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• W1973734543 abstract "Uropathogenic Escherichia coli(UPEC), the principal cause of urinary tract infection in women, attaches to the superficial facet cell layer of the bladder epithelium (urothelium) via its FimH adhesin. Attachment triggers exfoliation of bacteria-laden superficial facet cells, followed by rapid reconstitution of the urothelium through differentiation of underlying basal and intermediate cells. We have used DNA microarrays to define the molecular regulators of urothelial renewal and host defense expressed in adult C57Bl/6 female mice during the early phases of infection with isogenic virulent (FimH+) or avirulent (FimH−) UPEC strains. The temporal evolution and cellular origins of selected responses were then characterized by real time quantitative reverse transcriptase-PCR, in situ hybridization, and immunohistochemical analyses. Well before exfoliation is evident, FimH-mediated attachment suppresses transforming growth factor-β (Bmp4) and Wnt5a/Ca2+ signaling to promote subsequent differentiation of basal/intermediate cells. The early transcriptional responses to attachment also include induction of regulators of proliferation (e.g. epidermal growth factor family members), induction of the ETS transcription factor Elf3, which transactivates genes involved in epithelial differentiation and host defense (inducible nitric-oxide synthase), induction of modulators, and mediators of pro-inflammatory responses (e.g. Socs3, Cebp/δ, Bcl3, and CC/CXC chemokines), induction of modulators of apoptotic responses (A20), and induction of intermediate cell tight junction components (claudin-4). Both early and late phases of the host response exhibit remarkable specificity for the FimH+ strain and provide new insights about the molecular cascade mobilized to combat UPEC-associated urinary tract infection. Uropathogenic Escherichia coli(UPEC), the principal cause of urinary tract infection in women, attaches to the superficial facet cell layer of the bladder epithelium (urothelium) via its FimH adhesin. Attachment triggers exfoliation of bacteria-laden superficial facet cells, followed by rapid reconstitution of the urothelium through differentiation of underlying basal and intermediate cells. We have used DNA microarrays to define the molecular regulators of urothelial renewal and host defense expressed in adult C57Bl/6 female mice during the early phases of infection with isogenic virulent (FimH+) or avirulent (FimH−) UPEC strains. The temporal evolution and cellular origins of selected responses were then characterized by real time quantitative reverse transcriptase-PCR, in situ hybridization, and immunohistochemical analyses. Well before exfoliation is evident, FimH-mediated attachment suppresses transforming growth factor-β (Bmp4) and Wnt5a/Ca2+ signaling to promote subsequent differentiation of basal/intermediate cells. The early transcriptional responses to attachment also include induction of regulators of proliferation (e.g. epidermal growth factor family members), induction of the ETS transcription factor Elf3, which transactivates genes involved in epithelial differentiation and host defense (inducible nitric-oxide synthase), induction of modulators, and mediators of pro-inflammatory responses (e.g. Socs3, Cebp/δ, Bcl3, and CC/CXC chemokines), induction of modulators of apoptotic responses (A20), and induction of intermediate cell tight junction components (claudin-4). Both early and late phases of the host response exhibit remarkable specificity for the FimH+ strain and provide new insights about the molecular cascade mobilized to combat UPEC-associated urinary tract infection. Mucosal defense networks are characterized by a multifaceted