RESUMEN
Uroplakins are a family of membrane-spanning proteins highly specific to the urothelium. There are four uroplakin proteins in humans. These are encoded by the following UPK genes: UPK1A, UPK1B, UPK2 and UPK3 Uroplakin proteins span the apical membrane of umbrella cells of the urothelium, where they associate into urothelial plaques. This provides a barrier function to prevent passage of urine across the urothelium in the renal pelvis, ureters, and bladder. Uroplakins are also involved in developmental processes such as nephrogenesis. The specific localisation of uroplakins within the urothelium means that they are often expressed in primary and metastatic urothelial cell carcinoma and may be used as an immunohistochemical marker of urothelial malignancy.
Asunto(s)
Neoplasias de la Vejiga Urinaria , Uroplaquinas , Humanos , Uroplaquinas/genética , Uroplaquinas/metabolismo , Proteínas de la Membrana/genética , Vejiga Urinaria , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Urotelio/patologíaRESUMEN
The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.
Asunto(s)
Proteínas de la Membrana , Urotelio , Porcinos , Animales , Proteínas de la Membrana/metabolismo , Urotelio/química , Urotelio/metabolismo , Glicoproteínas de Membrana/metabolismo , Microscopía por Crioelectrón , Vejiga Urinaria , Uroplaquinas/análisis , Uroplaquinas/metabolismo , Escherichia coli/metabolismo , Lípidos/análisisRESUMEN
Uroplakins (UPKs) form physical and chemical barriers in the bladder and other urinary tract tissues. We previously reported the identification and localization of UPKs in the male reproductive tract of rat. In this study, we characterized Upk1a knockout mice and report a marginal reduction in fecundity associated with significant decrease in sperm count. Upk1a mice had lower bacterial clearance capacity when challenged with uropathogenic Escherichia coli for 1 to 5 days. High-throughput analyses of testicular transcriptome indicated that 1128 genes that are expressed in testis of wild-type mice were completely absent in the knockout, while 2330 genes were found to be expressed only in the testis of knockout mice. Furthermore, differential regulation of 148 (67 upregulated and 81 downregulated) was observed. Gene ontology analyses indicated that processes related to integral components of membrane (plasma membrane), G-protein receptor activity and signaling, olfactory receptor activity and perception of smell, organization of extracellular space/region, immune and inflammatory responses to pathogens, spermatid development, meiotic cell cycle, and formation of synaptonemal complex were affected. Results of this study provide evidence on the possible multi-functional role of Upk1a in male reproductive tract and in other tissues as well.
Asunto(s)
Testículo , Transcriptoma , Masculino , Ratones , Ratas , Animales , Testículo/metabolismo , Uroplaquina Ia/genética , Uroplaquina Ia/metabolismo , Ratones Noqueados , Semen/metabolismo , Fertilidad/genética , Uroplaquinas/genética , Uroplaquinas/metabolismoRESUMEN
The Golgi complex undergoes considerable structural remodeling during differentiation of urothelial cells in vivo and in vitro. It is known that in a healthy bladder the differentiation from the basal to the superficial cell layer leads to the formation of the tightest barrier in our body, i.e., the blood-urine barrier. In this process, urothelial cells start expressing tight junctional proteins, apical membrane lipids, surface glycans, and integral membrane proteins, the uroplakins (UPs). The latter are the most abundant membrane proteins in the apical plasma membrane of differentiated superficial urothelial cells (UCs) and, in addition to well-developed tight junctions, contribute to the permeability barrier by their structural organization and by hindering endocytosis from the apical plasma membrane. By studying the transport of UPs, we were able to demonstrate their differentiation-dependent effect on the Golgi architecture. Although fragmentation of the Golgi complex is known to be associated with mitosis and apoptosis, we found that the process of Golgi fragmentation is required for delivery of certain specific urothelial differentiation cargoes to the plasma membrane as well as for cell-cell communication. In this review, we will discuss the currently known contribution of the Golgi complex to the formation of the blood-urine barrier in normal UCs and how it may be involved in the loss of the blood-urine barrier in cancer. Some open questions related to the Golgi complex in the urothelium will be highlighted.
