Src and FAK are key early signalling intermediates required for neurite growth in NGF-responsive adult DRG neurons

Axonal regeneration is influenced by factors in the extracellular environment, including neurotrophins, such as NGF, and adhesion molecules, such as laminin. The provision of both NGF and a permissive substrate to cultured adult NGF-responsive DRG neurons results in enhanced levels of neurite growth not achievable by either factor alone. In this study, we have investigated the early signalling events that contribute to NGF and laminin-induced neurite growth. Adult NGF-responsive DRG neurons were plated on poly-d-lysine for 2 h then stimulated with NGF, laminin, or laminin plus NGF for 10 min, 1 h, or 6 h. Signalling pathways were subsequently analysed using Western blotting and pharmacological inhibition of specific signalling components. While activation of the various signalling intermediates (Src, FAK, Akt, MAPK) could be detected as early as 10 min-1 h after stimulation, significant neurite growth was observed mainly at the 6 h time point. The results of the time course experiments showed differential activation of the signalling intermediates. Src was activated by all treatments (NGF, laminin and the combination) at the earliest time point analysed, 10 min. NGF stimulation also resulted in detectable activation of FAK, Akt and MAPK by 10 min. However, laminin stimulation alone did not result in detectable activation of FAK, Akt or MAPK until the 1 h time point. Inhibition of either Src or FAK activity attenuated both laminin and/or NGF-induced PI 3-K/Akt and MEK/MAPK signalling pathways, as well as neurite growth. Downstream inhibition of Akt by Akt knockdown also blocked observed neurite growth, while inhibition of MEK/MAPK had no significant effect. Together, these results demonstrate that signalling underlying neurite growth can be detected within minutes of stimulation and provide a mechanism for the observed enhancement of neurite growth when both NGF and the permissive substrate, laminin, are provided.     

NGF activates the phosphorylation of MAP1B by GSK3beta through the TrkA receptor and not the p75(NTR) receptor

We have recently shown that nerve growth factor (NGF) induces the phosphorylation of the microtubule-associated protein 1B (MAP1B) by activating the serine/threonine kinase glycogen synthase kinase 3beta (GSK3beta) in a spatio-temporal pattern in PC12 cells that correlates tightly with neurite growth. PC12 cells express two types of membrane receptor for NGF: TrkA receptors and p75NTR receptors, and it was not clear from our studies which receptor was responsible. We show here that brain-derived neurotrophic factor, which activates p75NTR but not TrkA receptors, does not stimulate GSK3beta phosphorylation of MAP1B in PC12 cells. Similarly, NGF fails to activate GSK3beta phosphorylation of MAP1B in PC12 cells that lack TrkA receptors but express p75NTR receptors (PC12 nnr). Chick ciliary ganglion neurons in culture lack TrkA receptors but express p75NTR and also fail to show NGF-dependent GSK3beta phosphorylation of MAP1B, whereas in rat superior cervical ganglion neurons in culture, NGF activation of TrkA receptors elicits GSK3beta phosphorylation of MAP1B. Finally, inhibition of TrkA receptor tyrosine kinase activity in PC12 cells and superior cervical ganglion neurons with K252a potently and dose-dependently inhibits neurite elongation while concomitantly blocking GSK3beta phosphorylation of MAP1B. These results suggest that the activation of GSK3beta by NGF is mediated through the TrkA tyrosine kinase receptor and not through p75NTR receptors.     

In Vivo Regulation of Brain-Derived Neurotrophic Factor in Dorsal Root Ganglia Is Mediated by Nerve Growth Factor-Triggered Akt Activation during Cystitis

The role of brain-derived neurotrophic factor (BDNF) in sensory hypersensitivity has been suggested; however the molecular mechanisms and signal transduction that regulate BDNF expression in primary afferent neurons during visceral inflammation are not clear. Here we used a rat model of cystitis and found that the mRNA and protein levels of BDNF were increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. BDNF up-regulation in the L6 DRG was triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reduced BDNF levels during cystitis. The neutralizing NGF antibody also subsequently reduced cystitis-induced up-regulation of the serine/threonine kinase Akt activity in L6 DRG. To examine whether the NGF-induced Akt activation led to BDNF up-regulation in DRG in cystitis, we found that in cystitis the phospho-Akt immunoreactivity was co-localized with BDNF in L6 DRG, and prevention of the endogenous Akt activity in the L6 DRG by inhibition of phosphoinositide 3-kinase (PI3K) with a potent inhibitor LY294002 reversed cystitis-induced BDNF up-regulation. Further study showed that application of NGF to the nerve terminals of the ganglion-nerve two-compartmented preparation enhanced BDNF expression in the DRG neuronal soma; which was reduced by pre-treatment of the ganglia with the PI3K inhibitor LY294002 and wortmannin. These in vivo and in vitro experiments indicated that NGF played an important role in the activation of Akt and subsequent up-regulation of BDNF in the sensory neurons in visceral inflammation such as cystitis.     

