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Endocrinology Vol. 147, No. 9 4192-4204
Copyright © 2006 by The Endocrine Society

The Endocrine-Disrupting Compound, Nonylphenol, Inhibits Neurotrophin-Dependent Neurite Outgrowth

Cassandra L. Bevan, Donna M. Porter, Christopher R. Schumann, Elena Y. Bryleva, Tyler J. Hendershot, Hongbin Liu, Marthe J. Howard and Leslie P. Henderson

Departments of Physiology (C.L.B., D.M.P., C.R.S., L.P.H.) and Biochemistry (E.Y.B.), Dartmouth Medical School, Hanover, New Hampshire 03755; and Department of Neurosciences (T.J.H., H.L., M.J.H.), Medical University of Ohio, Toledo, Ohio 43614

Address all correspondence and requests for reprints to: Leslie P. Henderson, Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755. E-mail: Leslie.Henderson{at}Dartmouth.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endocrine-disrupting compounds (EDCs) may interfere with neuronal development due to high levels of accumulation in biological tissue and potentially aberrant steroid signaling. Treatment of dissociated embryonic Xenopus spinal cord neurons with the EDC, nonylphenol (NP), did not alter cell survival or neurite outgrowth but inhibited neurotrophin-induced neurite outgrowth, effects that were recapitulated by treatment with comparable concentrations of 17ß-estradiol (E2) and ß-estradiol 6-(O-carboxy-methyl)oxime: BSA (E2-BSA), but not a synthetic androgen. Effects of NP were not inhibited by the nuclear estrogen receptor antagonist, ICI 182,780, but were inhibited by the G protein antagonist, pertussis toxin. Nerve growth factor (NGF)-induced neurite outgrowth in Xenopus neurons was shown to require MAPK signaling. NP did not affect TrkA expression, MAPK signaling, or phosphatidylinositol 3' kinase-Akt-glycogen synthase kinase 3ß (PI3K-Akt-GSK3ß) signaling in Xenopus. The ability of NP to inhibit NGF-induced neurite outgrowth without altering survival was recapitulated in the rat pheochromocytoma (PC12) cell line. As with Xenopus neurons, the inhibitory actions of NP in PC12 cells were not antagonized by ICI 182,780 and did not involve alterations in signaling along either the MAPK or PI3K-Akt-GSK3ß pathways. NP did significantly inhibit the ability of NGF to increase protein kinase A activity in this cell line. These data have important implications with respect to potentially deleterious effects of NP exposure during early neural development and highlight the fact that bioaccumulation of EDCs, such as NP, may elicit very disparate effects along divergent signaling pathways than those that arise from the actions of physiological levels of endogenous estrogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STUDIES OF BOTH wildlife populations and laboratory animals indicate that exposure to a wide range of environmental chemicals, now numbering in the hundreds of thousands (1, 2), can elicit deleterious effects during development by disrupting hormone-sensitive processes (3). Nonylphenol (NP), one of the most prevalent and best-studied endocrine-disrupting compounds (EDCs), arises as a degradation product of the alkylphenol polyethoxylates; compounds widely used as nonionic surfactants in commercial production, as well as in herbicides, pesticides, polystyrene plastics, and paints (4, 5, 6). As with the majority of EDCs, NP is not itself a steroid but nonetheless acts as an agonist at nuclear estrogen receptors (ER) and thus can be defined as an environmental estrogen (6). EDCs have been shown to undergo significant bioaccumulation (7, 8, 9), and tissue concentrations of NP have been measured in the 1–20 µM range in aquatic organisms (10, 11, 12). Thus, EDCs may promote aberrant development both because these environmental toxins are present during inappropriate developmental epochs and because the high levels of these compounds may activate pathways not normally affected by physiological levels of hormones.

The developing nervous system is known to be particularly sensitive to the actions of endogenous steroids (for review, see Ref. 13) and may thus also be particularly sensitive to the deleterious effects of EDCs. Members of the neurotrophin family of growth factors, acting through their cognate receptors, Trks, are critical mediators of neural development, regulating neuronal survival, proliferation, and differentiation (for review, see Ref. 14). Expression of neurotrophins and Trks are regulated by estrogens (15, 16, 17), suggesting that exposure to environmental estrogens might perturb neuronal survival or differentiation by altering neurotrophin signaling. Xenopus laevis are aquatic amphibians that have proved to be highly useful sentinels for EDCs (18, 19, 20, 21). Dissociated Xenopus spinal cord neurons can be maintained in serum-free and defined medium where they undergo rapid differentiation that faithfully recapitulates in vivo development (for review, see22). These neurons respond to nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) with enhancement of neurite outgrowth (23, 24). Here, we have taken advantage of this tractable system to demonstrate that NP, at environmentally relevant concentrations, antagonizes neurotrophin-dependent differentiation of primary Xenopus neurons via a mechanism that is G protein-coupled. In addition, we show that the effects of NP on neurotrophin-induced differentiation do not involve changes in either Xenopus neurons or in the PC12 cell line in signaling pathways mediating neurotrophin actions (for review, see Refs. 14 and 25, 26, 27) that are also implicated in the rapid actions of estrogens (28, 29, 30, 31, 32, 33, 34). Our results suggest that this environmental estrogen, if present at elevated levels in the developing brain, may have deleterious effects on neuronal differentiation by interfering with neurotrophin effects and may do so by acting through signaling mechanisms that are distinct from those that represent known convergence points for the effects of physiological estrogens and neurotrophins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and maintenance
Matings of adult X. laevis (Nasco, Fort Atkinson, WI) were induced by injection of human chorionic gonadotropin (Sigma, St. Louis, MO) (35), and embryos were staged according to the normal table of Nieuwkoop and Faber (36). Animal care procedures were approved by the Institutional Animal Care and Use Committee at Dartmouth and adhere to both the National Institutes of Health and the American Veterinary Medical Association guidelines to minimize pain and discomfort and to minimize the numbers of animals used.

Cell culture and reagents
Primary cultures were prepared from neural plates of stage 15 or 22 (36) Xenopus embryos according to previously published procedures (35). Cells from a single neural plate were plated in phenol red-free Leibowitz-15 medium, supplemented with 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Mediatech, Herndon, VA). Primary motoneurons and neural crest-derived Rohon-Beard cells constitute more than 80% of the differentiated neurons in cultures prepared at stage 15 (37). All treatments were initiated at the time of plating, and cultures were assayed approximately 36 h later.

PC12 cells were plated at a density of 2.5 x 105/dish, maintained in a humidified CO2 environment in 100-mm plates for all signaling assays and in 35-mm plates for assays of neurite outgrowth in DMEM (Invitrogen, Carlsbad, CA) or in phenol red-free DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 5% horse serum (Invitrogen), 1% penicillin, 1% streptomycin, and 1% L-glutamine (all from Mediatech). In some experiments, serum was charcoal-stripped to deplete medium of steroids by preparing a slurry of 2 g charcoal/100 ml serum that was stirred on a rotator for 2 h at 4 C, centrifuged at 1000 rpm (10 min), and filtered (0.22 µm) before use. All treatments of cultures were initiated at the time of plating. For assays of neurite outgrowth and long-term protein kinase A (PKA) assays, cultures were examined after either 5 or 7 d (results were quantitatively and qualitatively similar). Medium and neurotrophins were replenished every other day. For all signaling assays, cultures were changed to a serum-free medium for 24 h, and assays were performed at 15, 30, or 60 min after addition of NGF and other reagents. All assays were performed on cells between the fourth and 12th passages.

