Faculty Bibliography

Cofactors modulate nuclear receptor activity and impact human health and disease, yet surprisingly little is known about their transcriptional regulation. Androgen receptor trapped clone-27 (ART-27) is a cofactor that binds to androgen receptor (AR) amino terminus and modulates AR-dependent transcription. Interestingly, ART-27 displays both a cell type- and developmental stage-specific expression pattern. However, the cis-acting elements and trans-acting factors affecting ART-27 gene expression have not been elucidated. We found that ART-27 gene expression is repressed and its promoter is histone H3-K27 tri-methylated in human embryonic kidney cells, but not prostate cells, and the histone deacetylase inhibitor, trichostatin A, relieves this inhibition. The DNA response elements that control the induction of ART-27 gene expression were also characterized. The major cis-acting element corresponds to a consensus cAMP-responsive element (CRE) and binds the CRE-binding protein (CREB) as shown by EMSA and chromatin immunoprecipitation assays. Furthermore, ART-27 promoter activity is induced upon CREB overexpression. Epidermal growth factor, which activates CREB via phosphorylation, also induces ART-27 expression, whereas a reduction in CREB phosphorylation or expression blocks this induction in prostate cells. In human prostate development, both epithelial and stromal cells express CREB; however, active phosphorylated CREB is restricted to epithelial cells where ART-27 is expressed. Based on these findings, we propose a transcriptional regulatory circuit for the developmental expression of ART-27 that includes repression by chromatin modification through a trichostatin A-sensitive factor and activation upon growth factor stimulation via CREB

The urinary tract is an outflow system that conducts urine from the kidneys to the bladder via the ureters that propel urine to the bladder via peristalsis. Once in the bladder, the ureteral valve, a mechanism that is not well understood, prevents backflow of urine to the kidney that can cause severe damage and induce end-stage renal disease. The upper and lower urinary tract compartments form independently, connecting at mid-gestation when the ureters move from their primary insertion site in the Wolffian ducts to the trigone, a muscular structure comprising the bladder floor just above the urethra. Precise connections between the ureters and the trigone are crucial for proper function of the ureteral valve mechanism; however, the developmental events underlying these connections and trigone formation are not well understood. According to established models, the trigone develops independently of the bladder, from the ureters, Wolffian ducts or a combination of both; however, these models have not been tested experimentally. Using the Cre-lox recombination system in lineage studies in mice, we find, unexpectedly, that the trigone is formed mostly from bladder smooth muscle with a more minor contribution from the ureter, and that trigone formation depends at least in part on intercalation of ureteral and bladder muscle. These studies suggest that urinary tract development occurs differently than previously thought, providing new insights into the mechanisms underlying normal and abnormal development

This study catalogs the cellular events underlying the formation of a human testis. These events were identified by immunocytochemistry using antibodies that served as markers for specific cell types, then contrasted with the events occurring in the developing mouse testis. The presence of germ cells in the embryonic gonadal ridge and of coelomic epithelial cells that give rise to Sertoli cells was observed at 7 weeks. This was followed by the appearance of Sertoli cells in testicular tubules and of Leydig cells at 9 weeks and by the appearance of vascular endothelial cells and peritubular myoid cells at 12 weeks. Overall the temporal sequence of events in humans was similar, albeit longer, than what occurs in mice. Notably, Leydig cell differentiation occurs earlier in the sequence of events and germ cell maturation occurs during fetal life. The candidate testis-determining genes, FGF9, GATA4, FOG2, EMX2, and CBX2 were expressed at 7 weeks suggesting a role in early gonadal development, such as that observed in mice. In addition, expression of FGF9 in germ cells following testis determination suggests a role in germ cell maturation

As the terminal differentiation products of human urothelium, uroplakins (UPs) would be expected to diminish during urothelial tumorigenesis. Surprisingly, recent studies found UPs to be retained even by well-advanced urothelial carcinomas, suggesting that the loss of UPs does not strictly parallel urothelial transformation. Little is known, however, about whether the status of UPs is associated with a particular pathologic parameter, the tumor's biological behavior, or patient outcome. Here we assessed UP expression by immunohistochemistry on tissue arrays from 285 patients with bladder urothelial carcinomas or nontumor conditions. UPs were expressed in all 9 normal urothelial specimens, 63 of 74 (85%) patients with non-muscle-invasive urothelial carcinomas on transurethral resection, 104 of 202 (51.5%) patients who underwent radical cystectomy for advanced urothelial carcinomas, and 33 of 50 (66%) lymph node metastases. Normally associated with urothelial apical surface, UPs were localized aberrantly in tumors, including microluminal, basal-laminal, cytoplasmic, or uniform patterns. In non-muscle-invasive diseases, there was no association between UP expression and disease recurrence, progression, or mortality. In contrast, in invasive diseases, absent UP expression was significantly associated with advanced pathologic stage, lymph node metastases, disease recurrence, and bladder cancer-specific mortality (P = .042, P = .035, P = .023, and P = .022, respectively) in univariate analyses. Furthermore, UP status was independent of key cell-cycle regulators, including p53, pRb, p27, and cyclin D1, thus excluding a functional link between these 2 groups of proteins. Our data demonstrate for the first time that persistent UP expression is associated with a favorable clinical outcome and that UPs may be used as adjunct markers for predicting the prognoses of patients with invasive and metastatic bladder carcinomas. Our results also suggest that UP-positive and -negative carcinomas have different clonal origins or may be derived from different cancer stem cells

Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%-90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity - a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system

Although it has been shown that mouse uroplakin (UP) Ia, a major glycoprotein of urothelial apical surface, can serve as the receptor for the FimH lectin adhesin of type 1-fimbriated Escherichia coli, the organism that causes a great majority of urinary tract infections, the glycan structure of this native receptor was unknown. Using a sensitive approach that combines in-gel glycosidase and protease digestions, permethylation of released glycans, and mass spectrometry, we have elucidated for the first time the native glycoform structures of the mouse UPIa receptor and those of its non-binding homolog, UPIb, and have determined the glycosylation site occupancy. UPIa presents a high level of terminally exposed mannose residues (located on Man(6)GlcNAc(2) to Man(9)GlcNAc(2)) that are capable of specifically interacting with FimH. We have shown that this property is conserved not only in the mouse uroplakins but also in cattle and, even more importantly, in human UPIa, thus establishing the concept that UPIa is a major urothelial receptor in humans and other mammals for the mannose-specific FimH variant. In contrast, our results indicate that most terminally exposed glycans of mouse UPIb are non-mannose residues, thus explaining the failure of FimH to bind to this UPIb. In cattle, on the other hand, complex carbohydrates constituted only about 20% of the UPIb N-linked glycans. Human UPIa contained exclusively high mannose glycans, and human UPIb contained only complex glycans. The drastically different carbohydrate processing of the UPIa and UPIb proteins, two closely related members of the tetraspanin family, may reflect differences in their folding and masking due to their interactions with their associated proteins, UPII and UPIIIa, respectively. Results from this study shed light on the molecular pathogenesis of urinary tract infections and may aid in the design of glyco-mimetic inhibitors for preventing and treating this disease

PURPOSE: Current American Urological Association treatment guidelines for vesicoureteral reflux do not include any recommendations pertaining to endoscopic therapy (subureteral injection of bulking agent). We performed a meta-analysis of the existing literature pertaining to endoscopic treatment to allow comparison with reports of open surgical correction. MATERIALS AND METHODS: We searched all peer reviewed articles published through 2003 pertaining to endoscopic treatment of vesicoureteral reflux. A total of 63 articles were double reviewed by 9 pediatric urologists, and the data were tabulated on data retrieval sheets. A mixed effects logistic regression model was used to obtain overall estimates of event probabilities (eg reflux resolution, ureteral obstruction) together with their 95% confidence intervals. Individual study estimates were obtained with overall estimate and observation characteristics using empirical Bayes calculations. Differences between or among specific groups were assessed using the F-test. RESULTS: The database included 5,527 patients and 8,101 renal units. Following 1 treatment the reflux resolution rate (by ureter) for grades I and II reflux was 78.5%, grade III 72%, grade IV 63% and grade V 51%. If the first injection was unsuccessful, the second treatment had a success rate of 68%, and the third treatment 34%. The aggregate success rate with 1 or more injections was 85%. The success rate was significantly lower for duplicated (50%) vs single systems (73%), and neuropathic (62%) vs normal bladders (74%). The success rate was similar among children and adults. Following a previous failed open reimplantation endoscopic treatment was successful in 65% of patients. After endoscopic treatment with variable followup pyelonephritis developed in 0.75% of patients and cystitis in 6%. There were few reports of renal scarring following treatment. CONCLUSIONS: Endoscopic treatment provides a high rate of success in children with reflux that decreases with increasing grade, although multiple treatments may be necessary. Future reports of endoscopic therapy should include rates of urinary tract infection and renal scarring

PURPOSE: Subureteral injection of dextranomer/hyaluronic acid copolymer is widely accepted for the treatment of primary vesicoureteral reflux. Few studies document the incidence of surgically relevant postoperative obstruction or the characteristics of patients at risk. MATERIALS AND METHODS: Four institutions had reported surgically relevant postoperative obstruction to representatives of Q-Med Scandinavia, the manufacturers of Deflux (dextranomer/hyaluronic acid). All children undergoing dextranomer/hyaluronic acid injection at these institutions were evaluated in this study. Patients requiring postoperative stenting were retrospectively reviewed for pertinent history, volume injected, technique of injection, duration of symptoms before intervention, duration of intervention and final outcome. RESULTS: A total of 745 patients (1,155 ureters) underwent injection. Five patients (6 renal units, 7 ureters) required stenting for obstructive symptoms and hydronephrosis, of whom 4 immediately became symptomatic. All patients had been injected with up to 1 ml dextranomer/hyaluronic acid. Four patients (80%) had either a neurogenic bladder or dysfunctional voiding. All stents were placed and removed without complications, with complete resolution of symptoms in all patients. Length of stenting ranged from 2 to 6 weeks. No patient required open surgery. One of 2 patients undergoing postoperative voiding studies had development of recurrent vesicoureteral reflux. CONCLUSIONS: Dextranomer/hyaluronic acid injection is associated with a small risk of postoperative ureteral obstruction requiring endoscopic intervention, with an overall incidence of less than 0.7% of patients injected. Patients with voiding dysfunction or neurogenic bladder may be at increased risk. Intervention with temporary ureteral stenting is effective, technically simple and curative