[Show abstract][Hide abstract] ABSTRACT: Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.
[Show abstract][Hide abstract] ABSTRACT: Previous studies on cancer cell invasion were primarily focused on its migration because these two events were often considered biologically equivalent. Here we found that T24T cells exhibited higher invasion but lower migration abilities than T24 cells. Expression of Rho-GDPases was much lower and expression of SOD2 was much higher in T24T cells than those in T24 cells. Indeed, knockdown of SOD2 in T24T cells can reverse the cell migration but without affecting cell invasion. We also found that SOD2 inhibited the JNK/c-Jun cascade, and the inhibition of c-Jun activation by ectopic expression of TAM67 impaired Rho-GDPases expression and cell migration in T24T shSOD2 cells. Further, we found that Sp1 can upregulate SOD2 transcription in T24T cells. Importantly, matrix metalloproteinase-2 (MMP-2) was overexpressed in T24T and participated in increasing its invasion, and MMP-2 overexpression was mediated by increasing nuclear transport of nucleolin, which enhanced mmp-2 mRNA stability. Taken together, our study unravels an inverse relationship between cell migration and invasion in human bladder cancer T24T cells and suggests a novel mechanism underlying the divergent roles of SOD2 and MMP-2 in regulating metastatic behaviors of human bladder T24T in cell migration and invasion.
[Show abstract][Hide abstract] ABSTRACT: p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1 to S cell-cycle transition and reduced p27Kip1 expression is prevalent in a wide range of human tumors, the exact mechanism(s) of p27Kip1-mediated tumor suppression remains obscure. In this study, we identified a close, inverse relationship between p27Kip1 and EGFR expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but alsocausal;depletion of p27Kip1 in MEF cells resulted in markedly elevated EGFR expression, a result reproducible with an egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found toregulate EGFR expression via the JNK/c-Jun transcriptional factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed egfr transcription. Furthermore, the proximal promoter of the egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1-/- cells than in p27Kip1+/+ cells. Introduction of GFP-p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. Our studies demonstrated that p27Kip1 suppressed JNK/c-Jun activation and EGFR expression in mouse MEFs and human bladder cancer cells, and obtained results consistent with those from human cancer specimens. This study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.
[Show abstract][Hide abstract] ABSTRACT: Genetically engineered mouse models (GEMMs) have been highly instrumental in elucidating gene functions and molecular pathogenesis of human diseases, although their use in studying kidney stone formation or nephrolithiasis remains relatively limited. This review intends to provide an overview of several knockout mouse models that develop interstitial calcinosis in the renal papillae. Included herein are mice deficient for Tamm-Horsfall protein (THP; also named uromodulin), osteopontin (OPN), both THP and OPN, Na(+)-phosphate cotransporter Type II (Npt2a) and Na(+)/H(+) exchanger regulatory factor (NHERF-1). The baseline information of each protein is summarized, along with key morphological features of the interstitial calcium deposits in mice lacking these proteins. Attempts are made to correlate the papillary interstitial deposits found in GEMMs with Randall's plaques, the latter considered precursors of idiopathic calcium stones in patients. The pathophysiology that underlies the renal calcinosis in the knockout mice is also discussed wherever information is available. Not all the knockout models are allocated equal space because some are more extensively characterized than others. Despite the inroads already made, the exact physiological underpinning, origin, evolution and fate of the papillary interstitial calcinosis in the GEMMs remain incompletely defined. Greater investigative efforts are warranted to pin down the precise role of the papillary interstitial calcinosis in nephrolithiasis using the existing models. Additionally, more sophisticated, second-generation GEMMs that allow gene inactivation in a time-controlled manner and "compound mice" that bear several genetic alterations are urgently needed, in light of mounting evidence that nephrolithiasis is a multifactorial, multi-stage and polygenic disease.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 06/2014; 9(6):e99644. DOI:10.1371/journal.pone.0099644 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Cell migration is a dynamic process that is central to a variety of physiological functions as well as disease pathogenesis. The modulation of cell migration by p27 has been reported, but the exact mechanism(s) whereby p27 intersects with downstream effectors that control cell migration have not been elucidated. By systematically comparing p27+/+ MEFs with genetically ablated p27-/- MEFs using wound healing, transwell and time-lapse microscopic analyses, we provide direct evidence demonstrating that p27 inhibits both directional and random cell migration. Identical results were obtained with normal and cancer epithelial cells using complementary knockdown and overexpression approaches. Additional studies revealed that overexpression of manganese superoxide dismutase (MnSOD) and reduced intracellular oxidation played a key role in increased cell migration in p27-deficient cells. Furthermore, we identified signal transducer and activator of transcription 3 (STAT3) as the transcription factor responsible for p27-regulated MnSOD expression which was further mediated by ERKs/ATF1-dependent transactivation of CRE within the stat3 promoter. Collectively, our data strongly indicate that p27 plays a crucially negative role in cell migration by inhibiting MnSOD expression in a STAT-3 dependent manner.