Asunto(s)
Uroplaquinas , Urotelio , Diferenciación Celular , Células Epiteliales/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Vejiga Urinaria , Uroplaquinas/metabolismoRESUMEN
Urinary bladder cancer is often multifocal; however, the intraluminal dissemination of the urothelial cancer cells is poorly understood. The involvement of N-cadherin in the adhesion of the cancer urothelial cells to the urothelium had not previously been studied. Therefore, we herein explore the possibility of the intraluminal dissemination of the urothelial cancer cells by evaluating the role of classical cadherins in the adhesion of urothelial cancer cells to the urothelium. We used E-cadherin negative T24 cells and established a T24 Ncadlow cell line with an additionally decreased expression of N-cadherin in the plasma membrane and a decreased secretion of proform of metalloproteinase 2. The labelled T24 and T24 Ncadlow cells were seeded onto urothelial in vitro models. After 24 h in co-culture, unattached cancer cells were rinsed and urothelia with attached cancer urothelial cells were processed for fluorescence and electron microscopy. Both the T24 and T24 Ncadlow cells attached to the urothelium, yet only to the uroplakin-negative urothelial cells. The ultrastructural analysis showed that T24 and T24 Ncadlow cells adhere to poorly differentiated urothelial cells by desmosomes. To achieve this, they first disrupt tight junctions of superficial urothelial cells. This study indicates that the lack of E-cadherin expression and decreased expression of N-cadherin in the plasma membrane of T24 cells does not interfere with their adhesion to the urothelium; therefore, our results suggest that intraluminal dissemination of cancer urothelial cells along the urothelium occurs on uroplakin-negative cells and is desmosome-mediated.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Vejiga Urinaria/inmunología , Uroplaquinas/metabolismo , Urotelio/metabolismo , Adhesión Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Humanos , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/patología , Urotelio/patologíaRESUMEN
The urothelium, an epithelium of the urinary bladder, primarily functions as blood-urine permeability barrier. The urothelium has a very slow turn-over under normal conditions but is capable of extremely fast response to injury. During regeneration urothelium either restores normal function or undergoes altered differentiation pathways, the latter being the cause of several bladder diseases. In this review, we describe the structure of the apical plasma membrane that enables barrier function, the role of urothelium specific proteins uroplakins and the machinery for polarized membrane transports in terminally differentiated superficial umbrella cells. We address key markers, such as keratins, cancer stem cell markers, retinoic acid signalling pathway proteins and transient receptor potential channels and purinergic receptors that drive normal and altered differentiation in bladder cancer and bladder pain syndrome. Finally, we discuss uncertainties regarding research, diagnosis and treatment of bladder pain syndrome. Throughout the review, we emphasise the contribution of immunohistochemistry in advancing our understanding of processes in normal and diseased bladder as well as the most promising possibilities for improved bladder cancer and bladder pain syndrome management.
Asunto(s)
Cistitis Intersticial/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Vejiga Urinaria/metabolismo , Urotelio/metabolismo , Animales , Humanos , Inmunohistoquímica , Receptores Purinérgicos P2X/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/patología , Uroplaquinas/metabolismoRESUMEN
Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named combined lectin- and immuno- histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryo-semithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 µm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.
Asunto(s)
Carcinoma Papilar/diagnóstico por imagen , Células Epiteliales/metabolismo , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Aglutininas/metabolismo , Amaranthus/química , Animales , Anticuerpos/inmunología , Artocarpus/química , Bovinos , Datura stramonium/química , Colorantes Fluorescentes/química , Cabras , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Lectinas de Plantas/metabolismo , Conejos , Rodaminas/química , Ácidos Sulfónicos/química , Vejiga Urinaria/patología , Uroplaquinas/inmunología , Uroplaquinas/metabolismo , Urotelio/metabolismo , Urotelio/patologíaRESUMEN
The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due to its coverage by urothelial plaques consisting of 2D crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature. Strikingly, Snx31 ablation also induces a massive accumulation of uroplakin-containing mitochondria-derived lipid droplets (LDs), which mediate uroplakin degradation via autophagy/lipophagy, leading to the loss of apical and fusiform vesicle plaques. These results suggest that MVBs play an active role in suppressing the excessive/wasteful endocytic degradation of uroplakins. Failure of this suppression mechanism triggers the formation of mitochondrial LDs so that excessive uroplakin membranes can be sequestered and degraded. Because mitochondrial LD formation, which occurs at a low level in normal urothelium, can also be induced by disturbance in uroplakin polymerization due to individual uroplakin knockout and by arsenite, a bladder carcinogen, this pathway may represent an inducible, versatile urothelial detoxification mechanism.