interplay between epithelial, inflammatory, and immune cells that works to control and eradicate pathogens encountered in the environment. Epithelial cells are typically the first host cell type to come into contact with potential microbial invaders. This places them in a unique position to contribute to innate defense. Each year, an estimated 150 million humans worldwide develop UTI 1UTIurinary tract infectionBmp4bone morphogenetic protein 4qRT-PCRreal time quantitative reverse transcriptase-PCRUPECuropathogenic E. colimAbmonoclonal antibodyiNOSinducible nitric-oxide synthaseFITCfluorescent isothiocyanateHB-EGFheparin-binding epidermal growth factor-like growth factorILinterleukinCamKIIcalmodulin-dependent protein kinase II 1UTIurinary tract infectionBmp4bone morphogenetic protein 4qRT-PCRreal time quantitative reverse transcriptase-PCRUPECuropathogenic E. colimAbmonoclonal antibodyiNOSinducible nitric-oxide synthaseFITCfluorescent isothiocyanateHB-EGFheparin-binding epidermal growth factor-like growth factorILinterleukinCamKIIcalmodulin-dependent protein kinase II (1Harding G.K.M. Ronald A.R. Int. J. Antimicrob. Agents. 1994; 4: 83-88Crossref PubMed Scopus (66) Google Scholar). The majority of UTIs are caused by uropathogenic strains of Escherichia coli(UPEC) (2Sobel J.D. Med. Clin. N. Am. 1991; 75: 253-273Crossref PubMed Scopus (44) Google Scholar). To establish disease in the urinary tract, UPEC must bind to bladder epithelial cells. Type 1 fimbriae are extracellular organelles expressed by UPEC that are critical for this interaction (3Langermann S. Palaszynski S. Barnhart M. Auguste G. Pinkner J.S. Burlein J. Barren P. Koenig S. Leath S. Jones C.H. Hultgren S.J. Science. 1997; 276: 607-611Crossref PubMed Scopus (475) Google Scholar). The distal tip of type 1 fimbriae contains FimH, an adhesin that binds to mannosylated receptors on the luminal surface of the bladder epithelium (urothelium) (4Wu X.R. Sun T.T. Medina J.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9630-9635Crossref PubMed Scopus (257) Google Scholar). Binding is followed by invasion (5Mulvey M.A. Lopez-Boado Y.S. Wilson C.L. Roth R. Parks W.C. Heuser J. Hultgren S.J. Science. 1998; 282: 1494-1497Crossref PubMed Scopus (762) Google Scholar, 6Martinez J.J. Mulvey M.A. Schilling J.D. Pinkner J.S. Hultgren S.J. EMBO J. 2000; 19: 2803-2812Crossref PubMed Scopus (553) Google Scholar). urinary tract infection bone morphogenetic protein 4 real time quantitative reverse transcriptase-PCR uropathogenic E. coli monoclonal antibody inducible nitric-oxide synthase fluorescent isothiocyanate heparin-binding epidermal growth factor-like growth factor interleukin calmodulin-dependent protein kinase II urinary tract infection bone morphogenetic protein 4 real time quantitative reverse transcriptase-PCR uropathogenic E. coli monoclonal antibody inducible nitric-oxide synthase fluorescent isothiocyanate heparin-binding epidermal growth factor-like growth factor interleukin calmodulin-dependent protein kinase II The consequences of FimH-mediated attachment have been explored in a mouse model. Transurethral inoculation of a FimH+ clinical isolate of UPEC (NU14) into the bladders of adult female C57Bl/6 mice results in rapid loss of the superficial facet cell layer of the urothelium. Scanning EM of mouse bladders harvested 2 h after inoculation reveals numerous bacteria attached to an intact urothelial surface. Six hours after inoculation, exfoliation of bacteria-laden superficial facet cells occurs, exposing underlying, less differentiated intermediate cells. Exfoliation is part of an innate host defense mechanism that helps clear bacteria from the bladder. Intermediate cells rapidly differentiate to superficial facet cells over the course of the next 12–48 h. After 7 days, the urothelium appears to be fully restored based on histologic criteria. In contrast, an isogenic FimH− UPEC mutant (NU14-1) fails to attach to the urothelium, is unable to colonize the bladders of adult female mice, and does not elicit exfoliation or significant inflammation (5Mulvey M.A. Lopez-Boado Y.S. Wilson C.L. Roth R. Parks W.C. Heuser J. Hultgren S.J. Science. 