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The Journal of Immunology, 2005, 175: 6271-6273.
Copyright © 2005 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Yin-Yang: Balancing Act of Prostaglandins with Opposing Functions to Regulate Inflammation

Asim K. Mandal1, Zhongjian Zhang, Sung-Jo Kim, Pei-Chih Tsai and Anil B. Mukherjee2

Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
For many years, cyclooxygenase-2 (COX-2), a critical enzyme for PG production, has been the favorite target for anti-inflammatory drug development. However, recent revelations regarding the adverse effects of selective COX-2 inhibitors have stimulated intense debate. Interestingly, in the early phase of inflammation, COX-2 facilitates inflammatory PG production while in the late phase it has anti-inflammatory effects. Moreover, although some PGs are proinflammatory, others have anti-inflammatory effects. Thus, it is likely that PGs with opposing effects maintain homeostasis, although the molecular mechanism(s) remains unclear. We report here that an inflammatory PG, PGD2, via its receptor, mediates the activation of NF-{kappa}B stimulating COX-2 gene expression. Most interestingly, an anti-inflammatory PG (PGA1) suppresses NF-{kappa}B activation and inhibits COX-2 gene expression. We propose that while pro- and anti-inflammatory PGs counteract each other to maintain homeostasis, selective COX-2 inhibitors may disrupt this balance, thereby resulting in reported adverse effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Prostaglandins, commonly known as lipid mediators of inflammation, are produced from arachidonic acid by cyclooxygenase (COX)3 enzymes. There are two isoforms of COX: COX-1 and COX-2. Although COX-1 facilitates the generation of PGs that are essential for maintaining physiological processes, COX-2 generates PGs that are linked to inflammation and cancer (1). Thus, for decades, inhibition of COX-2 has been the target of anti-inflammatory drug development (2). However, several adverse effects of selective COX-2 inhibitors (3) have become the subject of intense debate (4), although the molecular mechanism(s) that mediates these adverse effects remains unclear. Recent reports indicate that during the early phase of agonist-induced inflammation, COX-2 generates primarily inflammatory PGs such as PGD2 and PGF2{alpha}, while in the late phase, it may facilitate the production of anti-inflammatory PGs (5). It has been reported that cyclopentenone PGs, such as PGA1, play important roles in the resolution of inflammation (6). Although it is established that the biological effects of PGs, generated by inflammatory or allergic stimuli, are mediated via heterotrimeric G protein-coupled receptors, it is not clear 1) whether the effects of anti-inflammatory PGs counteract those of inflammatory PGs, and 2) if so, how might the anti-inflammatory PGs exert such effects. The answers to these questions may explain at least some of the adverse effects of selective COX-2 inhibitors (3). In the present study, we report that PGD2 and PGF2{alpha} mediate NF-{kappa}B activation stimulating the expression of COX-2, which is critical for the generation of proinflammatory PGs. PGA1, in contrast, suppresses NF-{kappa}B activation and inhibits COX-2 gene expression. Thus, the inflammatory and anti-inflammatory PGs appear to play a yin-yang role to maintain homeostasis.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Cell culture and treatment

The NIH 3T3 cells (American Type Culture Collection) were grown in DMEM supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C with 5% CO2. Before the treatment with various effectors (5 µM PGD2 and 5 µM PGF2{alpha} or PGA1), cells were grown in their respective recommended growth medium to 70–80% confluence, washed once with OptiMem-1 medium containing 2.5% FBS, and then treated with the indicated effectors in OptiMem-1 medium containing 2.5% FBS for 2 h at 37°C with 5% CO2.

RNA isolation and Northern blot analyses

Total RNAs were isolated using either RNazol B (Tel-Test) following the suppliers’ protocol. Thirty micrograms of the total RNA, loaded in each lane, was resolved by electrophoresis on 1.5% formaldehyde-agarose gels. Following electrophoresis, RNAs were transferred to Hybond N+ (Amersham Biosciences), cross-linked, and hybridized with [{alpha}-32P]deoxycytidine triphosphate-labeled cDNA probes at 68°C. Normalization of the amount of RNA loaded was achieved by hybridizing the same blots with GAPDH probe. Mouse and human cDNA probes, used to hybridize the Northern blots, were generated by RT-PCR.

