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Structural or functional defects of PTEN in urothelial cells lacking P53 drive basal/squamous-subtype muscle-invasive bladder cancer

He, Feng; Zhang, Fenglin; Liao, Yi; Tang, Moon-Shong; Wu, Xue-Ru
Muscle-invasive bladder cancer (MIBC) exhibits strong inter- and intra-tumor heterogeneity that affects biological behaviors, therapeutic responses, and prognoses. Mutations that activate RTK-RAS-PI3K and inactivate P19-P53-P21 coexist in 60-70% of MIBC. By time-controlled ablation of Tp53 and Pten, singly or combined, in adult mouse urothelium, we found that Tp53 loss alone produced no abnormality. While Pten loss elicited hyperplasia, it synergized with Tp53 loss to trigger 100% penetrant MIBC that exhibited basal/squamous features that resembled its human counterpart. Furthermore, PTEN was inactivated in human MIBC cell lines and specimens primarily by hyperphosphorylation of the C-terminus. Mutated or tailless PTEN incapable of C-terminal phosphorylation demonstrated increased inhibition of proliferation and invasion than full-length PTEN in cultured MIBC cells. In xenograft and transgenic mice, tailless PTEN, but not full-length PTEN, prevented further growth in established tumors. Collectively, deficiencies of both PTEN and P53 drive basal/squamous subtype MIBC. PTEN is inactivated by C-terminal hyperphosphorylation, and this modification may serve as a biomarker for subtyping MIBC and predicting tumor progression. Tailless PTEN is a potential molecular therapeutic for tumors, such as bladder cancer (BC), that can be readily accessed.
PMID: 36195293
ISSN: 1872-7980
CID: 5351542

Mitochondrial lipid droplet formation as a detoxification mechanism to sequester and degrade excessive urothelial membranes

Liao, Yi; Tham, Daniel K L; Liang, Feng-Xia; Chang, Jennifer; Wei, Yuan; Reddy, Sudhir Putty; Sall, Joseph; Ren, Sarah J; Chicote, Javier U; Arnold, Lora L; Hu, Chih-Chi Andrew; Romih, Rok; Andrade, Leonardo R; Rindler, Michael J; Cohen, Samuel M; DeSalle, Rob; Garcia-España, Antonio; Ding, Mingxiao; Wu, Xue-Ru; Sun, Tung-Tien
The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due 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. Since 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. [Media: see text] [Media: see text] [Media: see text].
PMID: 31577526
ISSN: 1939-4586
CID: 4116262

Uroplakins play conserved roles in egg fertilization and acquired additional urothelial functions during mammalian divergence

Liao, Yi; Chang, Hung-Chi; Liang, Feng-Xia; Chung, Pei-Jung; Wei, Yuan; Nguyen, Tuan-Phi; Zhou, Ge; Talebian, Sheeva; Krey, Lewis C; Deng, Fang-Ming; Wong, Tak-Wah; Chicote, Javier U; Grifo, James A; Keefe, David L; Shapiro, Ellen; Lepor, Herbert; Wu, Xue-Ru; DeSalle, Robert; Garcia-España, Antonio; Kim, Sang Yong; Sun, Tung-Tien
Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique 2D-crystals of 16-nm particles ("urothelial plaques") covering the apical urothelial surface. Although uroplakins are highly expressed only in mouse urothelium and are often referred to as being urothelium-specific, they are also expressed in several nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms and ovary/oocytes. In oocytes, uroplakins co-localize 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 2D-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form 2D- crystalline plaques during mammalian divergence enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, in order to protect/modify the apical surface of the modern-day mammalian urothelium.
PMID: 30303751
ISSN: 1939-4586
CID: 3335002

Sequential and compartmentalized action of Rabs, SNAREs and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells

