FGF-10 and its receptor exhibit bidirectional paracrine targeting to urothelial - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:38 |只看该作者 |倒序浏览 |打印

作者：Dianzhong Zhang, Jeffrey Kosman, Nicole Carmean, Richard Grady, and James A. Bassuk作者单位：Program in Human Urothelial Biology, Children?s Hospital and Regional Medical Center, Seattle, Washington , S5 W) z3 M+ N) g" Z1 B6 W5 ?



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      【摘要】
   Control of the regenerative properties of urothelial tissue would greatly aid the clinician in the management of urinary tract disease and disorders. Fibroblast growth factor 10 (FGF-10) is a mitogen which is particularly promising as a protein therapy for urothelial injury. The spatial synthesis, transport, targeting, and mechanistic pathway of FGF-10 and its receptor were studied in a human urothelial cell culture model and in fixed sections of lower urinary tract tissue. Synthesis of FGF-10 was restricted to mesenchymal fibroblasts, and secreted FGF-10 exhibited paracrine transport to two proximal sites, transitional epithelium and smooth muscle cell bundles, both of which were also the exclusive sites of FGF-10 receptor synthesis. The addition of recombinant FGF-10 to quiescent urothelial cells in vitro was sufficient to stimulate DNA synthesis. This stimulation was through a pathway independent of the epidermal growth factor receptor pathway. Deconvolution, light and transmission electron microscopic studies captured FGF-10 and its receptor in association with the urothelial cell surface, in cytoplasm, and within nuclei, observations that describe the mechanism that transduces the mitogenic signal in these tissues. Localization of the FGF-10 receptor to the superficial urothelial layer is clinically significant because intravesical administration of FGF-10 may provide the clinician a means to control the turnover of transitional epithelium in bladder disorders such as interstitial cystitis. ! j1 }( |6 `! l- s: D+ i; E4 i( v5 u; T
      【关键词】 transitional epithelium mesenchymal signaling growth factors* Q$ \' x- q/ ]
   EPITHELIAL - MESENCHYMAL INTERACTIONS play crucial roles during embryonic organ development and adult life. These interactions are mediated, in part, by paracrine factors produced by the mesenchyme but acting on the epithelium. Fibroblast growth factors (FGFs) and their tyrosine kinase receptors (FGFRs) are examples and their interactions are involved in multiple physiological and pathological processes such as cell proliferation, migration, and differentiation during embryonic development, and as homeostatic factors, they function in tissue repair and response to injury in the adult organism ( 40 ). Currently, the FGF family is comprised of at least 23 (FGF1-23) structurally related proteins that are expressed in specific spatial and temporal patterns ( 56 ). Four FGFR genes have been identified in mammals (FGFR1-4), with an additional receptor, FGF receptor-like protein-1 (FGFRL1) or FGFR5, also proposed ( 10, 29, 57, 69, 70 ). A typical FGFR is comprised of an extracellular domain containing two or three immunoglobulin-like (Ig) loops, a transmembrane segment, and an intracellular split tyrosine kinase domain ( 37 ). Signal transduction is mediated by FGFs interacting with low affinity with heparan sulfate proteoglycans ( 32, 33 ) and binding the receptors. This binding causes receptor dimerization, resulting in autophosphorylation and/or transphosphorylation of the tyrosine residues. These activated receptors then begin signal transduction through multiple pathways including phospholipase C (PLC ) ( 6, 39 ), phosphatidylinositol 3-kinase (PI3K) ( 20 ), mitogen-activated protein kinases (MAPK) ( 19 ), and signal transducers and activators of transcription (STATs) ( 12, 65 ). These effectors, in turn, disseminate the signals through signal transduction cascades ( 30, 47 ) by activating many target proteins, including transcription factors in the nucleus ( 31 ).+ O# `8 S( l! i) N* v

Although both loops II and III of the extracellular domain of the receptors are involved in ligand binding, ligand specificity is determined by the COOH-terminal portion of loop III ( 9, 73 ). Loop III undergoes alternative mRNA splicing that generates three different types of FGFR2 mRNAs designated as IIIa, IIIb, and IIIc. The variant IIIa encodes for a secreted FGF-binding protein, whereas the other two splicing variants, IIIb and IIIc, encode for membrane-bound receptors. These alternatively spliced FGFR isoforms have distinct ligand-binding properties and tissue-specific expression patterns ( 8, 14, 25, 67 ). Previous studies suggest that the IIIb isoform of FGFRs is expressed in many types of epithelial lineages, whereas the IIIc variant is restricted to mesenchymal origin ( 17, 42, 71 ).

FGFR2IIIb is essential for epithelial-mesenchymal interactions during the development of several organs ( 7, 11, 44, 51, 52, 68 ). FGFR2IIIb is activated by four known ligands (FGF-1, FGF-3, FGF-7, and FGF-10) that are synthesized predominantly in mesenchyme ( 15, 36, 41, 42, 45, 72 ). Both FGF-7 and FGF-10 bind FGFR2IIIb with similar high affinity and compete with each other for this binding ( 23, 34 ). However, striking phenotypic similarities between the FGF-10 and FGFR2IIIb knockout mice have established FGF-10 as the predominant ligand for FGFR2IIIb in developmental patterning and organogenesis ( 23 ). Both FGF10-null and FGFR2IIIb-null mice die at birth and show agenesis of the lungs and limbs ( 2, 38, 45, 55 ). The exceptional specificity and the pivotal role of FGF10-FGFR2IIIb signaling during development make this complex an ideal model system for studying mesenchymal- epithelial interactions.+ z& E2 _6 X& J, T  E

Epithelial-mesenchymal interactions in the lower urinary tract are related to many physiological and pathological processes including urothelial regrowth, injury recovery, and urinary tract diseases or disorders such as the obstructive uropathies of childhood or adult life. However, knowledge about urothelial-mesenchymal interactions remains limited and there are few mitogens known to act on human urothelial cells during normal homeostatic conditions or during the urothelial response to injury. Two potential mitogens are the fibroblast growth factors, FGF-7 and FGF-10. The collective evidence demonstrates an essential role for FGF-7 in development, growth, differentiation, and homeostasis of the mucosal lining of the urinary tract. Mice that contain a targeted disruption of the FGF-7 gene exhibit defects in ureteric bud outgrowth ( 49 ) and stratification of bladder urothelium ( 63 ). Except for these defects, however, FGF7-null mice appear developmentally normal and are fertile ( 18 ). Our stereological analysis of partially outlet obstructed bladders revealed that a statistically significant difference in the urothelial response to injury between wild-type and FGF7-null mice did not exist. One potential compensatory source is the FGF-10 pathway, and our previous work demonstrated that recombinant human FGF-10 induced proliferation of both human urothelial cells in vitro and transitional urothelium of wild-type and FGF7-null mice in vivo ( 4 ). FGF-10 was also found as a key regulator of epithelial growth in the prostate ( 64 ). Therefore, understanding the function of FGF-10 and its receptor in the cross talk between mesenchyme and urothelium is of considerable importance for the better management of urinary tract diseases and disorders. In this report, we describe the protein distribution and gene expression patterns of FGF-10 and its receptor (FGFR2IIIb). The mechanisms that urothelial cells use to transfer paracrine mitogenic signals to their nuclei are also described. These data provide the foundation for understanding mesenchymal-urothelial interactions and for determining the efficacy of using FGF-10 as a therapeutic tool in the management of some genitourinary conditions including infection and obstruction.5 }7 v: Y3 m1 d5 N8 v. K; x: }1 D# K
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MATERIALS AND METHODS

Human tissue. Intact mucosa, containing transitional epithelium adhered to its underlying lamina propria, was obtained from surgical explants of bladders, ureters, and renal pelvises of pediatric subjects. All tissues were obtained with informed consent and/or assent under approval of the Institutional Review Board of Children?s Hospital and Regional Medical Center.: ^1 y% W* h) u, |' w, r

In vitro propagation of urothelial cells. Cells were used between passages 2 and 6 and cultures were passaged between 70 and 90% confluence. Cells were cultured according to modifications to previously described procedures ( 4, 21, 58 ). Surgical explant tissue (0.5-4 cm 2 ) from renal pelvis, ureter, or urinary bladder was incubated in 0.1% (wt/vol) EDTA, 1 x HBSS, 10 mM HEPES (pH 7.4) at 4°C for 16-24 h. Following this incubation, the explant tissue either was washed twice with 1 x HBSS and minced, or scraped gently with fine forceps to separate sheets of urothelial cells from the underlying lamina propria. Minced tissues or urothelial sheets were then incubated for 10 min at 37°C in 1 x HBSS, 10 mM HEPES (pH 7.4), 0.0055 mM CaCl 2, and type IV collagenase from Clostridium (200 U/ml final concentration). Minced tissue was washed twice with warmed defined keratinocyte serum-free medium (DKSFM; Invitrogen, Carlsbad, CA), resuspended in 5 ml DKSFM that contained 0.03 µg/ml cholera toxin, and seeded onto a T25-cm 2 Primaria tissue culture flask (BD Biosciences, Franklin Lakes, NJ). Alternatively, urothelial sheets were washed once with warmed DKSFM, counted with a hemacytometer, and plated at an average cell density of 1 x 10 4 cells/cm 2 on Primaria tissue culture plates or flasks in DKSFM that contained 0.03 µg/ml cholera toxin. Cultures were maintained in a humidified water-jacketed incubator that contained 5% CO 2 at 37°C.! x/ J, r" [* v1 H
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In vitro propagation of human bladder smooth muscle cells. Bladder smooth muscle cells were obtained as a frozen vial from Cambrex BioScience (Walkersville, MD). Cells were thawed and plated in smooth muscle medium-2 (SmGM-2; Cambrex, Walkersville, MD) at a density of 3,500 cells/cm 2. When cells approached 80-90% confluency, they were trypsinized and passaged.