NP (Aldrich, St. Louis, MO), 17ß-estradiol (E2; 3,7ß-dihydroxy-1,3,5(10)-estratriene; Sigma), ß-estradiol 6-(O-carboxy-methyl)oxime: BSA (E2-BSA; Sigma), 17{alpha}-methyl-4-androstene-17ß-ol-3-one (17{alpha}-methyltestosterone; Sigma), the MAPK kinase (MEK) 1/2 inhibitor, U0126, and its inactive analog, U0124 (10 µM; Calbiochem, La Jolla, CA), and the pure antiestrogen, ICI 182,780 (38) (5 µM; Tocris, Ellisville, MO) were dissolved in 95% HPLC-grade ethanol or 100% dimethylsulfoxide (final vehicle concentration, 0.01%). The MEK1/2 inhibitor, PD98059, precipitated out of solution in the Xenopus culture medium and was therefore not suitable for use. Steroids, ICI 182,780, and NP were added at 5 µM except where indicated to the contrary. Five micromolar ICI 182,780 was used because this concentration is estimated to block 999 of 1000 available ER in the presence of 5 µM NP (21). Pertussis toxin (PTX; 200 ng/ml, Sigma) and the neurotrophins (NGF, BDNF, and NT-3; Upstate Cell Signaling Solutions, Lake Placid, NY) were rehydrated in sterile, distilled water. Neurotrophins were added at 50 ng/ml for neurite outgrowth assays and at 100 ng/ml for signaling assays in Xenopus and were added at 100 ng/ml for all PC12 assays.

Neurite outgrowth analysis
For each plating, images were captured from three to six dishes for each of the different incubation conditions indicated. Cell somata and neurite measurements were made using an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with a Dage-MTI (Michigan City, IN) CCD camera system and NIH Image software (National Institutes of Health, Bethesda, MD). Morphology was analyzed using NIH Image software augmented with a subroutine for collecting and tabulating the results of the analysis (courtesy of C. Daghlian, Dartmouth Medical School) or NIH Image J software (http://rsb.info.nih.gov/ij/). For Xenopus neurons, tracing of neurites emanating from individual cell somata were tabulated for primary neurites (those extending from the cell bodies), the numbers of branch points, and total neurite length. All experiments were performed for platings made from at least two separate batches of embryos.

For PC12 cells, the density of outgrowth did not permit identification of neurites as belonging to a given cell body. Therefore, measurements were made of total neurite length and numbers of cell somata per microscope field. Eight to 12 fields of view (each field contained on average 25 cell somata) were analyzed from each dish for mean neurite length and for cumulative neurite length per cell soma. Results were qualitatively similar from the two measurements, and data reported for PC12 cells are for mean neurite length.

Western blot analyses
Antibodies.
The following antibodies were used: an anti-TrkA affinity purified rabbit polyclonal antibody [TrkA(763), sc-118; Santa Cruz Biotechnologies, Santa Cruz, CA; 1:1000], a mouse monoclonal anti-{alpha}-tubulin antibody (DM1A; Sigma, 1:500), a mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (MAB374; Chemicon International, Temecula, CA; 1:5000), a rabbit polyclonal anti-ERK1/2 (Cell Signaling Technology; 1:500), a mouse monoclonal phospho44/42 MAPK(Erk1/Erk2) (Thr202/Tyr204) (E10; Cell Signaling Technology; 1:500), a rabbit polyclonal anti-ERK5 (3372; Cell Signaling Technology; 1:1000), a rabbit polyclonal anti-phosphoERK5 (Thr218/Tyr220) (3371; Cell Signaling Technology; 1:1000), a mouse monoclonal antiphosphoAkt(Ser 473) (587F11, Cell Signaling Technology; 1:1,000), a rabbit polyclonal antiphospho-glycogen synthase kinase 3ß (GSK3ß)(Ser9) (9336; Cell Signaling Technology; 1:1,000), a rabbit polyclonal anti-GSK3ß (9332; Cell Signaling Technology; 1:1,000), and a rabbit polyclonal antiphospho-cAMP-response element-binding protein (CREB) (Ser133) (9191; Cell Signaling Technology; 1:1000). Species-appropriate secondary antibodies (Pierce Biotechnology Inc., Rockford, IL) were used at 1:500 to 1:10,000. During the course of these experiments, we encountered problems with the anti-GSK3ß antibody that Cell Signaling later acknowledged arose due to unexpected cross-reactivity of this antibody with the phosphorylated molecule, thus giving spuriously high levels of GSK3ß in treatment conditions that augmented phosphoGSK3ß (Cell Signaling, personal communication). To ensure that problems arising from such cross-reactivity were not apparent with other antibodies, levels of phosphoproteins were also normalized to either {alpha}-tubulin or GAPDH. With the exception of the GSK3ß signals in PC12 cells where cross-reactivity was evident, all other experiments gave comparable results whether normalized to the unphosphorylated parent molecule or an independent housekeeping protein.

Xenopus.
For TrkA Westerns on primary Xenopus cultures, cells were lysed in 0.1 ml buffer containing 500 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 0.5% sodium deoxycholate, 0.02% sodium azide, and 10 mM sodium vanadate to which 10 µM aprotinin, 1 µM leupeptin and 1 mM phenylmethylsulfonyl fluoride were added just before use. Total protein concentration in the samples was determined using a BCA Protein Assay Reagent Kit (Pierce Biotechnologies). Equivalent amounts of protein for each treatment condition were separated by SDS-PAGE using 7.5% gels and electrophoretically transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). After washing with Tris-buffered saline [20 mM Tris-Cl and 150 mM NaCl (pH 7.4) and then TBST (20 mM Tris-Cl, 150 mM NaCl, 0.05% Tween 20], membranes were blocked overnight at 4 C in Tris-buffered saline + 5% milk and 2% BSA. Membranes were cut in half, and the higher molecular weight half incubated with the anti-TrkA antibody and the lower half with the mouse monoclonal anti-{alpha}-tubulin antibody, both at room temperature for 1 h. The membranes were washed and incubated with a secondary antibody for 1 h at room temperature. Antibody binding was detected using an enzyme-linked chemiluminescence detection kit (Supersignal West Femto, Pierce Biotechnologies) and visualized on autoradiographic film (Kodak, Rochester, NY). Cells from three to five dishes per each treatment condition were combined and assayed, and the experiment was repeated eight times.