Asunto(s)
Cuerpos Multivesiculares/metabolismo , Nexinas de Clasificación/metabolismo , Urotelio/metabolismo , Animales , Femenino , Gotas Lipídicas/metabolismo , Gotas Lipídicas/fisiología , Glicoproteínas de Membrana/metabolismo , Membranas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Vejiga Urinaria/metabolismo , Uroplaquinas/metabolismo , Uroplaquinas/fisiologíaRESUMEN
Congenital urinary tract obstruction (UTO) is the leading cause of chronic kidney disease in children; however, current management strategies do not safeguard against progression to end-stage renal disease, highlighting the need for interventions to limit or reverse obstructive nephropathy. Experimental UTO triggers renal urothelial remodeling that culminates in the redistribution of basal keratin 5-positive (Krt5+) renal urothelial cells (RUCs) and the generation of uroplakin-positive (Upk)+ RUCs that synthesize a protective apical urothelial plaque. The cellular source of Upk+ RUCs is currently unknown, limiting the development of strategies to promote renal urothelial remodeling as a therapeutic approach. In the present study, we traced the origins of adult Upk+ RUCs during normal development and in response to UTO. Fate mapping analysis demonstrated that adult Upk+ RUCs derive from embryonic and neonatal Krt5+ RUCs, whereas Krt5+ RUCs lose this progenitor capacity and become lineage restricted by postnatal day 14. However, in response to UTO, postnatal day 14-labeled adult Krt5+ RUCs break their lineage restriction and robustly differentiate into Upk+ RUCs. Thus, Krt5+ RUCs drive renal urothelial formation during normal ontogeny and after UTO by differentiating into Upk+ RUCs in a temporally restricted manner.
Asunto(s)
Diferenciación Celular , Células Epiteliales/metabolismo , Queratina-15/metabolismo , Enfermedades Renales/metabolismo , Riñón/metabolismo , Regeneración , Células Madre/metabolismo , Obstrucción Ureteral/complicaciones , Urotelio/metabolismo , Animales , Linaje de la Célula , Modelos Animales de Enfermedad , Células Epiteliales/patología , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Queratina-15/genética , Riñón/crecimiento & desarrollo , Riñón/patología , Enfermedades Renales/etiología , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Masculino , Ratones Noqueados , Organogénesis , Células Madre/patología , Uroplaquinas/metabolismo , Urotelio/crecimiento & desarrollo , Urotelio/patologíaRESUMEN
Uroplakins (UPKs) play an important role in the normal and pathophysiology of the urothelium. They protect the urothelium and play a crucial role during urothelial infections by Uropathogenic E. coli. However, their functions beyond this organ system remain unexplored. A wide variety of proteins secreted in the male reproductive tract tissues contribute to spermatogenesis, sperm maturation, fertilization and innate immunity. However, the presence of UPKs and their possible contribution to the male reproductive tract physiology is not yet reported. Hence, in this study, we characterized UPKs in the male reproductive tract of rats. To the best of our knowledge, for the first time, we report the expression of UPKs in the male reproductive system. Upk1a, Upk1b, Upk2 and Upk3b mRNA and their corresponding proteins were abundantly expressed in the caput, cauda, testis, seminal vesicles and the prostate. Their expression was not developmentally regulated. UPK protein expression was also localized on the spermatozoa, suggesting a role for these proteins in sperm function. To study the role of UPKs in innate immunity, Upk mRNA expression in response to endotoxin challenge was evaluated in vitro and in vivo. In the rat testicular and epididymal cell lines, Upk mRNA levels increased in response to lipopolysaccharide challenge. However, in the caput, cauda, testes, seminal vesicle and prostate obtained from LPS treated rats, Upk mRNA expression was significantly reduced. Results of this study indicate a role for UPKs in male reproductive physiology and innate immune responses.
