Guanosine enhances NGF-stimulated neurite outgrowth in PC12 cells.

Guanosine at 30 and 300 microM elicited the de novo extension of neurites from PC12 cells. With saturating concentrations of NGF, guanosine acted in a synergistic manner to enhance neuritogenesis. Adenosine alone also stimulated neurite outgrowth, but did not enhance NGF-induced neuritogenesis. 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog and A1/A2 receptor agonist, also alone had neuritogenic effects. It enhanced NGF-induced neuritic outgrowth but not to the same extent as guanosine. However, when NECA was added together with guanosine in the presence of NGF, these compounds elicited a greatly enhanced neuritogenic response. This suggested that the mechanisms through which NECA modulates the neuritogenic effects may be different from those of guanosine and NGF.     

Mitogen-activated protein kinase mediates purinergic-enhanced nerve growth factor-induced neurite outgrowth in PC12 cells

In 1999, we reported new observations that several compounds, including ATP, enhance neurite expression in PC12 cells when coapplied with nerve growth factor (NGF). Because purinergic and NGF signaling have several potential interfaces in PC12 cells, a series of experiments was conducted to elucidate the signal mediators contributing to the enhancement. Activities of selected kinases were measured and Western blots evaluated mitogen-activated protein kinase (MAPK) active and nonactive isoforms in lysates of the treated PC12 cells. In terms of purinergic potency, ATP and beta,gamma-methylene ATP elicited the greatest neurite-enhancing effect, whereas adenosine and alpha,beta-methylene ATP elicited the smallest. The effectiveness of a nonhydrolyzable analog such as beta,gamma-methylene ATP indicates that a nonmetabolic process is responsible. In response to ATP, NGF, or NGF + ATP, MAPK activity (measured by 32P incorporation) was maximal within 2 hr and remained statistically elevated over control levels throughout the 24 hr monitored. At maximal 32P incorporation, MAPK activity in response to ATP, NGF, and NGF + ATP was two-, four-, and sixfold higher, respectively, than control values; the observed increase was qualitatively confirmed using Western blots. Short-term inhibition experiments with protein kinase C and MAPK indicated that MAPK transduces the enhancing signal. We conclude from these experiments that ATP coapplied with NGF increases PC12 neurite expression by elevation of MAPK activity, likely by P2 receptor activation, and suggest that combination therapies with NGF and its enhancing adjunct compounds may be plausible for certain degenerative neurological disorders.     

Involvement of GPR12 in the induction of neurite outgrowth in PC12 cells

GPR12, an orphan G protein-coupled receptor, constitutively activates the Gs signaling pathway and further increases intracellular cyclic AMP. GPR12 overexpression has been reported to promote neurite extension in neurons or transform neuro2a neuroblastoma cells into neuron-like cells. However, the possible effects and mechanisms of GPR12 in the differentiation of PC12 cells are still unknown. The present study shows that GPR12 overexpression induced PC12 cells differentiation into neuron-like cells with enlarged cell sizes and neuritogenesis possibly via activation of Erk1/2 signaling and significantly increased the expression of several neurite outgrowth-related genes, including Bcl-xL, Bcl-2 and synaptophysin. These findings indicate that GPR12 may play a role in neurite outgrowth during PC12 cell differentiation.     

Methylmercury decreases NGF-induced TrkA autophosphorylation and neurite outgrowth in PC12 cells

Neurotrophin signaling through Trk receptors is important for differentiation and survival in the developing nervous system. The present study examined the effects of CH(3)Hg on (125)I-nerve growth factor (NGF) binding to the TrkA receptor, NGF-induced activation of the TrkA receptor, and neurite outgrowth in an in vitro model of differentiation using PC12 cells. Whole-cell binding assays using (125)I-NGF revealed a single binding site with a K(d) of approximately 1 nM. Methylmercury (CH(3)Hg) at 30 nM (EC(50) for neurite outgrowth inhibition) did not affect NGF binding to TrkA. TrkA autophosphorylation was measured by immunoblotting with a phospho-specific antibody. TrkA autophosphorylation peaked between 2.5 and 5 min of exposure and then decreased but was still detectable at 60 min. Concurrent exposure to CH(3)Hg and NGF for 2.5 min resulted in a concentration-dependent decrease in TrkA autophosphorylation, which was significant at 100 nM CH(3)Hg. To determine whether the observed inhibition of TrkA was sufficient to alter cell differentiation, NGF-stimulated neurite outgrowth was examined in PC12 cells after exposure to 30 nM CH(3)Hg, a concentration that inhibited TrkA autophosphorylation by approximately 50%. For comparison, a separate group of PC12 cells were exposed to a concentration of the selective Trk inhibitor K252a (30 nM), which had been shown to produce significant inhibition of TrkA autophosphorylation. Twenty-four hour exposure to either CH(3)Hg or K252a reduced neurite outgrowth to a similar degree. Our results suggest that CH(3)Hg may inhibit differentiation of PC12 cells by interfering with NGF-stimulated TrkA autophosphorylation.     

