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Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells

Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-Wen; Hu, Yu; Chen, Wei-Sheng; Chou, David; Liu, Yan; Donin, Nicholas; Huang, William C; Lepor, Herbert; Wu, Xue-Ru; Wang, Hailin; Beland, Frederick A; Tang, Moon-Shong
Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.
PMID: 24939871
ISSN: 1949-2553
CID: 1036762

Conformational inactivation induces immunogenicity of the receptor-binding pocket of a bacterial adhesin

Kisiela, Dagmara I; Rodriguez, Victoria B; Tchesnokova, Veronika; Avagyan, Hovhannes; Aprikian, Pavel; Liu, Yan; Wu, Xue-Ru; Thomas, Wendy E; Sokurenko, Evgeni V
Inhibiting antibodies targeting receptor-binding pockets in proteins is a major focus in the development of vaccines and in antibody-based therapeutic strategies. Here, by using a common mannose-specific fimbrial adhesin of Escherichia coli, FimH, we demonstrate that locking the adhesin in a low-binding conformation induces the production of binding pocket-specific, adhesion-inhibiting antibodies. A di-sulfide bridge was introduced into the conformationally dynamic FimH lectin domain, away from the mannose-binding pocket but rendering it defective with regard to mannose binding. Unlike the native, functionally active lectin domain, the functionally defective domain was potent in inducing inhibitory monoclonal antibodies that blocked FimH-mediated bacterial adhesion to epithelial cells and urinary bladder infection in mice. Inhibition of adhesion involved direct competition between the antibodies and mannose for the binding pocket. Binding pocket-specific inhibitory antibodies also were abundant in polyclonal immune serum raised against the functionally defective lectin domain. The monoclonal antibodies elicited against the binding-defective protein bound to the high-affinity conformation of the adhesin more avidly than to the low-affinity form. However, both soluble mannose and blood plasma more strongly inhibited antibody recognition of the high-affinity FimH conformation than the low-affinity form. We propose that in the functionally active conformation the binding-pocket epitopes are shielded from targeted antibody development by ligand masking and that strong immunogenicity of the binding pocket is unblocked when the adhesive domain is in the nonbinding conformation.
PMID: 24191044
ISSN: 0027-8424
CID: 600702

Tamm-Horsfall Protein Translocates to the Basolateral Domain of Thick Ascending Limbs, Interstitium and Circulation during Recovery from Acute Kidney Injury

El-Achkar, Tarek M; McCracken, Ruth; Liu, Yan; Heitmeier, Monique R; Bourgeois, Soline; Ryerse, Jan; Wu, Xue-Ru
Tamm-Horsfall protein (THP) is a glycoprotein normally targeted to the apical membrane domain of the kidney's thick ascending limbs (TAL). We previously showed that THP of TAL confers protection to proximal tubules against acute kidney injury (AKI) via a possible cross-talk between the two functionally distinct tubular segments. However, the extent, timing, specificity and functional effects of basolateral translocation of THP during AKI remain unclear. Using an ischemia-reperfusion (IRI) model of murine AKI, we show here that, while THP expression in TAL is down-regulated at the peak of injury, it is significantly upregulated 48 hours after IRI. Confocal immunofluorescence and immunoelectron microscopy reveal a major redirection of THP during recovery from the apical membrane domain of TAL towards the basolateral domain, interstitium and basal compartment of S3 segments. This corresponds with increased THP in the serum but not in the urine. The overall epithelial polarity of TAL cells does not change, as evidenced by correct apical targeting of NKCC2 and basolateral targeting of Na+-K+-ATPase. Compared to the wild-type, THP-/- mice show a significantly delayed renal recovery after IRI, due possibly to reduced suppression by THP of pro-inflammatory cytokines and chemokines such as MCP-1 during recovery. Taken together, our data suggest that THP redistribution in the TAL after AKI is a protein-specific event, and its increased interstitial presence negatively regulates the evolving inflammatory signaling in neighboring proximal tubules, thereby enhancing kidney recovery. The increase of serum THP may be used as a prognostic biomarker for recovery from AKI.
PMID: 23389456
ISSN: 1522-1466
CID: 265582

