Estrogen-induced proliferation of urothelial cells is modulated by nerve growth factor

Jian Teng1, Zun-Yi Wang1, and Dale E. Bjorling1,2

1 Department of Surgical Sciences, School of Veterinary Medicine; and 2 Division of Urology, Department of Surgery, School of Medicine, University of Wisconsin, Madison, Wisconsin 53706


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Both nerve growth factor (NGF) and estrogen have been shown to stimulate proliferation of various cell types. Human urothelial cells (HUC) express the alpha - and beta -subtypes of the estrogen receptor (ERalpha and ERbeta ) as well as tyrosine kinase A (trkA), the high-affinity receptor for NGF. We investigated interactions between estrogen and NGF relative to cell proliferation using primary cultures of HUC. 17beta -estradiol (E2) stimulated NGF synthesis by HUC, and E2 (50 nM), the ERalpha agonist 16alpha -iodo-17beta -estradiol (10 nM), or the ERbeta agonist genistein (50 nM) each stimulated HUC proliferation, an effect that was abolished by the estrogen antagonist ICI-182,780 (100 nM). NGF (1-100 ng/ml) stimulated HUC proliferation, and this was abolished by NGF antiserum (0.1 µl/ml) or the trkA antagonist K252a (100 nM). HUC proliferation stimulated by E2 was also abolished by NGF antiserum or K252a. Finally, we observed that treatment of HUC with NGF (50 ng/ml) or E2 (50 nM) stimulated trkA phosphorylation, and this was abolished by K252a (100 nM) or NGF antiserum (0.1 µl/ml). These data indicate that the effects of ER activation on HUC proliferation at least partly involve activation of trkA by NGF.

tyrosine kinase A; receptors; estradiol; genistein; ICI-182,780; K252a; p75


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NERVE GROWTH FACTOR (NGF) is not only a classical target-derived neurotrophic factor that is important in the development and maintenance of the peripheral and central nervous systems, it is also involved in growth and differentiation of a wide variety of tissues. For example, NGF has been demonstrated to be associated with an autocrine action that affects the proliferation of bladder smooth muscle cells (39) and corneal epithelial cells (27). Previous studies using pheochromacytoma (PC12) cells provided evidence that the majority of effects of NGF are mediated by binding to its high-affinity receptor, tyrosine kinase A (trkA), which induces trkA dimerization and autophosphorylation (20, 21, 23). The alkaloidlike compound K252a has been demonstrated to be a specific inhibitor of NGF-induced phosphorylation of trkA (5). Recently, Rende et al. (38) reported that exogenous NGF increased proliferation of normal myogenic cells but not cells that lack trkA. Moreover, NGF-overexpressing keratinocytes proliferated more rapidly than mock-transfected cells (37). In both cases, K252a blocked NGF-induced cell proliferation.

It has also been reported that estrogen enhances the proliferation of several cell types including epithelial cells (2). Two primary subtypes of estrogen receptors (ER), ERalpha and ERbeta , have been identified to date, and these receptors appear to play an important role in the estrogen-induced epithelial response (25). Intrahepatic biliary epithelial cells express both ERalpha and ERbeta , and estrogen stimulates proliferation of these cells in vivo and in vitro, an effect that is blocked by ICI-182,780, which is a specific antagonist of both ER subtypes (1). Buchanan et al. (10) also demonstrated that estrogen-stimulated vaginal epithelial proliferation was eliminated by ICI-182,780 and was not observed in ERalpha knockout mice.