Asunto(s)
Genitales Masculinos/metabolismo , Uroplaquinas/genética , Animales , Simulación por Computador , Endotoxinas/toxicidad , Epidídimo/efectos de los fármacos , Epidídimo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genitales Masculinos/efectos de los fármacos , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Testículo/efectos de los fármacos , Testículo/metabolismo , Distribución Tisular/efectos de los fármacos , Uroplaquinas/metabolismoRESUMEN
Bladder is affected by numerous pathologies which require augmentation or replacement of the organ. Currently, the gold standard is enterocystoplasty which causes many complications. Bioengineering techniques propose options to overcome these issues. The innovative and very simple tissue engineered three-dimensional spherical bladder model reported here mimics the bladder natural shape using collagen-derived scaffold. Bladder mesenchymal cells were embedded inside the scaffold and epithelial cells seeded at its surface. Therefore, the bladder mesenchymal and urothelial cells seeded in the model were subjected to tensions similar to what is found in the native tissue. Both cell types organize themselves simultaneously within a culture period of 15â¯days. Our spherical model was able to demonstrate characteristics of highly advanced urothelial maturity. Hematoxylin eosin staining, the uroplakins immunodetection and electron microscopy analysis showed the impressive degree of urothelial organization. In addition, collagen remodeling was observed and smooth muscle cells, expressing myosin, presented a tendency to realign parallel to the luminal surface. With properties comparable to native tissue, our three-dimensional spherical bladder model could offer the possibility to produce tissue-engineered bladder implants. This technique could be efficient for partial replacement of pathologic bladder sites.
Asunto(s)
Colágeno/química , Ingeniería de Tejidos/métodos , Vejiga Urinaria/fisiología , Células 3T3 , Animales , Bovinos , Diferenciación Celular , Matriz Extracelular/metabolismo , Mesodermo/citología , Ratones , Membrana Mucosa/fisiología , Porcinos , Vejiga Urinaria/ultraestructura , Uroplaquinas/metabolismo , Urotelio/citología , Urotelio/ultraestructuraRESUMEN
The isolation of a cell line, PICM-31D, with phenotypic characteristics like pancreatic duct cells is described. The PICM-31D cell line was derived from the previously described pig embryonic stem cell-derived exocrine pancreatic cell line, PICM-31. The PICM-31D cell line was morphologically distinct from the parental cells in growing as a monolayer rather than self-assembling into multicellular acinar-like structures. The PICM-31D cells were propagated for over a year at split ratios of 1:3 to 1:10 at each passage without change in phenotype or growth rate. Electron microscopy showed the cells to be a polarized epithelium of cuboidal cells joined by tight junction-like adhesions at their apical/lateral aspect. The cells contained numerous mucus-like secretory vesicles under their apical cell membrane. Proteomic analysis of the PICM-31D's cellular proteins detected MUC1 and MUC4, consistent with mucus vesicle morphology. Gene expression analysis showed the cells expressed pancreatic ductal cell-related transcription factors such as GATA4, GATA6, HES1, HNF1A, HNF1B, ONECUT1 (HNF6), PDX1, and SOX9, but little or no pancreas progenitor cell markers such as PTF1A, NKX6-1, SOX2, or NGN3. Pancreas ductal cell-associated genes including CA2, CFTR, MUC1, MUC5B, MUC13, SHH, TFF1, KRT8, and KRT19 were expressed by the PICM-31D cells, but the exocrine pancreas marker genes, CPA1 and PLA2G1B, were not expressed by the cells. However, the exocrine marker, AMY2A, was still expressed by the cells. Surprisingly, uroplakin proteins were prominent in the PICM-31D cell proteome, particularly UPK1A. Annexin A1 and A2 proteins were also relatively abundant in the cells. The expression of the uroplakin and annexin genes was detected in the cells, although only UPK1B, UPK3B, ANXA2, and ANXA4 were detected in fetal pig pancreatic duct tissue. In conclusion, the PICM-31D cell line models the mucus-secreting ductal cells of the fetal pig pancreas.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Conductos Pancreáticos/citología , Uroplaquinas/genética , Animales , Biomarcadores/metabolismo , Línea Celular , Proliferación Celular/genética , Separación Celular , Células Madre Embrionarias/ultraestructura , Proteómica , Porcinos , Uroplaquinas/metabolismoRESUMEN
