Differentiation-induced uroplakin III expression promotes urothelial cell death in response to uropathogenic E. coli

Uropathogenic E. coli (UPEC) expressing type 1 pili underlie most urinary tract infections (UTIs). UPEC adherence to the bladder urothelium induces a rapid apoptosis and exfoliation of terminally differentiated urothelial cells, a critical event in pathogenesis. Of the four major uroplakin proteins that are densely expressed on superficial urothelial cells, UPIa serves as the receptor for type 1-piliated UPEC, but the contributions of uroplakins to cell death are not known. We examined the role of differentiation and uroplakin expression on UPEC-induced cell death. Utilizing in vitro models of urothelial differentiation, we demonstrated induction of tissue-specific differentiation markers including uroplakins. UPEC-induced urothelial cell death was shown to increase with enhanced differentiation but required expression of uroplakin III: infection with an adenovirus encoding uroplakin III significantly increased cell death, while siRNA directed against uroplakin III abolished UPEC-induced cell death. In a murine model of UTI where superficial urothelial cells were selectively eroded to expose less differentiated cells, urothelial apoptosis was reduced, indicating a requirement for differentiation in UPEC-induced apoptosis in vivo. These data suggest that induction of uroplakin III during urothelial differentiation sensitizes cells to UPEC-induced death. Thus, uroplakin III plays a pivotal role in UTI pathogenesis.     

Urea Transporter UT-B Deletion Induces DNA Damage and Apoptosis in Mouse Bladder Urothelium

Background

Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line.

Methodology/Principal Findings

Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis.

Conclusions/Significance

UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders.     

Cellular basis of urothelial squamous metaplasia: roles of lineage heterogeneity and cell replacement

Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on their embryonic origin, uroplakin content, keratin expression pattern, in vitro growth potential, and propensity to keratinize during vitamin A deficiency. Moreover, these cells remain phenotypically distinct even after they have been serially passaged under identical culture conditions, thus ruling out local mesenchymal influence as the sole cause of their in vivo differences. During vitamin A deficiency, mouse urothelium form multiple keratinized foci in proximal urethra probably originating from scattered K14-positive basal cells, and the keratinized epithelium expands horizontally to replace the surrounding normal urothelium. These data suggest that the urothelium consists of multiple cell lineages, that trigone urothelium is closely related to the urothelium covering the rest of the bladder, and that lineage heterogeneity coupled with cell migration/replacement form the cellular basis for urothelial squamous metaplasia.     

Inactivation of p21WAF1 sensitizes cells to apoptosis via an increase of both p14ARF and p53 levels and an alteration of the Bax/Bcl-2 ratio

p21(WAF1) appears to be a major determinant of the cell fate in response to anticancer therapy. It was shown previously that HCT116 human colon cancer cells growing in vitro enter a stable arrest upon DNA damage, whereas cells with a defective p21(WAF1) response undergo apoptosis. Here we report that the enhanced sensitivity of HCT116/p21(-/-) cells to chemotherapeutic drug-induced apoptosis correlates with an increased expression of p53 and a modification of their Bax/Bcl-2 ratio in favor of the pro-apoptotic protein Bax. Treatment of HCT116/p21(-/-) cells with daunomycin resulted in a reduction of the mitochondrial membrane potential and in activation of caspase-9, whereas no such changes were observed in HCT116/p21(+/+) cells, providing evidence that p21(WAF1) exerts an antagonistic effect on the mitochondrial pathway of apoptosis. Moreover, the role of p53 in activation of this pathway was demonstrated by the fact that inhibition of p53 activity by pifithrin-alpha reduced the sensitivity of HCT116/p21(-/-) cells to daunomycin-induced apoptosis and restored a Bax/Bcl-2 ratio similar to that observed in HCT116p21(+/+) cells. Enhancement of p53 expression after disruption of p21(WAF1) resulted from a stabilization of p53, which correlated with an increased expression of the tumor suppressor p14(ARF), an inhibitor of the ubiquitin ligase activity of Mdm2. In accordance with the role of p14(ARF) in p53 stabilization, overexpression of p14(ARF) in HCT116/p21(+/+) cells resulted in a strong increase in p53 activity. Our results identify a novel mechanism for the anti-apoptotic effect of p21(WAF1) consisting in maintenance of mitochondrial homeostasis that occurs in consequence of a negative control of p14(ARF) expression.     

Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA₂ activation. Incubation with the membrane phospholipid-derived PLA₂ hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA₂ activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS.     