1998; 282: 1494-1497Crossref PubMed Scopus (762) Google Scholar). The rapid regenerative response to FimH+ UPEC-mediated injury contrasts with the slow turnover of the undamaged urothelium (estimates range up to 40 weeks) (7Jost S.P. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1989; 57: 27-36Crossref PubMed Scopus (72) Google Scholar). The pseudostratified transitional urothelium normally supports a continuum of differentiation as cells move from its basal, to intermediate, to surface layers. Differentiation is associated with repeated cell fusions and increasing ploidy; basal cells are diploid, whereas the nuclei of the large superficial facet cells are octoploid or higher (8Levi P.E. Cooper E.H. Anderson C.K. Williams R.E. Cancer (Phila.). 1969; 23: 1074-1085Crossref PubMed Scopus (79) Google Scholar). The basal layer functions as the proliferative compartment (mitotic labeling index = 0.11%) (7Jost S.P. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1989; 57: 27-36Crossref PubMed Scopus (72) Google Scholar). The remarkably slow pace of renewal of the undamaged urothelium has impeded efforts to identify the molecular factors that control this process. In this report, we describe how a functional genomics-based analysis of the host response to isogenic FimH+ and FimH− UPEC infection of the mouse bladder has revealed an intricate network of molecular regulators and effectors of urothelial regeneration, pro-inflammatory responses, and barrier functions. NU14 (fimH+) and NU14-1 (fimH−) strains were grown as described (3Langermann S. Palaszynski S. Barnhart M. Auguste G. Pinkner J.S. Burlein J. Barren P. Koenig S. Leath S. Jones C.H. Hultgren S.J. Science. 1997; 276: 607-611Crossref PubMed Scopus (475) Google Scholar). 8–10-week-old female C57Bl/6J mice (The Jackson Laboratories) were inoculated, via transurethral catheterization, with 50 μl of a suspension of 108 colony-forming units of NU14 or NU14-1 in phosphate-buffered saline (5Mulvey M.A. Lopez-Boado Y.S. Wilson C.L. Roth R. Parks W.C. Heuser J. Hultgren S.J. Science. 1998; 282: 1494-1497Crossref PubMed Scopus (762) Google Scholar). Total cellular RNA was isolated (RNeasy kit, Qiagen) from bladders of mice sacrificed prior to and 1.5 or 3.5 h after inoculation (n = 10 mice/time point/bacterial strain/experiment; n = 2 independent experiments). Equivalent amounts of RNA from each mouse in each group/experiment were pooled and biotinylated cRNA targets prepared (9Lee C.K. Klopp R.G. Weindruch R. Prolla T.A. Science. 1999; 285: 1390-1393Crossref PubMed Scopus (1269) Google Scholar). Each cRNA target was hybridized to Mu11K GeneChips (Affymetrix). GeneChip software was then used to compute an average fluorescence intensity across all probe sets on the GeneChips. The signal produced by each probe set on a chip was then multiplied using a “scaling factor” so that the average chip-wide intensity matched a specified intensity. As recommended by Affymetrix, we scaled the overall fluorescence intensity across each chip to an intensity of 1500 prior to conducting the pairwise chip-to-chip comparisons with GeneChip software. We followed the recommendation of the manufacturer to exclude genes whose expression changed less than 2-fold in single chip-to-chip comparisons. Several papers have established this threshold as a standard (10Claverie J.M. Hum. Mol. Genet. 