Epithelial cells that line the urinary tract also express both ERalpha and ERbeta (8). Increased NGF was observed in the mucosa of bladder biopsies obtained from female patients with painful bladder disorders as well as in the urine of interstitial cystitis and bladder-cancer patients (31, 34). It has been hypothesized that estrogen and NGF act synergistically by cross talk between receptors (43). However, interaction between estrogen and NGF in urothelium is poorly understood. Recently, we observed that trkA, tumor necrosis factor-2 receptor (p75), NGF, ERalpha , and ERbeta coexist within the bladder mucosa (7). The current study was undertaken to investigate interactions among estrogen, NGF, and proliferation of human urothelial cells (HUC). We used primary cultures of HUC to evaluate the effects of 17beta -estradiol (E2) as well as two other specific agonists of ERalpha and ERbeta and recombinant human NGF (rhNGF) on the proliferation of HUC. We also used NGF antiserum, the estrogen antagonist ICI-182,780, and the trkA antagonist K252a to investigate the interaction of estrogen and NGF relative to HUC proliferation. Our data indicate that the effects of ER activation on HUC proliferation at least partly involve modulation by NGF via its high-affinity receptor, trkA.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Primary HUC culture. All experimental protocols were reviewed and approved by the Health Sciences Human Subjects Committee of the University of Wisconsin. Ureter segments near the ureterovesical junction were obtained from healthy donors as byproducts of kidney transplant surgery. The mucosal layer was dissected from the underlying stroma, the urothelium was cut into 1-mm2 explants, and the explants were plated on 100-mm petri plates coated with type I rat-tail collagen gel substrate. The medium used was phenol red-free Ham's F-12 buffer (GIBCO, Grand Island, NY) supplemented with 0.1 µg/ml hydrocortisone, 5 µg/ml transferrin, 10 µg/ml insulin, 0.1 mM nonessential amino acid, 27 mg/ml dextrose, 2.0 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% charcoal-stripped fetal bovine serum (Sigma, St. Louis, MO) to constitute F-12+ buffer. Cultures were placed in a humidified incubator at 37°C in 5% CO2. Cells were dispersed for subculture after a 10-min incubation at 37°C in 0.05% trypsin-EDTA. Cell number and percent viability were determined by examination of singly dispersed cell samples that were stained for 5 min with 0.1% Trypan blue stain (Sigma). Cells were used between passages 3 and 5. Tissues used were obtained from two male and one female donor. Data reported here were generated using replicates of cells obtained from the same donor to allow for performance of contemporaneous studies. Studies were repeated three times, and results were consistent regardless of the source of cells. The results reported are representative of the experiments performed. In this study and preliminary experiments, no gender difference was observed among the responses.

AlamarBlue assay for cell proliferation. HUC were plated onto 96-well tissue-culture plates at an initial density of 500 cells/well and were incubated overnight at 37°C. The medium was changed to an F-12 mixture (without any other supplements except 100 U/ml penicillin and 100 µg/ml streptomycin) containing 5% AlmarBlue (TREK Diagnostic Systems, Westlake, OH), and cells were allowed to incubate in this medium for 4 h. E2 (1 nM-1 µM, dissolved in 95% ethanol; Sigma), 16alpha -iodo-17beta -estradiol (16IE2, 1-10 nM dissolved in 95% ethanol; kindly donated by Dr. Richard B. Hochberg, Yale University School of Medicine), genistein (10-50 nM dissolved in 95% ethanol; ICN Biomedicals, Costa Mesa, CA), ICI-182,780 (10 nM-1 µM dissolved in 10% DMSO; Tocris, Ballwin, MO), K252a (1-100 nM dissolved in 10% DMSO; Calbiochem, San Diego, CA), rhNGF (1-100 ng/ml; Genentech, San Francisco, CA), or NGF antiserum (0.1 µl/ml; Chemicon, Temecula, CA) were then added. Fluorescence was determined using a CytoFluor 4000 multiwell plate reader (PerSeptive Biosystems, Foster City, CA). An excitation wavelength of 530 nm (25-nm bandwidth filter) was used, and emission was read at 580 nm (50-nm bandwidth filter) on 30 units of sensitivity every 2 h. Results were expressed in arbitrary or relative fluorescence units (mean of 6-12 replicates). To confirm specificity of the effects of K252a and NGF antiserum, proliferation of HUC in response to recombinant human epidermal growth factor (rhEGF, 0.2-20 ng/ml; GIBCO) and phorbol-12-myristate-13-acetate (PMA, 10 nM-1 µM dissolved in 10% DMSO; ICN) were determined. Proliferation of HUC in response to NGF was evaluated in the presence or absence of NGF antiserum or normal rabbit serum (0.1 µl/ml; Vectashield, Vector Laboratories, Burlingame, CA) as a negative control for NGF antiserum.

