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Urogenital distribution of a mouse membrane-associated prostaglandin E2 synthase

Youfei Guan1, Yahua Zhang1, André Schneider1, Denis Riendeau2, Joseph A. Mancini2, Linda Davis1, Martin Kömhoff1, Richard M. Breyer1,3, and Matthew D. Breyer1,4

1 Division of Nephrology, Departments of Medicine, 4 Molecular Physiology and Biophysics, and 3 Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37212; and 2 Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada H9H 3L1


    ABSTRACT
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First published August 15, 2001; 10.1152/ajprenal.00116.2001.---PGE2 plays a critical role in regulating renal function and facilitating reproduction. One of the rate-limiting biosynthetic enzymes in PGE2 synthesis is the terminal PGE2 synthase (PGES). In the present studies, we report the functional expression of a membrane-associated murine PGES (mPGES) and its expression in urogenital tissues. Two independent cDNA clones sharing an identical open reading frame of 459 bp and encoding a peptide of 153 amino acids, but differing in the 3'-untranslated region, were identified. Assays for enzymatic activity, using microsomes prepared from cells transfected with mPGES cDNA, showed that these cDNA sequences encode a functional protein that catalyzes the conversion of PGH2 to PGE2. Constitutive expression of mPGES was highest in the mouse kidney, ovary, and urinary bladder but was also expressed at lower levels in uterus and testis. Renal mPGES expression was predominantly localized to epithelia of distal tubules and medullary collecting ducts. High expression was also seen in transitional epithelial cells of bladder and ureter and in the primary and secondary follicles in the ovary. In conclusion, mPGES is constitutively expressed along the urogenital tract, where it may have important roles in normal physiology and disease.

prostaglandin E2; membrane-associated prostaglandin E synthase; gene expression; urogenital tissue


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

PGE2 is an important regulator of many biological processes, including cell growth, inflammation, reproduction, and regulation of sodium homeostasis and blood pressure (2, 6). PGE2 synthase (PGES), a terminal prostanoid synthase, enzymatically converts the cyclooxygenase product PGH2 to PGE2. Recently, a microsomal membrane-associated GST-like enzyme that exhibits significant PGES activity has been identified (14). The expression of this enzyme can be induced on stimulation with inflammatory factors including lipopolysaccharide and interleukin-1 (14). On the basis of these findings, it has been suggested that this inducible membrane-associated PGE synthase (mPGES) could share a common gene-regulatory mechanism with cyclooxygenase-2 (COX-2) and may contribute to the increase in PGE2 biosynthesis associated with inflammatory and pyretic responses. Both COX-2 and PGE2 play critical roles in the kidney (3, 4) as well as in ovulation and reproduction (13, 17, 21). In the present studies, we report the characterization of a homologous mouse PGES and its localization along the mouse genitourinary tract.


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MATERIALS AND METHODS
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Materials and animals. Rabbit anti-human PGES antibody was purchased from Cayman Chemical (Ann Arbor, MI). A Tri Reagent RNA isolation kit was from Molecular Research Center (Cincinnati, OH). C57Bl/6 mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN).

Cloning of mouse mPGES cDNAs. A mouse expressed sequence tag (EST clone ID 621040, GenBank accession no. AA178132), showing high homology to human PGES, was purchased from Research Genetics (Huntsville, AL). The 806 bp of the insert were used as a probe to screen a mouse kidney cDNA library (Stratagene). Positive plaques were purified, and isolated cDNA-bearing phages were rescued to the plasmid form by using a M13 helper phage. Clones were analyzed by restriction digestion and sequenced.

Microsomal preparation and immunoblotting. A microsomal preparation was obtained from COS-1 cells transfected with a mouse PGES expression vector, as previously reported (14). Immunoblot analysis was used to examine protein levels of mPGES, using a rabbit anti-human PGES antibody (1:1,000).

Measurement of enzymatic activity of PGES. Enzymatic activity of PGES in microsomal fractions was measured by assessment of conversion of PGH2 to PGE2 in the presence of glutathione, as reported previously (18).

Northern hybridization and RNase protection assay. Northern hybridization and an RNase protection assay were performed to examine the tissue distribution of mPGES in mouse urogenital tissues, as described previously (10, 19). The full-length (831 bp) and EcoRI/NcoI fragment of mPGES1 cDNA (455 bp) were used as probes for Northern hybridization and the nuclease protection assay, respectively. Mouse beta -actin (250 bp) was utilized for RNA loading control.

