From the
Department of Biochemistry and Molecular Biology and the ¶Department of Neuroscience, Finch University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064 and the ||Departments of Physiology and Biophysics, and Anatomy and Neurobiology, University of California College of Medicine, Irvine, California 92697
Received for publication, December 3, 2002 , and in revised form, April 15, 2003.
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ABSTRACT |
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INTRODUCTION |
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Biochemical analyses have suggested that neurotrophin receptors can form three different types of receptor complexes: Trk homodimers, p75NTR homo-oligomers, and a mixed complex of uncertain stoichiometry containing both Trk and p75NTR. Demonstrating direct interactions between p75NTR and TrkA receptors has been difficult, hinting at the subtle and, perhaps, weak nature of the interactions. Evidence suggesting a direct physical interaction between the two receptors now includes cross-linking (8), co-patching (13), and immunoprecipitation (14). However, a critical question in neurotrophin signaling is whether heteromeric TrkA-p75NTR complexes possess signaling capacities that are inherently different from those of either TrkA or p75NTR alone. Furthermore, the contribution of each receptor oligomer to downstream signaling and cellular effects remains to be fully elucidated. Functionally, signaling by TrkA and p75NTR may be synergistic, independent, or antagonistic. How the neurotrophin receptors act individually and together to regulate the responses of cells to neurotrophins and the nature of the intracellular signals used by these receptors to exert their effects are key questions in neurotrophin signal transduction.
We sought to better understand the contribution of each receptor alone, as well as in combination, by examining differences in signal transduction and consequent cellular effects. A stable PC12 cell line expressing a chimeric PDGFR-TrkA receptor (abbreviated PTR cells) (15) has been used previously (16) to stimulate intracellular TrkA signaling alone and demonstrate activation of a TrkA to nuclear factor B pathway. The chimeric receptor contains the extracellular domain of the human PDGFR fused to the transmembrane and intracellular domains of TrkA (diagrammed in Ref. 16). Because the PC12 cell line for studying the effects of NGF does not normally express PDGF receptors (PDGFR), addition of PDGF to PTR cells allows selective activation of the PDGF/TrkA chimera and TrkA receptor-specific signaling programs but not the activation of endogenous TrkA (15, 16). In addition, the p75NTR-selective NGF mutant,
9/13 NGF, that lacks TrkA binding but retains normal binding to p75NTR (3, 17), allows specific activation of p75NTR signaling pathways alone.
In the present study, we compared the separate but simultaneous activation of TrkA and p75NTR oligomers versus functional analysis of NGF high affinity receptor formation. Thus, the contributions of p75NTR and TrkA receptor homomers and heteromers were separated in terms of two major downstream signaling pathways (Ras-MAPK and PI3-kinase-Akt) as well as specific cellular end points (e.g. differentiation and survival). Although a pre-formed NGF heteromeric TrkA-p75NTR complex was detected, the interaction between the two receptors was greatly stabilized upon NGF addition. In addition, activation of TrkA alone is sufficient for eliciting neurite outgrowth, as expected, with p75NTR signaling playing a modulatory role. The high affinity heteromeric NGF receptor site is necessary for integrating the MAPK and Akt pathways to result in complete neurite outgrowth. Although signaling through either TrkA or p75NTR alone facilitated increased cell survival, activation of the high affinity NGF receptor site was necessary for rapid and sustained activation of Akt and long term cell survival. Furthermore, a novel cross-talk mechanism between downstream receptor pathways exists, with negative regulation of the PI3-kinase-Akt pathway by the Ras-MAPK pathway.
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MATERIALS AND METHODS |
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Cell Culture and Cell LinesPC12 cells (TrkA+ and p75+) (a kind gift from L. A. Greene) and PTR cells (PDGFR-TrkA+, TrkA+, and p75+) (15) were grown in complete Dulbecco's modified Eagle's medium containing serum ("serum medium": 10% horse serum (Fisher), 5% fetal bovine serum (Fisher), 9 mM glutamine, 10 IU penicillin, 10 µg/ml streptomycin, 0.025 µg/ml amphotericin B (last three combined as antibiotic/antimycotic from Fisher)). PC12 and PTR cells were maintained as described (18). Briefly, the cells were kept in serum medium, split every 45 days at 90% confluency, and removed from that environment only 24 h before an experiment. Exponentially growing PC12 or PTR cells were split and plated onto poly-L-ornithine-coated plates at least 36 h before treatment with growth factor.
