Article |
Address correspondence to D. Kaplan, Cancer Research Program, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, M5G 1X8. Tel.: (416) 813-7654, ext. 1433. Fax: (416) 813-2212. email: dkaplan{at}sickkids.ca
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Abstract |
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Key Words: neurotrophins; NGF; sympathetic neurons; PC12 cells; apoptosis
H.N. Marsh's present address is Archemix Corporation, 1 Hampshire St., Cambridge, MA 02139.
Abbreviations used in this paper: Ad, adenovirus; MOI, multiplicity of infection; P, postnatal day; SCG, superior cervical ganglia; wt, wild-type.
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Introduction |
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The SHP family of protein tyrosine phosphatases includes SHP-1, SHP-2, and the Drosophila melanogaster homologue Corkscrew (Tonks and Neel, 2001). SHP-1 is expressed in the hematopoietic system, the nervous system, epithelial cells, and the NGF-responsive PC12 cell line (Tonks and Neel, 2001). The motheaten (me/me) mouse, which lacks SHP-1 expression, displays an array of hematopoietic abnormalities resulting in severe immunodeficiency and systemic autoimmunity (Tsui and Tsui, 1994). The pathology of the me/me mouse, which is caused by the overproduction of multiple hematopoietic cell lineages, initially suggested that SHP-1 was primarily a negative regulator of cell proliferation. In this regard, SHP-1 has been shown to interact with and dephosphorylate a number of growth factor receptors, including those for insulin-like growth factor-1, platelet-derived growth factor, EGF (Tonks and Neel, 2001), and Ros (Keilhack et al., 2001). In contrast, SHP-1, like SHP-2, has been shown to positively regulate MAPK signaling (Krautwald et al., 1996; Wright et al., 1997), as well as EGF, interferon-, and Ras signaling (Su et al., 1996; You and Zhao, 1997). The positive effects of SHP-1 signaling may explain why the absence of SHP-1 in me/me mice leads to decreased numbers of central nervous system glia (Wishcamper et al., 2001).
Whereas the survival and growth-promoting aspects of neurotrophin signaling are dependent on the levels of TrkA receptor autophosphorylation initiated by NGF binding; the existence of phosphatases that dephosphorylate TrkA would suggest an additional and important mechanism of neurotrophin receptor regulation. In this regard, our previous work in PC12 cells showed that SHP-1 was activated after NGF treatment of PC12 cells (Vambutas et al., 1995). Here, we have asked about the biological importance of this activation in two cell types that require TrkA signaling for their survival, developing sympathetic neurons, and PC12 cells (Greene and Tischler, 1976; Chun and Patterson, 1977). Our results indicate that SHP-1 functions as a TrkA phosphatase, controlling the level of TrkA activity in cultured neurons and PC12 cells and regulating the number of NGF-dependent sympathetic neurons during development.
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Results |
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SHP-1 overexpression suppresses the survival of sympathetic neurons and PC12 cells
The biochemical data indicate that SHP-1 negatively regulates TrkA tyrosine phosphorylation and activation. To determine whether this has functional consequences, we examined sympathetic neurons that require TrkA signaling for survival. Neurons were infected with Ad SHP-1 or a dominant-inhibitory SHP-1 mutant (SHP-1P; Neel and Tonks, 1997), and after 2 d, they were switched into media with or without 20 ng/ml NGF. After 72 h, survival was quantified using MTT, which measures mitochondrial function (Manthorpe et al., 1986; Fig. 5 A). SHP-1WT expression decreased sympathetic neuron survival by 70% (100 multiplicity of infection [MOI]), whereas SHP-1
P expression had little effect on survival in 20 ng/ml NGF relative to a LacZ-expressing control virus (Fig. 5 A). The specificity of the SHP-1 effect was demonstrated by coinfecting neurons with the Ad SHP-1
P; this completely rescued neurons from SHP-1WTinduced death (Fig. 5 A). Similarly, coexpression of Bcl-xL, a protein that prevents NGF withdrawal-induced apoptosis of sympathetic neurons (Gonzalez-Garcia et al., 1995), prevented SHP-1WTmediated suppression of neuronal survival. The expression of Ad SHP-1 proteins in sympathetic neurons was confirmed by Western blot analysis with antiSHP-1 (Fig. 5 B).