1999; 8: 1821-1832Crossref PubMed Scopus (233) Google Scholar, 11Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell. 1999; 97: 727-741Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 12Wang Y. Rea T. Bian J. Gray S. Sun Y. FEBS Lett. 1999; 445: 269-273Crossref PubMed Scopus (73) Google Scholar, 13Mills J.C. Gordon J.I. Nucleic Acids Res. 2001; 29: e72Crossref PubMed Scopus (67) Google Scholar). In addition, we required that the change be ≥2-fold in the same direction (increased or decreased) in FimH+ relative to FimH− UPEC-infected bladders in duplicate biological experiments. Additional groups of age-matched female C57Bl/6 mice were infected with FimH+ or FimH− UPEC strains for 6 h, 24 h, and 7 days (n = 4 animals/time point/strain). Bladder RNAs were pooled as above and used for qRT-PCR studies. For the 0-, 1.5-, and 3.5-h time points, qRT-PCR assays were performed using the same RNAs employed for the DNA microarray analysis. cDNAs, produced from the 0-, 1.5-, 3.5-, 6-, and 24-h and 7-day time points, were added to 25-μl qRT-PCRs containing 12.5 μl of 2× SYBR Green master mix (Applied Biosystems), 900 nm gene-specific primers (see on-line supplemental tables for a list), and 0.25 units of UDP-N-glycosidase (Invitrogen). A melting curve was used to identify a temperature where only the amplicon, and not primer dimers, accounted for the SYBR Green-bound fluorescence. Assays were performed in triplicate with an Applied Biosystems model 7700 instrument. All data were normalized to an internal standard (glyceraldehyde 3-phosphate dehydrogenase mRNA; ΔΔCT method, User Bulletin 2, Applied Biosystems). Bladders from uninfected mice, plus bladders from mice sacrificed 3.5 and 6 h after inoculation with NU14 or NU14-1, were fixed in Bouin's solution (overnight, 4 °C). Bladders from each group of mice were embedded together in paraffin, and serial 5-μm thick sections were prepared. For in situ hybridization analysis, sections were deparaffinized (Trilogy kit, CellMarque), incubated (15 min, 23 °C) with proteinase K (Roche Molecular Biochemicals; 5 μg/ml phosphate-buffered saline), and processed according to Ref. 14Zaidi A.U. Enomoto H. Milbrandt J. Roth K.A. J. Histochem. Cytochem. 2000; 48: 1369-1375Crossref PubMed Scopus (61) Google Scholar. Digoxigenin-UTP-labeled cRNA and control mRNA probes were prepared by T7 or SP6 RNA polymerase transcription of linearized plasmids containing double-stranded cDNAs encoding mouse Bmp4 (pBmp4), BmpRIa (pBmprIa; both from Brigid Hogan, Vanderbilt University), Wnt5a (pGEMwnt5a; Andrew McMahon, Harvard University), plus Dll1 (pCS2Delta, Raphael Kopan, Washington University). Protocols for probe hybridization and subsequent detection (tyramide signal amplification-direct method) are detailed in Ref. 14Zaidi A.U. Enomoto H. Milbrandt J. Roth K.A. J. Histochem. Cytochem. 2000; 48: 1369-1375Crossref PubMed Scopus (61) Google Scholar. Sections adjacent to those used for in situ hybridization were prepared for immunohistochemical studies by de-paraffinization with xylene, rehydration, and preincubation (15 min, 23 °C) with blocking buffer (1% bovine serum albumin, 0.3% Triton X-100 in phosphate-buffered saline). Sections were then stained with the following antibodies: (i) mouse monoclonal antibody (mAb) to uroplakin III (Research Diagnostics); (ii) rabbit polyclonal antibodies to aquaporin 3 (Ref. 15Matsuzaki T. Suzuki T. Koyama H. Tanaka S. Takata K. J. Histochem. Cytochem. 