Isolation of whole-cell lysate. To extract cell protein, HUC were washed in ice-cold PBS and 500 µl of lysis buffer [100 mM NaF, 120 mM NaCl, 0.5% Nonidet P-40, 50 mM Tris · HCl (pH 8.0), 2.0 mM Na3VO4] were added into each 100-mm petri plate. Individual plates were then rapidly frozen and thawed twice. Whole-cell lysate was obtained by removing cells using a cell scraper and then centrifuging the lysate in a microcentrifuge at 10,000 g for 10 min. Protease inhibitors [0.2 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml aprotinin, and 2 µg/ml leupeptin] were added to the buffer just before use, and protein content was determined by the Bradford assay.

Isolation of nuclear protein. To extract nuclear protein, HUC were dispersed by trypsin and washed in PBS. The final cell pellet was gently suspended in buffer A [10 mM HEPES (pH 8.0), 0.5 M sucrose, 50 mM NaCl, 2.5 mM MgCl2, 1 mM EDTA, 0.25 mM EGTA, 0.5% Triton X-100]. Nuclei were collected by centrifugation at 2,000 g for 5 min. Nuclei were then resuspended in buffer B [10 mM HEPES (pH 8.0), 12.5% glycerol, 530 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA] and allowed to incubate on ice for 20 min. The supernatant was collected by centrifugation at 2,000 g for 5 min. Protease inhibitors (0.2 mM PMSF, 2 µg/ml aprotinin, and 2 µg/ml leupeptin) were added to the buffer just before use, and protein content was determined by the Bradford assay.

Immunoblotting. Protein (30 µg/lane) was loaded, subjected to SDS gel electrophoresis using an 8% polyacrylamide gel, and transferred to nitrocellulose membrane (Micron Separations, Westborough, MA). Membranes were blocked overnight at 4°C in 10% fat-free dry milk in 1× TBST (20 mM Tris · HCl, 137 mM NaCl, pH 7.6, and 0.1% Tween 20). Membranes were washed free of blocker by TBST and incubated at room temperature for 1 h with primary antibody diluted in 1% BSA in TBST [1:10,000 dilution of rabbit anti-human ERalpha (Panvera, Madison, WI); 1:20,000 dilution of rabbit anti-human ERbeta (Panvera); 1:10,000 dilution of rabbit anti-human trkA (Santa Cruz, Santa Cruz, CA); 1:10,000 dilution of mouse anti-human phosphorylated trkA (p-trkA; Santa Cruz); and 1:10,000 dilution of rabbit anti-human p75 (Promega, Madison, WI)]. Membranes were then washed free of primary antibody and incubated at room temperature for 1 h in a 1:20,000 dilution of anti-rabbit or anti-mouse IgG with horseradish peroxidase tagged in 1% BSA in TBST. Finally, membranes were incubated in ECL detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 min and exposed to film for 10 s to 10 min.

Enzyme-linked immunosorbent assay. HUC were plated onto 100-mm petri plates in F-12+ buffer and incubated overnight at 37°C. Cells were exposed to test compounds for 24 h. Whole-cell lysates were prepared from individual plates. Enzyme-linked immunosorbent assays were performed using the NGF Emax immunoassay system (Promega) to determine NGF content. Briefly, flat-bottom 96-well plates were coated with anti-NGF polyclonal antibody and incubated for 14-18 h at 4°C. After 1 h of incubation with the blocking solution, the test solution was added and allowed to incubate for 6 h at room temperature while the plates were shaken. The plates were washed, monoclonal antibody was added, and the plates were incubated at 4°C for 14-18 h. After the plates were washed, the amount of bound monoclonal antibody was detected using antibody conjugated to horseradish peroxidase as a tertiary reactant. The unbound conjugate was removed by washing, chromogenic substrate was added, and color change was determined using an EL312e Microplate Reader (Bio-Tek Instruments) at 450 nm. All samples were run in duplicate on two separate occasions, and values were averaged.

