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Am J Physiol Renal Physiol 294: F1129-F1135, 2008. First published March 5, 2008; doi:10.1152/ajprenal.00572.2007
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Loss of prostaglandin E2 release from immortalized urothelial cells obtained from interstitial cystitis patient bladders

Prerna Rastogi,1 Alice Rickard,1 Nikolay Dorokhov,1 David J. Klumpp,2 and Jane McHowat1

1Department of Pathology, Saint Louis University School of Medicine, St. Louis, Missouri; and 2Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, Illinois

Submitted 2 December 2007 ; accepted in final form 4 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Interstitial cystitis (IC) is associated with increased activated mast cell numbers in the bladder and impairment of the barrier function of the urothelium. We stimulated immortalized urothelial cells derived from the inflamed region of IC bladders (SR22A or SM28 abn) or from healthy bladders (PD07i or PD08i) with tryptase and measured phospholipase A2 (PLA2) activity and the resultant release of arachidonic acid and prostaglandin E2 (PGE2). Tryptase stimulation of either PD07i or SR22A resulted in similar increases in PLA2 activity and arachidonic acid release. However, tryptase stimulation of SR22A and SM28 abn did not result in a significant increase in PGE2 release compared with the increase in PGE2 release from tryptase-stimulated PD07i and PD08i cells. Expression of mRNA for cyclooxygenase-2 and PGE synthase was lower and mRNA for 15-hydroxyprostaglandin dehydrogenase was higher in SR22A compared with PD07i, suggesting that both decreased synthesis and increased metabolism are responsible for the lack of a PGE2 response in tryptase-stimulated SR22A cells. Since PGE2 is a cytoprotective eicosanoid, the failure to produce this metabolite in cells isolated from the IC bladder may represent an increased susceptibility to damage by proinfammatory stimuli.

chronic painful bladder; activated mast cells; neurogenic inflammation

INTERSTITIAL CYSTITIS (IC) is a chronic painful bladder condition of unknown etiology that is characterized by an increase in the number of activated mast cells, urothelial permeability changes, and neurogenic inflammation (25). Mast cells are the primary effectors of type 1 IgE-mediated immune reaction and release a battery of stored and newly formed mediators upon activation including biogenic amines such as histamine, neutral proteases such as tryptase, chymase, cathepsin, and carboxypeptidase, arachidonic acid metabolites, chemokines, and cytokines (25). Bladder biopsies from patients diagnosed with IC have demonstrated an increase in the number of IL-6-positive mast cells (17a, 21) and mast cell mediators such as tryptase (1) and histamine (39) are detected in the urine of IC patients.

In a previous study, we demonstrated that activation of urothelial cell calcium-independent phospholipase A2 (iPLA2) by tryptase leads to membrane phospholipid hydrolysis and rapid release of arachidonic acid (22). Bisoxygenation of free arachidonic acid catalyzed by either cyclooxygenase (COX)-1 or COX-2 results in formation of prostaglandin H2 (PGH2), which is the precursor of all prostanoid products including prostaglandins, prostacyclin, and thromboxanes (30). Further metabolism of PGH2 by PGE synthase (PTGES) converts PGH2 to PGE2, which exerts its effects via a family of heterotrimeric G protein-coupled receptors. The actions of PGE2 can be pro- or anti-inflammatory depending on the expression pattern of the PGE receptors in each tissue. In the bladder, PGE2 has been shown to be increased in carcinogenesis, overactive bladder, and urinary tract infections (11, 12, 27, 35, 36). However, PGE2 is widely accepted to be cytoprotective in the epithelium and is involved in wound repair and cell motility. Since IC is associated with impairment of urothelial integrity, it is compelling to suggest that this prostaglandin may be beneficial in this disease.

