damage induced by ischemia. Furthermore, our finding that COX-2 protects neuronal cell damage induced by KA in vitro agrees with an earlier study that specific COX-2 inhibitors aggravated KA-induced neuronal DNA damage in an in vivo model. These findings suggest that enhanced COX-2 induction can contribute to postischemic signaling response in neurons and regulate an adaptive neuronal response to altered environmental conditions. Therefore, COX-2 is likely to be a homeostatic protein that mediates neuronal survival by preventing DNA damage under oxidative stress.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5957fje
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