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To determine whether antiproliferative factor (APF) or epidermal growth factor (EGF) can induce changes in purinergic signaling in normal bladder urothelial cells (BUC) and/or whether antagonizing EGF activity or blocking ATP-purinergic receptors can induce changes in purinergic signaling in interstitial cystitis (IC) cells.
IC and normal BUC were obtained from patients’ bladder biopsies. IC BUC were treated with genistein, which antagonizes EGF’s activity, while normal BUC were treated with EGF, mock APF, or APF. Suramin, which antagonizes ATP activity, was used to treat APF-treated normal BUC. ATP release was determined by stimulating BUC with 30μM ATP and then collecting supernatant over a 3-hour period. ATP quantification was measured by luciferin-luciferase assay. P2X3 expression on BUC was determined by fluorescence activated cell sorting (FACS).
Genistein treatment of IC BUC resulted in significantly decreased ATP release, thus reverting IC cells to a normal purinergic signaling phenotype. Conversely, normal BUC treated with EGF or APF resulted in significantly increased ATP release and P2X3 expression, converting normal BUC to an IC phenotype. Suramin treatment of APF-treated normal BUC significantly reduced ATP release.
Genistein and suramin reversed the augmented ATP release in IC BUC and APF-treated normal BUC respectively, suggesting the possibility of intravesical use of these agents in IC treatment. EGF and APF induced augmented purinergic signaling in normal BUC as determined by increased ATP release and increased P2X3 expression. These data suggest an association between cytokines and purinergic signaling in human BUC that should be explored further.
Symptoms of interstitial cystitis (IC) are characterized by urinary urgency, increased frequency, and bladder pain. Because the etiology is still unknown, treatment is empiric. Recent studies have indicated that explanted bladder urothelial cells (BUC) from IC patients have several stably heritable abnormalities compared to normal controls. These include unique expression of a small sialoglycopeptide, antiproliferative factor (APF) 1; altered expression of cytokines such as increased epidermal growth factor (EGF) and decreased heparin-binding epidermal growth factor-like growth factor (HB-EGF) 2,3; augmented ATP release in response to stretch and exogenous ATP stimulus 4,5; and increased expression of P2X3 and P2X2 puringeric receptors 6,7.
A new paradigm is that the bladder urothelium plays an active role in bladder sensory function and is not merely a barrier compartment. We previously demonstrated that exogenous ATP stimulation of IC BUC significantly increased ATP release by these cells in vitro 5. For the sake of brevity, this phenomenon will be referred to as ATP stimulated ATP release or ASAR. ASAR reflects the purinergic signaling pathway in BUC which is important for determining the apical urothelial cell surface area 8. We previously showed that treatment of IC BUC with HB-EGF decreased ASAR 5. We hypothesized that altering the cytokine milieu could affect ASAR in a parallel fashion. We therefore asked the questions: 1) could treatment of normal BUC with APF increase ASAR, thus converting normal BUC to IC phenotype? and 2) could treatment of normal BUC with EGF increase ASAR? Because the main downstream effect of EGF is protein phosphorylation, we also asked could genistein, a non-specific tyrosine kinase inhibitor, reduce ASAR in IC BUC? Also, could suramin, a non-specific purinergic P2X receptor antagonist, decrease the ASAR in APF-treated normal BUC (as it was previously shown to do in IC BUC) 5? Finally, could the expression of P2X3 in normal BUC be altered by treatment with APF or EGF? The measurement of the reciprocal relationship between cytokines and purinergic signaling will not only shed light into the pathophysiology of IC, but also establish a cell model by which potential novel agents for IC can be tested.
All chemicals were purchased from Sigma (Sigma, St. Louis, MO). P2X3 antibody was purchased from Neuromics (Neuromics, Edina, MN). Media were purchased from Invitrogen (Invitrogen, Carlsbad, CA).
IC patients met National Institute of Diabetes and Digestive and Kidney Diseases criteria9. Control patients had neither voiding nor pelvic pain symptoms. The ethics and study protocols were approved by the University of Maryland Baltimore Institutional Review Board.
Bladder biopsies were obtained from IC and normal subjects using the cold cup biopsy technique. The techniques of BUC culture from cystoscopic biopsies have been described in detail previously4, 11. After outgrowth became established, BUC were counted using a hemocytometer and evenly distributed onto 6-well cell culture plates at a density of 1×105 cells per well for each ASAR experiment.