In this study, we stimulated urothelial cells isolated from a normal bladder or an IC bladder with tryptase and examined activation of iPLA2 and the resultant arachidonic and PGE2 release. We now report that PGE2 release is impaired in urothelial cells isolated from an IC bladder and discuss the results in the context of a possible link between decreased PGE2 release and impaired cytoprotection and wound-healing properties of the urothelium in IC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Recombinant human skin β tryptase was obtained from Promega (Madison, WI). Antibody for PAR-2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PGE2 assay kit was obtained from R&D Systems (Minneapolis, MN). For RT-PCR, the Versagene RNA purification kit was obtained from Gentra Systems and Superscript was from Invitrogen. TaqMan gene expression arrays were from Applied Biosystems. We carried out sequence analysis and confirmed that the information given to us by Applied Biosystems will recognize our gene of interest.

Culture of the urothelial cell lines. Normal pediatric bladder epithelial (PD07i and PD08i) and inflamed region of bladder epithelium from a patient with IC (SR22A and SM28 abn) cell lines were generated by immortalization with a retrovirus encoding the oncoproteins E6 and E7 of human papillomavirus type 16 and selected for stable integration of the retroviral provirus with G418. Expanded cultures were grown in EpiLife Media (Cascade Biologics, Portland, OR) with calcium (0.06 mM), growth factor supplements provided by the manufacturer, and penicillin (20 U/ml)/streptomycin (100 µg/ml; Sigma, St. Louis, MO).

Characterization of epithelial cells by cytokeratins, ZO-1 and E-cadherin. PD07i and SR22A cell cultures were fixed and examined for an anti-epithelial keratin AE1/AE3 mixture and E-cadherin as previously described (22). To detect ZO-1 protein, cultured cells were washed in PBS, 1 mM calcium chloride, 37°C, fixed in cold methanol, washed, and refrigerated until the time of staining. Cells were treated on ice with 0.5% Triton X-100, 10 mM piperazine ethane sulfonic acid, 50 mM NaCl, 300 mM sucrose, and 3 mM MgCl2, pH 6.8 for 2 min, washed, blocked, and incubated with primary antibodies to ZO-1 (Invitrogen, Frederick, MD) or nonimmune serum. Following treatment with Alexa Fluor secondary antibody (Molecular Probes, Eugene, OR), samples were viewed by confocal microscopy (MRC 1024; Bio-Rad, Hercules, CA). All confocal images were analyzed using ImageJ software and Image Pro Plus (MediaCybernetics, Silver Spring, MD).

Measurement of phospholipase A2 activity. SR22A and PD07i were grown to confluence in 100-mm tissue culture dishes. The surrounding medium was removed and replaced with ice-cold buffer containing (in mmol/l) 250 sucrose, 10 KCl, 10 imidazole, 5 EDTA, and 2 DTT with 10% glycerol, pH 7.8 (PLA2 assay buffer). The cell suspension was sonicated and PLA2 activity was assessed by incubating enzyme (200 µg of cellular protein) with 100 µM (16:0; [3H] 18:1) plasmenylcholine substrate (specific activity ~150 dpm/pmol) in assay buffer containing 100 mM Tris, pH 7.0, and 10% glycerol with 4 mM EGTA at 37°C for 5 min in a total volume of 200 µl. Reactions were terminated by the addition of 100 µl butanol and released radiolabeled fatty acid was isolated by TLC and quantified by liquid scintillation spectrometry.

Measurement of arachidonic acid release. SR22A and PD07i were grown to confluence in 35-mm tissue culture dishes. Arachidonic acid release was determined by measuring [3H] arachidonic acid released into the surrounding medium from cells prelabeled with 3 µCi of [3H] arachidonic acid (specific activity 100 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA) per culture dish for 18 h. Cells were washed three times with HEPES buffer containing (in mmol/l) 133.5 NaCl, 4.8 KCl, 1.2 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 10 HEPES (pH 7.4), 10 glucose, and 0.36% bovine serum albumin and incubated at 37°C for 15 min before addition of inhibitors and/or tryptase. At the end of stimulation, radioactivity in the medium and cells was quantified by liquid scintillation spectrometry.