Biochemical and biological characterization of a neuroendocrine-associated phosphatase

The biochemical and biological properties of a novel neuroendocrine-associated phosphatase (NEAP) were characterized. NEAP had a sequence characteristic of a dual-specificity phosphatase (DSP), and was preferentially expressed in neuroendocrine cells/tissues as well as in skeletal muscle and heart. Expression of NEAP was up-regulated in nerve growth factor (NGF)-treated, differentiated PC12 cells. NEAP was cytosolic and did not apparently have effects against extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase activated by various stimuli. Although NEAP and MAPK phosphatase (MPK)-1 showed similar phosphatase activity towards p-nitro phenylphosphate (pNPP), in contrast to MKP-1, NEAP did not dephosphorylate JNK and p38-MAPK in vitro. Overexpression of NEAP, but not the C152S mutant, in PC12 cells suppressed NGF-induced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and Akt activation. Overexpression of NEAP also suppressed neurite outgrowth induced by NGF and sensitized PC12 cells to cisplatin-induced apoptosis. Suppression of NEAP by RNA interference enhanced NGF-induced neurite outgrowth and Akt activation. Our results indicated that, unlike other DSPs, down-regulation of conventional MAPKs was not the major function of NEAP. Furthermore, NEAP might be involved in neuronal differentiation via regulation of the PI3K/Akt signaling.     

Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival.

In this report, we have examined the mechanisms whereby neurotrophins and neural activity coordinately regulate neuronal survival, focussing on sympathetic neurons, which require target-derived NGF and neural activity for survival during development. When sympathetic neurons were maintained in suboptimal concentrations of NGF, coincident depolarization with concentrations of KCl that on their own had no survival effect, synergistically enhanced survival. Biochemical analysis revealed that depolarization was sufficient to activate a Ras-phosphatidylinositol 3-kinase-Akt pathway (Ras-PI3-kinase-Akt), and function-blocking experiments using recombinant adenovirus indicated that this pathway was essential for approximately 50% of depolarization-mediated neuronal survival. At concentrations of NGF and KCl that promoted synergistic survival, these two stimuli converged to promote increased PI3-kinase-dependent Akt phosphorylation. This convergent PI3-kinase-Akt pathway was essential for synergistic survival. In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl. Thus, NGF and depolarization together mediate survival of sympathetic neurons via intracellular convergence on a Ras-PI3-kinase-Akt pathway. This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.     

Two distinct regulatory mechanisms of neurotransmitter release by phosphatidylinositol 3-kinase

Recent studies have indicated that various growth factors are involved in synaptic functions; however, the precise mechanisms remain unclear. In order to elucidate the molecular mechanisms of the growth factor-mediated regulation of presynaptic functions, the effects of epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) on neurotransmitter release were studied in rat PC12 cells. Brief treatment with EGF and IGF-1 enhanced Ca2+-dependent dopamine release in a concentration-dependent manner. EGF activated both mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase) pathways, and the EGF-dependent enhancement of DA release was suppressed by a MAPK kinase inhibitor as well as by PI3-kinase inhibitors. In striking contrast, IGF-1 activated the PI3-kinase pathway but not the MAPK pathway, and IGF-1-dependent enhancement was suppressed by a PI3-kinase inhibitor but not by a MAPK kinase inhibitor. The enhanced green fluorescent protein-tagged pleckstrin homology (PH) domain of protein kinase B, which selectively binds to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate, was translocated to the plasma membrane after treatment with either EGF or NGF. By contrast, no significant redistribution was induced by IGF-1. These results indicate that PI3-kinase participates in the enhancement of neurotransmitter release by two distinct mechanisms: EGF and NGF activate PI3-kinase in the plasma membrane, whereas IGF-1 activates PI3-kinase possibly in the intracellular membrane, leading to enhancement of neurotransmitter release in a MAPK-dependent and -independent manner respectively.     