Growth Factor Stimulation and Cell Lysate PreparationPC12 and PTR cells were serum-deprived overnight in Dulbecco's modified Eagle's medium containing 0.5% FBS, 9 mM glutamine, 10 IU penicillin, 10 µg/ml streptomycin, 0.025 µg/ml amphotericin B (last three combined as antibiotic/antimycotic from Fisher) and then stimulated for the indicated time at 37 °C with neurotrophin. NGF was prepared and purified from male mouse submandibular gland (19). The p75-selective
9/13 NGF protein was overexpressed and purified from a baculovirus cell system as described previously (3). BDNF, NT-3, and NT-4/5 were kind gifts from Amgen (Thousand Oaks, CA) to KEN. Cells were stimulated for the indicated time with 50 ng/ml NGF,
9/13, BDNF, NT-3, or NT-4/5 in 0.5% serum medium. After stimulation, cells were washed twice with ice-cold phosphate-buffered saline (PBS) on ice, solubilized with lysis buffer as described below, followed by sonication. Lysates were clarified by centrifugation at 12,000 x g for 20 min at 4 °C. Protein concentration was determined by the BCA method (Pierce), and equal amounts of protein (ranging from 50 to 100 µg) were loaded in each experiment. Loading controls were done in most cases.
Immunoprecipitation and Western Blotting AnalysisAfter treatment with neurotrophin for the indicated time, cells were washed twice with ice-cold PBS and lysed at 4 °C in 300 µl of lysis buffer (50 mM HEPES, pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate). Lysates were then sonicated followed by centrifugation at 4 °C at 12,000 x g for 20 min. The supernatants were transferred to new tubes, and protein concentration was quantitated with the BCA reagent (Pierce). Equal amounts of protein (ranging from 500 µg to 1 mg) were then incubated with 2 µg of the appropriate immunoprecipitating antibody, e.g. TrkA or p75NTR antibody (Santa Cruz Biotechnology), and the remaining volume was raised to 500 µl with ice-cold PBS. This mixture was allowed to rock at 4 °C for 2 h, followed by addition of 20 µl of protein A/G slurry (Santa Cruz Biotechnology) overnight at 4 °C. Immunoprecipitates were then collected by centrifugation at 12,000 x g for 15 min, and the supernatants were removed by aspiration. The pellet of beads was then washed 4 times in ice-cold PBS, centrifuging between washes. After the final wash, the supernatant was aspirated, and the pellet was resuspended in 50 µl of SDS sample buffer. Samples were boiled for 3 min and clarified by centrifugation. 25 µl were loaded and separated by SDS-PAGE.
Proteins were then transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked in TBST (10 mM Tris base, pH 7.5, 137 mM NaCl and 0.1% Tween 20) containing 5% non-fat dry milk overnight at 4 °C. The blots were then washed thoroughly with TBST and probed with the indicated primary antibody in 5% bovine serum albumin/TBST overnight at 4 °C. Blots were then washed and incubated with either goat anti-rabbit (Kirkegaard & Perry Laboratories) or goat anti-mouse (Bio-Rad) horseradish peroxidase-conjugated secondary antibody in 5% milk/TBST for1hat room temperature. Blots were then extensively washed and visualized using the ECL chemiluminescence system (Amersham Biosciences) and Fuji XR film. Bands were quantified by densitometry using LKB image documentation system with Molecular Analyst software. Conclusions were drawn after experiments were repeated a minimum of three times.
Neurite Outgrowth BioassayNeurite outgrowth bioassays were done as described previously (20). Briefly, 96-well plates were coated with collagen (Vitrogen 100) and allowed to dry overnight. Collagen-coated plates were seeded with 5 x 103 cells/well either in 0.5% serum medium or defined medium (20 nM progesterone (Sigma), 100 µM putrescine (Sigma), 5 µg/ml insulin, 5 µg/ml selenium, 5 µg/ml transferring (last three combined as ITS from Sigma), 9 mM glutamine (Sigma), 10 IU penicillin, 10 µg/ml streptomycin, and 0.025 µg/ml amphotericin B (last three combined as antibiotic/antimycotic from Invitrogen)). Neurite outgrowth was allowed to proceed for 35 days in serum medium. The percentage of cells with neurites (those bearing processes at least one cell diameter in length) was determined. At least 200 cells were counted per field.
Survival AssayCells were assayed for survival using the sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid (XTT) assay (21). Cells were washed 5 times in serum-free medium and seeded into uncoated 96-well plates at a concentration of 5 x 103 cells/well. Various neurotrophin family members as well as the p75-selective NGF mutant (9/13 NGF) were added with and without inhibitors, and the cells were cultured for 5 days. The fraction of surviving cells was determined in triplicate by color formation of a formazan product using the Cell Proliferation Kit II (XTT; Roche Applied Science). This assay is based on the cleavage of the tetrazolium salt XTT in the presence of an electron coupling reagent by active mitochondria, only in viable cells, to a soluble formazan salt. The amount of orange color produced was measured using a scanning multiwell spectrophotometer (TECAN) measuring absorbance at 492 nm and subtracting out the background absorbance at 650 nm. The data are reported relative to untreated controls for each cell line. The amount of formazan dye produced directly correlates with the number of metabolically active cells surviving in the culture.