1999; 47: 1275-1286Crossref PubMed Scopus (143) Google Scholar; obtained from Kuniaki Takata, Gunma University; diluted 1:500 in blocking buffer); (iii) mouse mAb to cytokeratins 5/6 (clone D5/16B4; Chemicon; 1:500); (iv) rabbit antibodies to mouse claudin-4 (Ref. 16Morita K. Furuse M. Fujimoto K. Tsukita S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 511-516Crossref PubMed Scopus (960) Google Scholar; from Mikio Furuse, Kyoto University; 1:1000); (v) rabbit anti-human phospho-Smad1 (Upstate Biotechnology, Inc.; 1:500); (vi) rabbit antibodies to purified E. coli FimH adhesin (Ref. 3Langermann S. Palaszynski S. Barnhart M. Auguste G. Pinkner J.S. Burlein J. Barren P. Koenig S. Leath S. Jones C.H. Hultgren S.J. Science. 1997; 276: 607-611Crossref PubMed Scopus (475) Google Scholar; 1:500). Antigen retrieval protocols were used to detect cytokeratins 5/6 (17Riedel I. Czernobilsky B. Lifschitz-Mercer B. Roth L.M., Wu, X.R. Sun T.T. Moll R. Virchows Arch. 2001; 438: 181-191Crossref PubMed Scopus (89) Google Scholar). Antigen-antibody complexes were visualized with Cy3- or FITC-conjugated sheep anti-mouse Ig or FITC-labeled donkey anti-rabbit Ig (Jackson ImmunoResearch; 1:1000). High density oligo-based DNA microarrays, containing probe sets representing ∼11,000 mouse genes and expressed sequence tags, were used to compare gene expression in the bladders of adult female mice prior to and 1.5 and 3.5 h after exposure to isogenic FimH+ or FimH− strains of UPEC. We selected these early time points to assay how the urothelium prepares for exfoliation and subsequent replacement of its superficial facet cells. Furthermore, at these time points there is little change in the cellular composition of the urothelium; infiltration of inflammatory/immune cells is not a prominent feature of the host response until 6 h after inoculation of FimH+ UPEC (18Mulvey M.A. Schilling J.D. Martinez J.J. Hultgren S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8829-8835Crossref PubMed Scopus (351) Google Scholar). We chose DNA microarrays to initiate our analysis because (a) the outcomes of FimH+ and FimH− UPEC infection in this genetically defined mouse model are markedly divergent and predictable, and (b) there was scant prior information to guide investigation into the molecular determinants of the host response. RNA was prepared from the bladders of age-matched mice inoculated with 108 colony-forming units of either UPEC strain (n = 10 animals/group/time point). Equivalent amounts of RNA from each mouse per group per time point were pooled; cRNAs were generated and used to interrogate DNA microarrays. The biological experiment was repeated on two separate occasions so that replicate cRNAs for bladders infected with each bacterial strain at each time point could be hybridized to separate sets of microarrays. Comparisons of bladder RNAs from FimH+ versus FimH− UPEC-infected mice were performed with the FimH− UPEC microarrays designated as “Base line.” Data sets were generated by extracting all mRNAs that satisfied the following selection criteria: (a) called “Present” in either microarray (base line or partner); (b) levels changed ≥2-fold (increased or decreased) in FimH+ compared with FimH− UPEC-infected bladder RNA at the 1.5-h and/or 3.5-h time points; and (c) the increase or decrease in expression at a given time point was reproduced in duplicate experiments. A total of 56 genes satisfied all of these selection criteria. This includes five expressed sequence tags. They are grouped into functional categories in Table I. The largest category contained genes associated with cellular differentiation and proliferation. The table also presents DNA microarray-based comparisons of the expression of these 56 genes in FimH+ UPEC-infected versus uninfected bladder RNA. More information about these data sets, including additional annotation with hyperlinks to relevant literature citations, can be found in the on-line supplementary tables.Table IDNA microarray-based data set of genes that show changed expression in FimH+ relative to