IGF-1 increases laminin, cyclin D1, and p21Cip1 expression in glomerular mesangial cells: an investigation of the intracellular signaling pathway and cell-cycle progression

Insulin-like growth factor (IGF)-1 is accumulated in the diabetic kidney and is considered to be involved in the development of glomerular sclerosis. Here, we investigate IGF-1 regulation of laminin, an extracellular matrix (ECM) component, and cyclin D1 and p21Cip1, cell-cycle progression factor, expressions in glomerular mesangial cells. We show that IGF-1 increases the level of laminin gamma1 and beta1 subunits approximately 1.5- and 2.5-fold, respectively, in a time-dependent manner. IGF-1 also stimulates protein kinase Akt/PKB phosphorylation at Thr 308, which correlates with its activity, up to 24 h. The Akt activation is coupled with Ser 9 phosphorylation of its downstream target, glycogen synthase kinase-3beta (GSK-3beta), which inhibits its kinase activity. Laminin beta1 is reduced significantly (P < 0.03) by inhibitors of Akt and p38MAPK whereas laminin gamma1 is not affected. Surprisingly, IGF-1 activates the expression of both cyclin D1 and cell-cycle arrest factor, p21Cip1 parallely. Pharmacological inhibition of calcineurin by cyclosporin A blocks IGF-1-induced cyclin D1 and p21Cip1expression significantly (P < 0.05). IGF-1 enhances cellular metabolic activity and viability of rat mesangial cells; however, they are arrested at the G1 phase of cell cycle as revealed by the FACS analysis. These results indicate that IGF-1 mediates mesangial cell-cycle progression, hypertrophy, and ECM protein synthesis. The Akt/GSK-3beta, p38MAPK, and calcineurin pathways may play an important role in IGF-1 signaling, cell-cycle regulation, and matrix gene expression in mesangial cells leading to the development of diabetic glomerulopathy.     

PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease

Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.     

The tyrosine phosphatase inhibitor orthovanadate mimics NGF-induced neuroprotective signaling in rat hippocampal neurons

Activation of the high affinity neurotrophin receptor tropomyosin-related kinase A (TrkA) by nerve growth factor (NGF) leads to phosphorylation of intracellular tyrosine residues of the receptor with subsequent activation of signaling pathways involved in neuronal survival such as the phosphoinositide-3-kinase (PI3-K)/protein kinase B (PKB/Akt) pathway and the mitogen-activated protein kinase (MAPK) cascade. In the present study, we tested whether inhibition of protein-tyrosine phosphatases (PTP) by orthovanadate could enhance tyrosine phosphorylation of TrkA thereby stimulating NGF-like survival signaling in embryonic hippocampal neurons. We found that the PTP inhibitor orthovanadate (1 microM) enhanced TrkA phosphorylation and protected neurons against staurosporine (STS)-induced apoptosis in a time-and concentration-dependent manner. Inhibition of PTP enhanced TrkA phosphorylation also in the presence of NGF antibodies indicating that NGF binding to TrkA was not required for the effects of orthovanadate. Moreover, orthovanadate enhanced phosphorylation of Akt and the MAPK Erk1/2 suggesting that the signaling pathways involved in the protective effect were similar to those activated by NGF. Accordingly, inhibition of PI3-K by wortmannin and MAPK-kinase (MEK) inhibition by UO126 abolished the neuroprotective effects. In conclusion, the results indicate that orthovanadate mimics the effect of NGF on survival signaling pathways in hippocampal neurons. Thus, PTP inhibition appears to be an appropriate strategy to trigger neuroprotective signaling pathways downstream of neurotrophin receptors.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

p48 Ebp1 acts as a downstream mediator of Trk signaling in neurons, contributing neuronal differentiation