In situ hybridization. In situ hybridization was performed to examine mPGES expression in mouse ovary, uterus, testis, kidney, and bladder, as previously described (10). A full-length mPGES1 cDNA (831 bp) was utilized to synthesize 35S-UTP-labeled sense and antisense riboprobes.

Immunohistochemistry. To define the mPGES-positive nephron segments, immunostaining of renal tissue sections was performed, using rabbit anti-human PGES antibody (1:250). To better define the mPGES-positive nephron segments, in situ hybridization was followed by immunostaining of tissue sections, using a goat anti-human Tamm-Horsfall antibody (Organon-Technika) that specifically recognizes medullary and cortical thick ascending limb as well as the early portion of the distal tubule.


    RESULTS AND DISCUSSION
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Of the three cDNA clones isolated from kidney library screening, two were identical and composed of a 831-nucleotide sequence (mPGES1), whereas the third clone contained 1,314 nucleotides (mPGES2), sharing the 5'-untranslated region (UTR) and open reading frame and differing only by the presence of an additional 483 bp in the 3'-UTR (Fig. 1A). The full-length cDNA sequence was further confirmed by 5'- and 3'-rapid amplification of cDNA ends (RACE), using mRNA from kidney and ovary (Marathon-ready cDNA kit, Clonetech) (data not shown). The open reading frame of mouse PGES cDNA encodes a predicted 153-amino acid polypeptide that is 94 and 78% identical to rat and human sequences, respectively (14, 18, 19). A database search revealed that mPGES belongs to the MAPEG superfamily (membrane-associated proteins involved in eicosanoid and GSH metabolism), with 38% homology to human MGST-1 (5).


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Fig. 1.   A: nucleotide and deduced amino acid sequences of mouse membrane-associated PGE synthase (mPGES). The conserved amino acids among membrane-associated proteins involved in eicosanoid and GSH metabolism (MAPEG family) are highlighted in bold italics. The stop codon is indicated (*), and the polyadenylation (poly A+) signal is underlined. Note that 2 mPGES cDNA variants share the same 5'-untranslated region (UTR), coding region, and partial 3'-UTR (319 bp). B: Northern blot analysis of mPGES in mouse kidney and ovary. Note that 3 transcripts were observed. C: RNase protection assay was utilized to examine the mRNA expression levels along the urogenital tract, and beta -actin was used to control for RNA loading.

Northern blot analysis of mPGES expression in mouse kidney and ovary was performed, using the full-length sequence from mPGES1 cDNA. As seen in Fig. 1B, two bands of ~0.8 and 1.3 kb were detected in each tissue and corresponded to the two cDNA species cloned from the kidney library. A third band of ~3.0 kb was also present in both tissues, suggesting that an additional mPGES variant exists. The existence of multiple variants of mPGES cDNA suggests that additional 5' or 3' exons might exist in the mPGES gene.

To determine whether these cDNAs encode an enzymatically active PGES, the coding region of mPGES cDNA was cloned into the protein expression vector pCR3.1. The resultant construct, mPGES/pCR3.1, was transfected into COS-1 cells. As shown in Fig. 2A, transfection of COS-1 cells with the full-length mouse PGES cDNA resulted in a significant increase in immunoreactive protein, as recognized by immunoblot analysis using an anti-PGES antibody. Enzymatic activity was also significantly increased in the transfected cells compared with mock-transfected cells (Fig. 2B). In agreement with human PGES, mouse PGES was primarily expressed in the cell membrane fraction, as indicated by its enrichment in microsomes on immunoblot analysis (Fig. 2A). However, because overexpression of mPGES protein in COS-1 cells could result in inefficient protein trafficking and retention in the Golgi or endoplasmic reticulum apparatus, false-positive distribution in microsomes cannot be excluded. The above-mentioned data demonstrate that the cloned mPGES cDNA encodes a protein that is immunologically related to human mPGES and efficiently catalyzes the synthesis of PGE2 from PGH2.


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Fig. 2.   A: immunoblot analysis of mPGES expression in COS-1 cells transfected with mPGES1/pCR3.1 expression vector or pCR3.1 vector (mock). Ten micrograms of protein from either whole cell lysate or microsomal fraction were fractionated by SDS-PAGE and transferred to nylon membrane. The blot was then probed with rabbit anti-human mPGES antibody. Arrow, ~ 16-kDa band of mPGES protein. B: time course of PGE2 formation using microsomal fraction from COS-1 cells transfected with mPGES/pCR3.1 expression vector (open circle ) or pCR3.1 mock vector (black-lozenge ). Product formation after incubation with PGH2 was measured in the presence of GSH and quantified by RP-HPLC.