Immunocytochemistry. HUC were grown on chamber slides (Lab-Tek) for 12-24 h and then rinsed with PBS and fixed in cold acetone. After the slides were rinsed, the background was blocked with 10% normal goat serum for 1 h at room temperature, and the slides were then incubated with each antibody overnight at 4°C. Staining was revealed using a TSA fluorescence system (NEN Life Science Products, Boston, MA) in accordance with the manufacturer's instructions. This system uses tyramide reagents to amplify the staining signals. Slides were rinsed and coverslipped using an antifading solution (Vectashield). Slides were examined with a Nikon E600 microscope, and digital images were captured using a digital camera (Diagnostic Instruments, Sterling Heights, MI). For negative controls, slides were incubated with normal rabbit IgG instead of specific antibodies. The following antibodies were used: a 1:200 dilution of mouse anti-keratin AE1/AE3 (Chemicon), a 1:1,000 dilution of rabbit anti-trkA (Santa Cruz), 1:2,000 dilutions of both anti-ERalpha and anti-ERbeta (Santa Cruz), and a 1:1,000 dilution of rabbit anti-p75 (Promega).

Statistics. Data from immunoblotting and proliferation assays are presented as arithmetic means ± SE of 6-12 replicates. One-way or two-way ANOVA was performed to determine significant differences among one- or two-factor experimental treatments, respectively. Individual groups were compared using Fisher's (least-significance difference) test. A P value of <0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of rhNGF, E2, and ICI-182,780 on proliferation of primary cultures of HUC. rhNGF (100 ng/ml) and E2 (50 nM) both stimulated HUC proliferation (P < 0.01 for both; Fig. 1) in a time-dependent manner. The selective ERalpha agonist, 16IE2 (10 nM), and genistein (50 nM), which should act as an ERbeta agonist at this concentration (48), also stimulated proliferation of HUC (Fig. 2). Incubation of HUC with E2 (1 nM-1 µM) for 8 h demonstrated a concentration-dependent effect on proliferation, which was suppressed by the specific estrogen receptor antagonist ICI-182,780 (100 nM; P < 0.01; Fig. 3). These data provide direct evidence that estrogens stimulate proliferation of HUC by activating estrogen receptors.


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Fig. 1.   Effects of recombinant human nerve growth factor (rhNGF) and 17beta -estradiol (E2) on primary human urothelial cell (HUC) proliferation. Each point represents the mean ± SE of 6 replicates. Both rhNGF and E2 stimulated HUC proliferation (P < 0.01 and P < 0.01, respectively). **P < 0.01 compared with control at each time point.



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Fig. 2.   Effects of estrogen receptor (ER) agonists on proliferation of primary HUC. Cell proliferation was determined after incubation with drugs for 8 h. Each column represents the mean ± SE of 6 replicates. *P < 0.05, **P < 0.01 compared with control.



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Fig. 3.   Effects of estrogen antagonist ICI-182,780 on ER agonist-induced proliferation of primary HUC. ICI-182,780 suppressed increased HUC proliferation in response to E2 (P < 0.01). *P < 0.05, **P < 0.01 compared with E2 alone.

E2 stimulated NGF synthesis in primary cultures of HUC. Incubation of primary HUC with E2 (1 nM) for 24 h resulted in a 28% increase in NGF in whole-cell lysate (Fig. 4). ICI-182,780 (100 nM) alone had no effect on NGF content in cell lysate but inhibited estrogen-induced increase in cell-lysate NGF content. These data indicate that estrogen stimulates NGF synthesis by ligand binding to the estrogen receptor.


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Fig. 4.   Effects of E2 and ICI-182,780 on cell-lysate NGF content from primary HUC. Data are presented as means ± SE of 4 replicates. E2 stimulated NGF content in HUC, and this effect was blocked by ICI-182,780. *P < 0.05 compared with control; #P < 0.05 compared with E2 alone.