FimH-UPEC infected or uninfected mouse bladdersGeneGenBank™ accession numberFunctionFold change in expression relative to FimH-UPEC infected bladder RNAsFold change in expression relative to uninfected bladder RNAs1.5 h3.5 h1.5 h3.5 hDifferentiation Wnt5aNM009524Negative regulator of differentiation−10.9−7.2 Bone morphogenetic protein 4 (Bmp4)L47480Negative regulator of differentiation−2−2.1 Delta-like 1 (Dll1)X80903Cell fate regulation22.421.9 E74-like factor 3 (Elf3)AF016294Positive regulator of differentiation21.74.854.1126.8Proliferative and immediate-early responses Heparin-binding epidermal growth factor-like growth factor (HB-EGF)L07264Growth factor3.95.6 Epiregulin (Ereg)D30782Growth factor5.411.7 Serum-inducible kinase (Snk)M96163Early mitogenic response2.52.7 c-mycL00039Immediate-early gene2.42.3 FBJ osteosarcoma oncogene (c-fos)V00727Immediate-early gene8.913.541.1 junBU20735Immediate-early gene10.63.813.3 Early growth response 1 (Egr1)M22326Immediate-early gene2.75.72.65.7 Early growth response 2 (Egr2)X06746Immediate-early gene8.29.2 Immediate early response 2 (Ier2)M59821Immediate-early gene2.66.14.37.4 Fibroblast growth factor-regulated protein (Fgfrp)U04204Immediate-early gene2.22.2 Nuclear hormone-binding receptor (N10)X16995Immediate early gene3.43 Steroid/thyroid hormone nuclear receptor (Nurr1)U86783Immediate early gene11.717.0 B-cell translocation gene 2 (Btg2)M64292Immediate early gene3.23.1 Neurofibromatosis 2 (Nf2)L27090Regulator of proliferation4.7 Erythroid Krüppel-like transcription factor (Eklf)AA168164Transcriptional regulator6.75.4 Zinc finger protein 36/tristetraprolin (TTP)M58691Growth factor-inducible factor45.1 Retinoblastoma-like 1(p107)U27177Cell cycle regulator−3−2.8 T-lymphocyte-activated protein (Chx1)P17950Regulation of cell division2.13.6Pro-inflammatory responses Macrophage inflammatory protein-2 (Mip-2)X53798CXC chemokine5.310.219.7 Growth factor-inducible KC protein (Gro1)J04596CXC chemokine36.286.5 Monocyte chemotactic protein 1 (Mcp1)M19681CC chemokine1135.8 Small inducible cytokine A7 (Scya7,Mcp-3)Z12297CC chemokine2.55.4 Interleukin 6 (Il-6)X54542Cytokine12.526.3 Cytokine-inducible SH2-containing protein 3 (Socs3)U88328Regulator of cytokine signaling4.86.122.5 CCAAT/enhancer-binding protein, δ (C/ebpδ)X61800Regulator of IL-6 expression2.614.3 CCAAT/enhancer-binding protein, β (C/ebpβ)X62600Regulator of IL-6 expression2.74.4 GATA-binding protein 3 (Gata-3)X55123Regulates IL-4 activity−3−4.1 Bcl3M90397Transactivates NFκB p50/52 and AP-111.126.6 IκBU36277Inhibitor of NfκB2.62.9 RegIII, γD63361Anti-bacterial protein7.167.4 Endothelial cell-activated protein C receptor (Epcr)L39017Activation of protein C pathway3.16.4Apoptosis T-cell death-associated gene 51 (Tdag51)U44088Pro-apoptotic regulator2.93.33.1 A20 (Tnfaip3)U19463Inhibitor of apoptosis3.87.3 Phospholipid scramblase 1 (Tras1, Plscr1)D78354Phospholipid movement34.3Stress response Serum amyloid A 3 (SAA3)X03479Major acute phase reactant6.124.3 Activating transcription factor 3 (Atf3)U19118Stress inducible transcription factor6.54.811.0 Heat shock protein 40 (Hsp40)AA266407Protein aggregation2.43.62.7 Myeloid differentiation primary response gene 116 (Myd116)X51829DNA damage-inducible gene12.72213.3 Myeloid differentiation primary response gene 118 (Myd118)X54149DNA damage-inducible gene2.23.3 Growth arrest and DNA-damage-inducible gene (Gadd45)I28177DNA damage-inducible gene2.22.9Signal transduction PKCδX60304Signal transduction−2.2−2.0 CaMKIIδNM001221Calcium-dependent signal transduction−7.5 Gem GTPaseU10551Ras family GTPase2.44.2 Dual specificity protein phosphatase 6AA184871Non-receptor tyrosine phosphatase3.55.6 14-3-3 protein ςP31947Signaling adaptor4.12.8Cell-cell contacts Claudin-4AB000713Regulation