Frizzled 8-associated Antiproliferative Factor (APF) is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1) were treated with asialo-APF (as-APF), a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a) expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2), and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS.

Background

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder disorder with bladder epithelial thinning or ulceration, pain, urinary frequency and urgency. There is no reliably effective therapy for IC/PBS, and no generally accepted animal model for the disorder in which potential therapies can be tested. Bladder epithelial cells from IC/PBS patients make a small glycopeptide antiproliferative factor or "APF" that inhibits proliferation, decreases tight junction protein expression, increases paracellular permeability, and induces changes in gene expression of bladder epithelial cells in vitro that mimic abnormalities in IC/PBS patient biopsy specimens in vivo. We therefore determined the ability of a synthetic APF derivative to inhibit bladder epithelial repair in mice.

Methods

The bladder epithelium of female CBA/J mice was stripped by transurethral infusion of 3% acetic acid, and mice were subsequently treated daily with one of three intravesical treatments [synthetic as-APF, inactive unglycosylated control peptide, or phosphate buffered saline carrier (PBS)] for 1–21 days. Fixed bladder sections were either stained with haematoxylin and eosin for determination of epithelial area by image analysis, or incubated with anti-uroplakin III (UPIII) or anti-zonula occludens type 1 (ZO-1) antibodies for immunofluorescence microscopy. Epithelial measurement data were analyzed by a two-way analysis of variance (ANOVA); post hoc comparisons of multiple groups were carried out using the Tukey-Kramer method.

Results

Bladder epithelial repair was significantly attenuated in as-APF-treated mice as compared to control mice on days 3–21 (p < 0.05); the mean epithelial/total area over all measured days was also significantly lower in as-APF-treated mice vs. mice in either control group by post hoc analysis (p < 0.0001 for both comparisons). UPIII and ZO-1 expression was also decreased in as-APF-treated mice as compared to mice in either control group by day 7 (UPIII) or day 14 (ZO-1).

Conclusions

This model demonstrates in vivo effects of as-APF which abrogates bladder epithelial repair and expression of UPIII and ZO-1 in CBA/J mice following transurethral acetic acid infusion. As bladder epithelial thinning, decreased UPIII expression, and decreased ZO-1 expression are histopathologic features of IC/PBS patient biopsies, this model may be useful for studying the pathophysiology of IC/PBS and the effect of potential therapies.

Studies of the urothelium, the specialized epithelial lining of the urinary bladder, are critical for understanding diseases affecting the lower urinary tract, including interstitial cystitis, urinary tract infections and cancer. However, our understanding of urothelial pathophysiology has been hampered by a lack of appropriate model systems. Here, we describe the isolation and characterization of a non-transformed urothelial cell line (TRT-HU1), originally explanted from normal tissue and immortalized with hTERT, the catalytic subunit of telomerase. We demonstrate responsiveness of the cells to anti-proliferative factor (APF), a glycopeptide implicated in the pathogenesis of interstitial cystitis. TRT-HU1 carries a deletion on the short arm of chromosome 9, an early genetic lesion in development of bladder cancer. TRT-HU1 urothelial cells displayed growth and migration characteristics similar to the low-grade papilloma cell line RT4. In contrast, we observed marked differences in both phenotype and gene expression profiles between TRT-HU1 and the highly malignant T24 cell line. Together, these findings provide the first demonstration of a non-transformed, continuous urothelial cell line that responds to APF. This cell line will be valuable for studies of both benign and malignant urothelial cell biology.

Background

Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF) is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4). Because synthetic asialo-APF (as-APF) has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells.

Methods

T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled) double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β), β-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot.

Results

T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or β-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3β/β-catenin phosphorylation in response to APF treatment were all specifically abrogated following CKAP4 siRNA knockdown.

Conclusions

Synthetic as-APF inhibits cell proliferation in T24 bladder carcinoma cells via the CKAP4 receptor. The mechanism for this inhibition involves regulating phosphorylation of specific cell signaling molecules (Akt, GSK3β, and β-catenin) plus mRNA and protein expression of p53 and MMP2.

Objective

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.

Methods

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).

Results

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.

Conclusions

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.