Immunocytochemical Staining and Confocal MicroscopyPC12 and PTR cells were grown on collagen-coated 4-well chamber slides (NUNC) and placed in defined medium in the presence of 2 nM NGF,
9/13 NGF, PDGF, or PDGF plus 9/13 NGF for 48 h. Staining was done according to the manufacturer's protocol with washing in PBS at 37 °C, fixing in methanol at 20 °C, and blocking in 5% bovine serum albumin in PBS overnight at 4 °C. After washing with PBS, the primary antibodies rabbit anti-TrkA (1:200) (Santa Cruz Biotechnology), goat anti-p75NTR (1:200) (Santa Cruz Biotechnology), and mouse anti-
3-tubulin (1:5000) (Upstate Biotechnology, Inc.) were added in place of the PBS. Cells were kept overnight in the dark at room temperature, washed three times with PBS, and incubated with the appropriate mixture of fluorescein- (green), Cy3- (red), or Cy5-conjugated (blue) secondary antibodies (Molecular Probes) diluted in 1% bovine serum albumin for 2 h at room temperature in the dark. Coverslips were mounted and studied using confocal microscopy. Images were captured with an inverted Olympus Fluoview confocal microscope equipped with a krypton/argon laser. Imaging of negative controls was used to establish settings for subsequent qualitative analysis. Serial focal planes were collected using a x60 oil immersion objective and a confocal aperture sufficiently wide to image multiple immunofluorescence labels at high sensitivity with processes and cell bodies in the same focal plane, thereby increasing the optical section up to 2.5 µm. Figures were composed in Adobe Photoshop 6.0, and conclusions were drawn after experiments were repeated in duplicate.
Annexin V StainingAnnexin V binding was performed according to a modified protocol for the Vybrant Apoptosis Assay Kit Number 3 (fluorescein isothiocyanate/annexin V/propidium iodide (PI), Molecular Probes). Cells were plated on polyornithine-coated 4-well chamber slides and allowed to adhere overnight. Subsequently, cells were washed 3 times with serum-free medium, and apoptosis was induced by placing cells in serum-free medium (± growth factor) for a period of 8 h. Cells were washed 2 times with 1 ml of cold PBS and 2 times with Annexin V Binding Buffer. Cells were then fixed in 4% paraformaldehyde or 20 °C methanol and stained in 1x Annexin Binding Buffer (5x Annexin Binding Buffer (component C), 15 ml of 50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2, pH 7.4) supplemented with 25 µl of annexin V fluorescein isothiocyanate conjugate (component A, proprietary Molecular Probes) per 100 µl of binding buffer and 2 µl of PI (component B; 1 mg/ml solution in distilled H2O) per 100 µl of binding buffer for 15 min. Cells were washed twice with binding buffer and examined using a confocal microscope. The plasma membrane of cells in the early stages of apoptosis will have green annexin V-positive patches and no staining by PI. By contrast, healthy cells will lack any green annexin V-positive staining and again not demonstrate red nuclear staining with PI, typical of necrosis.
Molecular ModelingThe Wiesmann et al. (22) crystal structure (Protein Data Bank code 1WWW
[PDB]
) was used as a frame upon which the 9/13 NGF mutant was modeled. A model was constructed using QUANTA (Accelrys, San Diego), deleting residues 913, fixing residues 15115, and allowing the N terminus of NGF and the d5 subdomain of TrkA to undergo 500 cycles of energy minimization, such that it resulted in an energetically stable conformation for the mutated N terminus of
9/13 NGF.
Statistical AnalysisStatistical analysis was performed with GraphPad Prism (version 2.01). Differences between means were evaluated using one-way analysis of variance followed by a Tukey post hoc comparison. Differences were considered significant if p < 0.05. All results are expressed as mean ± S.D. Data were graphed using SigmaPlot 2000.
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RESULTS |
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Structural Modeling of the NGF 9/13 MutantBecause the p75NTR-selective NGF mutant (
9/13 NGF) (3, 17) was utilized in this study to examine the role of p75NTR alone, the structural basis for its selectivity was delineated by computer graphic modeling. The x-ray crystal structure of NGF in complex with the d5 subdomain of TrkA reported by Wiesmann et al. (22, 23) clearly illustrates the importance of the N terminus of NGF in binding to TrkA with residues 310 forming a one and one-half turn
-helix encompassing close interactions of NGF His-4, Leu-6, and Phe-7 with TrkA d5 (Fig. 1A). Deletion of the five residues from 913 (circled in Fig. 1B, left) has been modeled using Quanta software; the results (Fig. 1B, right) indicate that the N-terminal
-helix of NGF pulls away from the d5 subdomain of TrkA as expected, thereby abolishing extensive interactions with the NGF N terminus needed for complex formation with the TrkA receptor. The rest of the molecule remains the same, with those areas critical for p75NTR binding at the other end of the molecule (2, 22) left unchanged. This modeling result is in agreement with earlier comparison of CD spectra and denaturation studies of
9/13 NGF and wild-type NGF that revealed similar secondary structure and unfolding stability (3). Thus,
9/13 NGF is a useful tool allowing for the study of p75NTR-specific effects, both in the presence and absence of TrkA, and allows dissection of receptor-specific versus receptor-cross-talk effects.
