Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.     

Cell-cycle arrest in TrkA-expressing NIH3T3 cells involves nitric oxide synthase

We have examined nerve growth factor (NGF)-triggered signaling in two NIH3T3 cell lines exogenously expressing the NGF receptor, TrkA. TRK1 cells cease to proliferate and extend long processes in response to NGF, while E25 cells continue to proliferate in the presence of NGF. These two cell lines express similar levels of TrkA and respond to NGF with rapid elevation of mitogen-activated protein kinase (MAPK) activity. MAPK activation is slightly more sustained for E25 cells than for TRK1 cells, although sustained activation of MAPK has been suggested to cause cell-cycle arrest. As judged by NADPH-diaphorase staining, nitric oxide synthase (NOS) activity is increased in TRK1 cells upon exposure to NGF. In contrast, diaphorase staining in E25 cells is unaffected by NGF treatment. Immunocytochemistry shows that levels of the brain NOS (bNOS) isoform are increased in TRK1, but not E25, cells exposed to NGF. Furthermore, Western blots show that NGF elevated cyclin-dependent kinase inhibitor, p21(WAF1), in TRK1 cells only. NGF-induced p21(WAF1) expression, cell-cycle arrest and process extension are abolished by N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS. The inactive enantiomer, D-NAME, did not inhibit these responses. Furthermore, even though E25 cells do not respond to NGF or nitric oxide donors, they do undergo a morphological change in response to NGF plus a nitric oxide donor. Therefore, NOS and p21(WAF1) are induced only in the TrkA-expressing NIH3T3 cell line that undergoes cell-cycle arrest and morphological changes in response to NGF. These results demonstrate that sustained activation of MAPK is not the sole determining factor for NGF-induced cell-cycle arrest and implicate NO in the cascade of events leading to NGF-induced morphological changes and cell-cycle arrest.     

Individual and combined effects of TrkA and p75NTR nerve growth factor receptors. A role for the high affinity receptor site

A long-standing question in neurotrophin signal transduction is whether heteromeric TrkA-p75NTR complexes possess signaling capabilities that are significantly different from homo-oligomeric TrkA or p75NTR alone. To address this issue, various combinations of transfected PC12 cells expressing a platelet-derived growth factor receptor-TrkA chimera and the p75NTR-selective nerve growth factor mutant (Delta9/13 NGF) were utilized to selectively stimulate TrkA or p75NTR signaling, respectively. The contribution of individual and combined receptor effects was analyzed in terms of downstream signaling and certain end points. The results suggest two unique functions for the high affinity heteromeric NGF receptor site: (a) integration of both the MAPK and Akt pathways in the production of NGF-induced neurite outgrowth, and (b) rapid and sustained activation of the Akt pathway, with consequent long term cellular survival. Whereas activation of TrkA signaling is sufficient for eliciting neurite outgrowth in PC12 cells, signaling through p75NTR plays a modulatory role, especially in the increased formation of fine, synaptic "bouton-like" structures, in which both TrkA and p75NTR appear to co-localize. In addition, a new interaction in the TrkA/p75NTR heteromeric receptor signal transduction network was revealed, namely that NGF-induced activation of the MAPK pathway appears to inhibit the parallel NGF-induced Akt pathway.     

Effect of Cavtratin,a Caveolin-1 Scaffolding Domain Peptide,on Oligodendroglial Signaling Cascades

Caveolin and caveolin containing rafts are involved in the signaling of growth factors in various cell types. Previous reports of our lab indicated a co-localization of caveolin and the high affinity nerve growth factor (NGF) receptor tyrosine kinase A (TrkA). Mutual effects have been observed among which a caveolin-1 knock-down resulted in an impairment of the NGF signaling cascade rather than in an increase of activity as expected from other growth factor reports. On the other hand, an over-expression of caveolin-1 impaired the NGF stimulated activity of p42/44 mitogen activated protein kinases (MAPK). In this study, we used a caveolin-1 scaffolding domain (CSD) peptide (cavtratin) of which an inhibitory effect on growth factor receptors was reported. Our data showed that cavtratin suppresses the NGF-induced phosphorylation of TrkA as well as the activation of MAPK in porcine oligodendrocytes significantly.     

Nerve growth factor stimulates MAPK via the low affinity receptor p75(LNTR)

Apart from its high affinity receptor TrkA, nerve growth factor (NGF) can also stimulate the low affinity receptor p75(LNTR) and induce a Trk-independent signaling cascade. We examined the possible involvement of mitogen-activated protein kinase (MAPK) in this signaling pathway in neuronal cultures of the cerebellum of P2-aged rats and PCNA cells; both cell types express p75(LNTR) but not TrkA. We found a fast and transient phosphorylation of p42- and p44-MAPK after stimulation with NGF or C(2)-ceramide which proved to be sensitive to inhibition of MAPK kinase and protein kinase A (PKA). As stimulation with NGF also activated p21Ras it can be concluded that at least part of the observed MAPK activation was effected via p21Ras and via PKA.     