Preparation of tissue for immunolight microscopy and in situ hybridization. After surgical removal from patients, tissues were immediately fixed for 20-24 h at 4°C. Tissues embedded in paraffin were fixed in 4% paraformaldehyde (wt/vol; Electron Microscopy Sciences, Hatfield, PA), 50% (vol/vol) ethyl alcohol, and 5% (vol/vol) acetic acid. Fixed tissues were washed and dehydrated with a series of concentrations of alcohol that ranged from 50 to 100%, incubated with three 45-min changes of xylene substitute (Sigma, St. Louis, MO), and embedded in paraffin (Electron Microscopy Sciences) at 60-65°C. Six-micrometer-thick sections were cut and mounted on Superfrost Plus microscope slides (Erie Scientific, Portsmouth, NH). Sections destined for mRNA detection with synthesized RNA probes were processed with diethylpyrocarbonate-treated water.! s& v4 I' y- |" }
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Colorimetric immunolight microscopy. After removal of paraffin with three 15-min changes of xylene substitute, sections were rehydrated with 100, 80, 60, 40, 20% (vol/vol) ethyl alcohol and water. For detection of FGF-10, sections were blocked by incubation for 1 h with 5% (vol/vol) normal rabbit serum (Electron Microscopy Sciences) in TBST [10 mM Tris·HCl, pH 7.5, 250 mM NaCl, and 0.3% (vol/vol) Tween 20]. For detection of the FGF-10 receptor, sections were blocked by incubation for 1 h with TBST that contained 1% (wt/vol) BSA (fraction V, Sigma). Primary antibodies, goat anti-human FGF-10 IgG (catalog no. AF345) and mouse monoclonal anti-human FGF-10 receptor IgG (catalog no. MAB665), were purchased from R&D Systems. The antibody raised against FGF-10 was further affinity-purified as previously described ( 5 ). Sections were incubated for 4 h with primary antibodies in their respective blocking solutions at the following concentrations: 1.67 µg/ml for anti-FGF-10 and 20 µg/ml for the anti-FGF-10 receptor. Controls were defined by omission of primary antibodies on parallel slides. After a series of washes with TBST, the slides were treated for 1 h with the following secondary antibodies conjugated to alkaline phosphatase: 3 µg/ml rabbit anti-goat IgG (catalog no. 305-055-045, Jackson ImmunoResearch, West Grove, PA) and 1:100 sheep anti-mouse IgG (catalog no. 071K9185, Sigma). Sections were washed with TBST and developed with substrates 4-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Roche Diagnostics, Indianapolis, IN) at pH 9.5. Images were captured at ambient temperature at image resolutions of 1,300 x 1,030 with a Leica DC200 digital camera mounted on a Leica DMR light microscope equipped with N-Plan objectives with the following magnifications and numerical aperatures: 20 x air (0.40), 40 x air (0.65), and 100 x oil (1.25). The acquisition software was Leica DC Twain 4.1.8.0  running under the Import function of Photoshop 7.0.1, which was used to crop, size, and label images within figures.

Electron dense immunotransmission electron microscopy. Tissues destined for electron microscopy were cut into small pieces (1-2 mm) and fixed with 4% (wt/vol) formaldehyde and 0.2% (wt/vol) glutaraldehyde (Ted Pella, Redding, CA) in 100 mM PIPES buffer (pH 7.2). Fixed tissues were washed with the 100 mM PIPES buffer for four changes (20 min each) and dehydrated with 30, 50, 60, 70, and 80% ethyl alcohol (20 min each). Tissues were then sequentially infiltrated with a mixture of 80% ethyl alcohol and L. R. White resin (Ted Pella) at 1:1 and 1:2 ratios, followed by six incubations in pure resin. Each incubation lasted 10-12 h at room temperature. Tissues were then embedded in resin within gelatin capsules (Ted Pella) at 55-60°C for 24 h. Sections of 100 nm were cut and mounted on 200 mesh nickel grids for immunogold labeling. The grids were wetted with TBST [10 mM Tris·HCl (pH 7.5), 150 mM NaCl, and 0.3% (vol/vol) Tween 20], blocked with 1% BSA (fraction V) in TBST for 1.5 h, and incubated in primary antibodies for 4.5 h with gentle shaking. The concentrations of primary antibodies were 2.5 µg/ml for FGF-10 and 50 µg/ml for the FGF-10 receptor. Primary antibodies were eliminated in controls. After being washed with BSA/TBST and then TBST, grids were incubated for 1 h with secondary antibodies conjugated to gold particles at 1:20 dilution in BSA/TBST. The secondary antibodies were donkey anti-goat and donkey anti-mouse IgG tagged with 6- and 15-nm gold particles, respectively (Electron Microscopy Sciences, Ft. Washington, PA). Following washes with TBST and H 2 O, the grids were dried, fixed with 2% (wt/vol) osmium tetraoxide for 6 min, and sequentially stained for 5 min with 2% (wt/vol) uranyl acetate and 0.5% (wt/vol) lead citrate. A triple-wash with H 2 O accompanied each step. Finally, the grids were dried, visualized with a Zeiss 910 transmission electron microscope at x 25,000 for FGF-10 and x 16,000 for the FGF-10 receptor, and photographed with a Soft Imaging System Mega View III digital camera running under analysis FIVE software or with Kodak EM 4489 film.
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Fluorescent immunolight microscopy. Cultured urothelial cells were grown on 0.17-mm-thick coverslips (SecureSlips, Grace Biolabs) in DKSFM. At confluency, cells were fed DKSFM 5 µg/ml heparin with or without 1 µg/ml rFGF-10-His [recombinant human FGF-10 with a COOH-terminal His 6x tag, purified as previously described ( 4 )]. Cells were incubated with rFGF10-His for 22 h. On the next day, the overall health of the culture was evaluated by testing for uptake of the soluble fluorophore SYTOX (Molecular Probes, Eugene, OR). Cells with a damaged plasma membrane take up this probe from the medium and exhibit fluorescent nuclei. All experiments in this study were performed with cells that did not exhibit fluorescent nuclei, i.e., the cultures were healthy. Cells were fixed for 15 min with 4% paraformaldehyde in PBS (pH 7.4; Sigma), washed with PBS, permeabilized for 15 min with 0.1% (vol/vol) Triton X-100 (Sigma) in PBS, washed with PBS, and blocked for 1 h with 5% (vol/vol) normal goat serum (Electron Microscopy Sciences) in PBS. Cells were incubated for 1.5 h with the above-mentioned antibody for the FGF-10 receptor at 20 µg/ml in PBST (PBS with 0.05% Tween-20, Sigma). After three 15-min washes with PBST, cells were incubated for 1 h with 3.75 µg/ml goat anti-mouse IgG conjugated to the Cy3 fluor (Jackson ImmunoResearch) in PBST. Cell nuclei were counterstained for 20 min with 200 nM DNA stain DAPI (Molecular Probes). The coverslip was mounted on another 0.17-mm coverslip using ProLong Gold mounting media (Molecular Probes). Data sets of mounted cells were generated at ambient temperature with ImagePro v5.0 software (Media Cybernetics, Silver Spring, MD) that collected optical slices, 0.3-µm apart in the z -dimension, with a Leica DC500 digital camera mounted on a Leica DMIRE2 microscope using a Planapochromatic x 63 oil-immersion objective (numerical aperture 1.32). The resultant stack contained 137 slices at image resolutions of 1,300 x 1,030. Out-of-focus light and haze were removed from each image using deconvolution algorithms running under AutoDeblur v 9.3 software (AutoQuant, Troy, NY). Adjustments to contrast levels were equivalent across the image., Z) m' r' l5 S7 K8 ~+ J- q' I