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Detection of High Affinity TrkA-p75NTR Heteromeric Receptor ComplexThe presence of the NGF high affinity heteromeric receptor complex was detected using a set of co-immunoprecipitation experiments. Utilizing the Barde protocol (14), PC12 cells were treated with NGF or medium alone for 15 min, and lysates were normalized for protein content and immunoprecipitated with antibodies against either the intracellular (ICD) or extracellular domain (ECD) of the TrkA receptor. Samples were then examined by Western blotting with antibodies against p75NTR. Intriguingly, the data suggest the presence of a small amount of preformed TrkA-p75NTR complex that is stabilized upon NGF addition (Fig. 2A). Furthermore, the heteromeric complex is detected only when antibodies against the ICD of TrkA were used and is undetectable when antibodies against the ECD of TrkA were used for immunoprecipitation (Fig. 2B). Altogether, these findings suggest that NGF binding is important for stabilization of the heteromeric complex (see "Discussion"). These results are supported by recent findings that indicate that the transmembrane and cytoplasmic domains of TrkA and p75NTR are critical for high affinity site formation (7, 14).
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Specificity of the PDGFR-TrkA Chimeric SystemSelective activation of the chimeric PDGFR-TrkA receptor (PTR) upon addition of PDGF was verified in a series of immunoprecipitation experiments. PC12 and PTR cells were treated with 2 nM NGF or PDGF for 15 min at 37 °C. Cell lysates were subjected to immunoprecipitation with three different antibodies. Immunoprecipitation with an antibody to the ICD of TrkA pulled down both endogenous TrkA and the chimeric PTR receptor. As expected, subsequent blotting with anti-phosphotyrosine antibodies revealed activation of endogenous TrkA in both PC12 and PTR cells upon NGF addition (Fig. 3A). Furthermore, in PTR cells, selective activation of the PDGFR-TrkA chimera upon PDGF addition, but not NGF addition, was observed (Fig. 3A, lane 5). The extracellular domain of PTR consists of five immunoglobulin-like motifs, whereas TrkA contains only two such motifs. This results in a higher molecular weight and slower migration for the chimeric receptor upon electrophoresis as evidenced by the band at 180 kDa, compared with wild-type TrkA that migrates at 140 kDa (Fig. 3A, lane 5).
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To determine whether activation of the chimeric receptor resulted in nonspecific transactivation of endogenous TrkA, lysates were immunoprecipitated with an antibody to the ECD of TrkA, such that only endogenous TrkA was pulled down. Once again, activation of endogenous TrkA in PC12 and PTR cells occurred upon NGF addition as expected. However, the absence of this band upon PDGF addition (Fig. 3B, lane 5) indicated a lack of cross-activation of endogenous TrkA upon activation of the PTR chimera. Finally, to verify the specific activation of the PDGFR-TrkA chimeric receptor upon PDGF addition, lysates were immunoprecipitated with anti-PDGFR antibodies. Detection with an anti-phosphotyrosine antibody revealed a single band for the PTR cells upon PDGF stimulation (Fig. 3C, lane 5). These data corroborate previous results with PTR cells (15, 16) and suggest that specific activation of the chimeric receptor in these cells, upon PDGF addition, is a useful way for studying the effects of intracellular TrkA signaling alone.
To verify the TrkA phosphorylation data, neurite outgrowth bioassays comparing PC12 and PTR cells were performed. PC12 and PTR cells were treated with 2 nM NGF or PDGF for 3 days at 37 °C. Upon PDGF addition, PC12 cells (which lack any PDGF receptors) failed to demonstrate any neurite outgrowth. By contrast, PTR cells exhibited neurite outgrowth upon either PDGF or NGF addition, confirming previous observations that selective activation of PDGFR-TrkA chimeras are capable of activating intracellular TrkA signal transduction and neurite outgrowth (data not shown) (15, 16).
Differences in Neuritogenesis Induced by Various TrkA, p75NTR Receptor CombinationsActivation of TrkA signaling is well established as being necessary and sufficient for NGF-induced neurite outgrowth in PC12 cells. Neurite outgrowth bioassays, comparing the effects of competition of NGF with increasing concentrations of the p75NTR-selective mutant 9/13 NGF (up to 2 nM), were performed to verify this concept in the paradigm used in these studies. Indeed, increasing concentrations of
9/13 NGF did not diminish PC12 cell neurite outgrowth, in agreement with this view (data not shown).
To compare the effects of various receptor combinations on neurite outgrowth, PC12 and PTR cells were treated with 2 nM NGF, PDGF, and/or 9/13 NGF for 5 days at 37 °C. Phase contrast micrographs demonstrated morphological differences in neurite outgrowth (data not shown but summarized in Table II). Surprisingly, a visible difference was observed in the neurite outgrowth between the various receptor stimulations. Most strikingly, the combined activation of TrkA and p75NTR signaling (PTR cells plus PDGF plus
9/13 NGF) resulted in a significant increase in the number of synaptic "bouton-like" structures, similar to the appearance of PC12 cells treated with NGF but clearly different from the activation of TrkA signaling alone (PTR cells plus PDGF), which lacked any such structures. Altogether, these results suggest that activation of TrkA is essential for neurite elongation; however, p75NTR is also involved and plays a fine-tuning, modulatory role, especially with the increased formation of the bouton-like structures.