Understanding of mouse and human bladder at single‐cell resolution: integrated analysis of trajectory and cell‐cell interactive networks based on multiple scRNA‐seq datasets

ObjectivesTo elaborately decipher the mouse and human bladders at single‐cell levels.Materials and MethodsWe collected more than 50,000 cells from multiple datasets and created, up to date, the largest integrated bladder datasets. Pseudotime trajectory of urothelium and interstitial cells, as well as dynamic cell‐cell interactions, was investigated. Biological activity scores and different roles of signaling pathways between certain cell clusters were also identified.ResultsThe glucose score was significantly high in most urothelial cells, while the score of H3 acetylation was roughly equally distributed across all cell types. Several genes via a pseudotime pattern in mouse (Car3, Dkk2, Tnc, etc.) and human (FBLN1, S100A10, etc.) were discovered. S100A6, TMSB4X, and typical uroplakin genes seemed as shared pseudotime genes for urothelial cells in both human and mouse datasets. In combinational mouse (n = 16,688) and human (n = 22,080) bladders, we verified 1,330 and 1,449 interactive ligand‐receptor pairs, respectively. The distinct incoming and outgoing signaling was significantly associated with specific cell types. Collagen was the strongest signal from fibroblasts to urothelial basal cells in mouse, while laminin pathway for urothelial basal cells to smooth muscle cells (SMCs) in human. Fibronectin 1 pathway was intensely sent by myofibroblasts, received by urothelial cells, and almost exclusively mediated by SMCs in mouse bladder. Interestingly, the cell cluster of SMCs 2 was the dominant sender and mediator for Notch signaling in the human bladder, while SMCs 1 was not. The expression of integrin superfamily (the most common communicative pairs) was depicted, and their co‐expression patterns were located in certain cell types (eg, Itgb1 and Itgb4 in mouse and human basal cells).ConclusionsThis study provides a complete interpretation of the normal bladder at single‐cell levels, offering an in‐depth resource and foundation for future research.     

Inhibition of cell division by the human cytomegalovirus IE86 protein: role of the p53 pathway or cyclin-dependent kinase 1/cyclin B1

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G(0)/G(1) to G(1)/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser(15) p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G(2)/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53(+/+) and p53(-/-) cells.     

Adriamycin (ADM) induced apoptosis in Transitional Cell Cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1 , at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1 , Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.     

Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling

Fibroblast growth factor 10 (FGF10) is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation. In this study, we investigated the role of FGF10 as a lead induction factor for stem cell differentiation toward urothelial cell. To this end, human multipotent stem cell in vitro system was employed. Human amniotic fluid stem cells were co-cultured with immortalized bladder cancer lines to induce directed differentiation into urothelial cells. Urothelial markers, uroplakin II, III, and cytokeratin 8, were monitored by RT-PCR, immunocytochemistry, and Western blot analysis. Co-cultured stem cells began to express uroplakin II, III, and cytokeratin 8. Targeted FGF10 gene knockdown from bladder cancer cells abolished the directed differentiation. In addition, when FGF10 downstream signaling was blocked with the Mek inhibitor, the co-culture system lost the capacity to induce urothelial differentiation. Exogenous addition of recombinant FGF10 protein promoted stem cell differentiation into urothelium cell lineage. Together, this report suggests that paracrine FGF10 signaling stimulates the differentiation of human stem cell into urothelial cells. Current study provides insight into the potential role of FGF10 as a lead growth factor for bladder regeneration and its therapeutic application for bladder transplantation.     

Endocytotic activity of bladder superficial urothelial cells is inversely related to their differentiation stage

The composition of the apical plasma membrane of bladder superficial urothelial cells is dramatically modified during cell differentiation, which is accompanied by the change in the dynamics of endocytosis. We studied the expression of urothelial differentiation-related proteins uroplakins and consequently the apical plasma membrane molecular composition in relation to the membrane-bound and fluid-phase endocytosis in bladder superficial urothelial cells. By using primary urothelial cultures in the environment without mechanical stimuli, we studied the constitutive endocytosis. Four new findings emerge from our study. First, in highly differentiated superficial urothelial cells with strong uroplakin expression, the endocytosis of fluid-phase endocytotic markers was 43% lower and the endocytosis of membrane-bound markers was 86% lower compared to partially differentiated cells with weak uroplakin expression. Second, superficial urothelial cells have 5–15-times lower endocytotic activity than MDCK cells. Third, in superficial urothelial cells the membrane-bound markers are delivered to lysosomes, while fluid-phase markers are seen only in early endocytotic compartments, suggesting their kiss-and-run recycling. Finally, we provide the first evidence that in highly differentiated cells the uroplakin-positive membrane regions are excluded from internalization, suggesting that uroplakins hinder endocytosis from the apical plasma membrane in superficial urothelial cells and thus maintain optimal permeability barrier function.     