In situ hybridization. FGF-10 RNA probes were synthesized from pSTBlue1-FGF10 ( 4 ). The 3860N construct, a pGEM3Zf(-) plasmid containing a 148-bp insert of the mouse FGFR2 exon 8, was obtained from Dr. A. Farr, University of Washington; this insert codes for a portion of the unique exon of the FGFR2 gene that defines the FGFR2IIIb splice variant and differs from the human sequence by only three nucleotides, we refer to this splice variant as the mRNA encoding the FGF-10 receptor isoform. The identities of both constructs were confirmed by dideoxy sequencing. The pSTBlue1-FGF10 construct was linearized with Hin dIII and Pst 1, whereas the 3860N construct was linearized with Hin dIII and Eco R1. After digestion was confirmed by electrophoresis through a 1% (wt/vol) agarose gel, contaminants were removed from the linearized plasmids by phenol/chloroform extraction, and the DNA was collected by precipitation with ethyl alcohol. The sense and antisense single-strand RNA probes were synthesized from SP6 and T7 promoters, respectively, with a ribonucleotide mixture that contained digoxigenin-11-UTP (DIG-RNA Labeling SP6/T7 Kit, Roche Applied Science, Indianapolis, IN).% i9 E4 v9 u( g# i/ x' J% \) d: J" O
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Sections were dewaxed, rehydrated, treated for 15 min with 100 µg/ml proteinase K at 37°C, and prehybridized for 4 h with a solution that contained 50% (vol/vol) deionized formamide, 4 x SSC [600 mM NaCl, 60 mM sodium citrate (pH 7.0)], 5% dextran sulfate, 1 x Denhart?s solution, 0.2 mg/ml salmon sperm DNA, and 100 U/ml RNase inhibitor (Roche) at 58-60°C. Specimens were incubated for 15-18 h with 2 µg/ml of each probe in prehybridization solution at 58-60°C. Unbound probes were removed by a series of extensive washes with 2 x SSC that contained 50% formamide at 60°C and then sequential washes with 1, 0.25, 0.1, and 0.05 x SSC at room temperature. The slides were blocked with blocking solution (Roche) for 30 min and incubated for 1 h with 3.75 U/ml antidigoxigenin IgG conjugated to alkaline phosphatase (Roche). Signals were visualized by color development in the same manner as the immunostaining above.+ A3 p0 ~( b7 z* ]/ t6 C0 l( a

5-Bromo-2'-deoxyuridine assays of human urothelial and smooth muscle cell DNA synthesis. Primary cultures of human ureteric urothelial cells were grown in 96-well tissue culture plates (Costar, Corning, NY) in DKSFM. This medium was used with (DKSFM GF) or without (DKSFM-GF) the manufacturer?s growth factor (GF) supplement. Cultures at 80% confluency were washed twice with DKSFM-GF to remove GFs and fed either DKSFM GF or DKSFM-GF, with or without 1 µM PD-153035 (Roche), a potent epidermal growth factor (EGF) receptor (EGFR) inhibitor. PD-153035 blocks the autophosphorylation of EGFR, preventing any downstream signaling from this receptor ( 16 ). Thirty hours after addition of PD-153035, wells were drained, and cells were fed DKSFM that contained 5 µg/ml heparin (Sigma) with or without GF supplement, with or without PD-153035, and with or without rFGF10-His. Cells were incubated in these conditions for an additional 15 h, after which BrdU (Roche) was added to all wells. BrdU is an analog of thymidine and was used to facilitate the colorimetric quantification of DNA synthesis. Cells were exposed to 10 µM BrdU for 2 h, fixed for 0.5 h, and incubated with a monoclonal mouse anti-BrdU antibody conjugated to peroxidase for 1.5 h (Cell Proliferation ELISA, BrdU, Roche). The addition of tetramethyl-benzidine to each well produced a colorimetric reaction between the peroxidase and this substrate. Relative DNA synthesis correlated with the absorbance at 370 nm in each well.! S- ?* D! _3 l2 M

BrdU assays were performed similiarly with human bladder smooth muscle cells, with the exception of the growth medium. Smooth muscle cells were propagated in SmBM-20 (Clonetics) medium that contained 5 µg/ml heparin, the manufacturer?s GF supplement, and 5% (vol/vol) fetal bovine serum. Selected cells were rendered quiescent by feeding them a low-serum medium SmBM-20 which contained 5 µg/ml heparin, but which lacked the GF supplement and contained only 0.5% fetal bovine serum. PD-153035 was not used in any smooth muscle cell BrdU assays. Nevertheless, selected cells were maintained in the low-serum medium for 30 h before addition of rFGF-10-His to mimic the conditions experienced by quiescent urothelial cells. All other times, concentrations, and procedures were identical to those described for urothelial cells.
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Analysis of mRNA by RT and PCR analysis. RT-PCR technique was used to further determine whether FGF-10 and its receptor were expressed by cultured human ureteric urothelial cells. Cells were lysed and denatured with guanidium isothiocyanate and total RNA was recovered by affinity-chromatography (Micro-to-Midi Total RNA Purification System, Invitrogen). Isolated RNA was diluted to 50 ng/µl for use as a template in RT-PCR. Specific primers were designed across intron(s) according to the published sequences for human FGF-10 mRNA (GenBank accession NM_004465, forward: 5'-GCATCCTGGAGATAACATCAG, reverse: 5'-CTATGAGTGTACCACCATTGG) and for transcript variant 2 (IIIb type, encoding the FGF-10 receptor) of human FGFR2 (GenBank accession NM_022969, forward: 5'-CCAATGCAGAAGTGCTGGCT, reverse: 5'-CTTGCCCAGTGTCAGCTTAT). RT-PCR conditions were 30 min at 50°C for cDNA synthesis and 2 min at 94°C for denaturation, followed by 36 cycles of 94°C (15 s), 54°C (30 s), and 68°C (1 min; SuperScript III One-Step RT-PCR System). RT-PCR products were separated by electrophoresis on 1% (wt/vol) agarose gel and visualized by ethidium bromide staining with UV light. The identity of the product was verified by dideoxy sequence analysis.

RESULTS
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FGF-10 protein is detected within the urothelium and lamina propria. Immunostaining with polyclonal antibodies specific for human FGF-10 revealed that the GF was detected in fibroblasts of the lamina propria (submucosa) and in the urothelial layers of transitional epithelium (mucosa). 1 Strong signals were observed within fibroblasts of normal bladder, bladder exstrophy, and ureter ( Fig. 1, B, D, and F ). Transitional epithelium exhibited very strong signals with a similar pattern of distribution in normal bladder, bladder exstrophy, and ureter ( Fig. 1, B, D, and F ). The intensity of the FGF-10 signals was equivalent over the layers of transitional epithelium (urothelium) of normal bladder ( Fig. 1 B ) and ureter ( Fig. 1 F ). A similar distribution of even immunoreactivity was observed in bladder exstrophy ( Fig. 1 D; with the exception of the keratin layer). Classical bladder exstrophy is often characterized by an abnormal layer of keratinized squamous epithelium due to this birth defect?s herniated eversion of the epithelial layer, such parakeratosis is exhibited in Fig. 1 D (tissue labeled "K").
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Fig. 1. Fibroblast growth factor 10 (FGF-10) protein is detected on both sides of the urothelial basement membrane of bladder and ureter by immunostaining. The dark brown color shows FGF-10 signals. A, C, and E : controls in which primary antibody, goat anti-human FGF-10 IgG, was omitted. B, D, and F : are antibody treatments. A and B : normal bladder. C and D : bladder exstrophy. E and F : ureter. U, urothelium; LP, lamina propria; L, lumen; K, keratinized squamous epithelium. Bar = 20 µm.3 g, X; |3 k+ z2 P  O

Nuclei of urothelial cells in bladders and ureters were observed to exhibit immunoreactive signals for FGF-10 ( Fig. 1, B, D, and F ). FGF-10 nuclear signals were observed in all three urothelial cell layers that comprise transitional epithelia (basal, intermediate, and superficial). In bladder exstrophy ( Fig. 1 D ), the keratinized squamous epithelial cell layer also exhibited strong immunoreactive signals in their nuclei.- \/ ^7 A/ O% g# g7 c) e+ ^

FGF-10 mRNA is expressed in the lamina propria. In situ hybridization detected FGF-10 mRNA in the lamina propria (submucosa) but not in transitional epithelium (mucosa). Fibroblasts of the ureteric lamina propria just below the basement membrane exhibited strong FGF-10 mRNA signals ( Fig. 2 B ), a result consistent with our previous report on normal human bladder ( 4 ). Strong signals were also observed in the fibroblasts adjacent to blood vessels of the lamina propria ( Fig. 2 D ), also in agreement with our previous report on normal human bladder ( 4 ).: i2 I) X' h4 D7 }  Z8 [6 B+ L# E( u
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Fig. 2. Detection of FGF-10 mRNA in ureter tissue by in situ hybridization technique. The dark brown color represents FGF-10 mRNA signals in B, C, and D. A : sense control. B, C, and D : antisense probe. E and F : Masson?s trichrome-stained sections showing fibroblast cells surrounding smooth muscle bundles and blood vessels, respectively. SM, smooth muscle; BV, blood vessel. Bars = 20 µm.( ]2 C% l! c1 X5 H# A3 s