BUC were plated as described above. ATP (30 μM) was added to cell culture media5. Cell supernatants were collected periodically over a three hour period (0, 1, 2 and 3 hrs after addition of ATP with time 0 representing sample collected immediately after addition of ATP).
ATP assays were performed using a Sigma luciferin-luciferase assay kit as previously prescribed in detail4.
APF was harvested and purified from the supernatant of cultured IC BUC using molecular weight fractionation, ion exchange chromatography, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography (HPLC), as previously described1. Mock APF was prepared by using the supernatant of normal BUC and the same purification procedure.
IC BUC were pretreated with 100 μM genistein for 24 hours prior to ASAR measurement. Control experiments were normal BUC and IC BUC or vehicle underwent ASAR.
Normal BUC were pretreated with 10ng/ml or 20ng/ml EGF for 10 minutes prior to ASAR measurement. Control was normal BUC treated with medium alone.
Normal BUC were pretreated with 1 μM of AG1478, a selective tyrosine kinase inhibitor, for 2 hours before adding 20ng/ml EGF for 10 minutes prior to ASAR measurement. Controls were BUC underwent ASAR or treated with 20 ng/ml EGF for 10 minutes.
Normal BUC were plated as described above. The next day, cells were serum-starved and pretreated with 30 μl/cc media of HPLC-purified APF for 48 hours prior to ASAR measurement. As controls, normal BUC either underwent ASAR or treated with 30 μl/cc media of HPLC purified mock-APF for 48 hours prior to ASAR measurement.
Normal BUC were plated as described above, serum-starved, and treated with APF for 48 hours. After the first 24 hours’ of APF incubation, suramin (40 μM) was added to treat the cells for an additional 24 hours in the presence of APF prior to ASAR measurement.
FACS analysis was performed using 3-color FACScan™ Flow Cytometer (BD Bioscience, NJ) to determine expression of P2X3 receptor. The detailed methodology of P2X3 fluorescence labeling and data acquisition has been described previously 6.
For each patient-derived cell line, the experiment was performed in triplicate. Results were expressed as mean values plus or minus standard error of the mean (mean ± SEM). Analysis of variance (ANOVA) was used to compare the means and p-values were considered statistically significant if p < 0.05.
The IC BUC derived from six IC patients were treated with genistein for 24 hours and then underwent ASAR measurements. With exogenous ATP stimulation, IC BUC showed significantly augmented release of ATP compared to normal BUC (Fig. 1, line with open diamonds vs. line with open squares). Genistein treatment of IC BUC significantly decreased ASAR at 0 and 1 hour after addition of ATP (Fig. 1, line with closed squares).
Treatment of normal BUC from three control subjects with EGF at 10 ng/ml and 20ng/ml for 10 minutes, resulted in significantly increased ASAR (Fig. 2A, 0 hr time point), similar to the ASAR seen in IC cells. Pretreatment of normal cells with the selective tyrosine kinase EGFR inhibitor AG1478 significantly decreased ASAR in the presence of EGF (Fig. 2B, 0hr and 1hr time points, line with closed diamonds), suggesting that EGF stimulation of ASAR in normal BUC occurs through EGF/EGFR signaling.
Normal BUC from five control subjects were then treated by APF and normal BUC from three control subjects were treated with mock APF. As shown in Figure 2C, APF treatment of normal BUC resulted in an increased ASAR pattern similar to IC BUC. In comparison, mock APF had no effect on ASAR (Fig. 2C, line with closed squares).
We had previously published that suramin reduced the augmented ASAR detected in IC BUC5. In this study, normal BUC from were first treated by APF, followed by suramin, and then followed by ASAR. Suramin treatment also significantly decreased APF-stimulated ASAR in normal BUC (Fig. 2D, closed squares). In fact, suramin treatment reduced ASAR to a level even below that measured in untreated normal cells. (Fig. 2D, open diamonds).