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Subcellular Distribution of TrkA and p75 ReceptorsNeurotrophins predominantly act on nerve cells with highly specialized and polarized shape, thus the subcellular distribution of neurotrophin receptors in synapses, dendrites, axons, and boutons is functionally relevant. Therefore, confocal microscopy was utilized to compare receptor distribution and localization (Figs. 4 and 5) in order to characterize more precisely the differences in neurite morphology, with a specific focus on the bouton-like structures. PC12 and PTR cells were stimulated with various ligand combinations for 5 days at 37 °C, and the cells were fixed and stained with antibodies to TrkA, p75, PDGFR, or -tubulin and examined by confocal microscopy. PC12 cells were stained for TrkA (red), p75NTR (green), and
-tubulin (blue), whereas p75NTR was broadly distributed all across the cell surface, including the full-length of the neuritic processes; TrkA was largely restricted to the cell body. The bouton-like structures resulting from NGF stimulation of PC12 cells contained a mixture of TrkA and p75NTR (Fig. 4, EH, see arrowheads). In agreement with previous studies (3, 17), stimulation of PC12 cells with the p75NTR-selective mutant
9/13 NGF failed to elicit neurite outgrowth, resulting in an indistinguishable appearance from untreated cells (Fig. 4, AD and IL). When PTR cells were stained for PDGFR (red), p75NTR (green), and
-tubulin (blue), diffuse p75NTR staining was observed across the cell including neuritic processes, whereas a more restricted distribution of the PDGFR-TrkA chimera limited to the cell body and bouton-like structures was observed (Fig. 5, EH). Stimulation of both PTR and p75NTR, by addition of both PDGF plus
9/13 NGF to PTR cells, seemed to increase the number of bouton-like structures (Fig. 5, IL). Furthermore, the bouton-like structures produced upon PDGF addition alone or PDGF plus
9/13 NGF addition to PTR cells contain a mixture of both p75NTR and PTR, again indicating the co-localization of both receptors in these bouton-like structures. Significantly, both p75NTR and TrkA receptors have been implicated previously in playing a role in synaptic plasticity (24) and retrograde transport (25).
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Receptor-specific Contributions to Downstream Signaling To understand better the role of receptor homo-oligomers and heteromers in the activation of downstream signaling pathways, the contribution of each receptor and combinations thereof to two major downstream pathways known to be important in NGF signaling were examined, specifically (a) the Ras-MAP kinase pathway and (b) the PI3-kinase-Akt pathway. PC12 and PTR cells were stimulated with NGF, PDGF, and/or 9/13 NGF for varying times (0120 min) at 37 °C in 0.5% serum or platelet-poor serum medium, respectively. Lysates were processed as described under "Materials and Methods," and activation of MAPK and Akt was analyzed by Western blotting using phosphotyrosine-specific antibodies, with phosphorylation representing the activated form. The blots were stripped and reprobed for total MAPK and Akt to test for equal loading. Bands on the blots were quantified by densitometry.
In PC12 cells, stimulation with NGF resulted in a robust and sustained increase in MAPK (Fig. 6, A and E) and Akt phosphorylation (Fig. 7, A and E) within 5 min of stimulation that lasted for up to 2 h. By contrast, activation of p75NTR alone by treatment of PC12 cells with 9/13 NGF resulted in a transient activation of MAPK (Fig. 6, B and E) and no detectable Akt phosphorylation (Fig. 7, B and E). This somewhat unexpected MAPK activation is studied in detail elsewhere (54). Interestingly, activation of the PDGFR-TrkA chimera alone by treatment of PTR cells with PDGF resulted in a rapid and sustained increase in MAPK phosphorylation (Fig. 6, C and E), whereas Akt phosphorylation was delayed by
30 min and failed to reach levels induced by NGF treatment of PC12 cells (Fig. 7, C and E). Finally, activation of both PTR and p75NTR independently by stimulation of PTR cells with PDGF plus
9/13 NGF also resulted in a rapid and sustained increase in MAPK phosphorylation (Fig. 6, D and E), with Akt phosphorylation being detected after
30 min (Fig. 7, D and E). These results suggest that activation of TrkA alone is sufficient for a rapid and sustained activation of MAPK, whereas activation of p75 alone produces only a transient activation of MAPK. Interestingly, the data also suggest that, unlike NGF stimulation of PC12 cells, the independent activation of TrkA and p75NTR, by PDGF plus
9/13 NGF in PTR cells, is not sufficient to produce a rapid and sustained increase in Akt phosphorylation, implicating a potential role for the elusive NGF high affinity receptor site (see "Discussion").