Western blots for phosphorylated CREB (pCREB), pERK, and pGSK3ß in Xenopus were performed on explant tissue. Briefly, the spinal cord and underlying myotomal muscle tissue were dissected from stage 33 tadpoles (36). Two explants per sample were prepared, and tissue was exposed to vehicle [0.01% ethyl alcohol (EtOH)], NGF (100 ng/ml), NP (5 µM), or NGF + NP for 15 min. Tissue was subsequently lysed, and total protein concentration was determined using a BCA Protein Assay. Thirty micrograms of total protein were separated by SDS-PAGE using 7.5% gels and electrophoretically transferred to Immobilon-P polyvinylidene difluoride membrane. The membrane was blocked for 1 h, then incubated overnight with pCREB, pGSK3ß, or pERK antibodies at 1:1000, 1:1000, and 1:500, respectively; secondary at 1:5000. Blots were washed at room temperature before they were incubated for 1 h with secondary antibodies. Blots were washed, and antibody binding was detected using SuperSignal West Femto chemiluminescence detection kit. Subsequently, blots were stripped and reprobed with antibodies directed against unphosphorylated protein.

PC12 cells.
Western blots in PC12 cells were performed after serum deprivation and subsequent treatment with NGF for 15, 30, or 60 min in each treatment condition and carried out according to protocols described above with minor modifications in blocking and incubation parameters, as designated by the protocols provided with each antibody from the manufacturer. Proteins from lysates analyzed for ERK1/2 and GSK3ß were separated on 12% gels; 7.5% gels were used for analysis of ERK5 and Akt. All experiments were performed for at least three separate platings.

Ras activation assay
After serum deprivation and 15 min in each treatment condition, cultures were assayed according to the protocol provided by the manufacturer (Pierce Biotechnologies). Cells were harvested and lysed in 500 µl lysis buffer (provided with the kit), and lysates containing 500 µg total protein were incubated with a GST-Raf1-RBD fusion protein in the presence of an immobilized glutathione disc for 1 h in a spin column, and the mixtures were centrifuged at 7200 x g for 2 min to remove unbound proteins. Resins were washed three times with lysis buffer, centrifuged at 7200 x g for 2 min, and eluted with sodium dodecyl sulfate sample buffer containing ß-mercaptoethanol and boiling for 5 min. Samples were separated by SDS-PAGE on 12% gels. Active ras was detected by Western blot using an anti-ras antibody (1:200; Pierce Biotechnologies).

Immunocytochemical analysis of pCREB
Assessment of levels of phosphorylation of the transcription factor, CREB, were made by immunostaining PC12 cells using a modified version of a protocol obtained from Dr. Joseph Margiotta (Medical University of Ohio, personal communication). Briefly, PC12 cells were grown to approximately 60–70% confluency in 35-mm tissue culture dishes in phenol red-free DMEM. The cultures were serum-deprived, treated for 15 min, and fixed in 4% paraformaldehyde. Nonspecific binding was blocked by incubation in PBS containing 5% normal goat serum and 0.1% Triton-X-100 for 1 h at room temperature and incubated with a rabbit polyclonal antiphosphoCREB(Ser133) antibody (Cell Signaling Technology; 1:75) overnight at 4 C and with an AlexaFluor 594 goat antirabbit IgG (Molecular Probes, Eugene, OR; 1:200) followed by 4,6-diamidino-2-phenylindole (DAPI) nucleic acid stain (Molecular Probes; 300 nM). The cells were viewed on an Olympus BX51 microscope equipped with fluorescence optics using a x20 objective.

PKA assay
PKA activity was measured in PC12 cells grown under conditions described above. For short-term PKA assays, cells were grown for 1 d to reach 75% confluency. For long-term assays, cells were maintained for 7 d and were approximately 90% confluent at the time of assay. Cells were harvested on ice and homogenized in a modified radioimmunoprecipitation extraction buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 10 mM ß-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride. Each of the following protease inhibitors was added at 10 µg/ml: leupeptin, aprotinin, and sodium vanadate. The activity of PKA was determined according to manufacturer’s directions using the SignaTECT cAMP-Dependent PKA Assay System (Promega, Madison, WI).

Statistical analysis
For each experiment, values reported are means ± SEM. Statistical significance was determined by one- or two-way ANOVA using the General Linear Model procedure and the least squares means test of SAS (version 8.2, 2001; SAS Institute, Cary, NC). An {alpha} value of less than 0.05 was established as significant, and all data designated as significant met or exceeded this criterion. To facilitate the reading of the results, statistical values are reported in the text only if data are not shown in the figures. For all data presented in figures, statistical analysis is presented in the corresponding legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of NP on survival and differentiation of primary Xenopus spinal cord neurons in dissociated cell culture
Prior studies of Xenopus spinal cord neurons have demonstrated that only cells that have undergone their final round of DNA synthesis (birth date) will differentiate in dissociated cell culture (39). In cultures made from neural plate stage (stage 15) embryos, over 80% of the differentiated neurons are either Rohon-Beard primary sensory neurons or primary motoneurons (37). To determine whether NP altered the survival or differentiation of Xenopus spinal cord neurons, cultures were prepared from stage 15 embryos and treated with 1 or 10 µM NP or 1 or 10 µM E2. Neither compound at either concentration significantly altered the numbers of neurons that survived (Fig. 1AGo); relative percentages, 100% (EtOH), 103% (NP), and 87% (E2) or their morphological differentiation (neurite outgrowth), as assessed by the numbers of primary neurites and the total length of those neurites (Fig. 1Go, B and C). Both NP and E2 induced small, but significant, increases in the numbers of branch points in these Xenopus neurons (Fig. 1DGo).


Figure 1
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  		FIG. 1. Effects of NP and E2 on differentiation of dissociated Xenopus spinal cord neurons. Neither NP nor E2 altered survival and both had minimal effects on differentiation of Xenopus neurons. Dissociated cultures were prepared from the neural plate of stage 15 Xenopus embryos and maintained in defined, factor-free medium (control) for approximately 36 h, in this medium supplemented with vehicle or this medium supplemented with either NP or E2 at either 1 or 10 µM (data shown for 10 µM). The morphology of Xenopus neurons is variable even in control cultures; photograph (top) shows a typical neuron, alongside a myocyte and several undifferentiated cells. The numbers of differentiated neurons and the extent of neurite outgrowth were measured. No significant differences were observed between cultures maintained in control medium and in medium supplemented with vehicle alone (data not shown). Neither E2 nor NP significantly altered the numbers of neurons that survived and differentiated (A), the number of neurites (B), or total neurite length (C). D, Both E2 and NP slightly, but significantly, enhanced the number of branch points vs. cultures maintained in vehicle alone [F(2,405) = 3.717; P < 0.0251]. *, Post hoc analysis indicated that the number of branch points was significantly greater for NP (P < 0.027) and E2 (P < 0.0333) vs. vehicle. Data represent averages for eight separate dishes per condition.

 
Effects of NP on neurotrophin-induced morphological differentiation of primary Xenopus spinal cord neurons in dissociated cell culture
Cultures prepared from stage 15 embryos responded to treatment with NGF with significant increases in the numbers of primary neurites, the numbers of branch points, and total neurite length per cell. Surprisingly, although exposure of dissociated stage 15 cultures to NP alone did not alter survival and elicited only a modest effect on neurite outgrowth (an increase in the number of branch points, Fig. 1DGo), cotreatment of cultures with 5 µM NP completely blocked all aspects of the NGF-dependent enhancement of neurite outgrowth. In all subsequent morphological assays, comparable effects of NP on neurite length, branch points, and primary neurites were observed, and data are shown only for neurite length (Figs. 2Go, A, B, and D, and 3AGo). Although 5 µM NP reflects the range of tissue concentrations measured in aquatic organisms (10, 11, 12), significant inhibition of neurite length was also observed at 1 µM (P < 0.05), 500 nM (P < 0.01), and 100 nM (P < 0.01) NP (data not shown). For cultures prepared from stage 22 embryos, which contain neurons of more heterogeneous phenotype (40, 41) that respond to BDNF and NT-3 (24), NP also blocked the enhancement of neurite outgrowth induced by BDNF (BDNF vs. BDNF + NP; P < 0.05) and NT-3 (NT-3 vs. NT-3 and NP; P < 0.01) (data not shown).