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We turned to PC12 cells, whose survival can be promoted either by NGF or by serum factors such as IGF-1 (Greene and Tischler, 1976), to find out whether this suppressive effect of SHP-1 was specific for TrkA-mediated survival. PC12 cells were infected with Ad SHP-1WT, and cultured in serum or in NGF without serum for 72 h. MTT analysis demonstrated that overexpression of SHP-1WT decreased survival of PC12 cells maintained in NGF by 50% (100 MOI; Fig. 6 A), but had no effect on PC12 cells maintained in serum (Fig. 6 B). Thus, SHP-1 is specific for TrkA-mediated survival, and does not apparently inhibit survival promoted by other exogenous factors present in serum. Based on these results, together with the biochemical analysis, we hypothesized that SHP-1 directly inhibited TrkA and TrkA-mediated downstream survival signals.
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SHP-1 decreases the activity of NGF-stimulated signaling proteins
We first examined NGF-induced changes in tyrosine phosphorylation, to determine how overexpression of SHP-1 affects NGF-mediated TrkA survival signals. Sympathetic neurons were infected with Ad SHP-1WT, washed free of NGF, and then treated for 5 min with 100 ng/ml NGF. Western blot analysis with anti-pTyr revealed that SHP-1WT had little effect on overall basal tyrosine phosphorylation, but that it suppressed the tyrosine phosphorylation of NGF-stimulated signaling proteins (Fig. 7 A). Particularly evident was the decreased phosphorylation of two bands that migrated at the molecular weight of the MAPKs, Erk1, and Erk2.
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Finally, we examined whether overexpression of SHP-1 resulted in the activation of cell deathinducing proteins, as would be predicted if it induces apoptosis by suppressing Trk signaling. Two such proteins that are induced or activated by the lack of TrkA signaling are c-jun and Bim/Bod (Putcha et al., 2001; Whitfield et al., 2001). Western blot analysis of sympathetic neurons expressing SHP-1WT, and maintained in 20 ng/ml NGF, showed that SHP-1 overexpression caused increased levels of Bim/Bod and the phosphorylation (activation) of c-jun (Fig. 7 D), coincident with a decrease in Erk1/2 phosphorylation. In contrast, the levels of the p75NTR and the pro-survival protein XIAP (Wiese et al., 1999) were not affected by SHP-1 overexpression (Fig. 7 D).
Inhibition of endogenous SHP-1 rescues sympathetic neurons and PC12 cells from NGF withdrawal by increasing basal TrkA activation
These data indicate that overexpression of SHP-1 acts to decrease TrkA tyrosine phosphorylation, resulting in decreased downstream survival signals and subsequent neuronal apoptosis. To ask whether endogenous SHP-1 plays a similar role in regulating TrkA activity, we used the dominant-inhibitory mutant SHP-1P. Sympathetic neurons were cultured for 4 d, infected with Ad SHP-1
P or LacZ, and either withdrawn from NGF or treated with 10 ng/ml NGF. In the absence of NGF, SHP-1
P expression maintained the survival of 50% of the neurons, whereas survival of LacZ-infected neurons was similar to controls (Fig. 8 A). Similar results were obtained using PC12 cells (Fig. 8 B, left). PC12 cells maintained in serum were infected for 24 h with the SHP-1
P virus at various MOIs. After infection, the cells were washed free of serum and cell survival was assessed after 72 h using MTT (Manthorpe et al., 1986). Expression of SHP-1
P increased PC12 cell survival in the absence of serum or NGF in a dose-dependent manner (Fig. 8 B, left). Survival with 50 MOI of SHP-1
P was 50% of that seen with 50 ng/ml NGF.