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Role of p75NTR and TrkA Receptor-specific Pathways in Neurite OutgrowthTo compare downstream consequences of TrkA- and p75NTR-mediated regulation of MAPK and Akt activation with neuritogenesis, PC12 and PTR cells were stimulated with NGF, PDGF, and/or 9/13 NGF for 5 days at 37 °C in 0.5% serum or platelet-poor serum medium, respectively, and neurites greater than one cell body in length were scored as positive. Pretreatment for 1 h with PD98059 (an MEK1 selective inhibitor) and/or wortmannin (a PI3-kinase selective inhibitor) was used to block selectively the MAPK and Akt pathways, respectively.
In agreement with the work of others (26), NGF-mediated neurite outgrowth of PC12 cells was completely abolished upon inhibition of the MAPK pathway (PD in Fig. 8). By contrast, inhibition of Akt resulted in a relatively minor decrease (20%) in neurite outgrowth (W in Fig. 8). This suggests that both MAPK and Akt play a role in NGF-induced neurite outgrowth, with the MAPK pathway being absolutely necessary and Akt activation playing a secondary role. As expected, upon addition of
9/13 NGF to PC12 cells, neuritogenesis was absent (Fig. 8), supporting previous work (26, 27) that suggests that transient activation of MAPK (Fig. 6) is not sufficient for generation of neurite outgrowth. Activation of TrkA signaling alone, by treatment of PTR cells with PDGF, resulted in neurite outgrowth that was unaffected by inhibition of Akt (W in Fig. 8) but was altogether abolished by MAPK inhibition (PD in Fig. 8). Similarly, when both TrkA and p75NTR signalings were individually stimulated, by addition of PDGF plus
9/13 NGF to PTR cells, neuritogenesis was refractory to Akt inhibition but was completely negated upon inhibition of MAPK (PD in Fig. 8). Consistent with the "sustained versus transient" hypothesis proposed by Marshall and co-workers (26, 27), these results, along with the Western blot data (Fig. 6), support the concept that sustained activation of MAPK through TrkA is necessary for neurite outgrowth. Furthermore, as suggested previously, neurite outgrowth elicited by NGF seems to require both the MAPK (26) and Akt (28) pathways. However, neurite outgrowth generated from stimulation of TrkA signaling alone or simultaneous TrkA plus p75NTR signaling appears to require only sustained MAPK activation and is not affected by inhibition of Akt. This finding implicates a potential role for the NGF high affinity receptor site in facilitating the combined activation of both the MAPK and Akt pathways for production of NGF-induced neurite outgrowth.
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Differences in Cell Survival Induced by Various TrkA, p75NTR CombinationsIn addition to neuritogenesis, another important activity of neurotrophins is cell survival; therefore, the specific contributions of TrkA and p75NTR to cell survival were examined using the XTT assay. PC12 and PTR cells were placed in serum-free medium supplemented with either 2 nM NGF or PDGF and/or 9/13 for various times. In agreement with already well established results (1618), PC12 cells placed in serum-free medium alone rapidly die within 24 h secondary to lack of trophic support (Fig. 9). When PC12 cells are rescued by NGF, long term cell survival is evident with about 50% of cells surviving after 120 h (Fig. 9A). By contrast, when PC12 cells are rescued with
9/13 NGF, a significant but short-lived increase in the percentage of surviving cells is observed, remaining above the untreated control levels for about 24 h (Fig. 9A). These results clearly indicate a pro-survival function for
9/13 NGF through p75NTR, consistent with earlier findings (17, 2932) suggesting a role for p75NTR in cell survival. Interestingly, activation of TrkA signaling alone by addition of PDGF to PTR cells also resulted in increased cell survival for 72 h (Fig. 9B), a longer duration than that of
9/13 NGF (24 h) but significantly shorter than that of NGF-mediated survival (120 h). Furthermore, concomitant stimulation of both TrkA and p75NTR, by addition of both PDGF and
9/13 NGF to PTR cells, resulted in increased survival compared with PDGF treatment alone at intermediate time points, supporting the concept that both TrkA and p75NTR signalings contribute to cell survival. Intriguingly, however, unlike the increase in survival noted with NGF treatment, the level of survival after 5 days elicited by PDGF plus
9/13 NGF addition to PTR cells was not significantly different from that of the untreated control. These results suggest a possible role for the NGF high affinity site (TrkA/p75NTR) in regulating long term cellular survival and agree with differences in the kinetics of Akt activation noted earlier between the two treatments (Fig. 7; see "Discussion").
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Inhibition of Apoptosis by Various TrkA, p75NTR CombinationsIn addition to promoting survival of PC12 cells upon serum withdrawal, in a related process, NGF acts to inhibit the activation of programmed cell death (33). One of the earliest markers of apoptosis is disruption of the plasma membrane, with externalization of phosphatidylserine to the outer leaflet of the membrane. Annexin V is a calcium-dependent binding protein that has a high affinity for phosphatidylserine that is often used to measure the extent of plasma membrane disruption during the early stages of apoptosis (17). Annexin V staining of PC12 and PTR cells, performed 8 h after cells were placed in serum-free medium, was reduced upon treatment of cells with either 2 nM NGF or PDGF and/or 9/13 NGF, lending further support to pro-survival signaling through either TrkA or p75NTR (data not shown).