The combined data of Figs. 1 and 2 demonstrate that FGF-10 mRNA is synthesized and translated by fibroblasts of the lamina propria. Further support for this mechanism comes from our analysis of the FGF-10 gene and mRNA transcript. We identified a putative 37 residue NH 2 -terminal secretory signal peptide that is present within the nascent mRNA transcript. Because the FGF-10 protein has yet to be isolated and subjected to NH 2 -terminal Edman degradation, we speculate that this 4.131-kDa sequence is then removed by signal peptidases in the endoplasmic reticulum during posttranslational modifications, ultimately leading to secretion of the mature polypeptide into the extracellular space. The presence of FGF-10 protein in transitional epithelium is therefore a consequence of secretion by mesenchymal fibroblasts and transport across the urothelial basement membrane.) l+ U) P2 |$ E% ^- T8 `2 l
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FGF-10 mRNA is expressed in the detrusor muscle layer. FGF-10 mRNA was also observed in fibroblasts of the ureteric smooth muscle layer that forms the tunica muscularis ( Fig. 2 C ). The spatial pattern of FGF-10 mRNA signals in this layer was similar to the pattern observed in the submucosal layer. Both patterns of FGF-10 mRNA signals were consistent with the accepted spindle-shape morphotype of fibroblasts ( Fig. 2, E and F ). Strong patches of hybridization signals were observed throughout the lamina propria ( Fig. 2 B ) and the smooth muscle detrusor layer ( Fig. 2, C and D ), indicating that fibroblasts of both tissues were synthesizing FGF-10 mRNA. These signals were more intense in proximity to blood vessels ( Fig. 2 D ). Fibroblasts present between muscle bundles and blood vessels were identified by their localization to types I and III collagen fibrils, the latter being identified by a blue color after incubation with Mason?s Trichrome stain ( Fig. 2, E and F ).

FGF-10 protein is detected in fibroblasts and smooth muscle cells of the tunica muscularis. Immunoreactive signals for FGF-10 protein were observed within smooth muscle cell bundles and in fibroblasts that separated these bundles ( Fig. 3 B ). The pattern of FGF-10 immunoreactivity was such that observed signals were more intense inside bundles and that immunoreactivity was more intense within nuclei than in cytoplasms ( Fig. 3 B ). Because these bundles were negative for FGF-10 mRNA, the detection of FGF-10 protein inside the bundles was due to its transport from the surrounding fibroblasts. These observations document the mechanism of paracrine targeting within this tissue. The transport of FGF-10 into smooth muscle cells requires the polypeptide to traffic across collagen and elastin fibers that surround each bundle. Once inside smooth muscle cells, FGF-10 then exhibits translocation into nuclei, as observed by intense nuclear immunoreactivity ( Fig. 3 B ). The combined observations prove that FGF-10 exhibits a dual paracrine mode of targeting in the lower urinary tract, from mesenchymal fibroblasts into urothelial cells and from muscle layer fibroblasts into smooth muscle cells.

Fig. 3. FGF-10 and its receptor are present in smooth muscle cells of the ureter submucosa. A and C : control sections in which primary antibodies were omitted. The dark brown color in B and D depicts immunoreactive FGF-10 and the FGF-10 receptor signals, respectively. C and D were counterstained with hematoxylin. BV, arrowhead; I, interstitial cells. Bars = 40 µm.
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FGF-10 receptor is detected within transitional epithelium of the mucosal layer and smooth muscle cells of the detrusor layer. Immunostaining with monoclonal antibodies specific for the FGF-10 receptor detected strong signals in the urothelial layers of transitional epithelium from normal bladder, bladder exstrophy, and ureter ( Fig. 4, B, D, and F ). The intensity of the receptor signals was uniform across the basal, intermediate, and superficial urothelial layers ( Fig. 4, B, D, and F ), although a higher signal density was found near the basement membrane (not shown) and an even more intense signal was observed at the superficial layer ( Fig. 4, B and D, inset ).
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Fig. 4. FGF-10 receptor is detected in urothelial layers of bladder and ureter by immunostaining. The dark brown color shows the receptor signals. A, C, and E : control sections in which the primary monoclonal antibody was omitted. B, D, and F : sections that were treated with primary antibodies. A and B : normal bladder. C and D : bladder exstrophy. E and F : ureter. Bars = 20 µm. D : composite of 2 images taken at the same image resolution and magnification.8 v" T" `: Y2 q
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The nuclei of urothelial cells were observed to exhibit immunoreactivity to antibodies specific to the FGF-10 receptor ( Fig. 4, B, D, and F ), raising the possibility that the receptor is capable of undergoing translocation from the plasma membrane. All urothelial layers of transitional epithelium exhibited this nuclear immunoreactivity.

Strong immunoreactive signals for the FGF-10 receptor were also observed within smooth muscle cells of the ureteric detrusor layer ( Fig. 3 D ). Such signals were absent for adjacent endothelial cells ( Fig. 3 D, structure labeled "BV") and fibroblasts. While the FGF-10 receptor was observed within the cytoplasm and cell surface of smooth muscle cells, immunoreactivity was not observed within smooth muscle cell nuclei. This apparent lack of a nuclear FGF-10 receptor represents a characteristic that distinguishes smooth muscle cells from urothelial cells.7 K8 O" t3 c6 t4 B1 d9 i

mRNA encoding the FGF-10 receptor is expressed in transitional epithelium of the mucosal layer and smooth muscle cells of the detrusor layer. The mRNA splice variant encoding the FGF-10 receptor was detected in the basal, intermediate, and superficial layers of bladder and ureteric tissue by in situ hybridization ( Fig. 5, B and D ). This observation is consistent with the distribution of the protein by immunostaining ( Fig. 4 ), demonstrating that urothelial cells transcribe, splice, and translate RNA to form the FGF-10 receptor.

Fig. 5. mRNA encoding the FGF-10 receptor is detected in bladder exstrophy ( A and B ) and ureter ( C - F ) tissues by in situ hybridization. The dark brown color shows the receptor mRNA signals. A, C, and E : control sections that were hybridized with sense strand probes. B, D, and F : sections that were hybridized with antisense probes. All panels were counterstained with hematoxylin. Bars = 20 µm.
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Smooth muscle cells of the ureter ( Fig. 5 F ) were also observed to express the mRNA encoding the FGF-10 receptor. The area within the detrusor layer that exhibited positive hybridization signals was strictly limited to smooth muscle cells and was demarked by a clear and evident boundary ( Fig. 5 F ). The combined data prove that the FGF-10 receptor is synthesized in cells that are targeted by FGF-10, further validating our hypothesis of a dual targeting pathway for paracrine mitogens in the lower urinary tract.

Transmission electron microscopy demonstrates that FGF-10 and its receptor associate with each other on the surface of urothelial cells. As shown by immunogold electron microscopy, FGF-10 was observed to localize to the extracellular space and the urothelial cell surface ( Fig. 6 B; 6-nm gold particles, arrow). The detection of multiple signals at the cell surface is consistent with our hypothesis that FGF-10 interacts with its cognate receptor ( Fig. 6 C; 15-nm gold particles, arrowhead) and heparan sulfate proteoglycans in a paracrine mode of action. Signals for the receptor were observed both at the urothelial cell surface and in the cytoplasm ( Fig. 6, C and D ). Receptor signals were observed in basal (not shown), intermediate ( Fig. 6 C ), and superficial ( Fig. 6 D ) urothelial cells. Proof of colocalization of FGF-10 and its receptor at the urothelial cell surface was obtained with dual immunolabeling ( Fig. 6, E and F ) where multiple instances of colocalized 6- and 15-nm gold particles were observed. This consistent observation supports the concept that receptor dimerization, whether through the asymmetric ( 43 ) or symmetric models ( 22, 46, 54, 60 ), occurs as a consequence of ligand binding and provides further mechanistic details as to how paracrine mitogenic signals are transduced in the lower urinary tract.0 w6 q% J4 M7 ?; R
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Fig. 6. Localization of FGF-10 and its receptor to the urothelial cell surface as detected by immunoelectron microscopy. Antigen:antibody complexes were detected with secondary antibodies conjugated to gold particles and appear as black dots. A : no primary antibody control. B : FGF-10 was visualized at the cell surface by 6-nm gold particles (arrow). C and D : FGF-10 receptor was detected on the surface of intermediate and superficial cells, respectively, by 15-nm gold particles (arrowhead). E and F : colocalization of FGF-10 (arrow) and its receptor (arrowhead) on the cell surface. Ex, extracellular space; Cyt, cytoplasm. Original microscopic magnification: x 25,000 for A, B, E, and F; x 16,000 for C and D. The images in E and F were further magnified to distinguish between 6- and 15-nm gold particles.' O# Z5 f' p9 C4 \+ p