Effects of tunicamycin on NGF binding and neurite outgrowth in PC12 cells

The effects of inhibition of glycosylation on nerve growth factor (NGF) binding and neurite outgrowth response of PC12 cells have been examined. Exposure to tunicamycin (1-10 micrograms/ml) for 24-36 hr eliminates the rapidly dissociating component of NGF binding and decreases the proportion of PC12 cells capable of elaborating neurites in a dose-dependent manner. These decreased cellular responses are probably due to an underglycosylation of the NGF receptor, since the effects of tunicamycin are correlated with a decrease in 3H-fucose incorporation rather than a general decline in cellular metabolism as measured by viability and protein synthesis. These results suggest that carbohydrate side chains are important for the function and/or orientation of the NGF receptor in PC12 cells and that the rapidly dissociating component of NGF binding may be associated with a minimum concentration of functional receptors per cell required for the full biologic response.     

GTP and guanosine synergistically enhance NGF-induced neurite outgrowth from PC12 cells

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (processes greater than one cell diameter in length) in the presence of 300 μM extracellular GTP or 300 μM guanosine for 48 hr, compared to only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20–35% of PC12 cells had neurites after 48 hr, and the addition of 300 μM guanosine or GTP together with NGF synergistically increased the proportion of cells with neurites to 40–65%. GTP and guanosine also increased the average number of branches per neurite, from 0.6 in NGF-treated cultures to 1.2 (guanosine) or 1.5 (GTP). Neurites formed after exposure to NGF alone had axonal characteristics as determined by immunocytochemistry with antibody, SMI-31, against axonal-specific polyphosphorylated neurofilament epitopes. Neurites generated with the addition of both guanosine or GTP had the same characteristics.GTP probably did not exert its effects via the P_2X or P_2Y purinoceptors because the adenine nucleotides ATP, ATPγS, ADPβS, and ADP, which are all agonists of these receptors, inhibited rather than enhanced, NGF-induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P_2U-like nucleotide receptor. However, the response profile obtained, GTP > UTP ? ATP, does not fit the profile of any known P_2Y, P_2X or P_2U receptor. The poorly hydrolyzable GTP analogues, GTPγS and GDPβs were also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP-specific ectoenzyme or kinase. This idea was supported by results showing that another poorly hydrolyzable analogue, GMP-PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects after hydrolysis to guanosine since the metabolic intermediates GDP and GMP were also ineffective in enhancing the proportion of cells with neurites. Moreover, the effects of GTP and guanosine were mutually additive, implying that these two purines utilized different signal transduction mechanisms.The effects of guanosine were not affected by the nucleoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamole, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P₁ receptors, because the A2 receptor antagonists, 1, 3-dipropyl-7-methylxanthine (DPMX) or CGS15943, and the At receptor antagonist, 1, 3-dipropyl-8-(2-amino-4-chloro)xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enhanced neurite outgrowth by a signal transduction mechanism that does not include the activation of the Pt purinoceptors.The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiological implications in sprouting and functional recovery after neuronal injury in the CNS, due to the high levels of nucleosides and nucleotides released from dead or injured cells.     