Western blot analyses

For detection of proteins, cell lysates were prepared in presence of protease and protein phosphatase inhibitors. Forty micrograms of total protein from each sample was resolved by electrophoresis using 7.5% SDS-polyacrylamide gels under reducing conditions. Proteins were then electrotransferred to polyvinylidene fluoride membrane (Immobilon P; Millipore). Immunoblot analysis was performed using rabbit polyclonal anti-COX-1/COX-2 (Santa Cruz Biotechnology). HRP-conjugated anti-rabbit IgG was used as the secondary Ab. Chemiluminescent detection was performed by using ECL system (Amersham Biosciences) according to the manufacturer’s instructions.

Nuclear extract preparation, EMSA, and NF-{kappa}B assay

Cells were treated with 5 µM PGD2 or 5 µM PGF2{alpha} for 1 h in the presence and in the absence of 5 µM PGA1. Nuclear extracts were prepared using commercially available buffers (GENEKA Biotech) following the protocol provided by the supplier. EMSAs were performed using the nuclear extracts (20 µg protein) on a nondenaturing 5% polyacrylamide gel with the following oligonucleotides: mNF-{kappa}Bwt: 5'-GAG GGT GAG GGG ATT CCC TTA GTT AGG AC-3'. NF-{kappa}B double-stranded oligonucleotides were generated by annealing sense and antisense oligonucleotides. Specificity of protein-DNA complexes was verified by competing with cold NF-{kappa}B oligos or by immunoreactivity with polyclonal Abs specific for p65/p50 subunits of NF-{kappa}B. The supernatants were tested for the activity of NF-{kappa}B using TransAM Flexi NF-{kappa}B assay kit (Active motif), according to the manufacturers recommendations. Ten micrograms of nuclear protein was tested in each well.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
To determine the effects of the pro- and anti-inflammatory PGs on gene expression, we treated NIH 3T3 cells with various PGs and analyzed the level of COX-2 mRNA expression by Northern blot analysis. The results show that while PGD2 and PGF2{alpha} stimulate COX-2 mRNA expression at a high level, other PGs, including cyclopentenone PG, PGA1, and arachidonic acid, do not (Fig. 1A). To determine whether the anti-inflammatory cyclopentenone PG, PGA1, counteracts the effects of PGD2 and PGF2{alpha}, we treated the cells with these PGs in the presence and in the absence of PGA1. The results show that while in the absence of PGA1 both PGD2 and PGF2{alpha} stimulate the expression of COX-2 mRNA (Fig. 1, B and C) and COX-2 protein (Fig. 1, D and E), the presence of PGA1 suppresses the expression of COX-2 mRNA and protein expression.



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  		FIGURE 1. PGA1 inhibits PGD2- and PGF2{alpha}-mediated COX-2 expression. Northern blot analysis demonstrates PGD2 and PGF2{alpha} treatment (for 2 h at 37°C) stimulate COX-2 mRNA expression in NIH 3T3 cells (A). PGA1 inhibits the PGD2- and PGF2{alpha}-induced COX-2 mRNA expression in a dose-dependent manner (B and C). Western blot analysis demonstrates that PGA1 inhibits PGD2- and PGF2{alpha}-induced COX-2 protein expression (D and E) cells.