Expression of mRNA encoding the FGF-10 receptor in urothelial cells was further confirmed in vitro. A human cell culture model was developed to better understand the mechanistic pathways that urothelial cells use to respond to a FGF-10 mitogenic signal. The extent that cultured urothelial cells synthesize the mRNA for FGF-10 and its receptor was determined by RT-PCR. Specific primers designed across introns were predicted to generate RT-PCR DNA products of 500 and 302 bp for the receptor and GF, respectively. RT-PCR and agarose gel electrophoresis indicated that urothelial cells in vitro synthesized the FGFR2IIIb splice variant that encodes for the FGF-10 receptor isoform ( Fig. 7 A, lane 1 ) but not FGF-10 mRNA ( Fig. 7 A, lane 2 ). The observed RT-PCR DNA band in lane 1 of Fig. 7 A matched the expected size. This result is consistent with our in situ hybridization data from cultured urothelial cells (data not shown). Further evidence that urothelial cells synthesized the FGF-10 receptor comes from sequencing of the 500-bp RT-PCR product that confirmed the FGFR2 sequence and proves that this sequence contains exon 8 joined to exon 10 ( Fig. 7 B ). As expected, FGF-10 mRNA was not detectable by RT-PCR, demonstrating that urothelial cells in vitro do not synthesize FGF-10. The collective in vitro data are in complete agreement with our in vivo observations for these tissues.) m! V+ _+ {3 r' M) b4 O

Fig. 7. IIIb mRNA splice variant of the FGFR2 gene is expressed in urothelial cells in vitro. A : annealing of specific oligonucleotide primers to reverse transcribed cDNA and subsequent PCR amplification for FGFR2IIIb mRNA ( lane 1, *) and FGF-10 mRNA ( lane 2 ). B : sequence confirmation of ligation of exons 8 and 10 to form the FGFR2IIIb mRNA splice variant.4 o7 U0 L1 S* Y6 B$ C; C! s" ^5 g% ]
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FGF-10 is sufficient to induce urothelial cell DNA synthesis in vitro. The EGF and FGF signaling pathways represent the principal signaling mechanisms through which urothelial cell mitogens operate ( 4, 66 ). The mechanism of FGF-10 activity was studied through an assay designed to measure the polypeptide?s ability to stimulate DNA synthesis in vitro. Cells were maintained in DKSFM that contained growth supplements ( Fig. 8 A, bar with horizontal lines). The addition of rFGF10-His, up to 1 µg/ml, to actively growing cells failed to stimulate DNA synthesis (not shown), indicating that once the FGF signaling pathway was fully engaged, no further enhancement in the rate of DNA synthesis was observed. The specific FGF in the growth supplement is proprietary, although Western immunoblot analysis indicated that this supplement (and DKSFM) did not contain FGF-10 ( 4 ).6 M. y2 A, K1 b8 j% w# o

Fig. 8. A : rFGF10-His induces urothelial cell DNA synthesis via an EGF-independent pathway. Ureteric cells were fed DKSFM containing 5 µg/ml heparin with or without growth factor supplement, PD-153035, or rFGF10-His, as described in MATERIALS AND METHODS. *Statistical significance ( P
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The EGF pathway was blocked with the inhibitor PD-153035 ( 16 ), which suppresses autophosphorylation of the EGF receptor, isolating the FGF pathway for further study. The step to isolate the FGF pathway was necessary because urothelial cells synthesize their own EGF as part of an autocrine loop ( 66 ). Addition of PD-153035 to actively growing cells resulted in a 15% inhibition of DNA synthesis ( Fig. 8 A, gray bar), indicating that the FGF pathway was still active. Removal of growth supplements caused cells to synthesize 35% less DNA ( Fig. 8 A, gridded bar) compared with cells with growth supplements ( Fig. 8 A, bar with horizontal lines), cells remained healthy and grew slowly in culture for weeks (not shown). Cells were rendered quiescent by withdrawal of growth supplements and addition of PD-153035 ( Fig. 8 A, filled bar). The inclusion of 1 µM PD-153035 further inhibited DNA synthesis by a total of 88% ( Fig. 8 A, filled bar), compared with actively growing cells ( Fig. 8 A, bar with horizontal lines). The minor synthesis that remained may be due to residual GFs still bound to the cell or incomplete inhibition of the EGF pathway by PD-153035.6 g7 P1 H+ F% c# J
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Addition of rFGF10-His to quiescent cells demonstrated that this GF is a potent mitogen for urothelial cells in vitro. Concentrations of rFGF10-His as low as 1 ng/ml resulted in notable increases in DNA synthesis ( Fig. 8 A, open bars). This induction was dose dependent up through 0.1 µg/ml rFGF10-His. Concentrations that exceeded 0.1 µg/ml resulted in no change in BrdU incorporation, indicating that this pathway was maximally saturated. The stimulation of DNA synthesis by rFGF10-His was statistically significant; relative to quiescent cells ( Fig. 8 A, filled bar), P was calculated as 0.022 (0.001 µg/ml) and
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FGF-10 is insufficient to induce bladder smooth muscle cell DNA synthesis in vitro. Human bladder smooth muscle cells were assayed in parallel with urothelial cells to determine whether FGF-10 is a paracrine GF for smooth muscle cells. Cells were maintained in growth medium that contained 5% fetal bovine serum and growth supplements that included EGF. Under these conditions, cells actively synthesized DNA ( Fig. 8 B, bar with horizontal lines). The addition of rFGF10-His to actively growing cells did not elicit any further increase in DNA synthesis ( Fig. 8 B, gray bars). Propagation of cells in the absence of serum caused cell death within days (data not shown). Therefore, cells were synchronized to a quiescent state ( Fig. 8 B, filled bar) by reduction of the serum concentration to 0.5% and omission of growth supplements; this formulation permitted the maintenance of healthy, quiescent cells for up to a week (data not shown). The addition of exogenous rFGF10-His at concentrations of 0.01, 0.1, and 1.0 µg/ml did not elicit any significant increase in DNA synthesis ( p = 0.057, 0.029, and 0.385, respectively; Fig. 8 B, open bars); this observation is in contrast to urothelial cells which responded positively to rFGF10-His ( Fig. 8 A ).

FGF-10 receptor appears in the cytoplasm and nucleus of urothelial cells in vitro. The FGF-10 receptor was readily detected in urothelial cells cultured in vitro. The use of deconvolution microscopy and optical sectioning allowed the unambiguous interpretation of intracellular localization. In the montages presented in Fig. 9, fluorescently labeled FGF-10 receptor is present both in the cytoplasm and the nucleus. The majority of the FGF-10 receptor was observed to be perinuclear, an observation supported by its synthesis in the rough endoplasmic reticulum. Nuclei that exhibited receptor immunoreactivity ( Fig. 9 ) were detected by merging of the Cy3 fluorescence channel with the DAPI fluorescence channel, localization inside nuclei was visualized as a pink color (arrows, Fig. 9 ). This result is consistent with our in vivo data ( Fig. 4 D ). The detection of the FGF-10 receptor in the urothelial cell nucleus is consistent with what has been found for other members of the FGFR family ( 28, 59, 74 ) and describes a mechanism for how signal transduction pathways operate in the lower urinary tract.( L3 M5 O4 i- P, [" o

Fig. 9. Localization of the FGF-10 receptor to nuclei of urothelial cells in vitro. Cells were incubated with 1 µg/ml of rFGF10-His, as described in MATERIALS AND METHODS. Immunofluorescence labeling with monoclonal antibodies specific for the FGF-10 receptor with detection with secondary antibody conjugated to the Cy3 fluor is shown. Presented is a montage, with each panel representing a 0.3-µm-thick deconvolved optical slice 1.2 µm apart from the next panel. Arrows indicate intense localization of receptor (red) in DAPI-stained nuclei (blue). Bar = 10 µm.
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A novel pathway for the transport of FGF-10 and its receptor to the nucleus. FGF-10 was detected on the cell surface and plasma membrane, typically as immunoreactive clusters ( Fig. 10 A ). Once inside the cell, the GF was observed within the cytoplasm, also as immunoreactive clusters ( Fig. 10 B ) before detection in the nucleus ( Fig. 10 C ). Within nuclei, FGF-10 was found associated with high electron density areas or in the vicinity of nuclear pores ( Fig. 10 C ).

Fig. 10. Transport pathways of FGF-10 and its receptor within urothelial cells as detected by immunoelectron microscopy. Antigen:antibody complexes were detected with secondary antibodies conjugated to gold particles and appear as black dots. A, B, and C : transport of FGF-10 (arrows) at cell surface ( A ), through cytoplasm ( B ) and into the nucleus ( C ). D, E, and F : transport of the FGF-10 receptor (arrowheads) through cytoplasm ( D and E ) and into the nucleus ( F ). Ex, extracellular space; Cyt, cytoplasm; Nuc, nucleus; P, nuclear pore. Original amplification: x 25,000 for A, B, and C; x 16,000 for D, E, and F. The images in B, C, E, and F were further magnified for clarity.
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The FGF-10 receptor was also detected on the urothelial cell surface ( Fig. 6, C - F ), cytoplasm ( Fig. 6, C, D, and F; Fig. 10, D and E ), and nucleus ( Fig. 10 F ). This paranuclear detection of the FGF-10 receptor was characterized by clustering of immunoreactive signals.

