Nerve growth factor-induced differentiation of PC12 cells is accompanied by elevated adenylyl cyclase activity.

Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G(s)-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gα(s). These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation.

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

BACKGROUND: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis. METHODS: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro. RESULTS: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar. CONCLUSION: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

Neurotrophin-3: a neurotrophic factor related to NGF and BDNF.

The development and maintenance of the nervous system depends on proteins known as neurotrophic factors. Although the prototypical neurotrophic factor, nerve growth factor (NGF), has been intensively studied for decades, the discovery and characterization of additional such factors has been impeded by their low abundance. Sequence homologies between NGF and the recently cloned brain-derived neurotrophic factor (BDNF) were used to design a strategy that has now resulted in the cloning of a gene encoding a novel neurotrophic factor, termed neurotrophin-3 (NT-3). The distribution of NT-3 messenger RNA and its biological activity on a variety of neuronal populations clearly distinguish NT-3 from NGF and BDNF, and provide compelling evidence that NT-3 is an authentic neurotrophic factor that has its own characteristic role in vivo.

LIFR beta and gp130 as heterodimerizing signal transducers of the tripartite CNTF receptor.

The ciliary neurotrophic factor (CNTF) receptor complex is shown here to include the CNTF binding protein (CNTFR alpha) as well as the components of the leukemia inhibitory factor (LIF) receptor, LIFR beta (the LIF binding protein) and gp130 [the signal transducer of interleukin-6 (IL-6)]. Thus, the conversion of a bipartite LIF receptor into a tripartite CNTF receptor apparently occurs by the addition of the specificity-conferring element CNTFR alpha. Both CNTF and LIF trigger the association of initially separate receptor components, which in turn results in tyrosine phosphorylation of receptor subunits. Unlike the IL-6 receptor complex in which homodimerization of gp130 appears to be critical for signal initiation, signaling by the CNTF and LIF receptor complexes depends on the heterodimerization of gp130 with LIFR beta. Ligand-induced dimerization of signal-transducing receptor components, also seen with receptor tyrosine kinases, may provide a general mechanism for the transmission of a signal across the cell membrane.

Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components.

A recently defined family of cytokines, consisting of ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), and interleukin-6 (IL-6), utilize the Jak-Tyk family of cytoplasmic tyrosine kinases. The beta receptor components for this cytokine family, gp130 and LIF receptor beta, constitutively associate with Jak-Tyk kinases. Activation of these kinases occurs as a result of ligand-induced dimerization of the receptor beta components. Unlike other cytokine receptors studied to date, the receptors for the CNTF cytokine family utilize all known members of the Jak-Tyk family, but induce distinct patterns of Jak-Tyk phosphorylation in different cell lines.

Released form of CNTF receptor alpha component as a soluble mediator of CNTF responses.

The alpha component of the receptor for ciliary neurotrophic factor (CNTF) differs from other known growth factor receptors in that it is anchored to cell membranes by a glycosylphosphatidylinositol linkage. One possible function of this type of linkage is to allow for the regulated release of this receptor component. Cell lines not normally responsive to CNTF responded to treatment with a combination of CNTF and a soluble form of the CNTF alpha receptor component. These findings not only demonstrate that the CNTF receptor alpha chain is a required component of the functional CNTF receptor complex but also reveal that it can function in soluble form as part of a heterodimeric ligand. Potential physiological roles for the soluble CNTF receptor are suggested by its presence in cerebrospinal fluid and by its release from skeletal muscle in response to peripheral nerve injury.

CNTF, FGF, and NGF collaborate to drive the terminal differentiation of MAH cells into postmitotic neurons.

The differentiation of neuronal cell progenitors depends on complex interactions between intrinsic cellular programs and environmental cues. Such interactions have recently been explored using an immortalized sympathoadrenal progenitor cell line, MAH. These studies have revealed that depolarizing conditions, in combination with exposure to FGF, can induce responsiveness to NGF. Here we report that CNTF, which utilizes an intracellular signaling pathway distinct from that of both FGF and NGF, can collaborate with FGF to promote efficiently the differentiation of MAH progenitor cells to a stage remarkably reminiscent of NGF-dependent, postmitotic sympathetic neurons. We also find that similar collaborative interactions can occur during transdifferentiation of normal cultured chromaffin cells into sympathetic neurons.

Retrograde axonal transport of LIF is increased by peripheral nerve injury: correlation with increased LIF expression in distal nerve.