NGF antiserum and trkA inhibitor specifically block rhNGF-stimulated HUC proliferation. Incubation of HUC with rhNGF (1-100 ng/ml) for 8 h increased proliferation in a concentration-dependent manner, and this effect was not affected by nonimmune rabbit serum and was completely abolished by NGF antiserum (0.1 µl/ml; P < 0.01; Fig. 5A). K252a (100 nM), a specific trkA phosphorylation inhibitor, inhibited HUC proliferation in the absence of exogenous NGF (Fig. 5B). This effect suggests the presence of an autocrine/paracrine mechanism in the synthesis and release of NGF by HUC. K252a (100 nM) also suppressed HUC proliferation in response to rhNGF (1-100 ng/ml; P < 0.01; Fig. 5B). The specificity of the response of HUC to NGF was tested by evaluating proliferation of HUC in response to EGF (0.2-20 ng/ml) or PMA (10 nM-1 µM) in the absence or presence of K252a (100 nM) or NGF antiserum (0.1 µl/ml). Both EGF and PMA increased proliferation in a concentration-dependent manner (P < 0.01 for both; Fig. 6, A and B). Neither K252a nor NGF antiserum affected cell proliferation induced by EGF or PMA. These results indicate that NGF-induced HUC proliferation is mediated by the high-affinity receptor for NGF (trkA) and that NGF antiserum is not a nonspecific inhibitor of proliferation.


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Fig. 5.   Effects of NGF antiserum and K252a on NGF-stimulated proliferation of primary HUC. Each column represents the means ± SE of 6 replicates. A: no difference was observed between NGF and NGF + nonimmune rabbit serum. Increased proliferation of HUC in response to rhNGF was blocked by NGF antiserum (P < 0.01). **P < 0.01 compared with control (0 ng/ml rhNGF). B: K252a suppressed the proliferative effect of rhNGF (P < 0.01). NGF stimulated HUC proliferation in a concentration-dependent manner. K252a alone inhibited proliferation. **P < 0.01 compared with 0 ng/ml rhNGF; ##P < 0.05 compared with 0.1% DMSO control.



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Fig. 6.   Effects of NGF antiserum and K252a on epidermal growth factor (EGF)- or phorbol-12-myristate-13-acetate (PMA)-stimulated proliferation of primary HUC. Each column represents the means ± SE of 6 replicates. A: EGF stimulated concentration-dependent proliferation of HUC, and treatment with either K252a or NGF antiserum had no effect on this. **P < 0.01 compared with control (0 ng/ml EGF). B: PMA stimulated concentration-dependent proliferation of HUC, and treatment with either K252a or NGF antiserum had no effect on this. **P < 0.01 compared with control (0.1% DMSO).

NGF antiserum and trkA inhibitor block estrogen-stimulated HUC proliferation. HUC proliferation stimulated by E2, 16IE2, or genistein was abolished by NGF antiserum (0.1 µl/ml; Fig. 7). Incubation of HUC with E2 (1 nM-1 µM) for 8 h stimulated a concentration-dependent increase in proliferation, and this effect was abolished by K252a (100 nM; P < 0.01; Fig. 8). These results indicate that increased HUC proliferation stimulated by estrogen is at least partially mediated by the interaction of NGF and trkA.


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Fig. 7.   Effects of NGF antiserum on estrogen-induced proliferation of primary HUC. Data are presented as the means ± SE of 6 replicates. NGF antiserum abolished the proliferative effects of ER agonists on HUC. *P < 0.05, **P < 0.01 compared with control (absence of ER agonists or NGF antiserum); ##P < 0.01 compared with ER agonists alone.



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Fig. 8.   Effects of K252a on estrogen-induced proliferation of primary HUC. Data are presented as the means ± SE of 6 replicates. K252a suppressed the proliferative effects of E2 on HUC (P < 0.01). **P < 0.01 compared with E2 alone at corresponding time points.

Expression of ERalpha , ERbeta , trkA, and p75 receptors in primary cultures of HUC. Strong staining of keratin in the cytoplasm was observed in each cell, which confirms that the cells were of epithelial origin (Fig. 9). The networks of cytoskeletal fibers were clearly shown with keratin antibody. Strong staining of ERalpha and ERbeta was associated with the nuclei of HUC, and weaker staining was also observed in the cytoplasm. Specific staining of trkA and p75 was observed only in the cytoplasm of HUC. The presence of these receptors in HUC was further confirmed by immunoblotting (data not shown).


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Fig. 9.   Evaluation of ER and NGF receptors in primary HUC by immunocytochemistry. ERalpha -, ERbeta -, tyrosine kinase A (trkA)-, and tumor necrosis factor-2 (p75)-like immunoreactivities were observed in primary HUC. Note that staining of trkA and p75 was observed mainly in the cytoplasm of cells, whereas staining of ERalpha and ERbeta was observed primarily in the nuclei. Omitting primary antibodies abolished staining (Control). Staining of keratin confirmed the epithelial origin of urothelial cells.

