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* Department of Internal Medicine, Division of Rheumatology, Kentucky Clinic, University of Kentucky, Lexington, Kentucky, USA;
Ophthalmology and Visual Sciences, Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, USA
2Correspondence: Department of Internal Medicine, Rheumatology Division, Rm. J-509, Kentucky Clinic, University of Kentucky, Lexington, KY 40536-0284, USA. E-mail: lcrofford{at}email.uky.edu
SPECIFIC AIMS
Microsomal prostaglandin E synthase-1 (mPGES)-1 is the inducible enzyme that acts downstream of cyclooxygenase (COX) and catalyzes the conversion of prostaglandin (PG) H2 to PGE2. Specific inhibitors of mPGES-1 are not yet available, however, mice with genetic deletion of mPGES-1 have been generated that have given insight into the specific role of mPGES-1 in eicosanoid biosynthesis in vivo and in peritoneal macrophages. We hypothesized that creating mouse embryo fibroblast (MEF) cell lines would facilitate investigation of the role of mPGES-1 in fibroblast biology. We generated MEF from heterozygous matings to create mPGES-1 null, mPGES-1 Het, and mPGES-1 wild-type (WT) lines.
Aims of this study were to:
1. Evaluate the effect of genetic deletion of mPGES-1 on the expression of major prostanoid biosynthetic enzymes including COX-2, COX-1, mPGES-2, cytosolic PGES (cPGES), PGIS, hematopoietic-PGDS (h-PGDS), thromboxane synthase (TXS).
2. Evaluate the effect of genetic deletion of mPGES-1 on the production pattern of major prostanoids including PGE2, 6-keto PGF1
(stable metabolic product of PGI2), PGD2, and TXB2 (stable metabolic product of TXA2) in order to evaluate the shunting possibility within the arachidonic acid (AA) metabolic pathway.
3. Evaluate and compare the consequences of genetic deletion and pharmacological inhibition of COX-2 vs. genetic deletion of mPGES-1 on prostanoid biosynthesis in MEFs.
4. Evaluate the effect of genetic deletion of mPGES-1 and subsequent effect of prostanoid biosynthesis on the regulation of other inflammatory enzyme outside the arachidonic acid (AA) metabolic pathway, inducible nitric oxide synthase (NOS) (iNOS) and endothelial NOS (eNOS), and production profile of NOS stable metabolic product, nitrite.
PRINCIPAL FINDINGS
1. mPGES-1 is critical for PGE2 production
The present study for the first time demonstrates the role of mPGES-1 in fibroblast biology using mPGES-1 null MEFs. Results show that coordinated induction of COX-2 and mPGES-1 is concomitantly accompanied by the increase in PGE2 levels in MEFs. Using interleukin (IL)-1ßbeta; stimulated and unstimulated mPGES-1 Het and null MEFs, we show that PGE2 levels significantly drop as a result of mPGES-1 gene deletion in a gene-dose dependent fashion, further emphasizing the fact that mPGES-1 is essential for PGE2 synthesis. This is despite the fact that COX-1, cPGES, and mPGES-2 are expressed in these cells under basal and stimulated conditions and COX-2 is dramatically up-regulated by IL-1ßbeta;. These results support the studies by other groups that have also shown that mPGES-1 is critical for PGE2 production and deletion of mPGES-1 gene results in a dramatic decline in PGE2 levels in peritoneal macrophages and microglia.
2. Shunting of endogenous prostanoid biosynthesis with mPGES-1 gene deletion
Using mPGES-1 WT, Het, and null MEFs in the present study, we show that genetic deletion of mPGES-1 results in the diversion of the prostanoid production profile from predominant PGE2 to 6-keto PGF1 under basal as well as proinflammatory cytokine-stimulated conditions in a gene dose-dependent manner without any significant changes in the levels of other prostanoids, including PGD2 and TXB2. Furthermore, there is no evidence of shunting to PGD2 and TXB2 despite presence of PGDS, TXS, and other terminal biosynthetic enzymes in these cells. We also investigated whether PGIS was up-regulated in mPGES-1 null MEFs resulting in the redirection of PGE2 to 6-keto PGF1 following mPGES-1 gene deletion. However, no significant changes in the mRNA and protein levels of PGIS and other PG synthetic enzymes (mPGES-2, cPGES, PGIS, H-PGDS, TXS, and COX-1) were observed with mPGES-1 gene deletion under basal or IL-1ßbeta; stimulated conditions. These results indicate that in the absence of induced mPGES-1, elevation of COX-2 after inflammatory insult and the resultant increase in the availability of PGH2 as the common substrate for the generation of other prostanoids could account for increased levels of 6-keto PGF1 observed with mPGES-1 deletion in mPGES-1 Het and null MEFs.