DISCUSSION
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FGF-10 was first identified in a screen for molecules with homology to the FGF family in the lung ( 72 ). Since that time, the polypeptide has been implicated in broad mitogenic and cell survival activities, as well as a variety of biological processes that include embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion ( 48 ). The data provided in this report provide the mechanism by which FGF-10 functions in the context of mesenchymal-epithelial interactions in the lower urinary tract. We demonstrated that recombinant FGF-10 is a mitogen that stimulates DNA synthesis in urothelial cells in vitro, a critical process involved in control of growth, differentiation, and repair of urothelium. FGF-10 protein was detected in the lamina propria and transitional epithelium of bladder and ureteric tissues; in contrast, the FGF-10 receptor was localized to transitional epithelium. The cellular sources that synthesized FGF-10 and its receptor were identified by in situ hybridization as fibroblast cells of the lamina propria and urothelial cells of transitional epithelium, respectively. Because we did not perform immunostaining with fibroblast marker proteins, such as vimentin ( 13 ) or fibroblast-specific protein-1 ( 61 ), our data cannot rule out the remote possibility that another mesenchymal mast cell could be an additional source of FGF-10 protein ( 3 ).

The collective data demonstrate that the FGF-10 protein existing and functioning in urothelium must be transported from lamina propria. Transport of a 4- to 5-nm FGF-10 particle ( 73 ) through the pores of the primate urothelial basement membrane that have been measured as 82 ± 49 nm ( 1 ) is considered to be highly likely. These in vivo expression patterns demonstrate that FGF-10 is a paracrine mediator in urinary tract mesenchymal-epithelial interactions and that the GF plays a critical role in regulating urothelial proliferation, differentiation, and function. Support for the requirement of FGF-10 for proper development of the lower urinary tract comes from our unpublished observations that stratification of the urothelial cell layer is incomplete in FGF10-null mice.* r3 s: I( X' J& w
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The unexpected finding of this study was the paracrine targeting of FGF-10 to smooth muscle cells. The synthesis of FGF-10 was limited to interstitial fibroblasts that surround smooth muscles, whereas synthesis of the FGF-10 receptor was restricted to smooth muscle cells. The observation that FGF-10 protein was detected within smooth muscle cells documents that these cells were paracrine targets of the polypeptide GF. Our preparations of recombinant FGF-10, however, were unable to stimulate DNA synthesis of quiescent human bladder smooth muscle cells in vitro. When deprived of growth supplements, bladder smooth muscle cells displayed no significant response to FGF-10 at any concentration, despite our repeated attempts with a variety of serum concentrations. Bladder smooth muscle cells also lacked the nuclear form of the FGF-10 receptor and proved unresponsive to the EGFR inhibitor PD-153035 under all conditions, demonstrating additional differences between how smooth muscle and urothelial cells respond to exogenous GFs. It is likely that bladder smooth muscle cells exhibit a differential requirement for exogenous heparin ( 23 ), endogenous heparan- and chondroitin-sulfate proteoglycans ( 34, 53 ), and receptor isoforms with variable affinities for the FGF-10 ligand ( 34 ). This lack of response differentiates bladder smooth muscle cells from urothelial cells and would allow for selective targeting of cell types if FGF-10 were used as a clinical tool for treatment of urothelial disorders.
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The mechanism of how the FGF-10 mitogenic signal is transduced by epithelial cells has traditionally been viewed as initiation of the MAPK pathway and subsequent activation of nuclear transcription factors such as Elk1, c- myc, and c- fos ( 27, 62 ). In this context, both FGF-10 and its receptor function at cell surface. Our study not only agrees with this dogma but also documents the provocative concept that FGF-10 alone, or in concert with its receptor, undergoes translocation into urothelial cell nuclei. Nuclear translocations of other FGFs and receptors, such as FGF-1, FGF-2, FGFR1, and FGFR3, have been previously described ( 26, 35, 50 ). Two questions arise from these observations: why and how do FGFs and their receptors enter the nucleus? Regarding the first question, current opinion is that nuclear translocation may either play a direct role or serve as an alternative pathway in regulating gene transcription. It was demonstrated that a minimum of 12 h of FGF-1 treatment is required to achieve near-maximal DNA synthesis, which correlates with the continuous internalization of radiolabeled FGF-1 into the cytosol and nucleus ( 75 ). The function of nuclear translocation of both FGFs and the receptors is still not clear. To the second question, according to the emerging view, FGFs with nuclear localization sequence (NLS) may help nuclear transport of its receptor through "piggyback" because FGFs themselves do not need a NLS to enter the nucleus (

Taken together, our experiments demonstrate that FGF-10 functions as a mesenchymal paracrine regulator of normal epithelial growth in the lower urinary tract. It may also play a critical role in the urothelial response to injury that is secondary to infection, trauma, urinary tract obstruction, and/or ischemia. It is our observation that the expression of the FGF-10 receptor occurs not only in the basal cells of urothelium, but also in all urothelial layers. The expression of the receptor in the superficial layer is clinically significant and has led us to propose that installation of recombinant FGF-10 into the bladder lumen will trigger urothelial cell proliferation and turnover of transitional epithelium. Such FGF-10 therapy could assist the clinician in the management of a variety of urinary tract disorders including interstitial cystitis. Potential applications include the use of GFs to promote healing after genitourinary trauma or surgery. It may also be possible to use FGF-10 to abrogate the response to obstruction in the urinary tract seen with neurogenic bladder conditions, benign prostate hyperplasia, and other obstructive uropathies.
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 1-R01-DK-62251 to J. A. Bassuk.8 q7 E7 ~; n- i" \# ]
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ACKNOWLEDGMENTS- c6 `+ x9 z- k' B

We thank Dr. Jenny Southgate for suggesting the use of PD-15305.
      【参考文献】$ q/ J+ C( a% \/ U
Abrams GA, Murphy CJ, Wang ZY, Nealey PF, and Bjorling DE. Ultrastructural basement membrane topography of the bladder epithelium. Urol Res 31: 341-346, 2003.! g% I* @+ H3 r4 f' ]0 s# t% `$ M
  N# o6 E: c( k
. C* F1 d# m* Q. P1 Y

Arman E, Haffner-Krausz R, Gorivodsky M, and Lonai P. Fgfr2 is required for limb outgrowth and lung-branching morphogenesis. Proc Natl Acad Sci USA 96: 11895-11899, 1999.( g- o1 B: e8 a0 Q6 p. p

Artuc M, Steckelings UM, and Henz BM. Mast cell-fibroblast interactions: human mast cells as source and inducers of fibroblast and epithelial growth factors. J Invest Dermatol 118: 391-395, 2002.3 {3 R4 c  j) `4 z" A# m3 Y# |! ^! R

% i2 T% }% b! g5 J0 ^# _  u
$ h6 u0 [4 @% u" l( e( b
Bagai S, Rubio E, Cheng JF, Sweet R, Thomas R, Fuchs E, Grady R, Mitchell M, and Bassuk JA. Fibroblast growth factor-10 is a mitogen for urothelial cells. J Biol Chem 277: 23828-23837, 2002.

. G4 [  ]- N1 R% U' K8 X# A6 `
Bassuk JA, Birkebak T, Rothmier JD, Clark JM, Bradshaw A, Muchowski PJ, Howe CC, Clark JI, and Sage EH. Disruption of the Sparc locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation. Exp Eye Res 68: 321-331, 1999.

6 p  H1 d: V  J2 W; X

Burgess WH, Dionne CA, Kaplow J, Mudd R, Friesel R, Zilberstein A, Schlessinger J, and Jaye M. Characterization and cDNA cloning of phospholipase C-, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase. Mol Cell Biol 10: 4770-4777, 1990.

# C- r7 f' Z' m6 I& n
  F  k6 n  [. f, }4 l( l
Celli G, LaRochelle WJ, Mackem S, Sharp R, and Merlino G. Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multiorgan induction and patterning. EMBO J 17: 1642-1655, 1998.

" o+ c( {- ?: O" s2 m+ j; i6 l1 Y0 h

Chellaiah AT, McEwen DG, Werner S, Xu J, and Ornitz DM. Fibroblast growth factor receptor (FGFR) 3. Alternative splicing in immunoglobulin-like domain III creates a receptor highly specific for acidic FGF/FGF-1. J Biol Chem 269: 11620-11627, 1994.
1 g! ?: o- j0 |0 R) m
0 u1 f) j5 _+ S* ^, T) M7 ^
) h0 g' f, v/ i/ |
Cheon HG, LaRochelle WJ, Bottaro DP, Burgess WH, and Aaronson SA. High-affinity binding sites for related fibroblast growth factor ligands reside within different receptor immunoglobulin-like domains. Proc Natl Acad Sci USA 91: 989-993, 1994.
; j: M+ x& D7 j/ [; e9 z; U& I
' s! z/ z( _) T8 H, `8 u
/ ~* `4 c. W. F; S' x
Cornish EE, Natoli RC, Hendrickson A, and Provis JM. Differential distribution of fibroblast growth factor receptors (FGFRs) on foveal cones: FGFR-4 is an early marker of cone photoreceptors. Mol Vis 10: 1-14, 2004.

