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
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ABSTRACT |
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Both nerve growth factor (NGF) and
estrogen have been shown to stimulate proliferation of various cell
types. Human urothelial cells (HUC) express the - and
-subtypes
of the estrogen receptor (ER
and ER
) 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. 17
-estradiol (E2)
stimulated NGF synthesis by HUC, and E2 (50 nM), the ER
agonist 16
-iodo-17
-estradiol (10 nM), or the ER
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
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INTRODUCTION |
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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),
ER and ER
, 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 ER
and
ER
, 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 ER
knockout mice.
Epithelial cells that line the urinary tract also express both
ER and ER
(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, ER
, and ER
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 17
-estradiol (E2) as well as two
other specific agonists of ER
and ER
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.
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METHODS |
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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), 16-iodo-17
-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 ER (Panvera,
Madison, WI); 1:20,000 dilution of rabbit anti-human ER
(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-ER and anti-ER
(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.
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RESULTS |
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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 ER agonist, 16IE2 (10 nM), and genistein (50 nM), which should act as an ER
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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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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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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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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Expression of ER, ER
, 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
ER
and ER
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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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 -tubulin confirmed equal loading of protein in each preparation.
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DISCUSSION |
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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
ER 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, ER
and ER
, 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 ER
or ER
, homodimers of
either subtype interact with response elements in target gene promoters and influence transcription. In cells expressing both
ER
and ER
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 ER and ER
are found in the
epithelium, stroma, and myometrium of the uterus, where the
ER
isoform predominates. ER
and
ER
are also expressed in the epithelium and detrusor of
the human urinary bladder, but the ER
subtype is present in greater abundance (35, 42, 47). Via immunochemistry and immunoblotting, we found that HUC express both ER
and
ER
in the nucleus as well as the cytoplasm. We also
demonstrated that E2 as well as 16IE2 (which should act as a selective
ER
agonist at the concentrations used; see Ref.
40) and genistein (which should act as a selective
ER
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.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57258.
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FOOTNOTES |
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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.
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