TrkA signalling pathways in human airway smooth muscle cell proliferation

NGF may play a role in airway inflammation and hyperresponsiveness. We studied its possible involvement in airway remodelling and report here its proliferative effect and its receptor and signalling pathways in human airway smooth muscle cells in culture (HASMC). Proliferation of HASMC induced by NGF (0.1-10 pM) was assessed by the XTT and BrdU techniques with and without kinase inhibitors. Immunoprecipitation and Western blotting were used to study phosphorylation of TrkA and MAPK. NGF caused dose-dependent proliferation of HASMC and induced TrkA phosphorylation, both abolished by the tyrosine-kinase inhibitor K252a. PI3K and JNK inhibitors had no effect. PKC inhibitors partially inhibited NGF-induced proliferation and totally abolished p38 phosphorylation but did not affect ERK1/2 phosphorylation. The rafK inhibitor decreased NGF-induced proliferation, and totally abolished ERK1/2 phosphorylation, but did not affect p38 phosphorylation. This finding was confirmed by the decrease of NGF-induced proliferation after treatment with inhibitors of the p38 or of ERK1/2 pathways. In conclusion, NGF activation of the TrkA receptor involves two distinct signalling pathways: PKC selectively activates p38, and the ras/raf pathway selectively activates ERK1/2. Both are necessary to induce HASMC proliferation.     

SH2-B is required for nerve growth factor-induced neuronal differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.     

A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.     

Nerve growth factor-induced neurite outgrowth is potentiated by stabilization of TrkA receptors

Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK) and phosphatidylinositol- 3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the Ub(K63) chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors.     

Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases.

Treatment of PC12 cells with nerve growth factor (NGF) induces a rapid increase in tyrosine phosphorylation of multiple cellular proteins. Expression of a dominant inhibitory Ras mutant specifically blocked NGF- and TPA-induced tyrosine phosphorylation of two proteins of approximately 42 and 44 kd. Conversely, expression of an oncogenic variant of Ras induced tyrosine phosphorylation of the same 42 and 44 kd proteins. The 44 kd protein was immunoprecipitated with an antibody directed against extracellular signal-regulated kinase 1/mitogen-activated protein kinase (MAPK) and the 42 kd protein comigrated with a 42 kd MAPK, indicating that at least one and probably both Ras-regulated phosphoproteins are MAPKs. In addition, MAPK activation, as measured by in vitro phosphorylation of myelin basic protein, was also regulated by Ras. Ras was not required for NGF-induced activation of Trk or tyrosine phosphorylation of PLC-gamma 1. Thus, NGF-induced tyrosine phosphorylation occurs both prior to and following Ras action, and Ras plays a critical role in the NGF- and TPA-induced tyrosine phosphorylation of MAPKs.     

Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells.

The current paradigm for the role of nerve growth factor (NGF) or FGF-2 in the differentiation of neuronal cells implies their binding to specific receptors and activation of kinase cascades leading to the expression of differentiation specific genes. We examined herein the hypothesis that FGF receptors (FGFRs) are involved in NGF-induced neuritogenesis of pheochromocytoma-derived PC12 cells. We demonstrate that in PC12 cells, FGFR expression and activity are modulated upon NGF treatment and that a dominant negative FGFR-2 reduces NGF-induced neuritogenesis. Moreover, FGF-2 expression is modulated by NGF, and FGF-2 is detected at the cell surface. Oligonucleotides that specifically inhibit FGF-2 binding to its receptors are able to significantly reduce NGF-induced neurite outgrowth. Finally, the duration of mitogen-activated protein kinase (MAPK) activity upon FGF or NGF stimulation is shortened in FGFR-2 dominant negative cells through inactivation of signaling from the receptor to the Ras/MAPK pathway. In conclusion, these results demonstrate that FGFR activation is involved in neuritogenesis induced by NGF where it contributes to a sustained MAPK activity in response to NGF.     

Nerve growth factor-induced cell cycle reentry in newborn neurons is triggered by p38MAPK-dependent E2F4 phosphorylation

Cumulative evidence indicates that activation of cyclin D-dependent kinase 4/6 (cdk4/6) represents a major trigger of cell cycle reentry and apoptosis in vertebrate neurons. We show here the existence of another mechanism triggering cell cycle reentry in differentiating chick retinal neurons (DCRNs), based on phosphorylation of E2F4 by p38(MAPK). We demonstrate that the activation of p75(NTR) by nerve growth factor (NGF) induces nuclear p38(MAPK) kinase activity, which leads to Thr phosphorylation and subsequent recruitment of E2F4 to the E2F-responsive cdc2 promoter. Inhibition of p38(MAPK), but not of cdk4/6, specifically prevents NGF-dependent cell cycle reentry and apoptosis in DCRNs. Moreover, a constitutively active form of chick E2F4 (Thr261Glu/Thr263Glu) stimulates G(1)/S transition and apoptosis, even after inhibition of p38(MAPK) activity. In contrast, a dominant-negative E2F4 form (Thr261Ala/Thr263Ala) prevents NGF-induced cell cycle reactivation and cell death in DCRNs. These results indicate that NGF-induced cell cycle reentry in neurons depends on the activation of a novel, cdk4/6-independent pathway that may participate in neurodegeneration.     