Figure 2
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  		FIG. 2. NP inhibition of NGF-induced neurite outgrowth. A, NP, E2, and E2-BSA, but not the synthetic androgen, 17{alpha}-methyltestosterone, inhibited NGF-induced neurite outgrowth. All cultures were plated from stage 15 embryos and maintained for 36 h (5 µM NP, E2, or E2-BSA; 50 ng/ml NGF) [F(7,560) = 10.54, P < 0.0001]. *, Post hoc analysis indicated that neurite length was significantly less for vehicle (P < 0.0001), NGF + NP (P < 0.0001), NGF + E2 (P < 0.0001), and NGF + E2-BSA (P < 0.0001) vs. NGF. NGF-induced increases in neurite length were not significantly inhibited by 17{alpha}-methyltestosterone (NGF + 17{alpha}-MeT vs. vehicle; P < 0.0001; no significant difference between NGF and NGF + 17{alpha}-MeT). No differences in neurite length were evident among cultures treated with NGF + NP vs. NGF + E2 vs. NGF + E2-BSA. B, ER antagonist, ICI 182,780, did not inhibit NP’s antagonism of NGF-induced neurite outgrowth [F(5,741) = 17.13, P < 0.0001]. *, Post hoc analysis indicated that neurite length was significantly less for cultures maintained in vehicle, ICI 182,780, alone (5 µM), NGF + NP, and NGF + NP + ICI 182,780 vs. those maintained in NGF (all P < 0.0001). No significant differences were evident among cultures maintained in vehicle, ICI 182,780 alone, and NGF + NP + ICI 182,780. Neurite length was significantly less for cultures maintained in NGF + NP than for vehicle alone (P < 0.0024). C, ICI 182,780 had no effect on NGF-induced outgrowth or on E2-BSA’s antagonism of NGF-induced outgrowth [F(4,208) = 8.75, P < 0.0001]. *, Post hoc analysis indicated that neurite length was significantly less for control, E2-BSA + NGF, E2-BSA + NGF + ICI 182,780 (all P < 0.0001), and E2-BSA (P < 0.0012) vs. NGF alone. Values were not significantly different for vehicle, E2-BSA, E2-BSA + NGF, and E2-BSA + NGF + ICI 182,780. D, PTX had no effect on basal or NGF-induced outgrowth but significantly antagonized NP’s inhibition of NGF-induced neurite outgrowth [F(5,441) = 11.97, P < 0.0001]. *, Post hoc analysis indicated that values were significantly less for vehicle, NGF + NP, and PTX alone (all P < 0.001) vs. NGF. #, NGF + NP + PTX was significantly greater than NGF + NP (P < 0.0001). PTX antagonism was not complete because NGF + NP + PTX was significantly less than NGF alone (0.006).

 

Figure 3
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  		FIG. 3. Role of MAPK signaling in NGF-mediated neurite outgrowth and NP effects in Xenopus. A, NGF-induced neurite outgrowth in Xenopus spinal cord cultures is dependent upon MAPK signaling and is inhibited by the (MEK1/2) inhibitor, U0126. U0126 did not affect basal outgrowth [F(7,535) = 8.83, P < 0.0001]. *, Post hoc analysis indicates that all indicated values were significantly less than NGF (P < 0.0001). Although NP and U0126 appeared to have additive inhibitory effects on NGF-induced outgrowth, post hoc analysis indicated no significant difference for NGF + NP + U0126 vs. NGF + NP (P < 0.137) or NGF + NP + U0126 vs. NGF + U0126 (P < 0.08). B, Three Western blots demonstrating levels of pERK (only 44-kDa band was detected in Xenopus) for stages 32 and 33 tadpole explants treated with vehicle, 100 ng/ml NGF, 5 µM NP, or NGF and NP for 15 min. Fourth blot is for total ERK corresponding to the third of the pERK blots. Bar graphs, Averaged data representing the mean levels of phosphorylated ERK normalized to unphosphorylated ERK (vehicle, 100%). Two-way ANOVA revealed a significant effect of NGF [F(3,16) = 6.59, P = 0.026] but no effect of NP or NGF x NP interaction. *, Post hoc analysis indicated that pERK/ERK in vehicle cultures was significantly less than for NGF + NP (P = 0.0443) and that there was a comparable trend for vehicle vs. NGF (P = 0.0521).

 
Effects of NP on the expression of TrkA in Xenopus
Steroids have been shown to regulate the expression of Trks in neuronal tissue (for review, see Refs. 16 , 17). To assess whether exposure to NP results in a change in the expression of TrkA, Western blot analysis was performed with an anti-TrkA polyclonal antibody raised against the human receptor that recognizes TrkA across a wide range of species. This antibody detected a band of the predicted molecular weight (140 kDa) for mammalian TrkA (42) in mouse brain and a band of 98 kDa in both whole Xenopus embryos and in dissociated Xenopus cell cultures (Fig. 4AGo). No signal was detected in Xenopus tissue when the antibody was preadsorbed with the immunizing peptide (Fig. 4BGo). It is likely that the smaller size of the protein in Xenopus reflects differences in the extent of glycosylation of this protein (for discussion, see Ref. 43). Treatment of Xenopus embryos with NP (10 nM-5 µM; 36 h) had no effect on the signal corresponding to TrkA (normalized to {alpha}-tubulin) (Fig. 4CGo). Treatment of dissociated cultures with 5 µM NP revealed no effect of NGF alone, NP alone, or interaction of NP and NGF on the signal corresponding to TrkA (Fig. 4DGo). For Western blot analysis of these cultures, each culture was first assessed for effects of NGF and NP on neurite outgrowth. As expected from previous experiments (Fig. 1Go), NP alone in these cultures had no effect on neurite outgrowth, NGF promoted significant outgrowth, and the effects of NGF on neurite outgrowth were completely inhibited by NP in all cultures examined.


Figure 4
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  		FIG. 4. Effects of NGF and NP on TrkA expression. A, Representative Western blot on lysates from an adult mouse brain, stage 32 Xenopus tadpoles, and Xenopus cultures prepared from stage 15 embryos and maintained for 36 h. B, Blot on lysates from a mouse brain and a Xenopus embryo demonstrating that the signals were blocked when the antibody was preadsorbed by the immunizing peptide (right). TrkA antiserum detected a signal of the expected molecular weight (140 kDa) in mouse brain and a smaller band (98 kDa) in Xenopus tissue. Lines, Parts of the gel in which there were empty lanes were removed from the image. C, Representative Western blot for TrkA for lysates from stage 32 embryos treated with a range of NP concentrations (10 nM to 5 µM) and densitometric measurements demonstrating no apparent changes in TrkA levels (when normalized to {alpha}-tubulin). D, Representative Western blot from cultures prepared from stage 15 embryos and maintained for 36 h in medium alone (control), NGF, NP (5 µM), and NGF and NP (NGF + NP) and averaged data for TrkA expression normalized to {alpha}-tubulin. Two-way ANOVA revealed no significant effect of NGF, NP, or NGF x NP interaction on TrkA expression (normalized to {alpha}-tubulin; n = 9 separate platings).