Two Ebp1 isoproteins, p48 and p42, regulate cell survival and differentiation distinctively. Here we show that p48 is the major isoform in hippocampal neurons and is localized throughout the entire neuron. Notably, reduction of p48 Ebp1 expression inhibited BDNF-mediated neurite outgrowth in hippocampal neurons. The p48 protein acts as a downstream effector of the Trk receptor, which mediates the functions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in hippocampal cells. Trk receptor activation by both NGF and BDNF induced phosphorylation of Ebp1 at the S360 upon the activation of protein kinase Cδ (PKCδ) and triggered dissociation of p48 from retinoblastoma (Rb). Although both NGF and BDNF activate mitogen-activated protein kinase (MAPK; extracellular signal-related kinase (ERK)) as well as phosphatidylinositide 3-kinase (PI3K)/Akt, their activation is regulated in different time-frame upon growth factor specificity, especially, eliciting PKCδ mediated p48 S360 phosphorylation. Thus, p48 Ebp1 contributes to neuronal cell differentiation and growth factor specificity through the activation of PKCδ, acting as a crucial downstream effector of neurotrophin signaling.     

p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones

We have previously shown that nerve growth factor (NGF)-induced activation of nuclear factor-kappaB increased neuronal expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF-induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti-rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF-induced bcl-xL expression. Treatment with the blocking anti-p75 antibody also inhibited NGF-induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol-3-kinase (PI3 kinase) activity or overexpression of a dominant-negative mutant of Akt kinase inhibited NGF-induced nuclear factor-kappaB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti-p75 antibody inhibited constitutive and NGF-induced Bcl-xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF-induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3-kinase pathway.     

Insulin/PI3K signaling protects dentate neurons from oxygen–glucose deprivation in organotypic slice cultures

It is known that ischemia/reperfusion induces neurodegeneration in the hippocampus in a subregion‐dependent manner. This study investigated the mechanism of selective resistance/vulnerability to oxygen–glucose deprivation (OGD) using mouse organotypic hippocampal cultures. Analysis of propidium iodide uptake showed that OGD‐induced duration‐ and subregion‐dependent neuronal injury. When compared with the CA1–3 subregions, dentate neuronal survival was more sensitive to inhibition of phosphatidylinositol 3‐kinase (PI3K)/Akt signaling under basal conditions. Dentate neuronal sensitivity to PI3K/Akt signaling activation was inversely related to its vulnerability to OGD‐induced injury; insulin/insulin‐like growth factor 1 pre‐treatment conferred neuroprotection to dentate neurons via activation of PI3K/Akt signaling. In contrast, CA1 and CA3 neurons were less sensitive to disruptions of endogenous PI3K/Akt signaling and protective effects of insulin/insulin‐like growth factor 1, but more vulnerable to OGD. OGD‐induced injury in CA1 was reduced by inhibition of NMDA receptor or mitogen‐activated protein kinase signaling, and was prevented by blocking NMDA receptor in the presence of insulin. The CA2 subregion was distinctive in its response to glutamate, OGD, and insulin, compared with other CA subregions. CA2 neurons were sensitive to the protective effects of insulin against OGD‐induced injury, but more resistant to glutamate. Distinctive distribution of insulin receptor β and basal phospho‐Akt was detected in our slice cultures. Our results suggest a role for insulin signaling in subregional resistance/vulnerability to cerebral ischemia.     

Effects of the neurotrophins nerve growth factor,neurotrophin-3, and brain-derived neurotrophic factor (BDNF) on neurite growth from adult sensory neurons in compartmented cultures

We used compartmented cultures to study the regulation of adult sensory neurite growth by neurotrophins. We examined the effects of the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT3), and BDNF on distal neurite elongation from adult rat dorsal root ganglion (DRG) neurons. Neurons were plated in the center compartments of three-chambered dishes in the absence of neurotrophin, and neurite extension into the distal (side) compartments containing NGF, BDNF, or NT3 was quantitated. Initial proximal neurite growth did not require any of the neurotrophins, while subsequent elongation into distal compartments required NGF. After neurites had extended into NGF-containing distal compartments, removal of NGF by treatment with anti-NGF resulted in the cessation of growth with minimal neurite retraction. In contrast to the effects of NGF, no distal neurite elongation was observed into compartments with BDNF or NT3. To examine possible additive influences, neurite extension into compartments containing BDNF plus NGF or NT3 plus NGF was quantitated. There was no increased neurite extension into NGF plus NT3 compartments, while the combination of BDNF plus NGF resulted in an inhibition of neurite extension compared with NGF alone. We then investigated whether the regrowth of neurites that had originally grown into NGF subsequent to in vitro axotomy still required NGF. The results demonstrated that unlike adult sensory nerve regeneration in vivo, the in vitro regrowth did require NGF, and neither BDNF nor NT3 was able to substitute for NGF. Since the initial growth from neurons after dissociation (which is also a regenerative response) did not require NGF, it would appear that neuritic growth and regrowth of adult DRG neurons in vitro includes both NGF-independent and NGF-dependent components. The compartmented culture system provides a unique model to further study aspects of this differential regulation of neurite growth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 395–410, 1997     