of paracellular permeability3.22.43.74.7 Urokinase plasminogen activator receptor (Upar)C76481Matrix degradation/wound repair6.82.215.6 Hyaluronan synthaseD82964ECM/wound repair5.37.4 Heparan sulfated-glucosaminyl-3-O-sulfotransferase-1AF019385Heparan sulfate proteoglycan biosynthesis7.218.612.542.3 Filamin 1AA038347Actin cross-linking phosphoprotein2.416.7Metabolism Hexokinase IIY11666Glycolytic enzyme49.3Miscellaneous G7eU69488Viral envelope-like protein3.37.9The time points sampled in duplicate experiments are indicated. See on-line supplementary tables for additional details including gene-specific annotation and reference hyperlinks. Open table in a new tab The time points sampled in duplicate experiments are indicated. See on-line supplementary tables for additional details including gene-specific annotation and reference hyperlinks. Real time quantitative RT-PCR (qRT-PCR) was used to independently verify the change in levels of selected mRNAs from each functional category. To define the evolution of the host response, the qRT-PCR survey was extended to bladder RNAs prepared from mice sacrificed 6 h, 24 h, and 7 days after inoculation of the two UPEC strains (n = 4 animals/time point/experiment). The 6-h time point was selected because it represents the period of exfoliation of superficial facet cells and exposure of underlying intermediate cells. The 24-h time point represents the period of generalized differentiation of intermediate to superficial facet cells. Seven days represents the time of histologic restoration of the urothelium. As described below, these analyses have revealed a host response characterized by prominent changes in expression of molecular regulators and effectors of (a) epithelial differentiation and proliferation, (b) proinflammatory and apoptotic responses, and (c) barrier function. Bone morphogenetic protein 4 (Bmp4) is a member of the transforming growth factor-β (Tgf-β) superfamily of secreted signaling molecules and acts as a negative regulator of differentiation in a number of cellular systems (e.g. Refs. 19Treier M. Gleiberman A.S. O'Connell S.M. Szeto D.P. McMahon J.A. McMahon A.P. Rosenfeld M.G. Genes Dev. 1998; 12: 1691-1704Crossref PubMed Scopus (401) Google Scholar and 20Mathura J.R., Jr. Jafari N. Chang J.T. Hackett S.F. Wahlin K.J. Della N.G. Okamoto N. Zack D.J. Campochiaro P.A. Investig. Ophthalmol. Vis. Sci. 2000; 41: 592-600PubMed Google Scholar). Bmp4 is expressed in mesenchymal cells surrounding the ureteric bud stalk in the developing urinary tract and may act as an inhibitory factor to limit the sites of ureteral branch formation (21Miyazaki Y. Oshima K. Fogo A. Hogan B.L. Ichikawa I. J. Clin. Invest. 2000; 105: 863-873Crossref PubMed Scopus (347) Google Scholar). Bmp4 mRNA levels decline rapidly during the early phases of FimH+ UPEC infection, reaching a nadir at 6 h (Fig.1 A). In situhybridization studies revealed that Bmp4 mRNA is localized to the mesenchyme underlying the normal bladder urothelium and that FimH+ UPEC infection promotes a decline in mRNA levels within this cellular compartment (Fig. 1 B). Multilabel immunohistochemical studies indicated that bacteria are not detectable in the mesenchyme or in the overlying intermediate and basal cells (Fig. 1 C). Bmp4 binds to and signals through two serine/threonine kinase receptors, BmpRIa and BmpRIb (22ten Dijke P. Yamashita H. Sampath T.K. Reddi A.H. Estevez M. Riddle D.L. Ichijo H. Heldin C.H. Miyazono K. J. Biol. Chem. 1994; 269: 16985-16988Abstract Full Text PDF PubMed Google Scholar). Only BmpRIa mRNA was detectable by qRT-PCR in the bladders of normal uninfected animals as well as in mice infected with either strain of UPEC. In situhybridization disclosed that BmpRIa is expressed in the urothelium. qRT-PCR and in situ studies indicated that receptor mRNA levels did not change appreciably upon exposure to the FimH+ strain (data not shown). Bmp4 binding to the receptor leads to its activation, resulting in phosphorylation of a BMP signal transducer, Smad1, and subsequent transactivation of target genes (23Liu F. Hata A. Baker J.C. Doody J. Carcamo J. Harland R.M. Massague J. Nature. 