Quantitation of the growth-associated protein B-50/GAP-43 and neurite outgrowth in PC12 cells

A combined assay to measure neurite outgrowth and B-50/GAP-43 levels in PC12 cells is reported. During NGF-induced neuritogenesis, B-50/GAP-43 expression was monitored by enzyme-linked immunosorbent assay (ELISA). Neurite outgrowth was quantified at the same time by the use of video image analysis. Sensitivity and reliability of the methods are shown with a dose-response and time curve of beta-NGF-induced neuritogenesis. A linear increase in total length of neurites was induced by concentrations of beta-NGF greater than or equal to 5 ng/ml and was accompanied by a linear increase in the amount of B-50/GAP-43. The combined methods presented here can conveniently and reliably establish subtle changes in neurite outgrowth and intracellular protein contents.     

Enhancement of NGF- and cholera toxin-induced neurite outgrowth by butyrate in PC12 cells

It has been shown that sodium butyrate (NaBu) does not elicit neurite outgrowth of PC12, one of the most widely used cell lines as a model of neuronal differentiation. In this study, the effects of NaBu on nerve growth factor (NGF)- and cholera toxin-induced neurite outgrowth in PC12 cells were examined. NaBu dose-dependently enhanced neurite formation induced by both agents. The maximum responses obtained at 0.5 mM NaBu were nearly twice those of the inducers alone. Propionate and valerate were also effective, but acetate and caproate were ineffective. Among the butyrate analogs with a moiety of three to five carbon atoms tested, isobutyrate, isovalerate, vinylacetate and 3-chloropropionate enhanced neurite outgrowth promoted by both inducers. However, neither alpha-, beta-, and gamma-aminobutyrates nor alpha-, beta-, and gamma-hydroxybutyrates were effective. All of the effective short-chain fatty acids and their analogs increased the level of histone acetylation, while ineffective ones did not. Furthermore, Helminthosporium carbonum toxin (HC toxin), a structurally dissimilar inhibitor of histone deacetylase, mimicked the effect of butyrate. These results suggest that NaBu enhances neurite outgrowth induced by NGF and cholera toxin in PC12 cells through a mechanism involving an increase in the level of histone acetylation.     

Protein kinase B is involved in Nogo-66 inhibiting neurite outgrowth in PC12 cells

Nogo-A, a member of the reticulon family, is one of the most important myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the central nervous system. RhoA has been targeted pharmacologically to promote neurite outgrowth and functional recovery in the brain and spinal cord. However, the underlying mechanism of the inhibition of neurite outgrowth by Nogo-A has not yet been fully defined. Protein kinase B (PKB, also known as Akt) is a protein serine/threonine kinase that plays a key role in intracellular signaling and cellular homeostasis. This study reports the role of PKB signaling on Nogo-A-treated PC12 neuronal cells. An inhibitory fragment of Nogo-A (Nogo-66) activated RhoA and reduced the phosphorylation of PKB at Ser473 in a time-dependent manner. In contrast, pretreatment with Y27632, a specific inhibitor of Rho-A, resulted in an increase of the phosphorylation of PKB. Nogo-66 also inhibited the neurite outgrowth of PC12 cells, whereas pretreatment with LY294002, a specific inhibitor of PKB, ameliorated the neurite outgrowth. These data suggest that PKB is involved in the inhibition of neurite outgrowth by Nogo-A in PC12 cells.     

Heat shock induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway

We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.     

Sargaquinoic acid promotes neurite outgrowth via protein kinase A and MAP kinases-mediated signaling pathways in PC12D cells

We previously isolated a nerve growth factor (NGF)-dependent neurite outgrowth promoting substance MC14 (sargaquinoic acid) from a marine brown alga, Sargassum macrocarpum. In the present study, the NGF-potentiating activity of MC14 to neural differentiation of PC12D cells was investigated in detail. The treatment of cells with 3 microg/ml MC14 in the presence of 1.25-100 ng/ml NGF markedly enhanced the proportion of neurite-bearing cells compared with the NGF-only controls. In addition, MC14 significantly elevated the NGF-induced specific acetylcholinesterase (AchE) activity in PC12D cells, suggesting that MC14 could morphologically and biochemically promote the differentiation of PC12D cells. The mechanism of action of MC14 was further investigated by pharmacological inhibition of several intracellular signaling molecules. Results indicated that the neurite outgrowth promoting activity of MC14 was almost completely blocked by 10 microM PD98059, suggesting that a TrkA-dependent MAP kinases-mediated signaling pathway may play a crucial role in modulating the effect of MC14. Besides, the MC14-enhanced neurite outgrowth was substantially suppressed by the pretreatment with 10 ng/ml protein kinase A (PKA) inhibitor, demonstrating that the adenylate cyclase-PKA signaling cascade was also involved in the action of MC14. In contrast, a PKC inhibitor chelerythrine chloride did not inhibit the neurite outgrowth promoting activity of MC14. Altogether, these results demonstrate that MC14 enhances the neurite outgrowth by cooperating at least two separated signaling pathways, a TrkA-MAP kinases pathway and an adenylate cyclase-PKA pathway, in PC12D cells.     