Wankel, Bret; Ouyang, Jiangyong; Guo, Xuemei; Hadjiolova, Krassimira; Miller, Jeremy; Liao, Yi; Tham, Daniel Kai Long; Romih, Rok; Andrade, Leonardo R; Gumper, Iwona; Simon, Jean-Pierre; Sachdeva, Rakhee; Tolmachova, Tanya; Seabra, Miguel C; Fukuda, Mitsunori; Schaeren-Wiemers, Nicole; Hong, WanJin; Sabatini, David D; Wu, Xue-Ru; Kong, Xiangpeng; Kreibich, Gert; Rindler, Michael J; Sun, Tung-Tien
Uroplakins (UPs) are major differentiation products of urothelial umbrella cells, playing important roles in forming the permeability barrier, and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic E. coli receptor. While 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. Immuno-microscopy of normal and mutant mouse urothelia showed 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 showed that keratin 20 (K20), which formed a chicken-wire network 150-300 nm below the apical membrane and had hole sizes allowing FV passage, defined a subapical compartment containing FVs primed and strategically located for fusion. Finally, we showed that Rab8/11 and Rab27b function in the same pathway, that Rab27b-knockout leads to uroplakin and Slp2-a destabilization, and that 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.
PMID: 27009205
ISSN: 1939-4586
CID: 2052152

Angiotensin II type 2 receptor regulates ROMK-like K⁺ channel activity in the renal cortical collecting duct during high dietary K⁺ adaptation

Wei, Yuan; Liao, Yi; Zavilowitz, Beth; Ren, Jin; Liu, Wen; Chan, Pokman; Rohatgi, Rajeev; Estilo, Genevieve; Jackson, Edwin K; Wang, Wen-Hui; Satlin, Lisa M
The kidney adjusts K⁺ excretion to match intake in part by regulation of the activity of apical K⁺ secretory channels, including renal outer medullary K⁺ (ROMK)-like K⁺ channels, in the cortical collecting duct (CCD). ANG II inhibits ROMK channels via the ANG II type 1 receptor (AT1R) during dietary K⁺ restriction. Because AT1Rs and ANG II type 2 receptors (AT2Rs) generally function in an antagonistic manner, we sought to characterize the regulation of ROMK channels by the AT2R. Patch-clamp experiments revealed that ANG II increased ROMK channel activity in CCDs isolated from high-K⁺ (HK)-fed but not normal K⁺ (NK)-fed rats. This response was blocked by PD-123319, an AT2R antagonist, but not by losartan, an AT1R antagonist, and was mimicked by the AT2R agonist CGP-42112. Nitric oxide (NO) synthase is present in CCD cells that express ROMK channels. Blockade of NO synthase with N-nitro-l-arginine methyl ester and free NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt completely abolished ANG II-stimulated ROMK channel activity. NO enhances the synthesis of cGMP, which inhibits phosphodiesterases (PDEs) that normally degrade cAMP; cAMP increases ROMK channel activity. Pretreatment of CCDs with IBMX, a broad-spectrum PDE inhibitor, or cilostamide, a PDE3 inhibitor, abolished the stimulatory effect of ANG II on ROMK channels. Furthermore, PKA inhibitor peptide, but not an activator of the exchange protein directly activated by cAMP (Epac), also prevented the stimulatory effect of ANG II. We conclude that ANG II acts at the AT2R to stimulate ROMK channel activity in CCDs from HK-fed rats, a response opposite to that mediated by the AT1R in dietary K⁺-restricted animals, via a NO/cGMP pathway linked to a cAMP-PKA pathway.
PMID: 25100281
ISSN: 1522-1466
CID: 5288602

SNX31: A Novel Sorting Nexin Associated with the Uroplakin-Degrading Multivesicular Bodies in Terminally Differentiated Urothelial Cells