Measurement of PGE2 release. Cells were grown to confluence in 16-mm tissue culture dishes. Cells were washed twice with Hanks balanced salt solution (HBSS) containing (in mmol/l) 135 NaCl, 0.8 MgSO4, 10 HEPES (pH 7.6), 1.2 CaCl2, 5.4 KCl, 0.4 KH2PO4, 0.3 Na2HPO4, and 6.6 glucose. After being washed, 0.5 ml of HBSS with 0.36% bovine serum albumin was added to each culture well. Cells were then stimulated with the appropriate tryptase concentrations and PGE2 release was measured using an immunoassay kit (R&D Systems).

PAR-2 surface expression assay. SR22A and PD07i were cultured to confluence in 16-mm culture dishes. Cultures were fixed in a solution containing 127 mM NaCl, 5 mM KCl, 1.1 mM NaH2PO4, 2 mM MgCl2, 5.5 mM glucose, 20 mM PIPES, and 1% paraformaldehyde at 4°C overnight. Cells were washed with PBS, blocked in Tris-buffered saline containing 0.8% bovine serum albumin, 0.1% Tween, and 0.5% gelatin and incubated with primary antibody to PAR-2 (1:50, Santa Cruz Biotechnology) followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:5,000). At the end of incubation, secondary antibody was removed and 3,3',5,5'-tetramethyl benzidine was added. Cells were incubated for 30 min in the dark, the reaction was stopped by 8 N sulfuric acid, and color development was measured at 450 nm.

Real-time PCR. Total RNA from both PD07i and SR22A cells was extracted using the Versagene RNA purification kit (Gentra Systems), and first-strand cDNA was synthesized using Superscript II (Invitrogen). All PCRs were performed using TaqMan Gene Expression Assays (Applied Biosystems) on a 7300 Real-Time PCR System (Applied Biosystems). The cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min. Experiments were performed in triplicate for each data point. For all experiments, controls without templates were included. Reactions were then amplified with IQ SYBR Green supermix (Bio-Rad). The fold change in mRNA level for each protein of interest was calculated using the {Delta}{Delta}CT method against either 18s RNA or β glucuronidase.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To verify the epithelial origin of PD07i and SR22A cells used in this study, we examined the cell morphology and the presence of cytokeratins, ZO-1 tight junction-associated protein, and E-cadherin (Fig. 1). Taken together, the results confirmed the presence of these proteins in both cell lines and determine that the cell lines originated from epithelial cells of the bladder (Fig. 1).


Figure 1
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  		Fig. 1. Characterization of epithelial cells in PDO7i (A) and SR22A (B) cell cultures. 1: Confluent monolayer of cells exhibiting epithelial cobblestone appearance. 2: Subconfluent, single cells stained for AE1, AE3 epithelial specific cytokeratins. 3–4: Stratified cell cultures stained for ZO-1 and E-cadherin, respectively.

 
Confluent monolayers of PD07i and SR22A cells were stimulated with tryptase for up to 30 min and PLA2 activity was measured in the absence of calcium using (16:0, [3H] 18:1) plasmenylcholine as substrate. Tryptase stimulation resulted in an increase in PLA2 activity in both PD07i and SR22A cells that returned to unstimulated values by 15 min (Fig. 2). Significant increases in PLA2 activity occurred after 1 min of tryptase stimulation in PD07i, whereas the increase in activity was delayed and smaller in SR22A (Fig. 2). Since PLA2 activity was measured in the absence of calcium, this activity represents calcium-independent PLA2 (iPLA2). We pretreated PD07i and SR22A with bromoenol lactone (BEL, a selective iPLA2 inhibitor, 2 µM, 10 min) before tryptase stimulation and determined that the increase in PLA2 activity was inhibited (data not shown), further supporting our hypothesis that iPLA2 is involved.