Lead enhances NGF-induced neurite outgrowth in PC12 cells by potentiating ERK/MAPK activation

Although the neurotoxicity of lead exposure is well documented, the cellular and molecular mechanisms underlying lead neurotoxicity have not been well defined. We have investigated the effect of lead on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells and the role in this process of extracellular signal regulated protein kinase (ERK), a key component of NGF-induced differentiation. We found that exposure of cells to lead acetate (0.1-100 microM) resulted in enhanced NGF-induced neurite outgrowth. Lead exposure also promoted formation of multiple neurites per cell in NGF-treated cells. However, lead alone did not cause neurite outgrowth. Lead also enhanced NGF-induced ERK phosphorylation and activation, but lead alone did not stimulate ERK. The MAP kinase kinase (MEK) inhibitor, PD98059, significantly decreased the effect of lead on NGF-induced neurite outgrowth and ERK activation. These findings indicate that exposure of cells to low, toxic levels of lead amplifies growth factor-induced neurite outgrowth by means of an ERK-dependent signaling pathway.     

NGF induces neurite outgrowth via a decrease in phosphorylation of myosin light chain in PC12 cells.

The relationship between phosphorylation of myosin light chain (MLC) and neurite outgrowth induced by nerve growth factor (NGF) was studied in PC12 cells. Inhibitors of Rho kinase, HA-1077 or Y-27632 also induced neurite outgrowth. As already reported botulinum exoenzyme C3 which inactivates Rho protein also induced neurite outgrowth. Calyeulin A, an inhibitor of phosphatase counteracted both NGF- and C3- induced neurite outgrowth. Treatments of both NGF and C3 resulted in significant and transient decrease in phosphorylated MLC. These results suggest that NGF induces neurite outgrowth of PC12 by a transient decrease in phosphorylated MLC which is brought about by activation of MLC phosphatase via inhibition of Rho-Rho kinase pathway.     

Identification of a cell-surface protein involved in PC12 cell-substratum adhesion and neurite outgrowth on laminin and collagen

On substrata coated with laminin or native collagen (Types I/III), PC12 cells employ an active adhesion mechanism (i.e., one inhibited at low temperature, by azide or in the absence of divalent cations) to attach and extend neurites; on substrata coated with wheat germ agglutinin (WGA) or polylysine, by contrast, PC12 cells attach via a passive mechanism and fail to extend neurites (Turner et al., 1987). This paper reports the isolation of 2 monoclonal antibodies (3A3 and 1B1) that promote retraction of neurites extended on laminin and collagen. In studies of initial cell attachment, 3A3 inhibited active attachment to laminin or collagen but not passive attachment to WGA or polylysine, whereas 1B1 inhibited both active and passive attachment. The more potent of the antibodies, 3A3, precipitates 2 radioactive protein bands (of approximately 185 and 125 kDa) from 1% Nonidet P-40 extracts of metabolically labeled PC12 cells. The properties of these proteins suggest that the antigen recognized by 3A3 is a member of the integrin family of matrix receptors. The other monoclonal antibody, 1B1, reacts with many PC12 proteins, including both bands precipitated by 3A3. The available data strongly suggest that an integrin with specificity for both laminin and collagen mediates PC12 adhesion to the substratum at both the cell body and the neurite growth cone.     