Studies have demonstrated that ATP can stimulate the purinergic P2X3 signaling pathway in bladder urothelium and BUC 6,14. To determine whether the purinergic receptor P2X3 might be augmented as a result of increased ASAR, FACS analysis was performed on EGF-treated, APF-treated and mock APF-treated normal BUC 3 hours following treatment with ATP. Representative FACS histograms were shown in Figure 3. Regions of higher fluorescence (region M1) were seen after normal BUC were treated with EGF or APF (Fig. 3C, 3D), suggesting cytokine-stimulated ASAR can lead to higher purinergic receptor expression in BUC.
Table 1 shows the mean percentages of positive P2X3 receptor fluorescence density per 2,500 cells for each group. EGF and APF treatment both resulted in significantly increased expression of P2X3, (Table 1 rows C and D) whereas mock APF had no effect (Table 1 row E).
It was previously reported that BUC grown in vitro from IC patient bladder biopsies have abnormalities including altered cytokine production and augmented purinergic signaling1–7. Consistent with the in vitro cell culture phenotype, IC urine specimens also have increased EGF, decreased HB-EGF, increased APF activity1–3 and increased ATP levels4. Since changes in phenotypic expression persist in cultured IC BUC, it is possible to investigate the link between cytokine expression and purinergic signaling in IC BUC. In this report, we demonstrate that EGF treatment of normal BUC results in a significant increase in ASAR, while AG1478 pretreatment of normal BUC in response to EGF (Fig. 2B) and genistein treatment of IC BUC (Fig. 1) resulted in a significant decrease in ASAR, suggesting that EGF and mechanisms related to EGF-receptor activation/tyrosine phosphorylation may be able to modulate purinergic signaling. EGF was previously shown to change BUC behavior in several ways. It acutely suppressed the inward potassium current presumptively mediated by the Kir2.1 channel13, an effect that might result from increased tyrosine phosphorylation of Kir2.1 which closes the channel11
We showed previously that exposure to suramin changed the ASAR phenotype of IC BUC to normal5. Suramin has multiple mechanisms of action. It is a non-specific P2X and P2Y receptor blocker 15, 16; thus having an effect on purinergic signaling. Suramin has also been shown to inhibit the growth of urothelial carcinoma cells in vitro through inhibition of EGF binding 17 suggesting an effect on cytokine signaling. Although mechanisms underlying genistein and suramin treatment are different and are not clearly known, their effects on ASAR are similar, suggesting that intravesical genistein or suramin could be a potential therapeutic agent for IC.
The IC BUC phenotype includes decreased in vitro expression of HB-EGF, and possible decreased in vivo expression as manifested by decreased urine HB-EGF levels. Treatment of IC BUC with 20 ng/ml HB-EGF, which is the upper limit of normal concentration found in urine from asymptomatic normal subjects 2, was previously shown to lower the augmented ASAR in vitro 5. Our current data show that APF increases the ASAR in normal BUC suggesting a link between APF and purinergic signaling also.
In summary, this is the first report describing a link between the cytokines EGF and APF, and purinergic signaling, in bladder urothelial cells. To date, our studies have demonstrated that EGF, APF and HB-EGF alter the purinergic signaling phenotype of bladder urothelial cells in response to an ATP stimulus. Indeed, the ASAR characteristic of normal BUC can be converted to that of IC BUC after treatment of normal BUC with EGF or APF. Conversely, the ASAR characteristics of IC BUC can be converted to that of normal BUC by treating IC BUC with HB-EGF or genistein. Additional study should be focused on EGF- or HB-EGF-targeted pathways by using widely-available and well-studied specific inhibitors for these pathways (such as those that inhibit EGFR phosphorylation). Future treatments for IC could target urothelial abnormalities in the cytokine and/or purinergic pathways.
ASAR is augmented in IC BUC when compared to normal BUC. ASAR can be decreased in IC BUC by treating IC BUC with HB-EGF or genistein. Conversely, increased ASAR can be induced in normal BUC by treatment of normal BUC with EGF or APF. The expression of the purinergic receptor, P2X3, on normal BUC can be significantly increased by treating normal BUC with EGF or APF. These findings suggest a novel link between cytokine (EGF, HB-EGF, APF) and purinergic (ATP, P2X3) signaling pathways in human BUC. The ability to induce phenotype switching offers insight into therapeutic testing models for potential future treatments for IC.
Funding Sources: NIH R01-DK059441, R01 DK075728 and R01-DK52596.
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