Leukemia inhibitory factor (LIF) is a cytokine that affects the survival and differentiation of certain neuronal populations in vitro. To identify LIF-responsive neurons in the adult rat, we have demonstrated retrograde axonal transport of 125I-LIF to sensory and motor neurons. The accumulation of 125I-LIF by both cell types was significantly increased by prior sciatic nerve crush. Retrograde transport of 125I-LIF was inhibited by excess unlabeled LIF but not by related cytokines, indicating a specific receptor-mediated mechanism. Northern blot analysis revealed LIF expression in peripheral nerve that was increased in distal segments after axotomy. The correlation between LIF expression and increased retrograde transport following injury suggests that LIF plays a role in peripheral nerve regeneration.

The alpha component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development.

We recently proposed that ciliary neurotrophic factor (CNTF) shares two receptor components with a generally acting cytokine, leukemia inhibitory factor (LIF), but that CNTF also requires a third receptor component (CNTFR alpha) that is mostly restricted to the nervous system in its expression. Here we demonstrate that a transfected CNTFR alpha gene is sufficient to confer CNTF responsiveness upon hemopoietic cells normally responsive only to LIF, providing evidence that CNTFR alpha is a required receptor component that uniquely characterizes CNTF-responding cells. Consistent with this notion, CNTFR alpha expression could be localized to neurons within all known peripheral targets of CNTF. CNTFR alpha was also widely expressed within neurons of the CNS, suggesting that CNTF has broader CNS actions than previously appreciated. However, in vivo localization of CNTFR alpha, as well as of CNTF itself, is consistent with a particularly important role for CNTF in motor function as well as during neuropoiesis.

Alternative forms of rat TrkC with different functional capabilities.

We have identified transcripts encoding several different forms of rat TrkC, a member of the Trk family of receptor tyrosine kinases that serves as a receptor for neurotrophin-3. Some forms of TrkC lack the intracytoplasmic kinase domain and thus resemble previously defined truncated variants of TrkB. Other forms of TrkC contain variable-sized amino acid insertions within the tyrosine kinase domain. Transcripts encoding all forms of TrkC can be detected throughout the nervous system, displaying substantial overlap as well as mutually exclusive distribution patterns with transcripts for TrkB. Strikingly, only transcripts encoding the truncated forms of TrkB and TrkC are found in astrocytes, peripheral nerve, and nonneural tissues. Finally, forms of TrkC containing insertions within the kinase domain retain their ability to autophosphorylate in response to neurotrophin-3, but cannot mediate proliferation in fibroblasts or neuronal differentiation in PC12 cells.

Similarities and differences in the way neurotrophins interact with the Trk receptors in neuronal and nonneuronal cells.

We have exploited a battery of approaches to address several controversies that have accompanied the expansion of the nerve growth factor (NGF) family of neurotrophic factors and the identification of the Trk tyrosine kinases as receptors for these factors. For example, we find that a recently cloned mammalian neurotrophin, known as either neurotrophin-4 or neurotrophin-5 and assigned widely differing receptor specificities, represents the functional counterpart of Xenopus neurotrophin-4 and is a "preferred" ligand for TrkB. However, its interactions with TrkB can be distinguished from those of brain-derived neurotrophic factor (BDNF) with TrkB. We also find that all of the Trks display similar dose responses to their "preferred" ligands in neuronal as compared with nonneuronal cells (i.e., NGF for TrkA, BDNF and NT-4/5 for TrkB, and NT-3 for TrkC), providing evidence against a role for accessory molecules expressed in neurons in generating receptors that would allow for responses to lower concentrations of the neurotrophins. However, we find that a neuronal environment does restrict the Trks in their ability to respond to their "nonpreferred" neurotrophin ligands.

Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction.

Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

Expression of G protein beta subunits in rat skeletal muscle after nerve injury: Implication in the regulation of neuregulin signaling.

Tight regulation of gene transcription is critical in muscle development as well as during the formation and maintenance of the neuromuscular junction (NMJ). We previously demonstrated that the transcription of G protein beta1 (Gbeta1) is enhanced by treatment of cultured myotubes with neuregulin (NRG), a trophic factor that plays an important role in neural development. In the current study, we report that the transcript levels of Gbeta1 and Gbeta2 subunits in skeletal muscle are up-regulated following sciatic nerve injury or blockade of nerve activity. These observations prompted us to explore the possibility that G protein subunits regulate NRG-mediated signaling and gene transcription. We showed that overexpression of Gbeta1 or Gbeta2 in COS7 cells attenuates NRG-induced extracellular signal-regulated kinase (ERK) 1/2 activation, whereas suppression of Gbeta2 expression in C2C12 myotubes enhances NRG-mediated ERK1/2 activation and c-fos transcription. These results suggest that expression of Gbeta protein negatively regulates NRG-stimulated gene transcription in cultured myotubes. Taken together, our observations provide evidence that specific heterotrimeric G proteins regulate NRG-mediated signaling and gene transcription during rat muscle development.