To determine the tissue distribution of mPGES mRNA, an RNase protection assay and Northern blot analysis were performed. Mouse mPGES was highly expressed in certain urogenital tissues including kidney, bladder, and ovary, with lower levels of expression in testis and uterus (Fig. 1B). Tissue distribution of mPGES in the kidney and ovary was further explored by in situ hybridization and immunohistochemistry. In the kidney, mPGES mRNA and protein were mainly localized in the distal tubules and medullary collecting ducts (Fig. 3, A-D). This corresponds to previous studies showing that the collecting ducts exhibit the greatest rate of PGE2 synthesis of any segment along the nephron (1, 8). In the kidney, COX-2 expression is predominantly localized in macula densa, cortical ascending limb, and medullary interstitial cells, with little expression in collecting duct (9, 12, 16). In contrast, COX-1 is highly expressed in collecting ducts (11, 16), where mPGES was detected. These results raise the possibility of functionally coupling between COX-1 and mPGES in the kidney. This is somewhat different from the previous suggestion that mPGES functionally couples to COX-2 in macrophages (19).


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Fig. 3.   Photomicrograph showing in situ hybridization and immunohistochemistry of mouse mPGES in the kidney and ovary. A: autoradiogram demonstrating mPGES mRNA expression (white grains) in distal tubules in the cortex and collecting ducts in the medulla. B: immunohistochemical staining with an anti-PGES antibody showing mPGES expression over the distal tubules in renal cortex. Magnification: ×100. C: brown signals indicating immunoreactivity of mPGES in inner medullary collecting duct. Magnification: ×1,000. D: photomicrograph (×400) of mouse renal cortex showing mPGES mRNA (black grains) predominantly expressed in Tamm-Horsfall-negative distal tubules. The brown reaction product indicates regions labeling with ant-Tamm-Horsfall antibody. E: in situ hybridization demonstrating mPGES messenger RNA expression in the ovarian follicles. White grains (arrow) indicate areas of mPGES mRNA expression. Magnification: ×50 darkfield. F: photomicrograph (×200) showing mPGES mRNA expression in transitional epithelial cell of urinary bladder.

PG concentrations in the urine are among the highest of any body fluid, well above those in plasma (7, 20), suggesting active biosynthesis of PG along the urinary tract. The present study shows that mPGES mRNA is highly expressed in renal epithelia and urinary bladder (Fig. 1C). In situ hybridization further showed that mPGES mRNA labeling was particular intense in the transitional urothelium of mouse ureter (data not shown) and bladder (Fig. 3F). mPGES expression was restricted to the transitional epithelium, with no expression detected in surrounding smooth muscle. These studies demonstrate for the first time that the urothelium of the urinary bladder is a major site of mPGES expression.

PGs also play an important role in the physiological events influencing ovarian cellular function in all mammalian species, particularly during ovulation and luteolysis (13, 15, 17, 21). PGE2 plays a critical role in ovulation, and mice deficient in the PGE2 EP2 receptor exhibit a defect in ovulation with reduced litter size (13, 15). Recently, Hizaki et al. (13) showed that upregulation of the EP2 receptor in ovarian cumulus cells was necessary for normal reproduction. The present studies demonstrate that mPGES, the terminal enzyme for PGE2 synthesis, is highly expressed in mouse ovarian cells that coexpress the EP2 receptor, suggesting that mPGES could mediate synthesis of PGE2 functioning as an autocoid in these cells (Fig. 3E). These results provide clear evidence that the enzymatic machinery for PGE2 synthesis exists in the mouse ovarian follicle. At present, the factors regulating mPGES expression in the ovary remain uncharacterized.

In summary, we have cloned a functional mouse mPGES. Significant and localized expression of mPGES was found at several sites along the urogenital tract, including the kidney, bladder, and ovary. This expression pattern suggests that mPGES may play important roles in distal water and salt transport in the kidney and ovarian reproductive function. The development of mPGES inhibitors or gene-targeting studies will undoubtedly facilitate our understanding of the biological roles of mPGES.


    ACKNOWLEDGEMENTS

We thank for Drs. Scott Shappell and Richard Robert (Dept. of Pathology, Vanderbilt University Medical Center) for assistance in histological examination.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-39261 and DK-37097.

Address for reprint requests and other correspondence: Y. Guan, Div. of Nephrology, S-3223 MCN, Vanderbilt Univ. Medical Center, Nashville, TN 37232-2372 (E-mail:youfei.guan{at}mcmail.vanderbilt.edu).

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

First published August 15, 2001;10.1152/ajprenal.00116.2001

Received 10 April 2001; accepted in final form 23 July 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1173-F1177
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