Dose and age-dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target-derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF-dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax-/- mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA-positive, and TrkA-/- TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose- and age-dependent fashion, mediated by p75NTR signaling through TrkA expressing axons.     

EFA6A,an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.     

A Trifluoromethyl Analog of Verbenachalcone Promotes Neurite Outgrowth and Cell Proliferation of NeuroScreen-1 Cells

Past research has shown that natural products of plant and marine origins and their congeners enhance the actions of neuritogenic factors of the central nervous system (CNS) such as nerve growth factor (NGF). However, the role of fluorine substitutions in their structure–activity relationship (SAR) has not been explored. We have synthesized a trifluoromethyl analog of verbenachalcone (VC), a pharmacologically active natural compound previously shown to potentiate NGF activity. This analog, designated C278, enhances neurite outgrowth and proliferation of NeuroScreen-1™ (NS-1) cells, a subclone of PC12 pheochromocytoma cells. C278 increases the percentage of neurite bearing cells in the presence of suboptimal doses of NGF in comparison with controls treated with NGF alone. In addition, C278 stimulates cell growth in reduced serum and serum-free cell culture conditions based on our observation of increases in cell number and metabolic assessment with MTT reduction and resazurin assays. The addition of C278 partially restored inhibition of NGF-induced neurite outgrowth by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U0126. Short-term sequential exposure of cells to U0126, C278, and NGF enhanced phosphorylation of extracellular signal-regulated kinase (ERK) in comparison with cells treated with only the MEK inhibitor and NGF. C278 also attenuated cell growth arrest caused by exposure to PD98059, U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002 but did not alter phosphorylation of Akt, a classic downstream target of PI3K during cell survival. These data suggest that C278 promotes NGF-dependent neurite outgrowth in NS-1 cells through a MEK signaling pathway by a mechanism that alters short-term activation of ERK. In contrast, C278 promotes PI3K-mediated survival independently of Akt phosphorylation.     

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.     

Multiple G<Subscript>i</Subscript> Proteins Participate in Nerve Growth Factor-Induced Activation of c-Jun N-terminal Kinases in PC12 Cells

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of G_i/o proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Gα_z or the PTX-resistant mutants of Gα_i1–3 and Gα_oA. Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via G_i1–3 proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.     

Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein alpha subunits and thus inhibit signaling by many G protein-coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF-mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor-selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation.     

Development of sensory neurons in the absence of NGF/TrkA signaling in vivo

The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.     

Contribution of BDNF-mediated inhibition in patterning avian skin innervation

Multiple factors are involved in the development and regulation of sensory innervation in skin. The findings we report here suggest that brain-derived neurotrophic factor (BDNF)-mediated inhibition may play an important role in determining the pattern of sensory innervation in avian skin. In birds, cutaneous innervation is restricted to dermis, where axons form a ring of innervation around the base of each feather. Here we show that both BDNF message and protein are more abundant in avian epidermis than dermis when innervation is being established; the BDNF in dermis is localized to feather buds. In vitro, BDNF caused growth cones of NGF-dependent dorsal root ganglion neurons to collapse. Similarly, outgrowth of neurites toward BDNF-secreting fibroblasts was inhibited. The inhibitory effects of BDNF appear to be mediated by the low-affinity p75 neurotrophin receptor, rather than a trk receptor. Thus, the distribution of BDNF in embryonic avian skin and the inhibitory effects of BDNF on cutaneous neurites in vitro suggest that BDNF may be important in restricting axons from entering the epidermis and the core of feather buds during development in vivo.     

Dose and age‐dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target‐derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF‐dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax^−/− mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA‐positive, and TrkA^−/− TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75^NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose‐ and age‐dependent fashion, mediated by p75^NTR signaling through TrkA expressing axons. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

RhoA as a mediator of clinically relevant androgen action in prostate cancer cells

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.     