3. Absence of diversion of prostanoid biosynthesis from PGE2 to 6-keto PGF1 with COX-2 pharmacological inhibition and genetic deletion
In the present study we demonstrate that, unlike mPGES-1 deletion, COX-2 pharmacological inhibition and genetic deletion do not result in transition in the production pattern from PGE2 to 6-keto-PGF1. Using genetically deleted COX-2 MEFs and pharmacological inhibition with nonsteroidal antiinflammatory drugs (NSAID) such as selective COX-2 inhibitor (NS-398) and nonselective COX inhibitor (Indomethacin), we show that deletion/inhibition of COX-2 results in dramatic decrease in the levels of not only PGE2 but also 6-keto PGF1, thus showing clear differences between COX-2 vs. mPGES-1 inhibition on prostanoid synthesis profile in MEFs.
4. mPGES-1 and COX-2-derived PGE2 regulates iNOS expression and nitrite biosynthesis in mPGES-1 MEFs
In this study for the first time, we provide evidence that mPGES-1 deletion leads to up-regulation of iNOS expression and nitrite biosynthesis under proinflammatory cytokine-stimulated conditions. We observed that the levels of iNOS expression and nitrite biosynthesis in IL-1ßbeta; stimulated mPGES-1 null MEFs were significantly higher in comparison to low levels observed in mPGES-1 WT MEFs, which suggested that PGE2 may be involved in the regulation of iNOS and nitrite in these MEFs. To prove the involvement of PGE2 toward the regulation of iNOS and nitrite, we used NSAID (indomethacin and NS-398) treatment and exogenous PGE2 administration approaches in MEFs. Treatment with NSAIDs and resultant decrease in PGE2 levels up-regulated the levels of iNOS expression and nitrite synthesis in mPGES-1 WT MEFs, similar to the levels observed in mPGES-1 null MEFs. Similarly, exogenous administration of PGE2 reversed the enhanced iNOS expression and nitrite biosynthesis in mPGES-1 null MEFs, similar to the levels of mPGES-1 WT MEFs. Thus these results prove the fact that PGE2 derived from mPGES-1 and COX-2 negatively regulates iNOS expression and nitrite biosynthesis in MEFs.
CONCLUSION AND SIGNIFICANCE
PGE2 is a key proinflammatory mediator derived from COX-2 and mPGES-1, which has been shown to be elevated in various chronic inflammatory disorders, including reumatoid arthritis (RA), osteoarthritis (OA), atherosclerosis, Alzheimer’s disease, and various forms of cancer. COX-2 selective inhibitors exert their antiinflammatory pharmacological effects by inhibiting the excessive production of PGE2 at the site of inflammation. However, recent clinical studies have suggested serious cardiovascular side effects associated with the use of COX-2 inhibitors. One of the hypotheses put forward to explain the cardiovascular effects of COX-2 inhibition is the loss of antithrombotic PGI2 derived from endothelial COX-2, which plays a key role in the regulation of thrombogenesis.
In the present study we demonstrate that genetic deletion of mPGES-1 results in transition in the production pattern from PGE2 to 6-keto-PGF1, whereas pharmacological inhibition and genetic deletion of COX-2 results in dramatic decrease in the levels of not only PGE2 but also 6-keto-PGF1. In addition, our results demonstrate that mPGES-1 gene deletion and subsequent decrease in the levels of PGE2 causes elevation of nitrite levels. 6-keto-PGF1 and nitrite are stable metabolites of PGI2 and NO, which are involved in vasodilatation and maintenance of vascular homeostasis. Clinical studies have also indicated the beneficial effects of PGI2 and NO for the management of circulatory disorders. In view of the above facts, mPGES-1 pharmacological inhibition may escape the cardiovascular side effects seen with inhibition of COX-2. The efficacy of such a therapeutic strategy remains unclear since increased levels of PGI2 and NO could also exert proinflammatory effects as observed in the studies using PGI2 receptor and iNOS deficient mice. In light of our present results, however, inhibition of mPGES-1 continues to be an attractive therapeutic target for PGE2 inhibition in inflammatory conditions.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6366fje
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