mPGES-1 was the first enzyme identified and characterized as capable of catalyzing the conversion of PGH2
]. The generation of mice lacking this enzyme verified the role of mPGES-1 in PGE2
synthesis, both in its contribution to basal PGE2
levels and to increased PGE2
production during inflammatory responses [9
A number of observations made during the study of these mice suggested the existence of mPGES-1 independent PGE2
production. The expression levels of mPGES-1 in various tissues did not correspond well with the basal levels of PGE2
produced by these tissues. Furthermore, while mPGES-1 was responsible for the majority of PGE2
biosynthesis, measurable amounts of PGE2
could still be detected in mPGES-1 deficient cells and tissues. Moreover, contrary to expectation, some phenotypes of mice lacking COX-2 and the EP receptors were not evident in mPGES-1 deficient animals. Notably, the patent ductus observed in COX-2 deficient [27
] and EP4 deficient [28
] mice, was not recapitulated in mPGES-1 deficient mice [29
A possible explanation for these observations was suggested by the cloning of an enzyme, designated mPGES-2, initially purified from microsomal fractions of bovine heart [15
]. Over-expression of mPGES-2 in cell lines resulted in increased PGE2
]. Several subsequent publications furthered the supposition that mPGES-2 was a PGE2
synthase. Interestingly, mPges-2
is located in a cluster of genes, including Cox-1
, on mouse chromosome 2. To define the in vivo
role of mPges-2
biosynthesis, we have generated a mouse carrying a null allele of this gene. No mPges-2
mRNA can be detected in any of the tissues examined to date. However, surprisingly, analysis of PGE2
levels in these mice fails to support the classification and designation of mPges-2
as a prostaglandin E2
synthase. Basal PGE2
levels were not decreased in any of the mPGES-2 deficient tissues examined in this study. Ex vivo
studies of macrophages isolated from mPges-2
−/− mice also failed to support a role for mPGES-2 in PGE2
−/− macrophages produced amounts of PGE2
similar to wild-type controls in response to AA, Ca++
ionophore and LPS. Under identical conditions, mPGES-1 independent PGE2
production was measured as reported previously [9
]. These data suggest that mPGES-2 is not responsible for residual PGE2
production observed in mPGES-1 deficient mice.
These findings are in contrast with studies published by Tanikawa et al.
and Murakami et al.
], which demonstrate that mPGES-2 enzymatically promotes PGE2
biosynthesis in vitro
. In studies by Murakami et al.
, over-expression of mPGES-2 in cell lines, which express mPGES-1 at very low levels, resulted in a measurable increase in PGE2
production. The basis for the apparent discrepancy between these observations and our results is not obvious. It is possible that the ability of transfected cells to increase PGE2
was the indirect consequence of increased levels of this protein and not its function as a synthase. For instance, increases in PGE2
levels may potentially result from facilitating the non-enzymatic conversion of PGH2
or by alterations in the catabolism of PGE2
. It is possible that the ability of mPGES-2 to facilitate PGE2
production requires very high and non-physiological levels of expression, which are not observed in most healthy, normal tissues. However, at this time, we also cannot rule out the possibility that populations of cells or tissues not examined in this study can utilize this protein in the production of PGE2
Studies of mPGES-2 deficient mice also failed to support reports that this gene could act as GBF-1, a novel transactivator of IFN-γ dependent gene expression. Hu et al.
] demonstrated that mPGES-2/GBF-1 activated transcription of ISGF3g/p48/IRF-9 upon IFN-γ treatment of RAW264.7 murine macrophage cells. While a robust induction of Irf9
was measured, both in wild-type and mPges-2
−/− peritoneal macrophages stimulated with IFN-γ, Irf9
levels measured in mPGES-2 deficient macrophages were not significantly different from the wild-type macrophages (). Hu et al.
also demonstrate that Gbf-1/mPges-2
mRNA expression was induced in RAW cells after 8 hours of IFN-γ stimulation. In contrast, Gbf-1/mPges-2
mRNA levels are not up-regulated in peritoneal macrophages isolated from wild-type mice () after 8 or 16 hours of IFN-γ stimulation. In the context of these data, it is not surprising that Irf9
levels were not significantly reduced in mPges-2
−/− peritoneal macrophages. It is possible that peritoneal macrophages, unlike, RAW264.7 murine macrophage cells may not be dependent on GBF-1/mPGES-2, and these cells may have a different transcriptional program resulting from their immortalization.
Meng et al.
] recently reported that IFN-γ induces the interaction of GBF-1/mPGES-2 with CEBP-β, and that recruitment of GBF-1 to the GATE/ CEBP-β element is CEBP-β dependent. It is therefore possible that IRF-9 levels are not significantly decreased in the absence of mPGES-2 because IRF9
transcription is predominantly dependent on transcription factors such as CEBP-β in peritoneal macrophages. Alternately, it remains possible that steady state mRNA levels may not accurately represent changes in the rate of transcription of the IRF9
Because of the report that mPGES-2/GBF-1 may play a role in regulation of gene expression we carried out an additional line of inquiry into this potential function, using genome wide expression analysis of the heart. Surprisingly, few differences were found in the gene expression pattern of hearts lacking mPges-2. Of the genes with statistically significant differences in expression levels, only one of the five candidates tested could be validated by quantitative real-time PCR. We verified that expression levels of Trim13/RFP2/Leu5 (tripartite motif 13, Ret Finger Protein 2), were significantly down regulated in the heart, and interestingly, in the kidney and proximal small intestine, as well.
Trim13/ Leu5/ RFP2, a putative transcription factor, is a member of the tripartite motif family proteins containing RING finger, B-box type and coiled-coil domains [31
]. The mouse Trim13/RFP2/Leu5
gene locus located on chromosome 14 is not linked to mPges-2
and potentially encodes for two proteins, RFP2/Leu5 and DLTET, of unknown function [32
]. Loss of RFP2/Leu5 appears to be an early event in human B-cell CLL suggesting that RFP2/Leu5 is a tumor suppressor gene [34
]. To date, we have not observed any lymphoid tumors in mPges-2
−/− mice that are over 12 months old. Furthermore, FACS analysis of cells isolated from the spleen, thymus and lymph nodes has not revealed an abnormal increase in B-cells or any other lymphoid population. It is unclear at this time if the changes in Trim13
expression are directly caused by loss of mPges-2
dependent transcription or are indirect and potentially compensatory for the loss of mPGES-2 function. Thus, while a role for mPGES-2 in the regulation of IRF-9 is not supported by these studies, the gene expression profiles leave open the possibility that mPGES-2 can act as a transcription factor.
It was therefore of interest to determine if the increased basal levels of PGE2 observed in some tissues lacking mPGES-2 reflected altered expression of genes involved in PGE2 synthesis or metabolism. No differences were observed in expression levels of Cox-1, Cox-2 or mPges-1 mRNA, and while in some cases a decrease in expression of 15-Pgdh was observed, we failed to consistently observe increases in PGE2 levels. Furthermore, the potential increase in PGE2 levels was not observed after backcrossing of the mPges-2 mutation to the C57BL/6 genetic background.
In summary, our data indicates that, similar to cPGES, mPGES-2 does not encode a prostaglandin sythase. Thus mPGES-1 mediated conversion of cyclooxygenase-derived PGH2 to PGE2 may represent the only enzymatic pathway by which PGE2 is generated in vivo, with other non-enzymatic sources of production accounting for the differences in the phenotypes of the prostaglandin receptor-deficient mice and the mice lacking this enzyme.