De Moerlooze L, Spencer-Dene B, Revest J, Hajihosseini M, Rosewell I, and Dickson C. An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Dev Suppl 127: 483-492, 2000.' @9 m0 Q% o" j  D6 Y& s

0 t+ L' z, ^# H$ ^' E. b
Deo DD, Axelrad TW, Robert EG, Marcheselli V, Bazan NG, and Hunt JD. Phosphorylation of STAT-3 in response to basic fibroblast growth factor occurs through a mechanism involving platelet-activating factor, JAK-2, and Src in human umbilical vein endothelial cells. Evidence for a dual kinase mechanism. J Biol Chem 277: 21237-21245, 2002.# u- U" W! q7 P+ B
2 _, l6 F. }: z3 t9 k
6 M' K7 Y6 t1 g5 Y* R

Drake MJ, Hedlund P, Andersson KE, Brading AF, Hussain I, Fowler C, and Landon DN. Morphology, phenotype and ultrastructure of fibroblastic cells from normal and neuropathic human detrusor: absence of myofibroblast characteristics. J Urol 169: 1573-1576, 2003.% _# a9 ^- J8 r3 z" n& r9 k

: A0 q- X8 F2 H2 H
9 a/ O4 i* d% O% N& Z
Duan DS, Werner S, and Williams LT. A naturally occurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF. J Biol Chem 267: 16076-16080, 1992.

/ _: l6 B9 S! J4 i& g8 m$ v3 L) h
Finch PW, Cunha GR, Rubin JS, Wong J, and Ron D. Pattern of keratinocyte growth factor and keratinocyte growth factor receptor expression during mouse fetal development suggests a role in mediating morphogenetic mesenchymal-epithelial interactions. Dev Dyn 203: 223-240, 1995.

1 f0 T  E2 ]9 U0 z# H

Fry DW, Kraker AJ, McMichael A, Ambroso LA, Nelson JM, Leopold WR, Connors RW, and Bridges AJ. A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science 265: 1093-1095, 1994.
! }7 R* r, m$ L
% g, e2 p# k$ M! ]- s

Goldstrohm AC, Greenleaf AL, and Garcia-Blanco MA. Cotranscriptional splicing of premessenger RNAs: considerations for the mechanism of alternative splicing. Gene 277: 31-47, 2001.- ]# v+ K$ t. h; E, ^! h

! S$ t+ Y- [( y- G# s( c0 S
Guo L, Degenstein L, and Fuchs E. Keratinocyte growth factor is required for hair development but not for wound healing. Genes Dev 10: 165-175, 1996./ V- p/ u/ E1 Y

( P5 D; v* N9 [5 m$ v
Hadari YR, Gotoh N, Kouhara H, Lax I, and Schlessinger J. Critical role for the docking-protein FRS2 in FGF receptor-mediated signal transduction pathways. Proc Natl Acad Sci USA 98: 8578-8583, 2001.
4 `5 P8 j9 [: _+ ^

Hart KC, Robertson SC, and Donoghue DJ. Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation. Mol Biol Cell 12: 931-942, 2001.( D2 l0 ^* z- n! M8 f& v  S0 U
/ `0 K  k: X4 q3 p( X
# `8 h$ g% N# x7 I- J( q; L
5 E4 H$ q5 D% y6 E" P6 Y6 x- O
Hudson AE, Feng WC, Delostrinos CF, Carmean N, and Bassuk JA. Spreading of embryologically distinct urothelial cells is inhibited by SPARC. J Cell Physiol 202: 453-463, 2005.

- W$ d. N- j6 K$ c

Ibrahimi OA, Yeh BK, Eliseenkova AV, Zhang F, Olsen SK, Igarashi M, Aaronson SA, Linhardt RJ, and Mohammadi M. Analysis of mutations in fibroblast growth factor (FGF) and a pathogenic mutation in FGF receptor (FGFR) provides direct evidence for the symmetric two-end model for FGFR dimerization. Mol Cell Biol 25: 671-684, 2005.

% ]5 g+ B9 T! `/ b! z' `" z

Igarashi M, Finch PW, and Aaronson SA. Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7). J Biol Chem 273: 13230-13235, 1998.2 w' b. p8 ~8 \- I
! y/ ^* Z) ]4 B2 G

, t/ W. f; r0 ~# m( Q4 }( s. r) |
Jans DA. Nuclear signaling pathways for polypeptide ligands and their membrane receptors? FASEB J 8: 841-847, 1994.* @3 c# l3 X6 R9 x9 u: U! H! o

Johnson DE, Lu J, Chen H, Werner S, and Williams LT. The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain. Mol Cell Biol 11: 4627-4634, 1991.* R" J6 a" J; F# k& y
2 z; V  S% m+ N# l6 B# f
4 R/ q# k2 h2 G% y: ^/ c4 h1 z
2 B8 t+ _4 M+ P3 g
Johnston CL, Cox HC, Gomm JJ, and Coombes RC. Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus. J Biol Chem 270: 30643-30650, 1995.3 i$ Q/ F# H+ [6 I2 T. ]

Karin M and Hunter T. Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr Biol 5: 747-757, 1995.) u7 B2 f& z4 o( \4 x7 F" H$ I

Keresztes M and Boonstra J. Import(ance) of growth factors in(to) the nucleus. J Cell Biol 145: 421-424, 1999.
$ W4 |3 z8 ~" J5 O) R% W

Kim I, Moon S, Yu K, Kim U, and Koh GY. A novel fibroblast growth factor receptor-5 preferentially expressed in the pancreas(1). Biochim Biophys Acta 1518: 152-156, 2001.

6 f( T- \5 Y; u( l* j- S# g
* W# D3 S, o& t5 F3 ]' S7 }
Klint P and Claesson-Welsh L. Signal transduction by fibroblast growth factor receptors. Front Biosci 4: D165-D177, 1999.

$ l7 y+ `4 m$ {6 T, p  e6 T7 |0 o  c
Kwabi-Addo B, Ozen M, and Ittmann M. The role of fibroblast growth factors and their receptors in prostate cancer. Endocr Relat Cancer 11: 709-724, 2004.8 |! W& D8 M* {5 {% j

Lin X. Functions of heparan sulfate proteoglycans in cell signaling during development. Dev Suppl 131: 6009-6021, 2004.

Lin X, Buff EM, Perrimon N, and Michelson AM. Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development. Dev Suppl 126: 3715-3723, 1999.6 J0 d3 Z8 E$ S' l2 T

: M) H2 p' \3 U

Lu W, Luo Y, Kan M, and McKeehan WL. Fibroblast growth factor-10. A second candidate stromal to epithelial cell andromedin in prostate. J Biol Chem 274: 12827-12834, 1999.. i7 X! U# j2 p& o: p7 V+ \& B

: m' F" s+ R' j

Maher PA. Nuclear translocation of fibroblast growth factor (FGF) receptors in response to FGF-2. J Cell Biol 134: 529-536, 1996.

' R% M- Y7 [, e# I6 x! w  i

Mason IJ, Fuller-Pace F, Smith R, and Dickson C. FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial- mesenchymal interactions. Mech Dev 45: 15-30, 1994.# A8 b2 h7 Q0 p7 ?1 f9 D: q
. M7 S# v# k% A9 `) z* C

McKeehan WL, Wang F, and Kan M. The heparan sulfate-fibroblast growth factor family: diversity of structure and function. Prog Nucleic Acid Res Mol Biol 59: 135-176, 1998.

7 X8 {8 ~- [, r8 |

Min H, Danilenko DM, Scully SA, Bolon B, Ring BD, Tarpley JE, DeRose M, and Simonet WS. Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev 12: 3156-3161, 1998.
1 z' @; i7 y5 o8 r% T% u9 G* a  ?7 e

% T' @; `* q7 T+ A6 R- k
Mohammadi M, Honegger AM, Rotin D, Fischer R, Bellot F, Li W, Dionne CA, Jaye M, Rubinstein M, and Schlessinger J. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C- 1. Mol Cell Biol 11: 5068-5078, 1991.5 j7 _5 j0 V7 `+ s
8 T+ M- k# }: ~* Y" {( @. G5 P2 M1 h

Ornitz DM and Itoh N. Fibroblast growth factors. Genome Biol 2: S30051-S300512, 2001.- P3 X3 Q. d: v$ f
: q0 M* ~) e' f1 x/ ~

Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, and Goldfarb M. Receptor specificity of the fibroblast growth factor family. J Biol Chem 271: 15292-15297, 1996.
% M$ g; O# F  g
. F  M/ M! v$ c5 E
- n+ m; K$ L6 U
Orr-Urtreger A, Bedford MT, Burakova T, Arman E, Zimmer Y, Yayon A, Givol D, and Lonai P. Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev Biol 158: 475-486, 1993.# L2 l, ]) S: `1 }: }
' u; s( }# _" P* K/ s
( O* z+ V( ~" {& P6 p

Pellegrini L, Burke DF, von DF, Mulloy B, and Blundell TL. Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature 407: 1029-1034, 2000.

( C1 J5 m1 C4 {

Peters K, Werner S, Liao X, Wert S, Whitsett J, and Williams L. Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. EMBO J 13: 3296-3301, 1994.