Effect of K252a and NGF antiserum on phosphorylation of trkA in primary cultures of HUC. To study the possible mechanism of NGF-induced HUC proliferation, we assessed the effects of rhNGF and E2 on tyrosine phosphorylation of trkA. Using a monoclonal antibody for tyrosine-phosphorylated trkA, the expression of p-trkA after a 15-min, 30-min, or 2-h incubation of HUC with 50 ng/ml rhNGF or 50 ng/ml rhNGF combined with 100 nM K252a was evaluated (Fig. 10). Densitometric analysis revealed a three- to fourfold increase with rhNGF treatments, and this effect was completely inhibited by K252a. E2 and rhNGF both increased phosphorylation of trkA, which was abolished by K252a or NGF antiserum (Fig. 11). We also observed that expression of total trkA was unaffected by these treatments in both experiments (Figs. 10 and 11). Reprobing with monoclonal antibody for alpha -tubulin confirmed equal loading of protein in each preparation.


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Fig. 10.   Effects of K252a on NGF-stimulated phosphorylation of trkA in primary HUC. Each membrane was probed with a monoclonal antibody against phosphorylated trkA (p-trkA), a polyclonal antibody against trkA, and a monoclonal antibody against alpha -tubulin. Results presented are representative of 5 identical experiments. Expression of p-trkA quantitative data are means ± SE from 5 cell preparations. Band densities were evaluated by normalizing controls to 100. Incubation with K252a inhibited basal phosphorylation of trkA, and K252a inhibited NGF-induced phosphorylation of trkA (P < 0.01). *P < 0.05, **P < 0.01 compared with control (0.1% DMSO); ##P < 0.01 compared with rhNGF alone at the same time points.



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Fig. 11.   Effect of K252a and NGF antiserum on NGF- or E2-stimulated phosphorylation of trkA in primary HUC. Treatments were as follows: control (0.1% DMSO), E2 (10 nM), NGF (50 ng/ml rhNGF), ICI (100 nM ICI-182,780), K252a (100 nM), N-a (0.1 µl/ml NGF antiserum), E2 + I (10 nM E2 + 100 nM ICI-182,780), E2 + K (10 nM E2 + 100 nM K252a), E2 + N-a (10 nM E2 + 0.1 µl/ml NGF antiserum), and N + N-a (50 ng/ml rhNGF + 0.1 µl/ml NGF antiserum) for 8 h. Each membrane was probed by a monoclonal antibody against p-trkA, a polyclonal antibody against trkA, and a monoclonal antibody against alpha -tubulin. Results presented are representative of 5 identical experiments. Expression of p-trkA quantitative data are means ± SE from 5 cell preparations. Band densities were evaluated by normalizing controls to 100. E2 and rhNGF both increased phosphorylation of trkA, and these effects were abolished by K252a or NGF antiserum. Expression of total trkA was unaffected by these treatments. **P < 0.01 compared with control; ##P < 0.01 compared with E2; ++P < 0.01 compared with rhNGF.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study addresses the role of NGF in proliferation of HUC in response to estrogen. The data presented here contribute new observations to this area by demonstrating that 1) urothelial cells produce NGF, 2) estrogen stimulates proliferation of urothelial cells and this effect is attenuated by an ER antagonist, 3) NGF also exerts a proliferative effect on urothelial cells, and 4) the proliferative effects of both estrogen and NGF can be blocked by NGF antiserum or a trkA antagonist.