We performed comprehensive structure–activity relationship (SAR) studies on the peptide portion of antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition of 3H-thymidine incorporation in primary normal human bladder epithelial cells. Structural components of the peptide segment of APF that proved to be important for biological activity included the presence of at least eight of the nine N-terminal amino acids, a negative charge in the C-terminal amino acid, a free amino group at the N-terminus, maintenance of a specific amino acid sequence in the C-terminal tail, and trans conformation for the peptide bonds. These data provide critical guidelines for optimization of structure in design of APF analogues as potential therapeutic agents.

Purpose

To test for associations between urine markers, bladder biopsy features and bladder ulcers in interstitial cystitis/painful bladder syndrome (IC/PBS).

Materials and Methods

Subjects were 72 patients with IC/PBS undergoing bladder distention and biopsy. Urine was collected before the procedure. Urine marker levels were correlated with biopsy and cystoscopic findings. Patients with no previous IC/PBS treatments (n=47) were analyzed separately from previously treated patients (n=25).

Results

For untreated patients, urine IL-6 and cGMP were associated with urothelial EGF receptor staining (for IL-6 r=0.29, 95% CI (0.07, 0.51), p=0.01; for cGMP r=0.34, 95% CI (0.13, 0.55), p=0.002). Urine IL-8 was negatively associated with urothelial HB-EGF staining (r=-0.34, 95% CI (-0.55, -0.12), p=0.002) and positively associated with lamina propria mast cell count (r=0.29, 95% CI (0.06, 0.52), p=0.01). The latter association also was seen in treated patients (r=0.46, 95% CI (0.20, 0.73), p<0.001). None of the urine markers was significantly different for ulcer vs. nonulcer patients. All of the ulcer patients had extensive inflammation on bladder biopsy: severe mononuclear cell infiltration, moderate or strong IL-6 staining in the urothelium and lamina propria, and LCA staining in >10% of the lamina propria. However, these features also were seen in 24-76% of the nonulcer patients.

Conclusions

Overall, urine markers did not associate robustly with biopsy findings. The strongest association was a positive association between urine IL-8 levels and bladder mast cell count. Ulcer patients consistently had bladder inflammation, but the cystoscopic finding of ulcers was not a sensitive indicator of inflammation on bladder biopsy.

Previously, we identified cytoskeleton-associated protein 4 (CKAP4) as a major substrate of the palmitoyl acyltransferase, DHHC2, using a novel proteomic method called palmitoyl-cysteine identification, capture and analysis (PICA). CKAP4 is a reversibly palmitoylated and phosphorylated protein that links the ER to the cytoskeleton. It is also a high-affinity receptor for antiproliferative factor (APF), a small sialoglycopeptide secreted from bladder epithelial cells of patients with interstitial cystitis (IC). The role of DHHC2-mediated palmitoylation of CKAP4 in the antiproliferative response of HeLa and normal bladder epithelial cells to APF was investigated. Our data show that siRNA-mediated knockdown of DHHC2 and consequent suppression of CKAP4 palmitoylation inhibited the ability of APF to regulate cellular proliferation and blocked APF-induced changes in the expression of E-cadherin, vimentin, and ZO-1 (genes known to play a role in cellular proliferation and tumorigenesis). Immunocytochemistry revealed that CKAP4 palmitoylation by DHHC2 is required for its trafficking from the ER to the plasma membrane and for its nuclear localization. These data suggest an important role for DHHC2-mediated palmitoylation of CKAP4 in IC and in opposing cancer-related cellular behaviors and support the idea that DHHC2 is a tumor suppressor.

Purpose

To evaluate changes in urine markers and symptom scores after bladder distention in interstitial cystitis (IC) patients.

Materials and Methods

Subjects were 33 new patients with no prior IC treatments. Urine specimens were taken before and one month after bladder distention. University of Wisconsin (UW) symptom scores were done the same day as the urine specimen collection. Urine marker levels and symptom scores before and after distention were compared. Changes in markers were tested for associations with changes in symptom scores and other markers. Pre-distention markers and specific pre-distention symptoms were tested for their association with post-distention symptom improvement.