SHP-2 mediates target-regulated axonal termination and NGF-dependent neurite growth in sympathetic neurons

The tyrosine phosphatase SHP-2 has been implicated in a variety of signaling pathways, including those mediated by neurotrophins in neurons. To examine the role of SHP-2 in the development of sympathetic neurons, we inhibited the function of SHP-2 in transgenic mice by overexpressing a catalytically inactive SHP-2 mutant under the control of the human dopamine beta-hydroxylase promoter. Expression of mutant SHP-2 did not influence the survival, axon initiation, or pathfinding abilities of the sympathetic neurons. However, mutant SHP-2 expression resulted in an overproduction of sympathetic fibers in sympathetic target organs. This was due to interference with SHP-2 function, as overexpression of wild type SHP-2 had no such effect. In vitro, NGF-dependent neurite growth was inhibited in neurons expressing mutant SHP-2 but not in those expressing wild type SHP-2. Mutant (but not wt) SHP-2 expression also inhibited NGF-stimulated ERK activation. The NGF-dependent survival pathway was less affected than the neurite growth pathway. Our results suggest that NGF-regulated axon growth signals, and to a lesser degree survival signals, are mediated through a SHP-2-dependent pathway in sympathetic neurons. The increased sympathetic innervation in target tissues of neurons expressing mutant SHP-2 may result from interference with normal "stop" signals dependent on signaling by gradients of NGF.     

Evidence for Phosphatidylinositol 4-Kinase and Actin Involvement in the Regulation of 125I-β-Nerve Growth Factor Retrograde Axonal Transport

The signaling events regulating the retrograde axonal transport of neurotrophins are poorly understood, but a role for phosphatidylinositol kinases has been proposed. In this study, we used phenylarsine oxide (PAO) to examine the participation of phosphatidylinositol 4-kinases in nerve growth factor (NGF) retrograde axonal transport within sympathetic and sensory neurons. The retrograde transport of 125I-labeled betaNGF was inhibited by PAO (0.5-2 nmol/eye), and this effect was diminished by dilution. Coinjection of 2,3-dimercaptopropanol with PAO reduced its ability to inhibit 125I-betaNGF retrograde transport. PAO (20 nM to 200 microM) also inhibited NGF-dependent survival of both sympathetic and sensory neuronal populations. F-actin staining in sympathetic and sensory neuronal growth cones was disrupted by PAO at 10 and 2 nM, respectively, and occurred within 5 min of exposure to the drug. The actin inhibitor latrunculin A also rapidly affected F-actin staining in vitro and reduced 125I-betaNGF retrograde axonal transport in vivo to the same extent as PAO. These results suggest that both phosphatidylinositol 4-kinase isoforms and the actin cytoskeleton play significant roles in the regulation of 125I-betaNGF retrograde axonal transport in vivo.     

LAMTOR2-Mediated Modulation of NGF/MAPK Activation Kinetics during Differentiation of PC12 Cells

LAMTOR2 (p14), a part of the larger LAMTOR/Ragulator complex, plays a crucial role in EGF-dependent activation of p42/44 mitogen-activated protein kinases (MAPK, ERK1/2). In this study, we investigated the role of LAMTOR2 in nerve growth factor (NGF)-mediated neuronal differentiation. Stimulation of PC12 (rat adrenal pheochromocytoma) cells with NGF is known to activate the MAPK. Pharmacological inhibition of MEK1 as well as siRNA–mediated knockdown of both p42 and p44 MAPK resulted in inhibition of neurite outgrowth. Contrary to expectations, siRNA–mediated knockdown of LAMTOR2 effectively augmented neurite formation and neurite length of PC12 cells. Ectopic expression of a siRNA-resistant LAMTOR2 ortholog reversed this phenotype back to wildtype levels, ruling out nonspecific off-target effects of this LAMTOR2 siRNA approach. Mechanistically, LAMTOR2 siRNA treatment significantly enhanced NGF-dependent MAPK activity, and this effect again was reversed upon expression of the siRNA-resistant LAMTOR2 ortholog. Studies of intracellular trafficking of the NGF receptor TrkA revealed a rapid colocalization with early endosomes, which was modulated by LAMTOR2 siRNA. Inhibition of LAMTOR2 and concomitant destabilization of the remaining members of the LAMTOR complex apparently leads to a faster release of the TrkA/MAPK signaling module and nuclear increase of activated MAPK. These results suggest a modulatory role of the MEK1 adapter protein LAMTOR2 in NGF-mediated MAPK activation required for induction of neurite outgrowth in PC12 cells.