Recombinant adenovirus-mediated p14(ARF) overexpression sensitizes human breast cancer cells to cisplatin

p14(ARF), the alternative product from the human INK4a/ARF locus, is one of the major targets for alterations in the development of human cancers. Overexpression of p14(ARF) results in cell cycle arrest and apoptosis. To examine the potential therapeutic role of re-expressing p14(ARF) gene product in human breast cancer, a recombinant adenovirus expressing the human p14(ARF) cDNA (Adp14(ARF)) was constructed and used to infect breast cancer cells. Five days after infection, Adp14(ARF) had considerable cytotoxicity on p53-wild-type MCF-7 cells. A time-course study showed that Adp14(ARF) infection of MCF-7 cells at 100pfu/cell increased the number of cells in G0/G1 phase and decreased that in S and G2/M phases. The presence of apoptotic cells was confirmed using the TUNEL assay. Adp14(ARF)-mediated expression of p14(ARF) also resulted in a considerable increase in the amounts of p53 and its target proteins, p21(WAF1) and MDM2. Furthermore, the combination treatment of MCF-7 cells with Adp14(ARF) and cisplatin resulted in a significantly greater cell death. Together, we conclude that p14(ARF) plays an important role in the induction of cell cycle arrest and apoptosis in breast cancer cells and recombinant adenovirus-mediated p14(ARF) expression greatly increases the sensitivity of these cells to cisplatin. These results demonstrate that the proper combination of Adp14(ARF) with conventional chemotherapeutic drug(s) could have potential benefits in treating breast cancer that carries wild-type p53 gene.     

Expression of p21WAF1/CIP1 in bladder cancer: relation to schistosomiasis

Cell cycle regulation is mediated in part through expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Loss of p21WAF1/CIP1 expression may, therefore, contribute partially to schistosomal carcinogenesis in the urinary bladder. We compared p21WAF1/CIP1 expression in schistosomal and nonschistosomal bladder cancer to explore possible differences in p21WAF1/CIP1 expression between the two subtypes and the possible association between schistosomiasis and loss of p21WAF1/CIP1 expression. Tumor specimens were obtained from 130 patients who underwent transurethral biopsy or cystectomy. p21WAF1/CIP1 was determined by immunodot blot, Western blot, and enzyme immunoassay (EIA). We validated a highly sensitive quantitative EIA assay for determination of p21WAF1/CIP1 in cell lysates. Precision, analytical recovery, and linearity were all excellent. Our results did not show any correlation between p21WAF1/CIP1 expression and most clinicopathologic variables. Lower expression of p21WAF1/CIP1 was evident in squamous cell carcinoma (SCC) and schistosomal subtype than in transitional cell carcinoma and nonschistosomal tumors. Our data suggest a potential role for p21WAF1/CIP1 alteration in schistosomal carcinogenesis.     

Effect of inactivating various components of the signal pathways of the tumor suppressor p53 on genomic stability

To evaluate the role of different p53-regulated signaling pathways in the control of genomic integrity, we studied the frequency of changes in chromosome number and structure of cells of the sublines of mouse primary embryonic fibroblasts with the "knocked-out" genes for proteins p53, p21WAF, pRb, and p19ARF. Protein p21WAF is transactivated by p53 and is responsible for the cell block in the G1 phase of the damaged cells; protein pRb is a target for p21WAF which controls the G1-S-phase transition; and p19ARF protein is responsible for p53 activation in cells with certain anomalies. Inactivation of either of the studied genes proved to increase significantly the frequency of changes in the karyotype. However, the resultant chromosome instability differed: the frequency of the chromosome breaks, both spontaneous and induced with ethylmethane sulfonate (EMS), was in cells with inactivated p53 and lowest in cells with inactivated pRb. These distinctions were not caused by a different effect of various gene inactivation on the cell cycle progression: in all sublines, the cell block in G1 was abolished and the checkpoint function in G2 remained normal. However, the induction of apoptosis in EMS-treated cells differed in the studied sublines. The lowest number of apoptotic nuclei were determined in p53-/- cultures, whereas the highest were in the Rb-/- cultures. It is apparent that the degree of genetic instability is determined by a combined effect of apoptosis and abnormal regulation of the cell-cycle checkpoints.     

Functional TRPV4 channels and an absence of capsaicin-evoked currents in freshly-isolated,guinea-pig urothelial cells

Previously we have shown that the transient receptor potential vanilloid 4 (TRPV4) channel regulates urinary bladder function, and that TRPV4 is expressed in both smooth muscle and urothelial cell types within the bladder wall (Thorneloe et al. 2008). Urothelial cells have also been suggested to express TRPV1 channels (Birder et al., 2001). Therefore, we enzymatically isolated guinea-pig urothelial cells in an attempt to record TRPV4 and TRPV1-mediated currents. The identity of the isolated cells was confirmed by quantitative PCR for the urothelial marker uroplakin 1A. Whole-cell patch-clamp recordings with the TRPV4 agonist, GSK1016790A, activated urothelial currents with an EC50 of 11 nM that were completely inhibited by the TRPV4 inhibitor ruthenium red (5 µM). Urothelial currents were also activated by challenge with hypotonic extracellular solution (220 mOsm) known to activate TRPV4 channels. However, the TRPV1 agonist capsaicin, which activated TRPV1 currents in HEK cells expressing TRPV1, was unable to evoke current in these freshly-isolated guinea-pig urothelial cells. We demonstrate that TRPV4 channels are functionally expressed at the plasma membrane of freshly-isolated, guinea-pig urothelial cells, further supporting the important role of TRPV4 in urinary bladder physiology.