2 ~% m9 U! N4 E3 H' @
& ^+ L% J% T) A, i: e8 Z0 }
Peters KG, Werner S, Chen G, and Williams LT. Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Dev Suppl 114: 233-243, 1992.
" c8 j! y8 C5 Q- z& t7 S- q- ~

* I+ C1 K1 P1 [. G9 L- a
Plotnikov AN, Schlessinger J, Hubbard SR, and Mohammadi M. Structural basis for FGF receptor dimerization and activation. Cell 98: 641-650, 1999.

* P4 a5 c9 M" t# c5 s! }; M9 ^. S( o
Powers CJ, McLeskey SW, and Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 7: 165-197, 2000.3 j/ j* U! T7 q2 u1 p$ B
# a/ ]5 t4 C3 f/ r# j

Pruitt KD, Tatusova T, and Maglott DR. NCBI reference sequence (RefSeq): a curated nonredundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 33: D501-D504, 2005.) g$ c0 Q0 Z" I

6 h6 l- c. f/ U/ D1 n9 ~+ g
Qiao J, Uzzo R, Obara-Ishihara T, Degenstein L, Fuchs E, and Herzlinger D. FGF-7 modulates ureteric bud growth and nephron number in the developing kidney. Dev Suppl 126: 547-554, 1999.5 f- e6 m* p) F6 o' d: v
$ S( @+ p# ~5 ?& O3 p

: W* X5 ]% F, w$ g$ E; d1 ]- Y
Reilly JF and Maher PA. Importin -mediated nuclear import of fibroblast growth factor receptor: role in cell proliferation. J Cell Biol 152: 1307-1312, 2001.! N7 w3 J& [# O3 P6 [' ^: ^" M

  q: L, S% u0 D5 {5 f
2 f0 y1 P$ w% [& C& ?
Revest JM, Spencer-Dene B, Kerr K, De Moerlooze L, Rosewell I, and Dickson C. Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4. Dev Biol 231: 47-62, 2001.
4 t$ j8 n; N3 b* q+ m0 S

Revest JM, Suniara RK, Kerr K, Owen JJ, and Dickson C. Development of the thymus requires signaling through the fibroblast growth factor receptor R2-IIIb. J Immunol 167: 1954-1961, 2001.
7 u, I( S5 m% U9 L6 O

Sannes PL, Khosla J, Li CM, and Pagan I. Sulfation of extracellular matrices modifies growth factor effects on type II cells on laminin substrata. Am J Physiol Lung Cell Mol Physiol 275: L701-L708, 1998.* v% D1 I- N: _

3 K. N2 r4 n6 F& X, w+ k; _
Schlessinger J, Plotnikov AN, Ibrahimi OA, Eliseenkova AV, Yeh BK, Yayon A, Linhardt RJ, and Mohammadi M. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol Cell 6: 743-750, 2000.
4 J- V9 i3 Z2 k% I% F& [% ^& i
# f' p5 w0 Y( d/ O: Z
0 u% j8 h1 C& o
Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T, Sato T, Yagishita N, Matsui D, Koga Y, Itoh N, and Kato S. Fgf10 is essential for limb and lung formation. Nat Genet 21: 138-141, 1999.

; I( Q/ e+ o$ p: F! h  `

Shimada T, Mizutani S, Muto T, Yoneya T, Hino R, Takeda S, Takeuchi Y, Fujita T, Fukumoto S, and Yamashita T. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci USA 98: 6500-6505, 2001.0 \6 w' J- l* _1 p
: l. t; |$ U( u" g9 R0 |' @3 M/ y

: s- D! X2 N4 ]9 A6 Z, {
Sleeman M, Fraser J, McDonald M, Yuan S, White D, Grandison P, Kumble K, Watson JD, and Murison JG. Identification of a new fibroblast growth factor receptor, FGFR5. Gene 271: 171-182, 2001.

$ S9 e% `. G" {
Southgate J, Hutton KA, Thomas DF, and Trejdosiewicz LK. Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab Invest 71: 583-594, 1994.3 |# Z2 Z$ A. A0 v5 l8 g* |! e
: f9 g- C: i; \& {
3 ~1 @8 F5 U, G  X5 c% O# Q
+ U0 U- U( j! b( r$ U& e
Stachowiak MK, Fang X, Myers JM, Dunham SM, Berezney R, Maher PA, and Stachowiak EK. Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation. J Cell Biochem 90: 662-691, 2003.

8 O4 K& ^% y  \8 l) k

Stauber DJ, DiGabriele AD, and Hendrickson WA. Structural interactions of fibroblast growth factor receptor with its ligands. Proc Natl Acad Sci USA 97: 49-54, 2000.0 U. b1 O6 {/ D% q# o! M0 ~
1 n0 b0 m0 o, F. [

Strutz F, Okada H, Lo CW, Danoff T, Carone RL, Tomaszewski JE, and Neilson EG. Identification and characterization of a fibroblast marker: FSP1. J Cell Biol 130: 393-405, 1995.

Taniguchi F, Harada T, Sakamoto Y, Yamauchi N, Yoshida S, Iwabe T, and Terakawa N. Activation of mitogen-activated protein kinase pathway by keratinocyte growth factor or fibroblast growth factor-10 promotes cell proliferation in human endometrial carcinoma cells. J Clin Endocrinol Metab 88: 773-780, 2003.
5 W1 b5 ~' c" n% H5 k

Tash JA, David SG, Vaughan EDE, and Herzlinger DA. Fibroblast growth factor-7 regulates stratification of the bladder urothelium. J Urol 166: 2536-2541, 2001.  G- J) Z; O$ `" [( r$ T: i& v' V

, _& V4 f, F& J5 _
: ]& s7 s1 }$ P8 [7 V  }
Thomson AA and Cunha GR. Prostatic growth and development are regulated by FGF10. Dev Suppl 126: 3693-3701, 1999.
: o) r2 ~2 J8 D4 i6 t: J- }

Udayakumar TS, Stratton MS, Nagle RB, and Bowden GT. Fibroblast growth factor-1 induced promatrilysin expression through the activation of extracellular-regulated kinases and STAT3. Neoplasma 4: 60-67, 2002.
' z6 l& W( y5 f8 a

. t3 [2 B7 Q9 G( q: C
Varley C, Hill G, Pellegrin S, Shaw NJ, Selby PJ, Trejdosiewicz LK, and Southgate J. Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro. Exp Cell Res 306: 216-229, 2005.# n1 A) [/ j( M* ~  {! \

& E' [9 S/ e" `: T
Werner S, Duan DS, de Vries C, Peters KG, Johnson DE, and Williams LT. Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities. Mol Cell Biol 12: 82-88, 1992.$ Y( y. |) I& Z2 o+ [, R

' n& D3 v& r9 m! [% M2 i
Werner S, Smola H, Liao X, Longaker MT, Krieg T, Hofschneider PH, and Williams LT. The function of KGF in morphogenesis of epithelium and reepithelialization of wounds. Science 266: 819-822, 1994.
) S- u: O) A* _6 q' x

Wiedemann M and Trueb B. Characterization of a novel protein (FGFRL1) from human cartilage related to FGF receptors. Genomics 69: 275-279, 2000./ U0 J% Z$ P; x" o2 f7 U
1 }/ I2 s$ f- `+ b3 ?
! B  T) ]8 N  k& T, ?6 ^- P

Wiedemann M and Trueb B. The mouse Fgfrl1 gene coding for a novel FGF receptor-like protein. Biochim Biophys Acta 1520: 247-250, 2001.

6 [2 ^& M; ~! |  D6 f3 d
Wuechner C, Nordqvist AC, Winterpacht A, Zabel B, and Schalling M. Developmental expression of splicing variants of fibroblast growth factor receptor 3 (FGFR3) in mouse. Int J Dev Biol 40: 1185-1188, 1996.% `+ `+ Q; @& Z  w
3 v( K, R+ Q! P% w
* F# H& j- P& e  i1 M9 h
9 i4 ~9 F$ `) l+ g/ u( ~; Z
Yamasaki M, Miyake A, Tagashira S, and Itoh N. Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family. J Biol Chem 271: 15918-15921, 1996.

1 y" C! f4 x( d* `: S+ L
Yeh BK, Igarashi M, Eliseenkova AV, Plotnikov AN, Sher I, Ron D, Aaronson SA, and Mohammadi M. Structural basis by which alternative splicing confers specificity in fibroblast growth factor receptors. Proc Natl Acad Sci USA 100: 2266-2271, 2003.
8 @# E. t0 k0 ?9 k6 N, c
$ N- F+ A2 P1 v

Zammit C, Barnard R, Gomm J, Coope R, Shousha S, Coombes C, and Johnston C. Altered intracellular localization of fibroblast growth factor receptor 3 in human breast cancer. J Pathol 194: 27-34, 2001.3 {2 T9 O; c3 ^! i
! `1 Q8 r; r! j- o0 Y! K
7 @" l, L7 Z& l8 e9 {7 x
4 P( Y& G3 B+ v9 s( E$ s% g
Zhan X, Hu X, Friesel R, and Maciag T. Long-term growth factor exposure and differential tyrosine phosphorylation are required for DNA synthesis in BALB/c 3T3 cells. J Biol Chem 268: 9611-9620, 1993.

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