Figure 2
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  		Fig. 2. Activation of phospholipase A2 (PLA2) in PD07i ({blacksquare}) and SR22A ({circ}) following tryptase stimulation (20 ng/ml). PLA2 activity was determined by measurement of the release of radiolabeled fatty acid following incubation of enzyme (200 µg of cellular protein) with 100 µM (16:0; [3H] 18:1) plasmenylcholine substrate in assay buffer containing 4 mM EGTA at 37°C for 5 min. Data are shown as means ± SE for 3 separate cell cultures. *P < 0.05, **P < 0.01 compared with unstimulated values.

 
To investigate whether the delay in iPLA2 increased activity in SR22A cells was related to a difference in protease-activated receptor-2 (PAR-2) expression on the cell surface, the receptor density and cell number were determined in confluent monolayers of PD07i and SR22A. A measure of the cell surface expression of PAR-2 was achieved by developing an immunoassay that used an antibody to the extracellular NH2 terminus of the PAR-2 in nonpermeabilized monolayers. This allows selective assessment of the cell surface receptor expression independent of intracellular PAR-2 stores. Cell surface expression of PAR-2, as measured by absorbance at 450 nm, was found to be 40% less in SR22A compared with PD07i (Fig. 3). We fixed representative monolayers of SR22A and PD07i cells with methanol, stained them with hematoxylin and eosin, and counted the number of SR22A and PD07i in a defined area to estimate the number of cells per unit area in each culture. We found that there were 46 ± 4% (n = 3) more SR22A cells per unit area compared with PD07i. Taken together, these data suggest that the cell surface expression of PAR-2 per cell is lower in SR22A compared with PD07i and may explain the delay and attenuation of the tryptase-induced PLA2 response. However, although sequence analysis of the epitope of the PAR-2 antibody has been compared with the human protein database and shown to have no significant sequence similarity with any other protein (personal communication with Santa Cruz Biotechnology), we do not have experimental evidence that the antibody doesn't cross-react with other receptors. Therefore, our results should be taken in light that this antibody may have identified other PAR on the urothelial cell surface as well.


Figure 3
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  		Fig. 3. Expression of PAR-2 on the cell surface of confluent monolayers of PD07i and SR22A. Cell surface expression was determined on nonpermeabilized confluent monolayers using an antibody to the extracellular NH2 terminus of PAR-2 and an immunoassay as described in MATERIALS AND METHODS. Data are shown as means ± SE for 12 samples from 3 separate cell cultures. **P < 0.01 when comparing the 2 cell lines.

 
The delay in iPLA2 activity in SR22A compared with PD07i was reflected in the delay in [3H] arachidonic acid release from tryptase-stimulated SR22A and PD07i (Fig. 4). However, after 10 min of tryptase stimulation, there was no significant difference in the amount of arachidonic acid released from each cell line. The amount of [3H] arachidonic acid release from unstimulated PD07i or SR22A was not significantly different between time 0 and 30 min (data not shown).


Figure 4
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  		Fig. 4. [3H] arachidonic acid release from PD07i ({blacksquare}) and SR22A ({circ}) following tryptase stimulation (20 ng/ml). [3H] arachidonic acid released into the surrounding medium was measured after cells were prelabeled with 3 µCi of [3H] arachidonic acid per culture dish for 18 h. Radioactivity in the medium and cells was quantified by liquid scintillation spectrometry. Data are shown as means ± SE for 3 separate cell cultures. *P < 0.05, **P < 0.01 compared with unstimulated values.

 
Stimulation of confluent monolayers of PD07i with 20 ng/ml tryptase resulted in a fourfold increase in PGE2 release into the surrounding medium, whereas PGE2 release from SR22A was ~1.5-fold greater than unstimulated values (Fig. 5). This difference in the magnitude of PGE2 release was also observed at both 2 and 200 ng/ml tryptase (Fig. 5). To determine whether the increase in PGE2 release in immortalized urothelial cells from normal bladder was not affected by the immortalization process, we stimulated PD07i, PD08i, and primary human urothelial cells (HUR) with 20 ng/ml tryptase for up to 30 min. As shown in Fig. 6, increases in PGE2 release were similar between the three samples. Tryptase stimulation of an additional cell line obtained from IC bladder (SM28 abn) failed to show any increase in PGE2 release over 30 min (Fig. 7). Taken together, these data indicate that PGE2 release from immortalized cells isolated from IC bladders is significantly decreased compared with the responses from normal bladder urothelial cells and that the responses in normal immortalized cells are comparable to that observed in primary urothelial cell cultures.