50% of Caucasians carry a Thr300Ala variant (T300A) in the protein encoded by the macroautophagy/autophagy gene ATG16L1. Here, we show that the T300A variant confers protection against urinary tract infections (UTIs), the most common infectious disease in women. Using knockin mice carrying the human T300A variant, we show that the variant limits the UTI-causing bacteria, uropathogenic Escherichia coli (UPEC), from establishing persistent intracellular reservoirs, which can seed UTI recurrence. This phenotype is recapitulated in mice lacking Atg16l1 or Atg7 exclusively in the urothelium. We further show that mice with the T300A variant exhibit urothelial cellular abnormalities, including vesicular congestion and aberrant accumulation of UPK (uroplakin) proteins. Importantly, presence of the T300A variant in humans is associated with similar urothelial architectural abnormalities, indicating an evolutionarily conserved impact. Mechanistically, we show that the reduced bacterial persistence is independent of basal autophagic flux or proinflammatory cytokine responses and does not involve Atg14 or Epg5. However, the T300A variant is associated with increased expression of the small GTPase Rab33b; RAB33B interacts with ATG16L1, as well as other secretory RABs, RAB27B and RAB11A, important for UPEC exocytosis from the urothelium. Finally, inhibition of secretory RABs in bladder epithelial cells increases intracellular UPEC load. Together, our results reveal that UPEC selectively utilize genes important for autophagosome formation to persist in the urothelium, and that the presence of the T300A variant in ATG16L1 is associated with changes in urothelial vesicle trafficking, which disrupts the ability of UPEC to persist, thereby limiting the risk of recurrent UTIs. Abbreviations: 3-PEHPC: 3-pyridinyl ethylidene hydroxyl phosphonocarboxylate; ATG: autophagy; ATG16L1: autophagy related 16 like 1; BECs: bladder epithelial cells; dpi: days post infection; hpi: hours post infection; IF: immunofluorescence; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MVB: multivesicular bodies; T300A: Thr300Ala; TNF: tumor necrosis factor; QIR(s): quiescent intracellular reservoir(s); siRNA: short interfering RNA; UPEC: uropathogenic Escherichia coli; UTI(s): urinary tract infection(s); TEM: transmission electron microscopy; WT: wild type.
Asunto(s)
Autofagia/genética , Infecciones por Escherichia coli/metabolismo , Infecciones Urinarias/metabolismo , Escherichia coli Uropatógena , Urotelio/microbiología , Animales , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Línea Celular , Células Epiteliales/metabolismo , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Femenino , Variación Genética , Humanos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Cuerpos Multivesiculares/genética , Cuerpos Multivesiculares/microbiología , Cuerpos Multivesiculares/patología , Vejiga Urinaria/microbiología , Infecciones Urinarias/genética , Infecciones Urinarias/microbiología , Infecciones Urinarias/patología , Uroplaquinas/metabolismo , Urotelio/citología , Urotelio/metabolismo , Urotelio/ultraestructura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique two-dimensional crystals of 16-nm particles ("urothelial plaques") covering the apical urothelial surface. Although uroplakins are highly expressed only in mammalian urothelium and are often referred to as being urothelium specific, they are also expressed in several mouse nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms, and ovary/oocytes. In oocytes, uroplakins colocalize with CD9 on cell-surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs' fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require two-dimensional-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form two-dimensional-crystalline plaques during mammalian divergence, enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, to protect/modify the apical surface of the modern-day mammalian urothelium.
