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.     

Plasma 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are insensitive indicators of alpha 2-adrenoceptor mediated regulation of norepinephrine release in healthy human volunteers.

The usefulness of the plasma concentrations of two major metabolites of norepinephrine (NE), 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as indicators of neuronal NE release was investigated. The potent alpha 2-adrenoceptor agonist, dexmedetomidine, induced only about 15% maximal reductions in the metabolite concentrations, in spite of almost total inhibition of neuronal NE release, as evidenced by 90% reductions in plasma NE concentrations. Similarly, administration of the alpha 2-adrenoceptor antagonist atipamezole was followed by only small increases in plasma DHPG and no change in MHPG levels, in spite of almost six-fold, albeit short-lasting, increases in plasma NE. In contrast, a single dose of the reversible monoamine oxidase type A (MAO-A) inhibitor moclobemide reduced plasma DHPG levels by 78% and MHPG levels by 51%. It is concluded that the plasma concentrations of DHPG and MHPG are largely determined by intraneuronal, MAO-A-dependent metabolism of NE, and do not accurately reflect acute alterations in neuronal NE release. The concentration of NE in venous plasma is clearly a more sensitive indicator of alpha 2-adrenoceptor-mediated regulation of NE release.     

Increase in norepinephrine turnover after tyrosine or DL-threo-3,4-dihydroxyphenylserine (DL-threo-DOPS)

The amino acids tyrosine and DL-threo-3,4-dihydroxyphenylserine (DL-threo-DOPS) were compared for their effectiveness in increasing central nervous system norepinephrine (NE) turnover in both saline and DSP-4 pretreated mice. NE was decreased significantly in cortex, hippocampus and cerebellum, and only slightly in hypothalamus and brainstem two weeks after a single intraperitoneal injection of the neurotoxin DSP-4. Levels of the major NE metabolite, 3-methoxyl-4-hydroxyphenylethylene glycol (MHPG), decreased in parallel in these five brain regions. Neither administration of tyrosine (250 mg/kg, as the ethyl ester, i.p.) nor DL-threo-DOPS (200 mg/kg, i.p.) affected regional NE concentration. However, after tyrosine administration, MHPG levels increased significantly in cortex in control mice and in cortex and hippocampus of DSP-4 pretreated mice. In all five brain noradrenergic regions MHPG level increased after DL-threo-DOPS administration and this increase was enhanced (approximately doubled) in DSP-4 pretreated mice. Thus, both amino acids may be useful as precursors of central NE when its level is depleted (e.g. following administration of DSP-4); DL-threo-DOPS producing a generalized increase in brain NE turnover, while increases following tyrosine are specific to those areas in which neuronal activity is increased i.e. cortex and hippocampus.     

PACAP type I receptor transactivation is essential for IGF-1 receptor signalling and antiapoptotic activity in neurons

Insulin-like growth factor-1 (IGF-1) and pituitary adenylyl cyclase activating polypeptide (PACAP) are both potent neurotrophic and antiapoptotic factors, which exert their effects via phosphorylation cascades initiated by tyrosine kinase and G-protein-coupled receptors, respectively. Here, we have adapted a recently described phosphoproteomic approach to neuronal cultures to characterize the phosphoproteomes generated by these neurotrophic factors. Unexpectedly, IGF-1 and PACAP increased the phosphorylation state of a common set of proteins in neurons. Using PACAP type 1 receptor (PAC1R) null mice, we showed that IGF-1 transactivated PAC1Rs constitutively associated with IGF-1 receptors. This effect was mediated by Src family kinases, which induced PAC1R phosphorylation on tyrosine residues. PAC1R transactivation was responsible for a large fraction of the IGF-1-associated phosphoproteome and played a critical role in the antiapoptotic activity of IGF-1. Hence, in contrast to the general opinion that the trophic activity of IGF-1 is solely mediated by tyrosine kinase receptor-associated signalling, we show that it involves a more complex signalling network dependent on the PAC1 Gs-protein-coupled receptor in neurons.     