K-252a and staurosporine selectively block autophosphorylation of neurotrophin receptors and neurotrophin-mediated responses.

The same receptor tyrosine kinase (RTK) can mediate strikingly different biological responses in a fibroblast as opposed to a neuron. We have compared the rapidly induced tyrosine phosphorylations mediated by various RTKs in both NIH3T3 fibroblasts and in the PC12 neuronal precursor cell line and found that each RTK induces a distinct pattern of protein tyrosine phosphorylations in the two cell types. These findings are consistent with a model in which various cell types present a given RTK with different menus of signal transduction components, allowing the same RTK to elicit fundamentally distinct biological responses. Although there are obvious overlaps in the tyrosine phosphorylations induced by different RTKs in the same cell, there are also clear differences. The attempt to dissect these differences revealed that the kinase inhibitors K-252a and staurosporine inhibit RTK autophosphorylation and thus the biological consequences of receptor/ligand interaction. These inhibitors displayed substantially greater specificity for a subset of RTKs (including the neurotrophin receptors) than for other RTKs and acted as remarkably selective blockers of neurotrophin action in both neuronal and nonneuronal cells. A potential therapeutic application for these inhibitors is discussed.

Neurotrophic factor receptors: just like other growth factor and cytokine receptors?

As the actions of neurotrophic factors appear so strikingly different from those of growth factors and cytokines operating elsewhere in the body, it was long thought that neurotrophic factors might in some way be fundamentally different from traditional growth factors and cytokines. Recent advances in the understanding of the structure of the receptors for neurotrophic factors reveals them to be much more like the receptors used by other cytokines and growth factors than was perhaps first anticipated. These findings suggest that neurotrophic factors display distinctive actions not because they utilize novel receptor systems, but rather because they activate these receptors in neurons.

GABAB heterodimeric receptors promote Ca2+ influx via store-operated channels in rat cortical neurons and transfected Chinese hamster ovary cells.

The GABAB receptors are generally considered to be classical Gi-coupled receptors that lack the ability to mobilize intracellular Ca2+ without the aid of promiscuous G proteins. Here, we report the ability of GABAB receptors to promote calcium influx into primary cultures of rat cortical neurons and transfected Chinese hamster ovary cells. Chinese hamster ovary cells were transfected with GABAB1(a) or GABAB1(b) subunits along with GABAB2 subunits. In experiments using the fluorometric imaging plate reader platform, GABA and selective agonists promoted increases in intracellular Ca2+ levels in transfected Chinese hamster ovary cells and cortical neurons with the expected order of potency. These effects were fully antagonized by selective GABAB receptor antagonists. To investigate the intracellular pathways responsible for mediating these effects we employed several pharmacological inhibitors. Pertussis toxin abolished GABAB mediated Ca2+ increases, as did the phospholipase Cbeta inhibitor U73122. Inhibitor 2-aminethoxydiphenyl borane acts as an antagonist at inositol 1,4,5-trisphosphate receptors and at store-operated channels. In all cell types, 2-aminethoxydiphenyl borane prevented Ca2+ mobilization. The selective store-operated channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride prevented increases in intracellular Ca2+ levels as did performing the assays in Ca2+ free buffers. In conclusion, GABAB receptors expressed in Chinese hamster ovary cells and endogenously expressed in rat cortical neurons promote Ca2+ entry into the cell via the activation of store-operated channels, using a mechanism that is dependent on Gi/o heterotrimeric proteins and phospholipase Cbeta. These findings suggest that the neuronal effects mediated by GABAB receptors may, in part, rely on the receptor's ability to promote Ca2+ influx.

Identification of full-length and truncated forms of Ehk-3, a novel member of the Eph receptor tyrosine kinase family.