Tamm-Horsfall protein regulates circulating and renal cytokines by affecting glomerular filtration rate and acting as a urinary cytokine trap

Liu, Y; El-Achkar, TM; Wu, XR
Although few organ systems play a more important role than the kidneys in cytokine catabolism, the mechanism(s) regulating this pivotal physiological function and how its deficiency affects systemic cytokine homeostasis remain unclear. Here we show that elimination of Tamm-Horsfall protein (THP) expression from mouse kidneys caused a marked elevation of circulating IFN-gamma, IL1-alpha, TNF-alpha, IL6, CXCL1 and IL13. Accompanying this were enlarged spleens with prominent white-pulp macrophage infiltration. Lipopolysaccharide (LPS) exacerbated the increase of serum cytokines without a corresponding increase in their urinary excretion in THP knockout (KO) mice. This, along with the rise of serum cystatin C and the reduced inulin and creatinine clearance from the circulation, suggested that diminished glomerular filtration may contribute to reduced cytokine clearance in THP KO mice both at the baseline and under stress. Unlike wild-type mice where renal and urinary cytokines formed specific in vivo complexes with THP, this trapping effect was absent in THP KO mice, thus explaining why cytokine signaling pathways were activated in renal epithelial cells in such mice. Our study provides new evidence implicating an important role of THP in influencing cytokine clearance and acting as a decoy receptor for urinary cytokines. Based on these and other data, we present a unifying model that underscores the role of THP as a major regulator of renal and systemic immunity.
PMID: 22451664
ISSN: 0021-9258
CID: 162338

Molecular and cellular effects of Tamm-Horsfall protein mutations and their rescue by chemical chaperones

Ma L; Liu Y; El-Achkar TM; Wu XR
Correct folding of a nascent polypeptide in the lumen of endoplasmic reticulum (ER) into a three-dimensional conformation is a crucial step in the stability, intracellular trafficking and targeting to the final destination of a protein. By transiently and stably expressing human-relevant mutants of Tamm-Horsfall protein in polarized MDCK cells, we show here that a cysteine-altering mutation in evolutionally conserved cysteine-rich domain had more severe defects in ER exit, surface translocation and triggered more apoptosis than a cysteine-altering mutation outside the domain. Both mutants were able to specifically bind and trap the wild-type THP and prevent it from exiting the ER and translocating to the cell surface. This explains at least partly why in patients with THP-associated diseases there is a marked urinary reduction of both the mutant and the wild-type THP. Exposure of mutant-expressing cells to low temperature (30(0)C), osmolytes (glycerol, trimethylamine N-oxide and dimethyl sulfoxide) and Ca2+-ATP inhibitor, thapsigargin only slightly relieved ER retention and increased surface targeting of the mutants. In contrast, sodium 4-phenylbutyrate and probenecid, the latter a uricosuric drug used clinically to treat gout, markedly reduced ER retention of the mutants and increased their surface translocation and secretion into the culture media. The rescue of the THP mutants was associated with the restoration of the level and subcellular localization of cytosolic chaperone HSP70. Our results reveal intricate mechanistic details that may underlie THP-associated diseases, and suggest that novel therapeutics enhancing the refolding of THP mutants may be of important value in therapy
PMID: 22117067
ISSN: 1083-351x
CID: 145778

Ras mutation cooperates with beta-catenin activation to drive bladder tumourigenesis

Ahmad, I; Patel, R; Liu, Y; Singh, L B; Taketo, M M; Wu, X-R; Leung, H Y; Sansom, O J
Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras(Q61L) or K-Ras(G12D)) with an activating beta-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor beta-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo
PMID: 21368895
ISSN: 2041-4889
CID: 132242