Role of TrkA and p75NTR-specific Pathways in Cell SurvivalThe role of TrkA and p75-mediated MAPK and Akt activation of cell survival induced by serum withdrawal was also examined. The contribution of p75NTR and TrkA receptor-specific pathways to cell survival was determined with the XTT assay at 36 h. Pretreatment for 1 h with the inhibitors PD98059 and/or wortmannin was then used to block selectively the MAPK and/or Akt pathway, respectively.
Consistent with its role as an important component of cell survival signaling (34), inhibition of Akt resulted in almost complete negation of NGF-mediated PC12 cell survival (W in Fig. 10). By contrast, inhibition of MAPK resulted in a relatively minor decrease in NGF-mediated survival (PD in Fig. 10). When PC12 cells were treated with 9/13 NGF, a slight increase in survival was noted, which was not affected by pretreatment with wortmannin but was virtually abolished upon pretreatment with the MEK inhibitor PD98059. This result suggests the presence of a p75NTR-mediated, MAPK-dependent pathway that plays a role in cell survival and has been identified previously (54) in other cell systems. When PTR cells were treated with PDGF, no change in cell survival was observed upon inhibition of MAPK (PD in Fig. 10); however, a significant portion of the survival was abolished upon inhibition of the Akt pathway (W in Fig. 10). These data suggest a TrkA-mediated Akt-dependent regulation of cell survival. Surprisingly, when both PD98059 and wortmannin were added together, an increase in survival was observed from that of wortmannin treatment alone, suggesting an interaction between the MAPK and Akt pathways. Similar to PDGF treatment alone, when PTR cells were treated with a combination of PDGF plus
9/13 NGF, no decrease in cell survival was evident upon inhibition of MAPK; however, a large decrease was observed upon inhibition of the Akt pathway (Fig. 10). Intriguingly, a similar increase in survival was observed when both PD98059 and wortmannin were added in combination, when compared with wortmannin alone, bolstering the concept that the MAPK and Akt pathways may potentially interact.
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Interplay between MAPK and Akt PathwaysAlthough it is well established that the MAPK and Akt pathways are two major NGF signal transduction pathways (1, 35, 36), the concept of one pathway regulating the other has not been demonstrated previously. To examine possible interaction between these pathways, a dose response with increasing concentrations of the MEK inhibitor U0126 was obtained (Fig. 11; similar results were obtained with the MEK inhibitor PD98059 (data not shown)). NGF-induced activation of MAPK was abolished with increasing concentrations of U0126, as expected (Fig. 11, upper panel, from right to left). The signal was not affected at 1 µM, significantly decreased at 10 µM, and abolished at 25 µM, indicating a sharp sensitivity to U0126 concentration. When the same blot was stripped and reprobed for activated Akt, a dose-dependent increase in the levels of Akt activation was observed (Fig. 11, lower panel, from right to left) that correlated well with the sensitivity of inhibition of MAPK. These data suggest that MAPK, or the processes inducing its activation, negatively modulates the Akt pathway. By inhibiting the activation of MAPK, MAPK-related repression of the Akt pathway may be relieved, possibly accounting for the observed increase in intensity of phospho-Akt levels. This result helps to explain the survival data, in which addition of both MAPK and Akt pathway inhibitors resulted in an increase in survival compared with that of Akt inhibition alone (Fig. 10).
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DISCUSSION |
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The functional significance of the NGF high affinity binding site has been established previously. Examples include the following. (i) A reduced activation of TrkA at low NGF concentrations observed when binding to p75NTR was prevented in PC12 cells (12, 39). (ii) A TrkA-selective NGF mutant, unable to bind to p75NTR, was less active than wild-type NGF in supporting neuronal survival at low ligand concentrations (40). (iii) Caveolae and caveolae-related domains may focus TrkA/p75NTR interactions and effect internalization (41). (iv) An NGF-antibody complex, which interacts with TrkA but not p75NTR, was taken up faster than NGF alone and was accompanied by transient activation of MAPK and the absence of neuritogenesis (42). (v) Several adaptor proteins have been identified that may help mediate the interaction between p75NTR and TrkA (32, 4346).
Whereas activation of TrkA signaling is sufficient for eliciting neurite outgrowth, the results presented here indicate that signaling through p75NTR does play a modulatory role, especially in the increased formation of fine, synaptic bouton-like structures. Furthermore, the results suggest that both TrkA and p75NTR co-localize within these bouton-like structures (Figs. 4 and 5, see arrowheads). Interestingly, Yamashita et al. (47) recently demonstrated alteration of neurite outgrowth through p75NTR, as evidenced by inactivation of RhoA upon ligand binding to p75NTR, with subsequent modulation of actin filament assembly and decreased rigidity of the actin cytoskeleton (47).