Asunto(s)
Especiación Genética , Oocitos/metabolismo , Ovario/metabolismo , Uroplaquinas/genética , Urotelio/metabolismo , Cigoto/metabolismo , Animales , Diferenciación Celular , Femenino , Fertilización/genética , Regulación de la Expresión Génica , Tamaño de la Camada , Masculino , Ratones , Ratones Noqueados , Oocitos/citología , Ovario/citología , Partenogénesis/genética , Fosforilación , Filogenia , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Transducción de Señal , Testículo/citología , Testículo/metabolismo , Tetraspanina 29/genética , Tetraspanina 29/metabolismo , Uroplaquinas/clasificación , Uroplaquinas/metabolismo , Urotelio/citología , Xenopus laevis , Cigoto/citologíaRESUMEN
OBJECTIVES: To investigate the effect of cyclic hydrostatic pressure on the expression of uroplakins and the role of extracellular regulated protein kinases 1/2 (ERK1/2) in the hydrostatic pressure-induced uroplakin expression of human urothelial cells (UCs). METHODS: Human UCs were seeded into a cell culture flask and subjected to cyclic hydrodynamic pressures for 24â¯h. Pressure parameters were set as follows: static, 100â¯cmâ¯H2O, 200â¯cmâ¯H2O and 300â¯cmâ¯H2O pressure. Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect the expression of uroplakins. The role of the ERK1/2 was investigated using ERK1/2 inhibitor. RESULTS: Compared with the 0â¯cmâ¯H2O control group, 200â¯cmâ¯H2O hydrostatic pressure significantly increased the expression of uroplakins, however, 100â¯cm and 300â¯cm pressures could not promote uroplakin expression. Hence, ERK1/2 expression was also detected under 200â¯cmâ¯H2O hydrostatic pressure. Western blot showed that 200â¯cmâ¯H2O pressure promoted the expression of ERK1/2. ERK1/2 inhibitor decreased the pressure-induced ERK1/2 activivation and uroplakin expression. CONCLUSIONS: Cyclic hydrostatic pressure increases the expression of uroplakins via activating ERK1/2 signaling pathway in human UCs, and 200â¯cmâ¯H2O pressure may be an optimal stress parameter to promote the uroplakin expression.
Asunto(s)
Presión Hidrostática , Sistema de Señalización de MAP Quinasas , Uroplaquinas/metabolismo , Urotelio/citología , Células Cultivadas , Humanos , Transducción de Señal , Regulación hacia Arriba , Urotelio/metabolismoRESUMEN
Urinary tract obstruction represents a common cause of kidney injury across the human life span, resulting in chronic kidney disease and end-stage renal disease. Yet, the extent of obstructive renal damage can be heterogeneous between individuals, implying the existence of unknown mechanisms that protect against or accelerate kidney injury. In this study, we investigated the role of urothelial remodeling in renal adaptation during congenital and acquired obstruction. In the Megabladder ( Mgb-/-) model of congenital obstruction and unilateral ureteral ligation model of acute obstruction, progressive hydronephrosis is strongly associated with dynamic reorganization of the renal urothelium, which elaborates a continuous uroplakin (Upk) plaque. This led us to postulate that the Upk plaque prevents parenchymal injury during urinary tract obstruction. To test this hypothesis, we interbred Mgb-/- and Upk1b-/- mice, which lack the critical Upk1b subunit for Upk plaque formation. Upk1b-/-; Mgb-/- mice experienced an accelerated onset of bilateral hydronephrosis with severe (>67%) parenchymal loss, leading to renal failure and mortality in adolescence. To investigate the function of the renal Upk plaque during acute obstruction, we destabilized the Upk plaque by Upk1b deletion or genetically depleted Upk+ cells following unilateral ureteral obstruction. Both of these strategies accelerated renal parenchymal loss following ureteral ligation, attesting to a conserved, stabilizing role for Upk plaque deposition in the acutely obstructed kidney. In aggregate, these complementary experiments provide the first evidence that the Upk plaque confers an essential, protective adaptation to preserve renal parenchymal integrity during congenital and acquired urinary tract obstruction.
Asunto(s)
Riñón/patología , Obstrucción Ureteral/complicaciones , Uroplaquinas/metabolismo , Urotelio/patología , Animales , Modelos Animales de Enfermedad , Hidronefrosis/fisiopatología , Riñón/fisiopatología , Fallo Renal Crónico/complicaciones , Ratones Endogámicos C57BL , Ratones Transgénicos , Insuficiencia Renal/complicaciones , Insuficiencia Renal/patología , Urotelio/fisiopatologíaRESUMEN
Identification of transcription factors expressed by differentiated cells is informative not only of tissue-specific pathways, but to help identify master regulators for cellular reprogramming. If applied, such an approach could generate healthy autologous tissue-specific cells for clinical use where cells from the homologous tissue are unavailable due to disease. Normal human epithelial cells of buccal and urothelial derivation maintained in identical culture conditions that lacked significant instructive or permissive signaling cues were found to display inherent similarities and differences of phenotype. Investigation of transcription factors implicated in driving urothelial-type differentiation revealed buccal epithelial cells to have minimal or absent expression of PPARG, GATA3 and FOXA1 genes. Retroviral overexpression of protein coding sequences for GATA3 or PPARy1 in buccal epithelial cells resulted in nuclear immunolocalisation of the respective proteins, with both transductions also inducing expression of the urothelial differentiation-associated claudin 3 tight junction protein. PPARG1 overexpression alone entrained expression of nuclear FOXA1 and GATA3 proteins, providing objective evidence of its upstream positioning in a transcription factor network and identifying it as a candidate factor for urothelial-type transdifferentiation or reprogramming.