 
Role of ERs in mediating effects of NP on NGF-induced neurite outgrowth in Xenopus
To determine whether the ability of NP to block NGF effects on neurite outgrowth was dependent upon signaling mediated via nuclear ER, the effects of the antiestrogen, ICI 182,780 (38), were examined. ICI 182,780 has been shown to inhibit estrogen-mediated signaling at the Xenopus vitellogenin promoter (44, 45) and inhibit NP-induced alterations in Xenopus melanocytes in intact embryos (21), indicating that this antiestrogen is active in Xenopus. ICI 182,780 did not significantly alter cell survival (115 ± 11% of control cultures), basal outgrowth (Fig. 2BGo), NGF-induced outgrowth (Fig. 2CGo), or the ability of NP to inhibit NGF’s effects (Fig. 2BGo).

Estrogenic compounds, including NP, induce rapid membrane-initiated signaling (46, 47, 48, 49) that may arise from G protein-coupled membrane receptors (for review, see Refs. 50 , 51). Because conjugated forms of NP are not available, but NP effects are mimicked by E2, we tested whether E2-BSA (5 µM) would also inhibit NGF-induced outgrowth in Xenopus neurons. Although E2-BSA alone had no effect on basal neurite length (Fig. 2CGo), this conjugated estrogen, like NP and E2, significantly inhibited NGF-induced neurite outgrowth (Fig. 2Go, A and C). As with NP, inhibition of NGF-induced outgrowth by E2-BSA was also insensitive to ICI 182,780 (Fig. 2DGo). In contrast, cotreatment of Xenopus cultures with NGF and the synthetic androgen, 17{alpha}-methyltestosterone (5 µM), had no significant effect on the ability of NGF to enhance neurite outgrowth (Fig. 2AGo). These results indicate that the effects of NP can be mimicked by an estrogen whose actions are membrane-initiated and that they are not mimicked by a nonestrogenic steroid.

To test whether the effects of NP involved G protein-mediated signaling, cultures were coincubated with the Gi/o inhibitor, PTX (52). PTX alone had no significant effect on cultures maintained in vehicle-supplemented or control medium or on the ability of NGF to elicit neurite outgrowth in Xenopus neurons (Fig. 2DGo). Concomitant treatment with PTX significantly antagonized the ability of NP to inhibit NGF-dependent neurite outgrowth, although the antagonism of NP effects was not complete (Fig. 2DGo). Thus, although neurite outgrowth was significantly greater in cultures treated with NGF, NP, and PTX than in those treated with NGF and NP (Fig. 2DGo), it was nonetheless still significantly less than in cultures treated with NGF alone or NGF and PTX (Fig. 2DGo).

Effects of NP on NGF signaling pathways in Xenopus
In neurons, the membrane-initiated effects of E2 and those of NGF are known to converge on the MAPK pathway (28, 29, 30, 31, 32, 33, 34). To determine whether NGF-dependent neurite outgrowth in Xenopus neurons requires MAPK signaling, cultures were maintained in the presence of NGF or cotreated with NGF and the MEK1/2 inhibitor, U0126 (10 µM). U0126 alone was without effect on basal neurite outgrowth but significantly reduced NGF-dependent neurite outgrowth (Fig. 3AGo). Although concomitant treatment with U0126 and NP appeared to lead to a greater diminution in NGF-induced outgrowth than did either U0126 or NP with NGF, the differences did not attain significance (Fig. 3AGo).

To further determine whether NP altered signaling along the MAPK pathway, Western blot analysis for NGF-induced phosphorylation of ERK was performed. Because of the paucity of cells in Xenopus primary cultures, biochemical assays of cultures were not reliably successful. Therefore, assays were performed in tissue harvested from stages 32 and 33 embryos (approximately equivalent hours postfertilization as when determinations of neurite outgrowth were made for dissociated neurons in cell culture). A band corresponding to the 44-kDa ERK isoform was detected in all tissue, including basal levels of this phosphoprotein in vehicle-treated explants. Although results from batch of embryos to batch of embryos were variable (see three representative Western blots in Fig. 3BGo), averaged data indicated that NGF (15 min) elicited a significant increase in the level of pERK vs. vehicle-treated tissue (Fig. 3BGo). In contrast, NP (5 µM) alone did not increase basal levels of pERK and did not diminish NGF-induced phosphorylation (Fig. 3BGo). Western blot analysis was also performed for the effects of NP on phosphorylated levels of the transcription factor, CREB, a critical downstream effector in the NGF-MAPK pathway (for review, see Refs. 14 and 27) and a point of convergence for G protein-coupled receptor signaling pathways (27). NP alone did not significantly alter the levels of pCREB or alter NGF-induced phosphorylation of this factor. However, as with pERK analysis in Xenopus explants, results for pCREB from experiment to experiment were variable (data not shown). In addition to MAPK signaling, the phosphatidylinositol 3' (PI3) kinase-Akt kinase-GSK3ß pathway has also been implicated in mediating NGF effects on neurite outgrowth (53, 54, 55, 56 ; for review, see Ref. 26). Western blot analysis of explants of stages 32 and 33 Xenopus embryos did not demonstrate a significant effect of NP on either basal or NGF-induced levels of pAkt or pGSK3ß, but, as with pCREB and pERK, results on explant tissue were variable from experiment to experiment (data not shown).

Although none of the Western blot data on Xenopus explants supports a role for NP (at 5 µM) in eliciting changes in either the MAPK or PIK3-Akt-GSK3ß signaling or in interfering with NGF activation of these pathways, the variability from experiment to experiment for explant tissue made interpretation of this data difficult. Xenopus explants include a multiplicity of cell types, embryos of unknown sex, and potential differences from embryo to embryo with regard to access of NGF or NP through the tissue, all of which may have contributed to the observed variability. Thus, to address the biochemical mechanisms by which NP may be affecting NGF signaling in a more homogenous cell population, experiments were subsequently performed in PC12 cells, a cell line that has been thoroughly studied with respect to the biochemical pathways that mediate neurotrophin signaling, which can be maintained under conditions that minimize basal phosphorylation of phosphoproteins and optimize accessibility of reagents, and in which cell type variability is more limited.

The effects of NP on NGF signaling in PC12 cells
PC12 cells in their basal state express nuclear ER{alpha} and ERß (15, 57, 58, 59) and membrane ER (57). Although the rapid effects of estrogens on growth factor signaling pathways in PC12 cells have not been fully explored, E2 elicits rapid signaling via Akt (57) and interacts with IGF-I receptor signaling to promote neurite outgrowth in PC12 cells that express ER{alpha} (58).