These data are consistent with the hypothesis (26, 27) that a sustained but not a transient activation of MAPK is required to elicit neurite outgrowth in PC12 cells. However, no direct correlation can be drawn between the observed p75NTR-induced MAPK activation and the subsequent increase in the number of synaptic bouton-like structures, as addition of MAPK inhibitors abolish neurite outgrowth altogether. Furthermore, in addition to commitment to neuritogenesis based on the kinetics/duration of MAPK activation (26, 27), signaling strength is also likely to play a role. A certain threshold may be required for commitment to certain cellular end points, and the rate of responsiveness may be regulated by altering the signal intensity.
A long-standing question in neurotrophin signaling has been whether the heteromeric high affinity TrkA-p75NTR complex possesses signaling capacities that are inherently different from those of either TrkA or p75NTR alone or combined. The results presented herein suggest two unique functions for the high affinity heteromeric NGF receptor site as follows: (i) integration of both the MAPK and Akt pathways in the production of NGF-induced neurite outgrowth (Figs. 6, 7, 8), and (ii) rapid and sustained activation of the Akt pathway, with consequent long term cellular survival (Figs. 9, 10, 11).
Several previous studies indicate the potential for reciprocal receptor interactions between TrkA and p75NTR at the level of downstream signaling. Barker and colleagues (48) have provided evidence that signaling through p75NTR may inhibit TrkA activation. BDNF binding to p75NTR on PC12 cells markedly reduced TrkA activation and downstream signaling events produced by an NGF mutant that selectively bound TrkA, presumably by activation of sphingomyelinase and increased levels of ceramide. Furthermore, both BDNF and ceramide were shown to increase the phosphoserine content on the intracellular domain of TrkA, which corresponded with reduced TrkA activation (48, 49). In addition to regulation of TrkA signaling by p75NTR, the reverse has also been demonstrated, i.e. regulation of p75NTR signaling by TrkA. Although NGF has been shown previously to induce sphingomyelin hydrolysis through p75NTR, no such activation was seen in PC12 cells (TrkA+ and p75+). This effect is apparently mediated by TrkA signaling, since inhibition of TrkA tyrosine kinase activity with the inhibitor K252A restored the ability of NGF to elicit sphingomyelin hydrolysis (50).
A novel signaling interaction in the TrkA/p75NTR signal transduction network is revealed in this study, namely NGF-induced activation of MAPK inhibits the parallel NGF induction of Akt. Blockade of the MAPK activation pathway with specific inhibitors abolishes MAPK-dependent inhibition of the activation of Akt, resulting in increased NGF-induced activation of Akt, which facilitates increased cell survival. Similar results implicating cross-talk of the MAPK pathway with other pathways has been suggested by Yoon et al. (51), who used oligodendrocytes (TrkA and p75+) transfected with TrkA to demonstrate that NGF induction of the MAPK pathway could suppress p75NTR-mediated JNK activity, thereby preventing cell death. On the other hand, activation of ERK5, another member of the MAPK family, appears to be activated only by TrkA signaling and not p75NTR (52). Altogether, these results support the concept that a significant number of reciprocal interactions exist between TrkA and p75NTR signaling. Although the mechanism for this cross-talk is unknown, one possibility is through a TrkA-PI3-kinase-dependent mechanism that has been shown to generate cross-talk between TrkA and p75NTR pathways, with inhibition of p75NTR-mediated sphingomyelin hydrolysis (53).
An important and novel concept from these studies is the demonstration that a combination of receptor chimeras and receptor-selective ligand mutants can be utilized to dissect individual components of a multireceptor system. Description of a ternary complex between p75NTR, TrkA, and NGF by high resolution structural methods is necessary to better characterize the high affinity NGF receptor site and to fully answer a number of long-standing questions in the field of neurotrophin structure-function. Identification of the detailed molecular interactions that exist between the two classes of neurotrophin receptors will ultimately reveal how TrkA and p75NTR interact, how neurotrophin specificity is achieved, and shed light on the mechanism of action for the high affinity NGF-binding site.
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FOOTNOTES |
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Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy, Finch University of Health Sciences/The Chicago Medical School.
** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Finch University of Health Sciences/The Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064. Tel.: 847-578-3220; Fax: 847-578-3240; E-mail: neetk{at}finchcms.edu.
1 The abbreviations used are: p75NTR, common neurotrophin receptor; Akt, serine-threonine kinase (protein kinase B); NGF, mouse nerve growth factor (
-subunit); BDNF, brain-derived neurotrophic factor;
9/13 NGF, p75NTR-selective
NGF mutant with amino acid residues 913 deleted; ECD, extracellular domain; ICD, intracellular domain; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; NGF, nerve growth factor; PBS, phosphate-buffered saline; PC12, pheochromocytoma 12 cell line; PD98059, MEK 1,2-selective inhibitor; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PI, propidium iodide; PTR, PDGFR-TrkA chimeric receptor; TNF, tumor necrosis factor; TrkA, NGF receptor with tyrosine kinase activity; U0126, MEK 1,2-selective inhibitor; XTT, sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)-benzenesulfonic acid; PI3-kinase, phosphatidylinositol 3-kinase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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