Estrogen is highly mitogenic in hormone-sensitive tissues such as the uterus and breast, and estrogen modulates the growth of target cells expressing ER (30). In a series of experiments, Gollapudi et al. (15, 16) successfully transfected the ERalpha gene into rat PC12 cells. Estrogen treatment of these ER-expressing PC12 cells markedly increased cell survival. Estrogen also significantly stimulated bromodeoxy uridine incorporation into these cells, and this mitotic effect was completely abolished by the estrogen antagonist ICI-182,780. Activation of ER appears to be a multistep process that relies on a number of events including estrogen entering cells by simple or facilitated diffusion, binding to ER, dissociation of heat-shock proteins from ER, ER dimerization, ER phosphorylation, and binding to discrete DNA sequences termed estrogen-response elements (EREs) (18, 26, 36). Two primary subtypes of ER, ERalpha and ERbeta , which are encoded by genes located on different chromosomes, have been identified. Depending on the relative expression of ER subtypes, estrogens may activate different signaling pathways. In cells that express only ERalpha or ERbeta , homodimers of either subtype interact with response elements in target gene promoters and influence transcription. In cells expressing both ERalpha and ERbeta subtype proteins, heterodimers can be formed depending on the ratio of ER subtypes. EREs that interact preferentially with the heterodimer may exist (25, 26, 36).

Both ERalpha and ERbeta are found in the epithelium, stroma, and myometrium of the uterus, where the ERalpha isoform predominates. ERalpha and ERbeta are also expressed in the epithelium and detrusor of the human urinary bladder, but the ERbeta subtype is present in greater abundance (35, 42, 47). Via immunochemistry and immunoblotting, we found that HUC express both ERalpha and ERbeta in the nucleus as well as the cytoplasm. We also demonstrated that E2 as well as 16IE2 (which should act as a selective ERalpha agonist at the concentrations used; see Ref. 40) and genistein (which should act as a selective ERbeta agonist at the concentrations used; see Ref. 48) stimulate proliferation of urothelial cells. ICI-182,780, a 7-ackylamide analog of E2, seems to have pure antagonistic effects on both ER subtypes. The effects of ICI-182,780 binding have been attributed to impaired dimerization, increased ER turnover, and disrupted nuclear localization of ER. Not only are ER blocked functionally, but also cellular levels of ER are reduced markedly by ICI-182,780 (16). In our study, the concentration-dependent stimulatory effect of E2 was suppressed by ICI-182,780. This provides direct evidence that the proliferative effects of estrogen on urothelial cells are mediated by ER.

It has been known for some time that NGF strongly regulates growth, differentiation, and survival of cells by influencing cell-cycle proteins (14, 19, 33). NGF stimulated proliferation of MCF-7 breast-cancer cells in a concentration-dependent manner with an EC50 of 7.1 ng/ml (12). There are two kinds of transmembrane glycoprotein receptors that bind NGF: trkA and p75. TrkA has high affinity and specificity for NGF, whereas p75 binds to a variety of neurotrophins with lower affinity (3, 11). Early work using PC12 cells demonstrated that NGF mediates most of its effects by binding to trkA and stimulating trkA dimerization and the autophosphorylation of tyrosine residues (20, 21, 23). Although p75 may be involved in apoptosis (46), it has also been shown that p75 increases the affinity of trkA for NGF (4). Phosphorylation and internalization of trkA leads to activation of second-messenger cascades including mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase, two pathways that are important for cell differentiation and survival, respectively (23, 39, 49). In our study, immunocytochemical staining and immunoblotting also demonstrated expression of both trkA and p75 in HUC. rhNGF stimulates proliferation of these cells in a concentration-dependent manner, and this effect is abolished by antiserum directed against NGF.