Figure 5
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  		Fig. 5. PGE2 release from PD07i (A) and SR22A (B) following tryptase stimulation at 2 ng/ml (bullet), 20 ng/ml ({square}), or 200 ng/ml ({triangleup}). PGE2 release was measured using an immunoassay kit (R&D Systems). Data are shown as means ± SE of 6 separate cell cultures. **P < 0.01 compared with unstimulated values.

 

Figure 6
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  		Fig. 6. PGE2 release from PD07i, PD08i, and primary human urothelial cells (HUR) following 20 ng/ml tryptase stimulation. PGE2 release was measured using an immunoassay kit (R&D Systems). Corresponding PGE2 release in cell cultures incubated with the tryptase vehicle diluted to the same concentration as in the tryptase-stimulated cell cultures (Promega) is indicated by the dotted lines. Data are shown as means ± SE of 3 separate cell cultures. **P < 0.01 compared with unstimulated values.

 

Figure 7
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  		Fig. 7. PGE2 release from PD07i, PD08i, SR22A, and SM28abn following tryptase stimulation (20 ng/ml). Release from each unstimulated cell line is represented by the corresponding symbols and dotted lines. Data for stimulated samples are shown as means ± SE of 3 separate cell cultures. *P < 0.05, **P < 0.01 compared with unstimulated values. Data for unstimulated cell cultures are means from 3 separate cell cultures. For graphic clarity, SE for unstimulated cultures is not noted.

 
The low levels of PGE2 release seen with SR22A could be ascribed either to a decrease in synthesis of PGE2 by PGE synthase or an increase in degradation by 5-hydroxy prostaglandin dehydrogenase (15-HPGD) to 13,14 dihydro-15-keto PGE2. Using real-time PCR, we observed a decrease in mRNA for COX-2 and PGE synthase (PGES) and an increase in mRNA for 15-HPGD in SR22A compared with PD07i (Table 1). These data suggest that both decreased PGE2 synthesis by COX-2 and PGES and increased metabolism by 15-HPGD may contribute to the decreased PGE2 accumulation in tryptase-stimulated SR22A cells.


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  		Table 1. Fold change in gene expression in SR22A when compared with PD07i

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
IC is primarily a disease of the urothelium in which impaired urothelial cell barrier function can lead to penetration of urinary solutes into the bladder wall, leading to pain and inflammation (6, 20). Alterations in paracellular permeability and expression of specific tight junction proteins have been demonstrated in cultured IC bladder epithelial cells (40). Furthermore, bladder epithelial cells in IC patients produce anti-proliferative factor (APF) (11). APF treatment causes significant increases in paracellular permeability of normal bladder cells in culture and decreased expression of zonula occludens-1 and occludin (40). Thus, APF may contribute to the impaired urothelial cell barrier, leading to pain and inflammation. In addition, mast cells play a central role in the pathogenesis of IC. Several mediators that are indicative of mast cell activation, including IL-6 (13, 16), histamine (1), and tryptase (34), are elevated in the urine of IC patients and can come into contact with urothelial cells. Bladder mast cells can be activated as a result of the impaired barrier function and resultant leakage of urinary contents into the bladder wall, thus setting up a cycle of inflammation.