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Inhibition of endogenous SHP-1 differentially activates downstream TrkA signaling pathways in the absence of NGF
These experiments indicated that endogenous SHP-1 normally functions to keep basal TrkA activation low, and thereby to maintain the NGF dependence of PC12 cells and sympathetic neurons. However, SHP-1P did not cause PC12 cells to extend neurites in the presence or absence of serum (unpublished data), suggesting that in the absence of NGF, SHP-1 inhibition might only activate a subset of TrkA signaling pathways. In PC12 cells, survival is an Akt-dependent process, whereas neurite outgrowth is a MEK/MAPK-dependent process (Klesse et al., 1999; Kaplan and Miller, 2000). Therefore, we examined the effect of SHP-1
P on MAPK1/2 and Akt activity. PC12 cells were infected with Ad SHP-1
P, and the phosphorylation state of Akt and MAPK1/2 in the absence of NGF was examined in Western blots with phosphospecific antibodies. SHP-1
P caused an increase in Akt (Fig. 8 E, bottom panel) but not of MAPK (Erk1/2) phosphorylation (Fig. 8 E, top panel), which is consistent with the cell biology data.
Because TrkA forms complexes with and activates SHP-1 after NGF treatment of PC12 cells, we asked whether endogenous SHP-1 plays a role in attenuating TrkA activity in the continued presence of its NGF ligand. In sympathetic neurons, TrkA tyrosine phosphorylation is maximal after 5 min of exposure to NGF, with this phosphorylation being largely attenuated by 48 h, even in the continued presence of NGF (Belliveau et al., 1997; Fig. 8 F). Suppression of SHP-1 activity after SHP-1P expression led to sustained and elevated TrkA tyrosine phosphorylation in the continuous presence of 100 ng/ml NGF, with the levels at 48 h being similar to those seen at 5 min in neurons infected with Ad LacZ (Fig. 8 F). These results suggest that, in the presence of NGF, TrkA activates SHP-1, which in turn functions to attenuate TrkA activity and downstream signaling, thereby participating in a negative feedback loop.
Sympathetic neuron number is increased in the me/me mouse
These experiments using cultured cells indicate that endogenous SHP-1 functions both to keep basal levels of TrkA activity low in the absence of NGF and to attenuate TrkA activity in the presence of NGF. If SHP-1 plays a similar role in vivo, then sympathetic neurons in the me/me mouse, which is genetically deficient in SHP-1, should have up-regulated TrkA activity, and should not die appropriately during naturally occurring cell death. To test this hypothesis, we analyzed the number of neurons in SCGs taken from P15 me/me mice; the major period of sympathetic neuron death occurs in the first few weeks postnatally, and SCG neuron number decreases from 25,000 at birth to
15,000 at P15. SCGs from me/me and wt mice were removed and sectioned at 7-µm thickness, and neuronal numbers were determined by counting all neuronal profiles with nucleoli on every fourth section, as described by Coggeshall et al. (1984). This analysis demonstrated a statistically significant increase of 35% in the relative number of sympathetic neurons in me/me (21,289 ± 452; n = 4) relative to wt mice (15,813 ± 1033; n = 9; Fig. 9 A). Therefore, this analysis suggests that in vivo, SHP-1 regulates sympathetic neuron apoptosis. To ascertain whether increased TrkA expression could account for the increases in neuron number, we determined the levels of TrkA protein in sympathetic ganglia at P3, soon after the commencement of naturally occurring cell death, and at P10 and P15, when cell death is maximal or complete. TrkA protein levels were equivalent in wt and me/me SCG at P3, but were reduced in the me/me SCG by
50% at P10 and P15 (Fig. 9, B and C). Thus, the increase in neuron number could not be accounted for by an increase in TrkA expression level. Due to the low levels of TrkA, we could not assess TrkA autophosphorylation in me/me SCGs.