Progressive renal papillary calcification and ureteral stone formation in mice deficient for Tamm-Horsfall protein

Liu, Yan; Mo, Lan; Goldfarb, David S; Evan, Andrew P; Liang, Fengxia; Khan, Saeed R; Lieske, John C; Wu, Xue-Ru
Mammalian urine contains a range of macromolecule proteins that play critical roles in renal stone formation, among which Tamm-Horsfall protein (THP) is by far the most abundant. While THP is a potent inhibitor of crystal aggregation in vitro and its ablation in vivo predisposes one of the two existing mouse models to spontaneous intrarenal calcium crystallization, key controversies remain regarding the role of THP in nephrolithiasis. By carrying out a long-range follow-up of more than 250 THP-null mice and their wild-type controls, we demonstrate here that renal calcification is a highly consistent phenotype of the THP-null mice that is age and partially gene dosage dependent, but is gender and genetic background independent. Renal calcification in THP-null mice is progressive, and by 15 mo over 85% of all the THP-null mice develop spontaneous intrarenal crystals. The crystals consist primarily of calcium phosphate in the form of hydroxyapatite, are located more frequently in the interstitial space of the renal papillae than intratubularly, particularly in older animals, and lack accompanying inflammatory cell infiltration. The interstitial deposits of hydroxyapatite observed in THP-null mice bear strong resemblances to the renal crystals found in human kidneys bearing idiopathic calcium oxalate stones. Compared with 24-h urine from the wild-type mice, that of THP-null mice is supersaturated with brushite (calcium phosphate), a stone precursor, and has reduced urinary excretion of citrate, a stone inhibitor. While less frequent than renal calcinosis, renal pelvic and ureteral stones and hydronephrosis occur in the aged THP-null mice. These results provide direct in vivo evidence indicating that normal THP plays an important role in defending the urinary system against calcification and suggest that reduced expression and/or decreased function of THP could contribute to nephrolithiasis
PMID: 20591941
ISSN: 1522-1466
CID: 138204

Temporally and spatially controllable gene expression and knockout in mouse urothelium

Zhou, Haiping; Liu, Yan; He, Feng; Mo, Lan; Sun, Tung-Tien; Wu, Xue-Ru
Urothelium that lines almost the entire urinary tract performs important functions and is prone to assaults by urinary microbials, metabolites, and carcinogens. To improve our understanding of urothelial physiology and disease pathogenesis, we sought to develop two novel transgenic systems, one that would allow inducible and urothelium-specific gene expression, and another that would allow inducible and urothelium-specific knockout. Toward this end, we combined the ability of the mouse uroplakin II promoter (mUPII) to drive urothelium-specific gene expression with a versatile tetracycline-mediated inducible system. We found that, when constructed under the control of mUPII, only a modified, reverse tetracycline trans-activator (rtTA-M2), but not its original version (rtTA), could efficiently trans-activate reporter gene expression in mouse urothelium on doxycycline (Dox) induction. The mUPII/rtTA-M2-inducible system retained its strict urothelial specificity, had no background activity in the absence of Dox, and responded rapidly to Dox administration. Using a reporter gene whose expression was secondarily controlled by histone remodeling, we were able to identify, colocalize with 5-bromo-2-deoxyuridine incorporation, and semiquantify newly divided urothelial cells. Finally, we established that, when combined with a Cre recombinase under the control of the tetracycline operon, the mUPII-driven rtTA-M2 could inducibly inactivate any gene of interest in mouse urothelium. The establishment of these two new transgenic mouse systems enables the manipulation of gene expression and/or inactivation in adult mouse urothelium at any given time, thus minimizing potential compensatory effects due to gene overexpression or loss and allowing more accurate modeling of urothelial diseases than previously reported constitutive systems
PMID: 20427471
ISSN: 1522-1466
CID: 138180