Inhibitory effects of brain chondroitin sulfate proteoglycans on neurite outgrowth from PC12D cells

Soluble chondroitin sulfate proteoglycans (CSPGs), prepared from 10-d-old rat brain, were added to the culture medium of PC12D cells containing NGF to examine the effects on NGF-induced neurite outgrowth from the cells. PC12D cells, a flat-shaped variant of PC12 pheochromocytoma cells, are characteristic of prompt neurite formation in response not only to NGF, but also to cAMP-enhancing reagents such as forskolin. Brain CSPGs inhibited the neurite elongation irreversibly in a dose-dependent manner; complete inhibition was observed at a concentration of 50 nmol uronic acid/ml. Closely similar dose-dependent inhibition was observed in the forskolin-induced neurite outgrowth from PC12D cells. NGF-induced neurite outgrowth from conventional PC12 cells was also inhibited completely by 50 nmol uronic acid/ml CSPGs. Some brain CSPGs seemed to be inhibitory, but the cartilage-unique CSPG did not show any inhibitory effect. Chondroitin sulfate, a polysaccharide moiety of CSPGs, did not show any inhibitory effect even at a concentration of 250 nmol uronic acid/ml, while core proteins prepared from brain CSPGs by digestion with chondroitinase ABC exhibited inhibitory activity similar to that of intact CSPGs. This indicates that the site of the inhibitory activity exists in the core protein moiety of brain CSPGs. From these observations, it is conceivable that brain CSPGs are involved in the regulation of neuronal differentiation.     

Manganese induces neurite outgrowth in PC12 cells via upregulation of alpha(v) integrins

Previous studies have demonstrated that the divalent cation manganese (Mn) causes PC12 cells to form neurites in the absence of NGF. Since divalent cations modulate the binding affinity and specificity of integrins, and integrin function affects neurite outgrowth, we tested the hypothesis that Mn induces neurite outgrowth through an integrin-dependent signaling pathway. Our studies support this hypothesis. Function-blocking antisera specific for beta(1) integrins block the neurite-promoting activity of Mn by 90-95%. Bioassays and biochemical studies with antisera specific for the alpha(v), alpha(5), or alpha(8) integrin subunit suggest that the alpha(v)beta(1) heterodimer is one of the principal beta(1) integrins mediating the response of PC12 cells to Mn. This is corroborated by studies in which Mn failed to induce neurite outgrowth in a clone of PC12 cells that does not express alpha(v) at levels detectable by immunoprecipitation or immunocytochemistry. SDS-PAGE analysis of biotinylated surface proteins immunoprecipitated from Mn-responsive PC12 cells, as well as confocal laser microscopy of PC12 immunostained for surface alpha(v) indicate that Mn increases the surface expression of alpha(v) integrins. This increase appears to be due in part to synthesis of alpha(v) since specific inhibitors of RNA and protein synthesis block the neurite-promoting activity of Mn. These data indicate that Mn induces neurite outgrowth in PC12 cells by upregulating alpha(v) integrins, suggesting that Mn potentially represents an additional mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.     

Role of Rho kinase pathway in chondroitin sulfate proteoglycan-mediated inhibition of neurite outgrowth in PC12 cells

Activation of the Rho kinase (ROCK) pathway has been associated with inhibition of neurite regeneration and outgrowth in spinal cord injury. Growth-inhibitory substances present in the glial scar such as chondroitin sulfate proteoglycans (CSPGs) have been shown to create a nonpermissive environment for axon regeneration that results in growth cone collapse. In this study, an in vitro model was developed in nerve growth factor-differentiated PC12 cells where the Rho/ROCK pathway was modulated by CSPG. CSPG elicited concentration-dependent inhibition of neurite outgrowth in PC12 cells, which was reversed by ROCK inhibitors such as fasudil, dimethylfasudil, and Y27632. Further studies on the interactions of CSPG with ROCK inhibitors revealed that the modulation of ROCK by CSPG is noncompetitive in nature. It was also observed that ROCK inhibitors increased neurite outgrowth in undifferentiated PC12 cells, indicating constitutive ROCK activity in the cells. Analysis of signaling pathways demonstrated that the effect of CSPG increases the phosphorylation of myosin phosphatase, a substrate immediately downstream of ROCK activation. Fasudil, dimethylfasudil, and Y27632 inhibited the phosphorylation of myosin phosphatase induced by CSPG with rank order potencies comparable to those observed in the neurite outgrowth assay. In addition, ROCK inhibitors reversed cofilin phosphorylation induced by CSPG with similar rank order potencies. Taken together, our data demonstrate that the interaction of CSPG with the ROCK pathway involves downstream effectors of ROCK such as myosin phosphatase and cofilin.