In these studies, we used urothelial cells from normal and IC patients that were immortalized using HPV E6E7, which endows epithelial cells with extended life span in vitro. While E6 and E7 are known to interact with cell cycle regulators, the resulting perturbations are nonetheless permissive for epithelial differentiation. Indeed, the HPV life cycle requires epithelial differentiation for amplification of the viral genome and induction of late gene expression. Furthermore, the immortalization induced by E6E7 is distinct from the transformed phenotype associated with carcinoma cell lines that are capable of substrate-independent growth in soft agar and tumor formation in nude mice. Immortalization of urogenital epithelial cultures results in cell lines that retain the capacity to express tissue-specific differentiation markers in multiple culture models of epithelial differentiation, including three-dimensional organotypic raft cultures (15, 18). Immortalized urothelial cell lines also undergo responses to pathogenic insult that mimics the in vivo kinetics of the murine urothelium (16, 17). Most importantly, immortalized urothelial cells retained the same pattern of differentiation-induced expression of the cyclin-CDK inhibitors p21 and p27 as the parental, primary urothelial cultures (18). In this study, we demonstrated that immortalized cells derived from normal bladder biopsies demonstrate a similar PGE2 release response to tryptase stimulation compared with primary human urothelial cells, suggesting that the biochemical responses to tryptase stimulation are not changed by the immortalization process. Zhang et al. (40) showed cultured IC cells have significantly less ZO-1 expression compared with cultured normal cells. In contrast, our results showed similar staining in the normal and IC-derived cell lines (Fig. 1). This difference in observations between the two laboratories could possibly be due to sample variablilty. IC patient sample variablity may be due to several factors, including severity of the disease, duration of the disease, initiating etiology, and hormonal status of the patient at the time the original sample was taken.

In a previously published study, we demonstrated iPLA2 activation and increased release of arachidonic acid and PGE2 from human urothelial cells stimulated with thrombin or tryptase (22). In the present study, activation of iPLA2 and the subsequent release of arachidonic acid from tryptase-stimulated PD07i and SR22A were similar to the results we obtained previously with primary cells from human adult urothelium (22). However, the time course of iPLA2 activation and arachidonic acid release was slightly delayed in SR22A compared with that of PD07i. Further studies suggested that there is a smaller number of PAR-2 expressed on the cell surface of SR22A compared with PD07i. In our previous study (22), immunoblot analysis demonstrated the presence of both PAR-1 and PAR-2 in isolated human urothelial cells and all four PAR are present in the J82 human urothelial cell line (3). Saban et al. (23) demonstrated the presence of all four PAR in the mouse bladder and observed that urothelial PAR-2 is downregulated in inflammation (3). The authors suggested that the downregulation of PAR-2 may be due to continued PAR activation and that the downregulation may contribute to inflammation since PAR-2 ligands have been proposed to be anti-inflammatory in inflammatory bowel disease (4, 5). We now report that PAR-2 cell surface expression is significantly lower in urothelial cells isolated from an IC bladder compared with those from a normal bladder in the absence of receptor activation. Real-time PCR data indicated that mRNA expression for both PAR-1 and PAR-2 was significantly decreased in SR22A compared with PD07i (Table 1), indicating that there is not increased production and turnover of these receptors in SR22A cells. Thus, our data indicate that in urothelial cells isolated from an IC bladder, there may be a permanent change in the downregulation and cell surface expression of PAR-2 that is independent of receptor activation. Whether the initiating event is long-term cleavage of PAR-2 by increased mast cell tryptase in the early stages of this disease remains to be determined.

Despite the downregulation of cell surface expression of PAR-2, tryptase-stimulated increases in iPLA2 activity and arachidonic acid release remained similar between the two cell types. Resting PLA2 activity in SR22A was slightly higher than that in PD07i, but did not reach significance. Bladder instillation of PAR activating peptides has been shown to result in upregulation of iPLA2 in cytoplasmic extracts from the mouse bladder mucosa (24). Resting iPLA2 activity and arachidonic acid release were higher in SR22A and activation of iPLA2 and arachidonic acid was delayed in SR22A compared with PD07i, suggesting that there may be some long-term alteration in PLA2 activation or expression in SR22A.