Factors that bind and activate receptor tyrosine kinases are known to play key roles during development and in the adult. The Eph-related receptors constitute the largest known family of receptor tyrosine kinases. Members of the Eph family exhibit intriguing patterns of expression in the embryo, implicating them in a variety of developmental processes, and their expression is often restricted to particular subpopulations of postmitotic neurons in the adult. We describe the identification and characterization of a novel member of the Eph receptor family, which we have termed Ehk-3 for Eph Homologous Kinase 3. Ehk-3 displays all the major structural features shared by other members of the Eph family, including a cysteine-rich region and tandem fibronectin type-III domains in its extracellular portion. Ehk-3 is expressed in two forms in a developmentally-regulated fashion: a conventional full-length version containing the intracellular tyrosine kinase domain, as well as a truncated form that lacks this domain. Both forms of Ehk-3 are quite restricted to the nervous system in the adult, but Ehk-3 is more widely expressed in the embryo, suggesting that Ehk-3 mediates different functions during development and in the adult.

Expression of the P2Y1 nucleotide receptor in chick muscle: its functional role in the regulation of acetylcholinesterase and acetylcholine receptor.

In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y(1) receptor. The activation of the P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca(2+) mobilization in cultured chick myotubes. P2Y(1) receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y(1) receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus, and rat) but not in the chick embryo. In chicks after hatching, this P2Y(1) localization develops over approximately 3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y(1) transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR alpha-subunit were induced when the P2Y(1) receptors were activated by specific agonists or by overexpression of P2Y(1) receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter-reporter gene constructs for those subunits, actions that were blocked by a P2Y(1)-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.

NRC-interacting factor directs neurite outgrowth in an activity-dependent manner.

Nuclear hormone receptor coregulator-interacting factor 1 (NIF-1) is a zinc finger nuclear protein that was initially identified to enhance nuclear hormone receptor transcription via its interaction with nuclear hormone receptor coregulator (NRC). NIF-1 may regulate gene transcription either by modulating general transcriptional machinery or remodeling chromatin structure through interactions with specific protein partners. We previously reported that the cytoplasmic/nuclear localization of NIF-1 is regulated by the neuronal Cdk5 activator p35, suggesting potential neuronal functions for NIF-1. The present study reveals that NIF-1 plays critical roles in regulating neuronal morphogenesis at early stages. NIF-1 was prominently expressed in the nuclei of developing rat cortical neurons. Knockdown of NIF-1 expression attenuated both neurite outgrowth in cultured cortical neurons and retinoic acid (RA)-treated Neuro-2a neuroblastoma cells. Furthermore, activity-induced Ca(2+) influx, which is critical for neuronal morphogenesis, stimulated the nuclear localization of NIF-1 in cortical neurons. Suppression of NIF-1 expression reduced the up-regulation of neuronal activity-dependent gene transcription. These findings collectively suggest that NIF-1 directs neuronal morphogenesis during early developmental stages through modulating activity-dependent gene transcription.

Characterization and regulation of receptors for epidermal growth factor in mouse calvaria.

Epidermal growth factor (EGF) has been shown to stimulate bone resorption in mouse calvaria in organ culture by a prostaglandin-mediated mechanism. In this report we demonstrate and characterize specific receptors for EGF in mouse bone. Binding of tracer quantities of [125I]iodo-EGF reached a maximum after 2 h of incubation at 37 C and began to decrease only after 8 h of continuous exposure to EGF. Scatchard analysis of equilibrium binding data showed a single class of binding sites with a Kd = 2 X 10(-9) M present at a concentration of 3.8 X 10(10) sites/calvarium. The association (ki = 4.4 X 10(6) M-1 min-1) and dissociation (k2 = 0.015 min-1) rate constants for the approach to equilibrium were calculated, and there was no evidence of cooperativity between binding sites. Pretreatment of bones with EGF for 12 h produced a decrease in EGF binding of about 20%, and a maximum decrease to 50-60% of control binding after 48 h of treatment. Decreased binding was due solely to a decrease in the number of receptors rather than to a change in receptor affinity. This down-modulation of receptors occurred even when the bone resorption-stimulating action of EGF was blocked completely by indomethacin, and thus seemed to be triggered only by occupancy of receptors by EGF. Treatment of calvaria with parathyroid hormone, 1,25-dihydroxyvitamin D3, calcitonin, and prostaglandin E2 produced no net change in the number or affinity of EGF receptors in bone. EGF also produced no change in the total DNA content of mouse calvaria. We conclude that mouse bone contains specific high affinity receptors for EGF, and that this experimental system is useful for investigations on EGF receptor modulation and the biological actions of EGF on bone.