As in primary cultures of Xenopus neurons, NP alone (5 µM) did not affect survival or promote neurite extension in PC12 cultures in either serum-containing or -stripped (steroid-depleted and phenol red-free) medium. Moreover, treatment of PC12 cells maintained in serum-stripped medium with the ER antagonist, ICI 182,780, was without effect on neuron survival, suggesting minimal levels of estrogens in this steroid-depleted medium. Specifically, under these culture conditions, the average number of cells per field (60 total fields examined per condition) was 24 ± 3 (NGF), 26 ± 3 (NGF and NP), 29 ± 3 (NGF and ICI 182,780), and 28 ± 3 (NGF, NP, and ICI 182,780). In addition, NP did not elicit phosphorylation of ERK5 or abrogate NGF-induced phosphorylation of ERK5, a signaling molecule implicated as critical for mediating NGF effects on survival (60) (Fig. 5AGo). Thus, as with Xenopus neurons, treatment with micromolar concentrations of NP was without noted effect on PC12 cell survival or basal neurite outgrowth.


Figure 5
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  		FIG. 5. NP does not alter NGF-induced MAPK or PI3-Akt-GSK3ß signaling in PC12 cells. A, Representative blots showing that NGF (100 ng/ml; 15 min) increased levels of activated ras and phosphorylated pERK1/2 and pERK5 but that NP did not elicit activation of the MAPK pathway nor antagonize NGF-induced signaling. Two-way ANOVA revealed a trend toward a significant effect of NGF on levels of activated ras [F(3,12) = 4.56, P = 0.0541] and a significant effect of NGF on levels of pERK1/2 [F(3,11) = 28.21, P = 0.0002] but no effect of NP or NGF x NP interaction. Analysis of pERK5 revealed comparable activation by NGF and lack of effect of NP as shown for pERK1/2. For ERK blots, comparable data were obtained for cells exposed to NGF for 30 or 60 min (data not shown). B, Representative Western blots showing that NGF (15 min) increased levels of phosphorylated Akt and GSK3ß. NP alone did not induce phosphorylation of either Akt or GSK3ß nor did NP inhibit NGF-induced signaling along the PI3 kinase-Akt-GSK3ß pathway. Two-way ANOVA of averaged data from different platings revealed a significant effect of NGF on levels of pAkt [F(3,16) = 13.70, P = 0.0019] and pGSK3ß [F(3,28) = 10.49, P = 0.0031] but no effect of NP and no NGF x NP interaction. Post hoc analysis indicated values for NGF and NGF + NP were significantly greater than control for pAkt (P < 0.0169 and 0.0087, respectively) and pGSK3ß (P < 0.026 and P < 0.0334, respectively). For pAkt, comparable results were obtained when blots were normalized to unphosphorylated Akt or GAPDH. Problems with cross-reactivity for the GSK3ß antibodies precluded accurate normalization to GSK3ß (see Materials and Methods).

 
NGF elicited robust neurite outgrowth for cells maintained in serum-supplemented or -stripped medium (Fig. 6Go). As with primary Xenopus neurons, cotreatment with NP significantly decreased NGF-elicited neurite outgrowth in either medium (Fig. 6Go), Moreover, as with Xenopus neurons, significant inhibitory effects of NP on NGF-induced outgrowth were evident for concentrations as low as 50 nM (P < 0.0290; data not shown). Finally, as with Xenopus neurons, for PC12 cells maintained in serum-stripped medium, ICI 182,780 was without effect on NGF-induced outgrowth and did not antagonize the inhibitory effects of NP (Fig. 6CGo). Taken together, these results indicate that NP did not alter cell survival or elicit neurite outgrowth in the absence of NGF in PC12 cells but did inhibit NGF-induced outgrowth via a mechanism that is independent of nuclear ER and thus has parallel effects in this cell line and in primary Xenopus neurons. We did observe that ICI 182,780 significantly inhibited NGF-induced outgrowth in PC12 cells maintained in serum-supplemented medium (P < 0.0013 for NGF and ICI 182,780 vs. NGF alone; data not shown), an effect that most likely reflects the antagonistic action of ICI 182,780 on the nuclear ER-mediated outgrowth-promoting effects of estrogens present in serum (16, 59).


Figure 6
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  		FIG. 6. NP effects on NGF-dependent neurite outgrowth in the PC12 cell line. A, Representative micrographs of PC12 cells maintained in serum-stripped medium in the presence of NGF (100 ng/ml; left), NGF and NP (5 µM) (center), or NGF, NP, and ICI 182,780 (5 µM) (right). B, NP has no effect on basal neurite outgrowth but significantly inhibited NGF-induced outgrowth in cells maintained in serum-supplemented medium [F(4,45) = 97.66, P < 0.0001]. *, Post hoc analysis indicated all other conditions were significantly less than NGF (all P < 0.0001); however, NP inhibition of NGF-induced outgrowth was not complete; NGF + NP was significantly greater than control, vehicle, and NP alone (all P < 0.0001). B, NGF also elicited significant neurite outgrowth in cultures maintained in serum-stripped and phenol red-free medium, which was inhibited by NP. ICI 182, 780 did not antagonize NGF-induced outgrowth in serum-stripped medium, nor did it antagonize NP’s inhibition of NGF-induced outgrowth [F(>4,266) = 25.88, P < 0.0001]. *, Post hoc analysis indicated that outgrowth was significantly less for vehicle, NGF + NP, and NGF + NP + ICI 182,780 than for cultures treated with NGF or NGF + ICI 182,780 (all P < 0.0001). As with PC12 cells in serum-containing medium, NP’s inhibitory effects were not complete, and values for NGF + NP and NGF + nonylphenol + ICI 182,780 were significantly greater than vehicle (both P < 0.0001).

 
The effects of PTX and E2-BSA were also tested for PC12 cells; however, both compounds had unexpected effects that did not permit conclusions to be drawn from these experiments. Specifically, PTX had a dramatic inhibitory effect on NGF-induced neurite outgrowth in PC12 cells; neurite length in cultures supplemented with NGF and PTX was only 38% of that observed for NGF alone (P < 0.0008). Because long-term PTX treatment reduced NGF-dependent outgrowth to nearly basal levels, it was not possible to assess whether inhibitory effects of NP were affected by cotreatment with PTX. With respect to E2-BSA, long-term treatment with this BSA-conjugated steroid induced notable cell death, and the remaining cells had grossly altered morphology, in which most cells were characterized as flat, large, and fibroblast-like.

Interactions of NP with the NGF-mediated MAPK and PI3 kinase-Akt signaling cascades in PC12 cells
Consistent with previous reports (61, 62, 63), NGF treatment of PC12 cells resulted in an increase in the levels of activated ras, as detected by a pull-down reaction using a GST-Raf1-RBD fusion protein and subsequent Western blot analysis, but NP alone had no effect on basal ras levels, nor did it alter NGF-induced activation of ras (Fig. 5AGo). Similarly, NGF elicited significant increases in the level of phosphorylated ERK1/2, whereas NP alone was without effect and did not abrogate the ability of NGF to induce phosphorylation of ERK1/2 (Fig. 5AGo). With respect to NGF-induced signaling along the PI3 kinase-Akt-GSK3ß pathway, results were the same; NGF enhanced phosphorylation of both Akt and GSK3ß, but NP neither promoted phosphorylation of either Akt or GSK3ß nor diminished NGF-dependent phosphorylation of these molecules (Fig. 5BGo).

