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FGF-10 and its receptor exhibit bidirectional paracrine targeting to urothelial and smooth muscle cells in the lower urinary tract

Program in Human Urothelial Biology, Children’s Hospital and Regional Medical Center, Seattle, Washington

Submitted 24 January 2006 ; accepted in final form 19 March 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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.

transitional epithelium; mesenchymal signaling; growth factors

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.

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.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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.

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 cm²) from renal pelvis, ureter, or urinary bladder was incubated in 0.1% (wt/vol) EDTA, 1x 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 1x 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 1x HBSS, 10 mM HEPES (pH 7.4), 0.0055 mM CaCl₂, 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-cm² 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 10⁴ cells/cm² 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% CO₂ at 37°C.

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/cm². 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.

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: 20x air (0.40), 40x air (0.65), and 100x oil (1.25). The acquisition software was Leica DC Twain 4.1.8.0 [EC] 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 H₂O, 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 H₂O accompanied each step. Finally, the grids were dried, visualized with a Zeiss 910 transmission electron microscope at x25,000 for FGF-10 and x16,000 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.

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 x63 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.

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 HindIII and Pst1, whereas the 3860N construct was linearized with HindIII and EcoR1. 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).

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, 4x SSC [600 mM NaCl, 60 mM sodium citrate (pH 7.0)], 5% dextran sulfate, 1x 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 2x SSC that contained 50% formamide at 60°C and then sequential washes with 1, 0.25, 0.1, and 0.05x 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.

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.

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.

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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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. 1B) and ureter (Fig. 1F). A similar distribution of even immunoreactivity was observed in bladder exstrophy (Fig. 1D; 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. 1D (tissue labeled "K").

View larger version (124K): [in this window] [in a new window]   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.

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. 2B), 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. 2D), also in agreement with our previous report on normal human bladder (4).

View larger version (124K): [in this window] [in a new window]   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.

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. 2C). 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. 2B) 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. 2D). 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. 3B). 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. 3B). 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. 3B). 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.

View larger version (135K): [in this window] [in a new window]   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.

View larger version (125K): [in this window] [in a new window]   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.

Strong immunoreactive signals for the FGF-10 receptor were also observed within smooth muscle cells of the ureteric detrusor layer (Fig. 3D). Such signals were absent for adjacent endothelial cells (Fig. 3D, 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.

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.

View larger version (131K): [in this window] [in a new window]   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.

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. 6B; 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. 6C; 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. 6C), and superficial (Fig. 6D) 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.

View larger version (131K): [in this window] [in a new window]   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: x25,000 for A, B, E, and F; x16,000 for C and D. The images in E and F were further magnified to distinguish between 6- and 15-nm gold particles.

View larger version (46K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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 < 0.025). B: rFGF10-His does not induce bladder smooth muscle cell DNA synthesis. Cells were fed SmGM-20 containing 5 µg/ml heparin and varying concentrations of serum (see MATERIALS AND METHODS). The absorbance at 370 nm represents BrdU incorporation into cellular DNA.

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. 8A, 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. 8A, filled bar), P was calculated as 0.022 (0.001 µg/ml) and <0.002 for concentrations of rFGF10-His from 0.01 to 10.0 µg/ml. The collective evidence demonstrates that FGF-10 is an effective mitogen for urothelial cells and functions through an EGF-independent pathway.

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. 8B, bar with horizontal lines). The addition of rFGF10-His to actively growing cells did not elicit any further increase in DNA synthesis (Fig. 8B, 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. 8B, 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. 8B, open bars); this observation is in contrast to urothelial cells which responded positively to rFGF10-His (Fig. 8A).

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. 4D). 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.

View larger version (68K): [in this window] [in a new window]   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.

View larger version (156K): [in this window] [in a new window]   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: x25,000 for A, B, and C; x16,000 for D, E, and F. The images in B, C, E, and F were further magnified for clarity.

	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.

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.

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 (<40–45 kDa) (24, 28). In our study, NLS have been identified in both FGF-10 and the receptor (Kosman J, Zhang D, and Bassuk JA; unpublished observations), which implies that the nuclear transport of the receptor does not need the help from FGF-10 and that instead a specific nuclear transport mechanism must exist. Our transmission electron microscopic data have indicated that FGF-10 and its receptor may be transported to the nucleus separately through vesicles or clusters.

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.

	GRANTS

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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.

	ACKNOWLEDGMENTS

 
We thank Dr. Jenny Southgate for suggesting the use of PD-15305.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. A. Bassuk, Program in Human Urothelial Biology, Children’s Hospital and Regional Medical Center, 4800 NE Sand Point Way, Mail Stop A8938, Seattle, WA 98105 (e-mail: james.bassuk{at}seattlechildrens.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ In this manuscript, the term fibroblast is used to describe spindle-shaped cells that are associated with the Masson’s trichrome staining of collagen in the lamina propria and the tunica muscularis.

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