The most dramatic difference between the two cell types is clearly the lack of PGE2 accumulation in SR22A and SM28 abn cells. Despite increased arachidonic acid production in response to tryptase, there was little subsequent metabolism to PGE2. Real-time PCR data demonstrate that this may be a result of a combination of downregulation of COX-2 and PGES and upregulation of HPGD, resulting in both decreased synthesis and increased metabolism of PGE2 in SR22A cells. Among prostaglandins, PGE2 is the most widely produced in the body and exhibits the most versatile actions. It can have both pro- and anti-inflammatory actions that are regulated by the expression of PGE receptors in the relevant tissues (for a review, see Ref. 32). PGE2 is involved in a wide spectrum of biological activities, including pain, pyrexia, platelet aggregation, airway contraction and bronchodilation, bone resorption, ovulation and fertilization, vasodilation, modulation of cytokine and chemokine production, and gastric and duodenal cytoprotection. Increased PGE2 release in the bladder has been associated with several diseases, including bladder carcinogenesis (27, 29), overactive bladder (11, 12), cyclophosphamide-induced cystitis (19), and urinary tract infections (35). This is the first study to describe a decrease in PGE2 release in bladder disease.

PGE2 is widely considered to have a cytoprotective role in epithelium (2, 26, 31, 37, 38). Bladder biopsies from patients with IC can demonstrate epithelial denudation. Following injury, the process of regeneration involves spreading and migration of cells at the wound edge into the denuded area to re-epithelialize the wound site. Extracellular matrix components, growth factors, and eicosanoids all play a role in the process of wound healing and repair. Prostaglandins are known to favor cell migration and wound healing in a variety of tissues including corneal endothelium (9), umbilical cord endothelium (8), keratinocytes (10), skin (33), intestinal epithelium (41), and airway epithelium (26). Our finding that SR22A and SM28 abn cells fail to release PGE2 in response to tryptase may represent a mechanism whereby the urothelium in the IC bladder is not protected in settings where there is mast cell activation and suggests that the failure to produce PGE2 may contribute to delayed wound healing in the IC bladder.

The decrease in PGE2 release from SR22A appears to be a result of both decreased synthesis by COX-2 and/or PGES and increased metabolism by PGDH. Increased PGE2 production associated with increased COX-2 expression has been observed in bladder cancer and in rat bladder inflammation (27, 35, 36). Membrane-bound PGE2 synthase-1 mRNA is increased in bladder carcinogenesis (27). However, we observed significant decreases in both COX-2 and PGES mRNA levels in SR22A compared with PD07i. Prostaglandin dehydrogenase has been identified previously in the urothelium and shown to be decreased in the ureter during obstruction, contributing to the accumulation of prostaglandins (7). More recently, PGDH has been implicated in maintaining urothelial differentiation (34). Urothelial cells treated with siRNA for PGDH showed lower expression of E-cadherin (34), although whether this was a direct result of increased PGE2 accumulation remains to be evaluated. It is becoming increasingly apparent that IC may be associated with distinctive changes in the urothelium at both the structural and molecular level. A recent study suggested that urothelial cells in the IC bladder may follow an aberrant differentiation program leading to altered synthesis of several proteins involved in barrier function (28). Our study suggests that in addition to these proteins, alterations in COX-2, PGES, or PGDH and the resultant loss of PGE2 production in IC could further contribute to loss of protection and wound repair of the urothelium.

In conclusion, this study demonstrates that urothelial cells derived from the inflamed area of the bladder of IC patients fail to release PGE2 in response to tryptase stimulation. This may represent a key impairment in the normal protection and repair of the urothelium during inflammatory events in the progression of this disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by DK-66119 (J. McHowat).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. McHowat, Dept. of Pathology, Saint Louis Univ. School of Medicine, Doisy Research Bldg., 3rd Floor, 1110 S. Grand Blvd., St. Louis, MO 63104 (e-mail: jane.mchowat{at}tenethealth.com)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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