Role of protein kinase B and the MAP kinase cascade in mediating the EGF-dependent inhibition of glycogen synthase kinase 3 in Swiss 3T3 cells

Epidermal growth factor (EGF), insulin-like growth factor 1 (IGF1) and phorbol myristate acetate (PMA) induce the inhibition of glycogen synthase kinase 3 (GSK3) by stimulating the phosphorylation of an N-terminal serine. Here, we show that protein kinase B (PKB) plays a key role in mediating EGF-induced inhibition of GSK3alpha and that the classical MAP kinase (MAPK) cascade has two functions in this process. Firstly, it makes a transient contribution to EGF-induced inhibition of GSK3alpha. Secondly, it shortens the duration of PKB activation and GSK3alpha inhibition. In contrast, PKB alone mediates the IGF1-induced inhibition of GSK3alpha, while the MAPK cascade mediates the inhibition of GSK3alpha by PMA.     

Comparative signaling pathways of insulin-like growth factor-1 and brain-derived neurotrophic factor in hippocampal neurons and the role of the PI3 kinase pathway in cell survival

Insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are trophic factors required for the viability and normal functions of various neuronal cells. However, the detailed intracellular mechanism(s) involved in these effects in neuronal cells remains to be fully elucidated. In present study, the respective intracellular signaling pathway induced by IGF-1 and BDNF and their possible role in neuronal survival were investigated. Both IGF-1 and BDNF protected hippocampal neurons from serum deprivation-induced death with IGF-1 apparently being more potent. Western blot analyses showed that both IGF-1 and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3)/Akt (protein kinase B) kinase and the mitogen-activated protein kinase (MAPK) pathways. The phosphorylation of Akt and its downstream target, FKHRL1, induced by IGF-1 was rapid and sustained while that of MAPK was transient. The reverse situation was observed for BDNF. Moreover, IGF-1 potently induced the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with PI3 kinase while BDNF was weak in these assays. In contrast, the tyrosine phosphorylation of Shc proteins was dramatically stimulated by BDNF, with IGF-1 having only a minimal effect. Most interestingly, only the inhibitor of the PI3K/Akt pathway, LY294002, was able to block the survival effects of both IGF-1 and BDNF; an inhibitor of the MAPK pathway inhibitor, PD98059, being ineffective. Taken together, these data reveal that the survival properties of both IGF-1 and BDNF against serum deprivation are mediated by the activation of the PI3K/Akt, but not the MAPK, pathway in hippocampal neurons.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Artemin activates axonal growth via SFK and ERK-dependent signalling pathways in mature dorsal root ganglia neurons

Artemin, one of the glial cell line-derived neurotrophic factor (GDNF) family, enhances the generation and survival of early sympathetic neurons and superior cervical ganglion (SCG) neurons. Src-family kinases (SFK) are involved in the growth and differentiation of cells, which are composed of unique Src homology 2 (SH2), Src homology 3 (SH3) and kinase domains. Various extra-cellular molecules containing growth factors and G-protein coupled receptors stimulate SFK. In this report, artemin is shown to have a significant effect on the neurite growth of dorsal root ganglia (DRG) neurons. Also, artemin triggers Src-family kinase activation and the phosphorylation of extra-cellular signal-regulated kinases (ERK) mitogen-activated protein kinase (MAPK). Artemin also regulated actin polymerization. There are several indications that another SH3-containing protein, Hck, and an SH3-containing adaptor protein, Nck1, play an important role in the organization of the actin cytoskeleton by cellular signalling. These findings suggest that the exploration of binding partners for the SH3 domain could provide an insight into regulation between the microtubule and actin networks. The binding partners for the SH3 domains of Nck, Src and Hck that we identified were Smc chromosome segregation ATPases, FOG Zn-finger protein and the FYVE zinc-binding domain, respectively.