Glutamate acting on N-methyl-D-aspartate receptors attenuates insulin-like growth factor-1 receptor tyrosine phosphorylation and its survival signaling properties in rat hippocampal neurons

Impairing intracellular signaling induced by survival factors and excess glutamate have recently been suggested to play important role in neurodegenerative processes. However, the underlying mechanism(s) and interrelationships between these factors mostly remain to be established. In the present study, we show that glutamate attenuates the tyrosine phosphorylation of the insulin-like growth factor-1 (IGF-1) receptor and the survival effect of IGF-1 (100 nm) in hippocampal cultured neurons. Pretreatment of cultured hippocampal neurons with glutamate concentration dependently inhibited the tyrosine phosphorylation of IGF-1 receptors as well as that of IRS-1 and Shc, two IGF-1 receptor adapter proteins. The effect of glutamate was also evident on the phosphorylation of Akt, as well as its upstream kinase PI3K/PDK1 and downstream targets, GSK3beta and FOXO3a. The inhibitory effect of glutamate (1 mm) was blocked by antagonists of the N-methyl-d-aspartate (NMDA) receptor, including MK801 (20 microm) and AP5 (100 microm), but not by blockers of other ionotropic or metabotropic glutamate receptor sub-types demonstrating the involvement of the NMDA receptor. This hypothesis is supported further by the observation that treatment with NMDA concentration dependently inhibited the activation and phosphorylation of IGF-1 receptors and downstream targets induced by IGF-1 (100 nm). These findings demonstrate that glutamate can block the effect of IGF-1 by decreasing IGF-1 receptor signaling and responsiveness, hence attenuating the survival properties of this trophic factor in neuronal cells. Our results also suggest a novel mechanism by which glutamate can reduce cell viability and induce neurotoxicity.     

Potentiation of neuronal L calcium channels by IGF-1 requires phosphorylation of the alpha1 subunit on a specific tyrosine residue

Insulin-like growth factor 1 (IGF-1) rapidly potentiates N and L calcium channel currents in cerebellar granule neurons by an unknown mechanism. Here, we show that the L channel alpha1C subunit is tyrosine phosphorylated in response to IGF-1. Moreover, expression of kinase-dead c-Src in neurons or acute block of Src family kinases with a cell-permeable inhibitor specifically blocks L channel potentiation. Purified Src kinase phosphorylates tyrosine residue Y2122 of the C terminus of neuronal alpha1C in vitro, and c- and v-Src directly bind the C terminus. When expressed in neuroblastoma cells, point mutation of Y2122 prevents both tyrosine phosphorylation of alpha1C and IGF-1 potentiation. Our data provide a biochemical mechanism whereby phosphorylation of a single specific tyrosine residue rapidly modifies ion channel physiology.     

The involvement of GABA(A) receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the ventromedial-infundibular region of hypothalamus in anestrous ewes.

To examine the role of the GABA(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/IN) of anestrous ewes, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), 4-hydroxy-3-methoxy-phenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC) were quantified during local stimulation or blockade of GABA(A) receptors with muscimol or bicuculline respectively. In most animals stimulation of GABA(A) receptors significantly attenuates GnRH release with concomitant increase of beta-endorphin and DA release, and MHPG and DOPAC levels. Blockade of the GABA(A) receptors generally did not affect GnRH and NE release but inhibited in most animals beta-endorphin release and decreased dopaminergic activity. These results suggest, that GABA may suppress GnRH release directly by GABA(A) receptor mechanism on the axon terminal of GnRH neurons or indirectly by GABA(A) receptor processes activating beta-endorphin-ergic and dopaminergic neurons in the VEN/NI. On the basis of these results in could not be distinguish between these two events. The decrease in extracellular beta-endorphin and dopamine concentration without evident changes in the GnRH level during GABA(A) receptor blockade may suggest that other neuronal systems are involved in this effect.     