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Discussion |
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How does SHP-1 regulate TrkA activity? We propose that SHP-1 regulates both basal and NGF-stimulated TrkA activity. Because inhibition of endogenous SHP-1 stimulates the NGF-independent phosphorylation of TrkA, SHP-1 can regulate the basal, nonliganded activity of TrkA. TrkA activity, in the absence of NGF, is thus normally controlled and suppressed by SHP-1 activity. In the presence of NGF, TrkA is efficiently dimerized and hyperactivated, and TrkA tyrosine kinase activity predominates over basal SHP-1 tyrosine phosphatase activity. The enhanced TrkA activity results in receptor transphosphorylation, followed by recruitment of cytoplasmic signaling proteins to TrkA transphosphorylation sites, and TrkA-induced tyrosine phosphorylation of these substrates that in turn stimulates survival and growth pathways. However, SHP-1 is also recruited to and stimulated by NGF-bound TrkA, resulting in an increase in SHP-1 tyrosine phosphatase activity. The increase in SHP-1 tyrosine phosphatase activity would result in an attenuation of TrkA activity. Thus, we propose a model whereby SHP-1 either directly or indirectly associates with TrkA, resulting in an increase in SHP-1 activity followed by dephosphorylation of TrkA at the Y674 and Y675 sites; a similar mechanism is used by the tyrosine phosphatase PTP1B to regulate the insulin receptor (Salmeen et al., 2000). The dephosphorylation of these sites results in decreased TrkA biochemical and biological activity (Cunningham et al., 1997) and subsequent decreased activation of NGF-signaling proteins. Therefore, we suggest that SHP-1 has two functions: (1) to keep TrkA in an "off" state in the absence of ligand, and (2) to modulate TrkA activity after dimerization and activation of TrkA by NGF.
What is the role of SHP-1 during sympathetic neuron development? We propose that SHP-1 has two functions: (1) to control TrkA activity in the absence of NGF, and (2) to "fine-tune" TrkA-mediated survival signals in the presence of NGF. Correct neuron number during sympathetic development is dependent on the functional interplay of TrkA-induced survival signals and p75NTR-induced apoptotic signals (Kaplan and Miller, 2000; Majdan et al., 2001). Mice deficient in TrkA lack most sympathetic neurons, whereas mice deficient in p75NTR have twice the number of sympathetic neurons per ganglia in the SCG at P20. me/me mice that lack SHP-1 have 35% more neurons than wt mice (Fig. 9 A), indicating that SHP-1 functions during development to either suppress TrkA activity or the activity of other apoptotic signals. On the basis of our work in cultured SCG neurons, we favor the former hypothesis. In particular, we propose that SHP-1 is essential to keep TrkA off in neurons that have not contacted the correct targets and/or are late arriving, and subsequently, have not sequestered sufficient levels of NGF. Any basal TrkA activation in these neurons would serve to undermine the biological purpose of the cell death period, which is to ensure that only those neurons that are appropriately connected are maintained. Moreover, even in neurons that have sequestered limited NGF, SHP-1 regulation of TrkA signaling may well serve to regulate the precise balance between "positive" TrkA and "negative" p75NTR signaling, a balance that is essential for establishment of appropriate neuron numbers.
Until recently, SHP-1 expression was largely thought to be restricted to the hematopoietic system. However, recent studies have demonstrated that SHP-1 is expressed throughout the central nervous system in both neurons (Jena et al., 1997; Horvat et al., 2001) and glia (Massa et al., 2000). SHP-1 plays a key role in oligodendrocyte and glial development, as me/me mice display decreased numbers of central nervous system glia and dysmyelination (Massa et al., 2000; Wishcamper et al., 2001). Together, these observations suggest an important role for SHP-1 in the development and maintenance of the nervous system, a role that we propose is mediated at least partially via regulation of the TrkA neurotrophin receptor.
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Materials and methods |
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SCG neuronal cultures
Mass cultures of pure sympathetic neurons derived from the SCG of P1 rats (Sprague-Dawley) or me/me mice were prepared and cultured as described previously (Ma et al., 1992; Bamji et al., 1998).
Expression of recombinant TrkA in Sf9 cells
TrkA association assays were performed as described previously (Kaplan et al., 1990; Stephens et al., 1994) with the following modifications. Wild-type and phosphorylation site mutant human TrkA proteins (Stephens et al., 1994) immunoprecipitated from Sf9 cells and autophosphorylated in vitro were resuspended in 1 ml of lysate prepared from 107 PC12 cells lysed in NP-40 lysis buffer. The immune complexes were incubated with the lysate for 3 h at 4°C and washed three times with NP-40 lysis buffer and once with 10 mM Tris, pH 7.4.