Interactions of NP with the NGF-mediated enhancement of PKA activity and phosphorylation of CREB in PC12 cells
The first described rapid effect of estrogen, demonstrated nearly 40 yr ago, was a rise of cAMP in uterine tissue (64), later shown to result from nongenomic actions of estrogen on membrane adenylate cyclase and increased levels of cAMP (65). NP alone was without effect on either PKA activity or levels of pCREB (Fig. 7Go). However, NGF treatment (15 min) elicited a significant increase in PKA activity, and cotreatment with NP significantly antagonized this action of NGF (Fig. 7AGo). Analysis of averaged data indicated that treatment with NGF also elicited a significant increase in the level of pCREB vs. vehicle-treated cultures (Fig. 7BGo), but the stimulatory effect of NGF was not inhibited by NP (Fig. 7BGo). Immunostaining corresponding to the phosphorylated form of CREB was negligible in untreated PC12 cells, in PC12 cells treated with vehicle (Fig. 7DGo, bottom), or in PC12 cells treated with NP alone (Fig. 7EGo, bottom). NGF promoted marked phosphorylation and nuclear localization of CREB (Fig. 7Go, B and C, bottom). Consistent with Western blot analysis, the ability of NGF to phosphorylate CREB and promote nuclear localization of this phosphoprotein was not blocked by coincubation with NP (Fig. 7FGo, bottom). The lack of demonstrable effect of NP on phosphorylation of CREB is not necessarily inconsistent with the observed NP-mediated decreases in PKA activity because CREB is downstream of a host of kinase pathways, including the MAPK (for review, see Ref. 66).


Figure 7
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  		FIG. 7. NP inhibits the ability of NGF to increase PKA activity but does not alter CREB phosphorylation in PC12 cells. Top, A, Average data demonstrating that NGF (100 ng/ml; 15 min) increased PKA activity (normalized to control at 100%), NP alone had no effect, and NP decreased the NGF-induced increase. Two-way ANOVA revealed a significant effect of NGF [F(3,21) = 6.93, P = 0.0045], no significant effect of NP, and a significant NGF x NP interaction [F(3,21) = 7.95, P = 0.0010]. *, Post hoc analysis indicated that values were significantly less than for NGF alone for control (P = 0.0002), NP alone (P = 0.0026), and NGF + NP (P = 0.0046). No significant differences were evident between control, NP, and NGF + NP. Prolonged (7 d) exposure of PC12 cells to NGF, NP alone, or NP + NGF did not promote any change in PKA activity (data not shown). B, Averaged data for Western blots assessing levels of pCREB normalized to loading control and expressed relative to vehicle alone (100%), demonstrating that NGF (100 ng/ml; 15 min) increased levels of pCREB, whereas NP alone had no effect, and NP did not antagonize the NGF-induced increase. Two-way ANOVA revealed a significant effect of NGF [F(3,12) = 6.93. P = 0.0218] but no effect of NP or NGF x NP interaction. *, Post hoc analysis indicated that outgrowth was significantly less in cultures maintained in vehicle than in NGF (P = 0.0310). Outgrowth was also less in vehicle than for NGF + NP (P = 0.0302). Bottom, Representative bright-field (A) and fluorescence (B and C) micrographs of a single field of PC12 cells treated with NGF (15 min) and incubated with an antibody directed against pCREB and the nuclear stain, DAPI. B, Immunostaining for pCREB. C, Coincident staining for pCREB and DAPI indicating nuclear localization. D to F, Representative fluorescence micrographs of PC12 cells treated with vehicle alone (EtOH; D), NP (E), or NGF and NP (F) demonstrating that only background levels of immunostaining were evident in cultures treated with vehicle alone or with NP alone and that NP did not prevent NGF-dependent phosphorylation of CREB. Results were repeated in three to five dishes from three separate platings.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown previously that exposure to NP during early embryonic development in Xenopus induces a wide range of deficits, some of which may be consistent with NP interfering with neurotrophin-induced differentiation (21). Specifically, Xenopus embryos treated with NP in early gastrula and examined as free-swimming tadpoles exhibited significant aberrations in the migration and differentiation of NGF-sensitive neural crest-derived melanocytes. In addition, NP-treated tadpoles exhibited behavioral manifestations (abnormal swimming patterns and spasticity when touched) that may reflect aberrant patterns of sensory or motor inputs. NP’s effects on melanocyte differentiation in intact embryos were blocked by ICI 182,780 (21), indicating that NP can interfere with nuclear ER-mediated signaling in Xenopus and that ICI 182,780 is an active antagonist in this species, consistent with prior studies (44, 45). Here, we demonstrate that NP itself did not alter the survival of dissociated Xenopus spinal cord neurons, including neural crest-derived Rohon-Beard cells (67), or of the PC12 cell line. Moreover, NP alone at micromolar concentrations was without effect (positive or negative) on outgrowth in PC12 cells and had minimal effects in Xenopus neurons, promoting only a slight enhancement of the number of branch points but no change in neurite length or the number of primary neurites. These data indicate that NP does not have a general inhibitory effect on cell metabolism, nor does it, by itself, either enhance or inhibit neurite initiation or alter fundamental processes associated with elongation, such as microtubule assembly. Surprisingly, however, NP completely inhibited the ability of NGF to enhance neurite outgrowth in stage 15 Xenopus cultures and significantly inhibited the neurite-promoting effects of BDNF and NT-3 in cultures prepared from older embryos. Significant inhibitory effects of NP on neurite outgrowth for both Xenopus and PC12 cells were observed at concentrations ranging from high nanomolar to low micromolar levels. Tissue concentrations of NP in aquatic organisms have been estimated to be in the 1–20 µM range (10, 11, 12). Thus, the effects of NP described here occur at environmentally relevant concentrations. Moreover, because NP bioaccumulates (7, 8, 9), our results may be broadly applicable to a wide range of not just aquatic, but terrestrial, species that are higher in the food chain.

There is a wealth of literature demonstrating that NP is an agonist of nuclear ER-mediated transcription, and classical activity of this nuclear ER mediates many of its reported biological actions (for review, see Ref. 68). It was thus surprising that the nuclear ER antagonist, ICI 182,780, did not prohibit the inhibitory effects of NP on NGF-mediated process outgrowth in either primary Xenopus neurons or in PC12 cells maintained in serum-stripped (steroid-depleted) medium. Our data also demonstrate that inhibitory effects of NP on NGF-induced outgrowth in Xenopus neurons were mimicked by the membrane-impermeant estrogen, E2-BSA, but not by the synthetic androgen, 17{alpha}-methyltestosterone, and that effects of E2-BSA were also insensitive to ICI 182,780. Finally, our data demonstrate that the inhibitory effects of NP on NGF-induced outgrowth in Xenopus could be significantly antagonized by the Gi/o inhibitor, PTX. Although free E2 arising from dissociation of the BSA-conjugated estrogen is likely to be present in cultures incubated with E2-BSA for 36 h (69), and we cannot categorically rule out a role for free E2 in these experiments, when taken in conjunction with the lack of antagonism by ICI 182, 780 for either NP or E2-BSA and the antagonism of NP’s action by PTX, our data support the hypothesis that NP acts via a nonclassical mechanism of estrogenic signaling (membrane ER- or intracellular ER-mediated, nonnuclear signaling) to suppress the growth-promoting effects of NGF without eliciting global effects on cell viability or energy homeostasis.