Relationship between norepinephrine release in the hypothalamic paraventricular nucleus and circulating prolactin levels in the Siberian hamster: role of photoperiod and the pineal gland

The impact of norepinephrine (NE) and its metabolite, 3-methoxy4-hydroxyphenylglycol (MHPG), on circulating prolactin (PRL) was evaluated in the paraventricular region of the hypothalamus as a function of photoperiod and integrity of the pineal gland. In Experiment 1, whole tissue content of NE and MHPG was assessed in male and female hamsters that had been pinealectomized or sham-pinealectomized and exposed to long or short photoperiods for 5 weeks. The results revealed a marginal effect of photoperiod in males, but no overall effects of surgery. Because analysis of whole tissue content can be complicated by concurrent changes in synthesis and storage rates, Experiment 2 was conducted using microdialysis to assess extracellular levels of NE and MHPG in female hamsters. Pinealectomy completely prevented the short-day-induced suppression of luteinizing hormone, but it only partially prevented the effects of short days on PRL. Furthermore, both NE and MHPG levels were significantly elevated in short-day-exposed pinealectomized and sham-operated animals. These results suggest that NE release within the paraventricular nucleus inhibits the circulating PRL levels and is one mechanism by which direct photic information can influence the neuroendocrine axis independently of the pineal melatonin signal.     

The luteinizing hormone-releasing hormone inhibits the anti-apoptotic activity of insulin-like growth factor-1 in pituitary alphaT3 cells by protein kinase Calpha-mediated negative regulation of Akt

The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the alphaT3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in alphaT3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) alpha-specific inhibitor, and by a dominant negative form of PKCalpha. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKCalpha-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue alphaT3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells.     

Activation of the Insulin-like Growth Factor Type 1 Receptor by Deletion of Amino Acids 870–905

We have created a deletion mutant of the insulin-like growth factor type 1 receptor (IGF-1 R) which lacks the 36 amino acids (aa) immediately N-terminal to the transmembrane domain (Δ870–905 IGF-1 R). This region has been reported to have a negative effect on the transforming potential of an avian sarcoma virus gag-IGF-1 R fusion protein. We have sought to determine whether this region plays a similar role in the intact IGF-1 R. Analysis of the tyrosine kinase activity of the Δ870–905 IGF-1 R shows that the mutant receptor is autophosphorylated without IGF-1 stimulation, indicating that the tyrosine kinase domain is constitutively active. In addition, processing of the receptor is decreased, resulting in accumulation of a high molecular weight proreceptor containing both α and β-subunits. A well-characterized substrate of the IGF-1 R, IRS-1, is constitutively phosphorylated by the Δ870–905 IGF-1 R and phosphoinositide (PI) 3-kinase activity, which is normally activated by the phosphorylation of IRS-1 following IGF-1 stimulation, is increased even in the absence of IGF-1. A second intracellular signal pathway normally activated by IGF-1, the MAP kinase pathway, showed no increase in activity in the absence of IGF-1. The Δ870–905 IGF-1 R promoted cell proliferation only in the presence of IGF-1. We conclude that this deletion increases the basal activity of the IGF-1 receptor tyrosine kinase and activates PI 3-kinase, but is unable to stimulate MAP kinase in the absence of ligand. These results confirm those seen in the gag-IGF-1 R fusion protein and indicate that aa 870–905 exert a negative effect on the tyrosine kinase domain of the β-subunit of the IGF-1 R.     

Mechanisms of 4-hydroxytamoxifen anti-growth factor activity in breast cancer cells: alterations of growth factor receptor binding sites and tyrosine kinase activity

We previously demonstrated that antiestrogen 4-hydroxytamoxifen (OH-Tam) blocks the mitogenic activity of growth factors in breast cancer. We now investigate this mechanism by evaluating how OH-Tam affects growth factor binding and receptor tyrosine kinase activity. We show here that OH-Tam has an opposite effect on epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) binding in estrogen receptor (ER) positive cells. A decrease in IGF-1 binding sites may explain the reduced IGF-I mitogenic effect, whereas an increase in high affinity EGF binding associated with a decrease in in vitro receptor autophosphorylation rather favors the possibility of an alteration in EGF receptor tyrosine kinase activity. We conclude that OH-Tam may prevent growth factor action in ER+ cells both by modulating the concentration of growth factor binding sites and by altering growth factor receptor functionality.     

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.     

Tyrosine kinase-independent activation of extracellular-regulated kinase (ERK) 1/2 by the insulin-like growth factor-1 receptor

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gβγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.     