Vieira, Neide; Deng, Fang-Ming; Liang, Feng-Xia; Liao, Yi; Chang, Jennifer; Zhou, Ge; Zheng, Weiyue; Simon, Jean-Pierre; Ding, Mingxiao; Wu, Xue-Ru; Romih, Rok; Kreibich, Gert; Sun, Tung-Tien
Uroplakins (UP), a group of integral membrane proteins, are major urothelial differentiation products that form 2D crystals of 16-nm particles (urothelial plaques) covering the apical surface of mammalian bladder urothelium. They contribute to the urothelial barrier function and, one of them, UPIa, serves as the receptor for uropathogenic Escherichia coli. It is therefore important to understand the mechanism by which these surface-associated uroplakins are degraded. While it is known that endocytosed uroplakin plaques are targeted to and line the multivesicular bodies (MVBs), it is unclear how these rigid-looking plaques can go to the highly curved membranes of intraluminal vesicles (ILVs). From a cDNA subtraction library, we identified a highly urothelium-specific sorting nexin, SNX31. SNX31 is expressed, like uroplakins, in terminally differentiated urothelial umbrella cells where it is predominantly associated with MVBs. Apical membrane proteins including uroplakins that are surface biotin-tagged are endocytosed and targeted to the SNX31-positive MVBs. EM localization demonstrated that SNX31 and uroplakins are both associated not only with the limiting membranes of MVBs containing uroplakin plaques, but also with ILVs. SNX31 can bind, on one hand, the PtdIns3P-enriched lipids via its N-terminal PX-domain, and, on the other hand, it binds uroplakins as demonstrated by co-immunoprecipitation and proximity ligation assay, and by its reduced membrane association in uroplakin II-deficient urothelium. The fact that in urothelial umbrella cells MVBs are the only major intracellular organelles enriched in both PtdIns3P and uroplakins may explain SNX31's MVB-specificity in these cells. However, in MDCK and other cultured cells transfected SNX31 can bind to early endosomes possibly via lipids. These data support a model in which SNX31 mediates the endocytic degradation of uroplakins by disassembling/collapsing the MVB-associated uroplakin plaques, thus enabling the uroplakin-containing (but 'softened') membranes to bud and form the ILVs for lysosomal degradation and/or exosome formation.
PMID: 24914955
ISSN: 1932-6203
CID: 1033592

Retinoid signaling in progenitors controls specification and regeneration of the urothelium

Gandhi, Devangini; Molotkov, Andrei; Batourina, Ekatherina; Schneider, Kerry; Dan, Hanbin; Reiley, Maia; Laufer, Ed; Metzger, Daniel; Liang, Fengxia; Liao, Yi; Sun, Tung-Tien; Aronow, Bruce; Rosen, Roni; Mauney, Josh; Adam, Rosalyn; Rosselot, Carolina; Van Batavia, Jason; McMahon, Andrew; McMahon, Jill; Guo, Jin-Jin; Mendelsohn, Cathy
The urothelium is a multilayered epithelium that serves as a barrier between the urinary tract and blood, preventing the exchange of water and toxic substances. It consists of superficial cells specialized for synthesis and transport of uroplakins that assemble into a tough apical plaque, one or more layers of intermediate cells, and keratin 5-expressing basal cells (K5-BCs), which are considered to be progenitors in the urothelium and other specialized epithelia. Fate mapping, however, reveals that intermediate cells rather than K5-BCs are progenitors in the adult regenerating urothelium, that P cells, a transient population, are progenitors in the embryo, and that retinoids are critical in P cells and intermediate cells, respectively, for their specification during development and regeneration. These observations have important implications for tissue engineering and repair and, ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.
PMID: 23993789
ISSN: 1534-5807
CID: 549622

Uroplakins do not restrict CO2 transport through urothelium

Zocher, Florian; Zeidel, Mark L; Missner, Andreas; Sun, Tung-Tien; Zhou, Ge; Liao, Yi; von Bodungen, Maximilian; Hill, Warren G; Meyers, Susan; Pohl, Peter; Mathai, John C
Lipid bilayers and biological membranes are freely permeable to CO(2), and yet partial CO(2) pressure in the urine is 3-4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO(2) resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder P(CO2) ( approximately 1.6 x 10(-4) cm/s). To test whether the observed permeability barrier to CO(2) was due to an unstirred layer effect or due to kinetics of CO(2) hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that P(CO2) was unstirred layer limited ( approximately 7 x 10(-3) cm/s). However, in the total absence of CA activity P(CO2) decreased 14-fold ( approximately 5.1 x 10(-4) cm/s), indicating that now CO(2) transport is limited by the kinetics of CO(2) hydration. Expression of aquaporin-1 did not alter P(CO2) (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low P(CO2) is due to the lack of CA. The observed dependence of P(CO2) on CA activity suggests that the tightness of biological membranes to CO(2) may uniquely be regulated via CA expression.
PMID: 22315218
ISSN: 0021-9258
CID: 240542