With respect to common and divergent effects in the two different cell types, we do note that although the actions of NP on cell survival and on NGF-induced outgrowth were comparable for Xenopus and PC12 cells, the effects of E2-BSA and PTX were not. Specifically, although neither E2-BSA nor PTX affected survival, basal outgrowth, or NGF-induced outgrowth in Xenopus neurons, E2-BSA decreased survival of PC12 cells, and PTX antagonized NGF-induced outgrowth in this cell line. These differences may reflect a general difference between Xenopus cultures, in which only postmitotic neurons differentiate, and the PC12 cell line, in which cells are not postmitotic until NGF supplementation. Conversely, these differences may reflect that these agents may activate multiple and/or distinct downstream pathways in a cell type-specific manner.

Sustained signaling mediated by the MAPK cascade is essential for NGF-mediated process outgrowth in neural crest-derived sympathetic and sensory neurons and in the PC12 cell line (for review, see Refs. 26 and 70). We show here that NGF-induced neurite outgrowth in cultured Xenopus neurons that include crest-derived Rohon-Beard cells is also dependent on MAPK signaling. Previous reports in nonneuronal cells have demonstrated that, like physiological E2, lower concentrations of NP can promote signaling along the MAPK cascade (47, 48, 49). Thus, we pursued as a likely hypothesis that the ability of micromolar concentrations of NP to inhibit NGF-induced outgrowth might arise if higher levels of this EDC blocked NGF-dependent MAPK signaling. Contrary to expectations, NP at micromolar concentrations neither activated MAPK signaling nor interfered with NGF-induced MAPK signaling in either Xenopus neurons or PC12 cells. Activation of the PI3 kinase-Akt pathway has also been shown to play an important role in promoting NGF-induced process outgrowth and elongation (54, 55, 56 ; for review, see Ref. 26). However, contrary to expectations, here again, micromolar concentrations of NP neither promoted phosphorylation of Akt or GSK3ß nor interfered with the ability of NGF to do so. Interestingly, overexpression of Akt inhibits NGF-dependent outgrowth in PC12 cells (71), underscoring the relevance of concentrations of signaling molecules in eliciting biological effects. In summary, our data suggest that the inhibitory actions of NP (at environmentally relevant concentrations) on NGF-induced outgrowth do not arise from interference of NGF signaling along these critical pathways.

Our data from PC12 cells indicate that NP does elicit a significant inhibition of NGF-induced increases in PKA activity. The roles of cAMP and PKA in NGF-mediated neurite outgrowth have been controversial (72, 73, 74, 75). Brief treatments with NGF elicit increases in PKA activity/cAMP in PC12 cells (73, 75), and this increase has been suggested to be PTX-sensitive (75). However, use of a dominant negative construct in PC12 cells has shown that NGF can promote differentiation of PC12 cells, including neurite outgrowth, by pathways that are distinct from cAMP-activated PKA (74). Although these data indicate that PKA activity is not required for NGF-induced differentiation, the authors note that PKA may nonetheless play an indirect role in mediating the responses of PC12 cells to NGF (74). Specifically, NGF-dependent changes in cAMP/PKA may interact with other signaling pathways to alter neurite outgrowth. Recent studies in primary neurons indicate that the ultimate endpoint of process outgrowth reflects the assimilation by growth cones of signaling events arising from an array of environmental cues, many of which may act through common effectors, such as PKA. Thus, NP could promote signaling events that countermand the neurite-promoting actions of NGF without directly blocking the major signaling pathways believed to underlie NGF’s actions. Of particular interest are experiments in Xenopus neurons demonstrating that alterations in cAMP and PKA activity have profound effects on the attractant/repulsive properties of a number of environmental cues, including the neurotrophins (24, 76, 77). The complexity of potential interactions between neurotrophin- and G protein-mediated pathways is also illustrated by Cai et al. (78), who demonstrate that priming of sensory neurons with BDNF or NGF activates PKA and by doing so can block the inhibitory effects on axon outgrowth of myelin-associated glycoprotein, a protein that inhibits PKA activity via a G{alpha}i-dependent mechanism. NGF has also been shown to reduce growth cone collapse of sensory neurons induced by semaphorin 3A, and this effect requires PKA activation (79). NGF-dependent increases in PKA activity may enhance the attractant effects of other environmental cues. NP, by countermanding this NGF-dependent enhancement, may blunt the growth-promoting actions of these other exogenous signals.

In summary, although the mechanisms underlying the inhibitory action of NP are not well understood, our data demonstrate that this environmental estrogen, at concentrations evident in tissue of aquatic organisms, acts via a G protein-sensitive, nuclear ER-independent mechanism to interfere with neurotrophin-induced differentiation. The ability of environmental estrogens, when present during inappropriate developmental epochs, to disrupt the formation of reproductive structures is well documented. Our results highlight the fact that these ubiquitous contaminants may also have widespread effects on the development of the nervous system arising from their actions via both classical and nonclassical mechanisms that may contribute to impaired cognitive dysfunction that has been reported with in utero EDC exposure (for review, see Ref. 80). Most important, our results underscore the fact that the actions of EDCs such as NP, even though categorized as estrogen mimetics, may dramatically diverge from the known effects of physiological estrogens due to their markedly greater degree of bioaccumulation and elevated tissue levels.


   Acknowledgments
 
This work partially fulfills the requirements for the Ph.D. for C.L.B. We thank Dr. Robert Maue for many hours of advice and assistance, Dr. William Snider for helpful suggestions, Dr. Robert Campenot and Dr. John Bixby for comments on the manuscript, and Dr. Beth Costine for statistical analysis.


   Footnotes
 
This work was supported by the National Institutes of Health (Grants ES10143 to L.P.H., NS40644 to M.J.H., and T32 DK07508 for C.L.B.).

Present address for C.L.B.: Biology Department, Colorado State University, Pueblo, Colorado 81001.

Disclosure summary: all authors have nothing to declare.

First Published Online June 15, 2006

Abbreviations: BDNF, Brain-derived neurotrophic factor; CREB, cAMP-response element-binding protein; DAPI, 4,6-diamidino-2-phenylindole; E2, 17ß-estradiol; E2-BSA, ß-estradiol 6-(O-carboxy-methyl)oxime: BSA; EDC, endocrine-disrupting compound; ER, estrogen receptor; EtOH, ethyl alcohol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK3ß, glycogen synthase kinase 3ß; MEK, MAPK kinase; 17{alpha}-methyltestosterone, 17{alpha}-methyl-4-androstene-17ß-ol-3-one; NGF, nerve growth factor; NP, nonylphenol; NT-3, neurotrophin-3; pCREB, phosphorylated CREB; PI3, phosphatidylinositol 3'; PKA, protein kinase A; PTX, pertussis toxin.

Received May 2, 2006.

Accepted for publication June 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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