A Highly Selective Dual Insulin Receptor (IR)/Insulin-like Growth Factor 1 Receptor (IGF-1R) Inhibitor Derived from an Extracellular Signal-regulated Kinase (ERK) Inhibitor

Dual inhibitors of the closely related receptor tyrosine kinases insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) are promising therapeutic agents in cancer. Here, we report an unusually selective class of dual inhibitors of IGF-1R and IR identified in a parallel screen of known kinase inhibitors against a panel of 300 human protein kinases. Biochemical and structural studies indicate that this class achieves its high selectivity by binding to the ATP-binding pocket of inactive, unphosphorylated IGF-1R/IR and stabilizing the activation loop in a native-like inactive conformation. One member of this compound family was originally reported as an inhibitor of the serine/threonine kinase ERK, a kinase that is distinct in the structure of its unphosphorylated/inactive form from IR/IGF-1R. Remarkably, this compound binds to the ATP-binding pocket of ERK in an entirely different conformation to that of IGF-1R/IR, explaining the potency against these two structurally distinct kinase families. These findings suggest a novel approach to polypharmacology in which two or more unrelated kinases are inhibited by a single compound that targets different conformations of each target kinase.     

Beta-arrestin1 mediates insulin-like growth factor 1 (IGF-1) activation of phosphatidylinositol 3-kinase (PI3K) and anti-apoptosis

beta-arrestins (1 and 2) are widely expressed cytosolic proteins that play central roles in G protein-coupled receptor signaling. beta-arrestin1 is also recruited to the insulin-like growth factor 1 (IGF-1) receptor, a receptor tyrosine kinase, upon agonist binding. Here we report that, in response to IGF-1 stimulation, beta-arrestin1 mediates activation of phosphatidylinositol 3-kinase in a pathway that leads to the subsequent activation of Akt and anti-apoptosis. This process is independent of both Gi and ERK activity. The pathway fails in mouse embryo fibroblasts lacking both beta-arrestins and is restored by stable transfection of beta-arrestin1. Remarkably, this pathway is insensitive to chemical inhibition of IGF-1 receptor tyrosine kinase activity. These results suggest that, in addition to their roles in G protein-coupled receptor signaling, beta-arrestins couple the IGF-1 receptor tyrosine kinase to the phosphatidylinositol 3-kinase system and suggest that this mechanism is operative independently of the tyrosine kinase activity of the receptor.     

Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells.

A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie-infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in the level of IGF-1R in two independently scrapie-infected neuroblastomas, ScN2a and ScN1E-115 cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In contrast to the elevated IGF-1R expression in ScN2a, receptor binding studies revealed an 80% decrease in specific (125)I-IGF-1-binding sites compared with N2a cells. This decrease in IGF-1R-binding sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1 stimulated IGF-1Rbeta tyrosine phosphorylation showed no major change in the dose-response between the cell types, possibly due to altered tyrosine kinase signaling in scrapie-infected neuroblastoma cells. Altogether these data indicate that scrapie infection affects the expression, binding affinity, and signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and function may weaken the trophic support in scrapie-infected neurons and thereby contribute to neurodegeneration in prion diseases.     

Mechanisms of ADRF release from rat aortic adventitial adipose tissue

Blood vessels are surrounded by variable amounts of adipose tissue. We showed earlier that adventitial adipose tissue inhibits rat aortic contraction by release of a transferable factor, adventitium-derived relaxing factor (ADRF), which activates smooth muscle K(+) channels. However, little is known about the mechanisms of ADRF release. Using isolated rat aortic rings and isometric contraction measurements, we show that ADRF release depends on extracellular [Ca(2+)] (EC(50) approximately 4.7 mM). ADRF effects do not involve neuronal presynaptic N-type Ca(2+) and Na(+) channels or vanilloid, cannabinoid, and CGRP receptors. ADRF release is strongly inhibited by the protein tyrosine kinase inhibitors genistein and tyrphostin A25. In contrast, daidzein, an inactive genistein analog, and the protein tyrosine kinase inhibitor ST638 had no effect. Protein kinase A inhibition by H89 also inhibited ADRF release, whereas the protein kinase G inhibitor KT-5823 had no effect. We propose that ADRF release is Ca(2+) dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. Furthermore, ADRF release does not depend on perivascular nerve endings.