 
It has been reported that PGD2 and PGF2{alpha} exert their biological effects via G protein-coupled receptors, DP and FP, respectively (reviewed in Ref.7). Because activation of NF-{kappa}B by PGD2 and PGF2{alpha} via these receptors stimulates COX-2 gene expression (Ref. 8; A. K. Mandal et al., manuscript in preparation), we performed EMSA to determine whether PGA1 treatment of the cells inhibits PGD2– and/or PGF2{alpha}–mediated NF-{kappa}B activation. The results show that in both cell lines PGA1 suppresses NF-{kappa}B activation (Fig. 2A). We further performed a NF-{kappa}B activity assay, and our results demonstrate that PGA1 suppresses NF-{kappa}B activity in the cells (Fig. 2B). Taken together, these results strongly suggest that inflammatory PGs via receptor-mediated pathways mediate the activation of NF-{kappa}B, thereby stimulating the expression of COX-2, a critical enzyme for the production of proinflammatory lipid mediators. In this scenario, the anti-inflammatory PGA1 counteracts these effects by suppressing the activation of NF-{kappa}B, which is essential for COX-2 gene expression. The physiological roles of cyclopentenone PGs such as PGA1 are not clearly established as the levels of these PGs are somewhat difficult to determine because conversion of PGE2, PGE1, and PGD2 to PGA1 may occur in vitro by dehydration within the cyclopeptane ring (9). Undoubtedly, further investigations establishing the levels of PGA1 by mass spectrometry may allow quantitation of the physiological levels of this PG and its role in suppressing inflammation. Nevertheless, our results at least in part provide a proof of principle that PGA1 inhibits COX-2 gene expression via suppression of NF-{kappa}B activation. We propose that a balance between the levels of pro- and anti-inflammatory PGs maintains homeostasis and disruption of this balance, which may occur due to preferential inhibition of COX-2 activity by selective COX-2 inhibitors, may underlie the adverse effects of these agents.



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  		FIGURE 2. PGA1 inhibits DP- and FP-mediated NF-{kappa}B activation. An EMSA using nuclear extracts from control and PGD2- or PGF2{alpha}-treated (for 1 h at 37°C) cells and 32P-labeled double-stranded NF-{kappa}B in the absence and in the presence of respective double-stranded competitor unlabeled oligo, demonstrating the effect of DP- and FP-signaling on the activation of NF-{kappa}B. This activation of NF-{kappa}B by PGD2 and PGF2{alpha} is inhibited in presence of PGA1 (A). PGA1 inhibits the PGD2-induced NF-{kappa}B activity in the cells. The control bar represents the non-PGD2-induced NF-{kappa}B activity (B).

 

   Acknowledgments
 
We thank J. Chou, I. Owens, and S. W. Levin for critical review of the manuscript and for helpful suggestions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Current address: Department of Medicine/Renal Unit, Harvard Medical School and Massachusetts General Hospital-East, Charlestown, MA 02129. Back

2 Address correspondence and reprint requests to Dr. Anil B. Mukherjee, Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 9D42, Bethesda, MD 20892-1830. E-mail address: mukherja{at}exchange.nih.gov Back

3 Abbreviation used in this paper: COX, cyclooxygenase. Back

Received for publication May 6, 2005. Accepted for publication September 14, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 

  1. Warner, T. D., J. A. Mitchell. 2004. Cellular mechanisms of acetaminophen: role of cyclooxygenase. FASEB J. 18:790.-804. [Abstract/Free Full Text]
  2. Vane, J. R., Y. S. Bakhle, R. M. Botting. 1998. Cyclooxygenases 1 and 2. Annu. Rev. Pharmacol. Toxicol. 38:97.-120. [Medline]
  3. Mukherjee, D., S. E. Nissen, E. J. Topol. 2001. Risk of cardiovascular events associated with selective COX-2 inhibitors. J. Am. Med. Assoc. 286:954.-959. [Abstract/Free Full Text]
  4. Wadman, M.. 2005. Drug safety special: the safety catch. Nature 434:554.-556. [Medline]
  5. Gilroy, D. W., P. R. Colvile-Nash, D. Willis, J. Chivers, M. J. Paul-Clark, D. A. Willoughby. 1999. Inducible cyclooxygenase may have anti-inflammatory properties. Nat. Med. 5:698.-701. [Medline]
  6. Rossi, A., P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin, M. G. Santoro. 2000. Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of I{kappa}B kinase. Nature 403:103.-108. [Medline]
  7. Hata, A. N., R. M. Breyer. 2004. Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation. Pharmacol. Ther. 103:147.-166. [Medline]
  8. Mandal, A. K., Z. Zhang, R. Ray, M. S. Choi, B. Chowdhury, N. Pattabiraman, A. B. Mukherjee. 2004. Uteroglobin represses allergen-induced inflammatory response by blocking PGD2 receptor-mediated functions. J. Exp. Med. 199:1317.-1330. [Abstract/Free Full Text]
  9. Straus, D. S., C. K. Glass. 2001. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Med. Res. Rev. 21:185.-210. [Medline]



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