Asunto(s)
Mucosa Bucal/citología , Mucosa Bucal/metabolismo , Factores de Transcripción/metabolismo , Urotelio/citología , Urotelio/metabolismo , Diferenciación Celular , Transdiferenciación Celular , Células Cultivadas , Reprogramación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , PPAR gamma/genética , PPAR gamma/metabolismo , Fenotipo , Ingeniería de Tejidos , Factores de Transcripción/genética , Uroplaquinas/genética , Uroplaquinas/metabolismoRESUMEN
Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking from the endoplasmic reticulum to the GA in UCs. Moreover, UPIb, UPIIIa or UPIb/UPIIIa expressing UCs revealed fragmentation and peripheral redistribution of Golgi-units. Notably, expression of UPIb or UPIb/UPIIIa triggered similar GA fragmentation in MDCK and HeLa cells that do not express UPs endogenously. The colocalization analysis of UPIb/UPIIIa-EGFP and COPI, COPII or clathrin suggested that UPs follow constitutively the post-Golgi route to the apical PM. Depolymerisation of microtubules leads to complete blockade of the UPIb/UPIIIa-EGFP post-Golgi transport, while disassembly of actin filaments shows significantly reduced delivery of UPIb/UPIIIa-EGFP to the PM. Our findings show the significant effect of the UPs expression on the GA fragmentation, which enables secretory Golgi-outpost to be distributed as close as possible to the sites of cargo delivery at the PM.
Asunto(s)
Aparato de Golgi/metabolismo , Modelos Biológicos , Uroplaquinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Membrana Celular/metabolismo , Polaridad Celular , Clatrina/metabolismo , Perros , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Microtúbulos/metabolismo , Transporte de Proteínas , Porcinos , Urotelio/citología , Urotelio/metabolismo , Urotelio/ultraestructuraRESUMEN
Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network â¼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.
Asunto(s)
Queratina-20/metabolismo , Proteínas con Dominio MARVEL/metabolismo , Proteínas SNARE/metabolismo , Urotelio/citología , Urotelio/metabolismo , Animales , Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Liso/metabolismo , Transporte de Proteínas , Uroplaquinas/genética , Uroplaquinas/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
An unique element of bladder urothelium is a multilayer membrane, which extends from the renal pelvis to the urethra. Urotelial membrane covers more than 90% of the inner portion of the bladder and is in direct contact with urine. Urothelium is composed of characteristic two-dimensional, asymmetric plaques, composed of uroplakins (UP), differentiated, hexagonally arranged proteins. The unique structure of the urothelial plaques determines the tightness, integrity and strength of the urothelium, prevent rupture of the walls of the bladder during the build-up of urine in the bladder and protects against the toxic ingredients. Uroplakins are tissue-specific, heterogeneous glycoproteins whose oligosaccharide part plays a specific role in the structure and function of urothelium. Disorders of normal expression of uroplakins are highly associated with the pathogenesis in infection and urinary tract malignancies, primary vesico-urinary reflux, hydronephrosis and renal impairment. The emergence of uroplakins in urine and / or plasma may have a potential role in the early detection of bladder tumors. In this paper, the structure and function of uroplakins types Ia, Ib, II, IIIa, their natural oligomerization into heterodimers, tetramers and hexamers, and the role in the construction of asymmetric and flexible urothelial epithelium is presented. We discuss the potential role of uroplakins in laboratory diagnosis of umbrella cell differentiation and in the screening analysis of urinary bladder disorders. The possibilities of using the knowledge of uroplakins in clinical settings as well as in modern strategies for treatment of infectious diseases and cancer of the urinary tract are highlighted.