Nerve Growth Factor-Induced Differentiation in Neuroblastoma Cells Expressing TrkA but Lacking p75NGFR

Abstract: Nerve growth factor (NGF) binds to two distinct cell surface receptors, TrkA, which is a receptor tyrosine kinase, and p75^NGFR, whose role in NGF-induced signal transduction remains unclear. We have found that human neuroblastoma IMR-32 cells express TrkA, but p75^NGFR expression was not detectable in these cells by northern blot analysis, immunoblotting, or chemical crosslinking experiments. Despite the lack of p75^NGFR expression, subnanomolar concentrations of recombinant human NGF induced neurite outgrowth, tyrosine phosphorylation, and immediate early gene expression in these cells. These results strongly suggest that NGF-induced neuronal differentiation in IMR-32 cells is initiated through TrkA in the absence of p75^NGFR. Thus, IMR-32 cells may provide a model for studying neurotrophic effects of NGF on adult striatal cholinergic neurons, which also lack p75^NGFR expression.     

p185HER2 signal transduction in breast cancer cells.

A partially agonistic monoclonal antibody, 4D5, known to bind to the extracellular domain of p185HER2 and shown to inhibit long term growth of p185HER2-overexpressing breast cancer cells, was used to study signal transduction and phosphotyrosyl protein substrates associated with this receptor. Normal breast epithelial cells and breast carcinoma cells expressing low levels of p185HER2 were not affected by 4D5. HER2/neu-overexpressing breast cancer cells (BT-474 and SK-Br-3) exposed to 4D5 exhibited rapid phosphorylation of both p185HER2 and an associated 56-kDa phosphotyrosyl protein (ptyr56). Paralleling the 4D5- stimulated phosphorylation of p185HER2 and ptyr56 was a 5-10-fold induction of c-fos mRNA and phosphatidylinositol 4-kinase activity and a 2-fold induction of inositol 1,4,5-trisphosphate 3'-kinase activity. The increased phosphatidylinositol 4-kinase activity immunoprecipitated with p185HER2 and also co-eluted with ptyr56 from an antiphosphotyrosine immunoaffinity column. These results indicate that short term (less than 6 h) 4D5 activation of p185HER2 in overexpressing breast cancer cells produces agonistic-like signaling typical of homologous tyrosine kinase growth factor receptors such as epidermal growth factor receptor. The data also suggest that ptyr56 represents a novel phosphorylated substrate associated with 4D5-stimulated p185HER2.     

Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells.

Monoclonal antibody (MAb) 4D5 was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4D5 may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%, and addition of 4D5 further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in tyrosine-specific phosphorylation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of p185HER2 in the presence of newborn calf serum was not attributable to stimulation of the epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-alpha. Extension of these observations to two other mammary carcinoma cell lines. MDA-MB-453 and BT-474, also demonstrated a significant capacity of serum to induce p185HER2 phosphorylation. The demonstration of antibody-mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate p185HER2. Our observation that growth-inhibitory MAb 4D5 is able to reduce the phosphorylation of p185HER2 by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate cell proliferation.     

PC12 cells have caveolae that contain TrkA. Caveolae-disrupting drugs inhibit nerve growth factor-induced, but not epidermal growth factor-induced, MAPK phosphorylation

Nerve growth factor (NGF) induces survival and differentiation of the neural crest-derived PC12 cell line. Caveolae are cholesterol-enriched, caveolin-containing plasma membrane microdomains involved in vesicular transport and signal transduction. Here we demonstrate the presence of caveolae in PC12 cells and their involvement in NGF signaling. Our results showed the expression of caveolin-1 by Western blot and confocal immuno-microscopy. The presence of plasma membrane caveolae was directly shown by rapid-freeze deep-etching electron microscopy. Moreover, combined deep-etching and immunogold techniques revealed the presence of the NGF receptor TrkA in the caveolae of PC12 cells. These data together with the cofractionation of Shc, Ras, caveolin, and TrkA in the caveolae fraction supported a role for these plasma membrane microdomains in NGF signaling. To approach this hypothesis, caveolae were disrupted by treatment of PC12 cells with cholesterol binding drugs. Either filipin or cyclodextrin treatment increased basal levels of MAPK phosphorylation. In contrast, pretreatment of PC12 cells with these drugs inhibited the NGF- but not the epidermal growth factor-induced MAPK phosphorylation without affecting the TrkA autophosphorylation. Taken together, our results demonstrate the presence of caveolae in PC12 cells, which contain the high affinity NGF receptor TrkA, and the specific involvement of these cholesterol-enriched plasma membrane microdomains in the propagation of the NGF-induced signal.