TrkA dephosphorylation assays
Sf9 insect cells infected with SHP-1, SHP-2, or TrkA baculoviruses for 48 h were lysed in NP-40 lysis buffer, and SHP or TrkA proteins were immunoprecipitated. TrkA immunoprecipitates were washed once with RIPA, twice with NP-40 lysis buffer, and once with phosphatase buffer (25 mM Hepes, pH 7.3, 5 mM EDTA, and 10 mM DTT). Washed TrkA immunoprecipitates were incubated for 30 min at 30°C in phosphatase buffer containing 5 µCi -[32P]ATP (5 µM ATP final) per reaction. SHP immunoprecipitates were washed once with RIPA, twice with NP-40 lysis buffer, and once with phosphatase buffer. To detect TrkA dephosphorylation, TrkA and SHP immunoprecipitates were incubated together for 30 min at 30°C. The reaction was stopped with Laemmli SDS sample buffer and boiled for 5 min.
Recombinant adenoviruses and viral infections
Replication-defective recombinant Ad SHP-1 WT and P were prepared and purified as described previously (Slack et al., 1996; Mazzoni et al., 1999). Recombinant adenoviruses were amplified and purified on CsCl gradients and titered by plaque assay. GFP (Aegera Therapeutics Inc.) or Escherichia coli ß-galactosidase (LacZ)expressing recombinant adenoviruses (provided by F. Graham, McMaster University, Hamilton, ON) were prepared in the same backbone as the SHP-1 adenoviruses. Viral infections of sympathetic neurons were performed as described previously (Mazzoni et al., 1999; Wartiovaara et al., 2002). For PC12 infections, cells plated on poly-L-lysine were infected with adenoviruses for 48 h.
Immunoprecipitation, immunoblotting, and immunocytochemistry
Cells were treated with NGF, lysates were prepared, and immunoprecipitations and Western blotting were performed as described previously (Kaplan et al., 1991; Vambutas et al., 1995; Mazzoni et al., 1999). Lysates were prepared from normal and me/me SCGs as follows. Freshly dissected ganglia were homogenized in 100 µl of lysis buffer (Kaplan et al., 1991), transferred to microfuge tubes, rocked gently at 4°C for 30 min, and microcentrifuged at 13,000 rpm for 10 min at 4°C. Immunoprecipitations and Western blots were performed as described by Majdan et al. (2001).
Immunocytochemistry
Fluorescence immunocytochemistry was performed essentially as described previously (Wartiovaara et al., 2002). Cells were double labeled with a polyclonal antibody to neurofilament (1:200; Chemicon) and an mAb to SHP-1 (1:50; Transduction Laboratories). All the secondary antibodies were obtained from Alexa (goat Rb Alexa-488 and goat
ms Alexa-555, both 1:1,000; Molecular Probes, Inc.). The pictures were taken using a digital camera (model Retiga Exi; Q-Imaging) mounted directly on a microscope (model Axioplan 2; Carl Zeiss MicroImaging, Inc.) in a room with a temperature between 21 and 24°C and with an air objective (model Plan Neofluor, 20x/0,5; Carl Zeiss MicroImaging, Inc.). The acquisition software used was Northern Eclipse, and the pictures were assembled in Adobe Photoshop.
Cell survival assays and analysis of the me/me mice
Survival assays were performed 72 h after NGF withdrawal as described previously using MTT (Slack et al., 1996), TUNEL (Wartiovaara et al., 2002), or by counting phase-bright cells. Survival assays with me/me SCG neurons were performed by counting phase-bright (live) cells in premarked, randomly selected fields at 24-h intervals. The same fields were followed for the entire experiment with five to seven fields counted per well (30300 cells/field). The percentage of survival was calculated by dividing the number of phase-bright cells remaining at each time point by the number of phase-bright cells at 0 h. Control neurons used for these experiments were obtained from either wt littermate or wt C3H mice. The number of sympathetic neurons per ganglia of the me/me was determined as described previously (Bamji et al., 1998).
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Acknowledgments |
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This work was supported by operating grants to F.D. Miller and D.R. Kaplan and by fellowships to H.N. Marsh, M. Majdan, and A. Lee from the Canadian Institutes for Health Research (CIHR). D.R. Kaplan is a recipient of a Canada Research Chair, and F.D. Miller and K. Siminovitch are CIHR Senior Scientist.
F. Said is an employee, and F. Miller and D. Kaplan are founders of Aegera Therapeutics.
Submitted: 5 September 2003
Accepted: 13 October 2003
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