Results

After distention, the median total UW score decreased significantly (28.5 before, 10 after, p<0.001). Twelve patients (36%) had at least 30% improvement in UW score, and eight patients (24%) had at least 50% improvement. No pre-distention markers or symptoms predicted which patients would have a good response. Two of the urine markers improved significantly after distention: anti-proliferative factor (APF) activity (median −96% before, −17% after, p< 0.001) and heparin binding-epidermal growth factor-like growth factor (HB-EGF) levels (median 0.34 ng/mg creatinine before, 4.1 after, p<0.001). None of the changes in urine markers associated with changes in symptom scores.

Conclusions

The median symptom score for newly diagnosed IC patients decreased after distention, but only a minority of patients had at least 30% symptom improvement. Bladder distention altered urine APF activity and HB-EGF levels towards normal, but the mechanism of symptom relief after distention is still unknown.

Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis, a urinary bladder disorder of unknown etiology that is characterized by chronic pelvic pain. The present study was directed toward uncovering a pathway through which APF signals. Treatment of human urothelial cells with native APF resulted in growth inhibition accompanied by blockade of cell cycle transit and increased p53. Reduced expression of p53 by RNA interference diminished, while ectopic expression of p53 mimicked, the effects of APF. These are the first findings implicating the network of p53 target genes in urothelial defects associated with interstitial cystitis.

Background

Interstitial cystitis (IC) is a chronic bladder disorder of unknown etiology. Antiproliferative factor (APF), a peptide found in the urine of IC patients, has previously been shown to decrease incorporation of thymidine by normal bladder epithelial cells. This study was performed to determine the effect of APF on the cell cycle of bladder epithelial cells so as to better understand its antiproliferative activity.

Methods

Explant cultures from normal bladder biopsy specimens were exposed to APF or mock control. DNA cytometry was performed using an automated image analysis system. Cell cycle phase fractions were calculated from the DNA frequency distributions and compared by two-way analysis of variance (ANOVA).

Results

APF exposure produced statistically significant increases in the proportion of tetraploid and hypertetraploid cells compared to mock control preparations, suggesting a G2 and/or M phase cell cycle block and the production of polyploidy.

Conclusions

APF has a specific effect on cell cycle distributions. The presence of a peptide with this activity may contribute to the pathogenesis of interstitial cystitis through disruption of normal urothelial proliferation and repair processes.

Type 1 fimbriae, expressed by most Escherichia coli strains, are thought to attach to human uroepithelium as an initial step in the pathogenesis of urinary tract infections (UTI). Numerous reports using both in vitro and murine models support this role for type 1 fimbriae in colonization. Unfortunately, only a limited number of studies have directly examined the expression of fimbriae in vivo. To determine whether type 1 fimbrial genes are transcribed during an acute UTI, we employed a modification of an established method. The orientation (ON or OFF) of the invertible promoter element, which drives transcription of type 1 fimbrial genes, was determined by PCR amplification using primers that flank the invertible element, followed by SnaBI digestion. The orientation of the type 1 fimbrial switch was determined under three experimental conditions. First, E. coli strains from different clinical sources (acute pyelonephritis patients, cystitis patients, and fecal controls) were tested under different in vitro culture conditions (agar versus broth; aerated versus static). The genes in the more-virulent strains (those causing acute pyelonephritis) demonstrated a resistance, in aerated broth, to switching from OFF to ON, while those in fecal strains readily switched from OFF to ON. Second, bladder and kidney tissue from CBA mice transurethrally inoculated with E. coli CFT073 (an established murine model of ascending UTI) was assayed. The switches directly amplified from infected bladder and kidney tissues were estimated to be 33 and 39% ON, respectively, by using a standard curve. Finally, bacteria present in urine samples collected from women with cystitis were tested for type 1 fimbria switch orientation. For all 11 cases, an average of only 4% of the switches in the bacteria in the urine were ON. In 7 of the 11 cases, we found that all of the visible type 1 fimbrial switches were in the OFF position (upper limit of detection of assay, 98% OFF). Strains recovered from these urine samples, however, were shown after culture in vitro to be capable of switching the fimbrial gene to the ON position and expressing mannose-sensitive hemagglutinin. The results from experimental infections and cases of cystitis in women suggest that type 1 fimbrial genes are transcribed both in the bladder and in the kidney. However, those bacteria found in the urine and not attached to the uroepithelium are not transcriptionally active for type 1 fimbrial genes.