K252a, an alkaloidlike compound isolated from Nocardiopsis, was originally characterized as an inhibitor of protein kinase C (PKC) and cyclic nucleotide-dependent kinase (5). Previous investigations have shown that most of the known biochemical events induced by NGF could be inhibited by K252a; however, at nanomolar concentrations (0.1-200 nM), inhibition of the effects of NGF by K252a was not due to inhibition of PKC or protein kinase A (PKA), but was rather due to a direct and specific inhibition on trkA tyrosine phosphorylation (5, 24). Pharmacological inhibition of trkA signal transduction with K252a (in the nanomolar range) resulted in a dramatic concentration-dependent decrease in proliferation of myogenic cells (38) and a significant inhibition of NGF-stimulated MCF-7-cell proliferation (12). EGF has been demonstrated to stimulate proliferation of urothelial cells (6). Certain types of phorbol ester have also been shown to induce cell proliferation by activating PKC (17). To verify the specificity of K252a and NGF antiserum at the concentrations used in this study, we observed the effects of K252a as well as NGF antiserum on EGF- or PMA-stimulated HUC proliferation. Neither K252a nor NGF antiserum affected EGF- or PMA-stimulated HUC proliferation. This confirms the specificity of K252a and NGF antiserum in urothelial cells. K252a inhibited HUC proliferation in the absence of exogenous NGF, which suggests an autocrine/paracrine action of NGF. Proliferation of HUC in response to exogenous NGF was also suppressed by K252a. These results indicate that NGF-induced proliferation of HUC is mediated by trkA. To investigate the role of NGF in trkA autophosphorylation, we evaluated tyrosine phosphorylation of trkA in response to NGF in the presence or absence of K252a after 15 min, 30 min, and 2 h. Phosphorylation of trkA was significantly stimulated by NGF, and this effect was abolished by K252a. The total trkA expression was unchanged, which indicates that proliferation of HUC stimulated by NGF involves phosphorylation of trkA.

The interaction of steroid hormones with membrane-associated receptor tyrosine kinase signaling pathways is currently under investigation but is still poorly understood. There is increasing evidence that reproductive hormones modulate NGF synthesis. In the guinea pig uterus, NGF protein levels showed no significant change during pregnancy, but they showed a marked increase during the early postpartum period (9). In the zebra finch telecephalon, reduced estrogen significantly decreased both trkA and p75, and E2 treatment increased trkA (13). Furthermore, Lara et al. (29) observed that a single injection of estrogen resulted in increased intraovarian synthesis of NGF and p75 in rats. In addition, it has been observed that ER, trkA, and p75 are colocalized in a subpopulation of dorsal-root ganglion neurons, and E2 directly upregulated trkA and p75 NGF receptors in a time- and concentration-dependent manner (26). NGF also significantly enhanced cell survival and reduced cell apoptosis in both ER-expressing and control PC12 cells, but E2 stimulated a further significant enhancement of cell survival and reduction in apoptosis only in ER-expressing cells. Estrogen treatment was also observed to increase trkA mRNA levels in ER-expressing PC12 cells (15, 16). It is therefore quite possible that the synergistic effects of estrogen and NGF operate at the NGF receptor level. In our study, we observed a significant increase in the NGF content of HUCs in response to E2, and this effect was completely abolished by ICI-182,780.

It has been speculated that estrogen may act synergistically with NGF through mechanisms that involve the regulation of EREs (43). Support for this hypothesis comes from the observation that motifs that mimic classic EREs are found in the promoter and 5' flanking regions of the genes for human NGF, trkA, and p75 (22, 44). We tested the possibility that NGF and trkA participate in estrogen-induced HUC proliferation using NGF antiserum and K252a. Either NGF antiserum or K252a abolished HUC proliferation induced by E2, 16IE2, or genistein. Therefore, the effect of ER activation is likely to be NGF and/or trkA related. It should also be noted that NGF was found to increase expression of ER in the developing cerebral cortex and basal forebrain in a post-transcriptional manner, which suggests that NGF may also affect the abundance of ER (32, 44). Certain membrane receptors have been shown to interact with liganded ER as coactivators or cosuppressors of transcription (45). Thus estrogen and NGF may interact reciprocally by regulation of gene transcription, signal transduction, or receptor/ligand availability.

In summary, this study reports the presence of NGF and trkA in HUC. Both NGF and estrogen stimulate HUC proliferation by activation on the respective receptors. Moreover, we also demonstrate that estrogen stimulates NGF synthesis. Thus the proliferative effect of estrogen is at least partially mediated by NGF and especially by phosphorylation of the trkA receptor. These findings may have relevance to development or repair of the lining of the urinary tract. Future work will assess signal transduction involved in this mechanism.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57258.


    FOOTNOTES

Address for reprint requests and other correspondence: D. E. Bjorling, Dept. of Surgical Sciences, School of Veterinary Medicine, 2015 Linden Dr., Madison, WI 53706 (E-mail: bjorlind{at}svm.vetmed.wisc.edu).

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.

First published January 2, 2002;10.1152/ajprenal.00215.2001

Received 9 July 2001; accepted in final form 28 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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