1996; 381: 620-623Crossref PubMed Scopus (587) Google Scholar, 24Graff J.M. Bansal A. Melton D.A. Cell. 1996; 85: 479-487Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Immunohistochemical studies revealed that 6 h after infection, phospho-Smad1 levels were decreased in intermediate and basal cells (Fig. 1 D), providing direct evidence for reduced signaling. The FimH− mutant produced negligible alterations in expression of components of the Bmp4 pathway (Fig. 1, A, B, and D). Genetic studies in Drosophila have demonstrated an important role for heparan sulfate proteoglycans in regulating Tgf-β/Bmp activity (25Jackson S.M. Nakato H. Sugiura M. Jannuzi A. Oakes R. Kaluza V. Golden C. Selleck S.B. Development. 1997; 124: 4113-4120Crossref PubMed Google Scholar). Bmp4 binds to heparin with high affinity in vitro (26Sampath T.K. Muthukumaran N. Reddi A.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7109-7113Crossref PubMed Scopus (289) Google Scholar). Moreover, a loss-of-function mutation in the heparan sulfate proteoglycan, glypican-3, appears to potentiate cellular responses to Bmp4 in the developing mouse limb (27Paine-Saunders S. Viviano B.L. Zupicich J. Skarnes W.C. Saunders S. Dev. Biol. 2000; 225: 179-187Crossref PubMed Scopus (154) Google Scholar). Intriguingly, DNA microarray and qRT-PCR analyses disclosed that FimH+ but not FimH− UPEC induces a pronounced increase in expression of heparan sulfated-glucosaminyl-3-O-sulfotransferase 1 (3-Ost1), an enzyme that catalyzes terminal modification of the heparan sulfate chain (28Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (656) Google Scholar). This response peaks 6 h after infection (18-fold rise relative to uninfected bladders; Fig. 1 E). Increased 3-Ost1 expression may serve to further promote the effects of reduced Bmp4 expression in enhancing intermediate cell differentiation. The Wnt family of secreted glycoproteins binds to frizzled (Fz) receptors. Two Wnt pathways are known: a “classical” pathway that signals through β-catenin and high modality group box transcription factors, and a more recently described, non-canonical pathway (29Miller J.R. Hocking A.M. Brown J.D. Moon R.T. Oncogene. 1999; 18: 7860-7872Crossref PubMed Scopus (601) Google Scholar) where Wnt5a:Fz(3/4/6)-stimulated increases in intracellular Ca2+ leads to activation of protein kinase C and Ca2+/calmodulin-dependent protein kinase II (CamKII) (30Sheldahl L.C. Park M. Malbon C.C. Moon R.T. Curr. Biol. 1999; 9: 695-698Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 31Kuhl M. Sheldahl L.C. Malbon C.C. Moon R.T. J. Biol. Chem. 2000; 275: 12701-12711Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Forced expression of Wnt5a delays chondrocyte differentiation in the developing chick limb (32Hartmann C. Tabin C.J. Development. 2000; 127: 3141-3159Crossref PubMed Google Scholar). qRT-PCR revealed that the normal mouse bladder expresses Fz receptors 3, 4, and 6. Although levels of these" @default.
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• W1973734543 date "2002-03-01" @default.
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• W1973734543 title "Molecular Regulation of Urothelial Renewal and Host Defenses during Infection with Uropathogenic Escherichia coli" @default.
• W1973734543 cites W1502075862 @default.
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