PC12h-R cell,a subclone of PC12 cells,shows EGF-induced neuronal differentiation and sustained signaling

Unlike nerve growth factor (NGF), epidermal growth factor (EGF) does not induce neuronal differentiation but promotes proliferation of the rat pheochromocytoma PC12 cells. We found that PC12h-R, a subclone of PC12 cells, differentiated into neuron-like cells in response to EGF as well as to NGF. PC12h-R cells treated with EGF extended neurites, attenuated cell proliferation, and increased the levels of tyrosine hydroxylase protein synthesis and of acetylcholinesterase activity as those treated with NGF. The EGF-induced differentiation of PC12h-R cells was not mediated by the indirect activation of p140^trkA by EGF. In addition, EGF induced the sustained tyrosine phosphorylation of the EGF receptor, mitogen-activated protein (MAP) kinases, and 46 and 52 kDa proteins, and the prolonged activation of MAP kinases in PC12h-R cells compared with the parent PC12h, which does not show EGF-induced differentiation. The response of PC12h-R cells to EGF was not simply due to an increase in the level of EGF receptor protein. These results indicated that the duration of EGF-induced signaling might determine the cellular response of PC12 cells between cell proliferation and neuronal differentiation. © 1996 Wiley-Liss, Inc.     

Differentiation of PC12 cells with v-src: comparison with nerve growth factor

The PC12 rat pheochromocytoma cell line is used extensively as a model to study neuronal differentiation. These cells resemble adrenal chromaffin cells, differentiating both morphologically and biochemically when cultured in the presence of dexamethasone, but develop a sympathetic neuron-like phenotype when cultured in the presence of nerve growth factor. Expression of the protein product of the v-src oncogene in PC12 cells also induces neurite outgrowth similar to that resulting from nerve growth factor treatment (Alema et al: Nature 316:557-559, 1985). It is thus possible that c-src or a src-like tyrosine kinase participates in the signal transduction pathway by which nerve growth factor acts on PC12 cells. In this study a temperature-sensitive v-src gene has been introduced into PC12 cells. When cultures of these src-transformed cells are switched from the nonpermissive (40 degrees C) to the permissive (37 degrees C) temperature they elaborate neurites. The differentiation induced by src has been compared with that induced by nerve growth factor by determining whether src-transformed PC12 cells at 37 degrees C exhibit the same biochemical alterations as those induced in PC12 cells treated with nerve growth factor. Neurite extension at 37 degrees C in v-src-transformed cells, like NGF-induced differentiation, is accompanied by an increase in the nerve growth factor-inducible large external (NILE) protein. However, neurite extension in v-src-transformed cells is not blocked by the protein kinase inhibitor K-252a, which completely blocks NGF-induced neurite extension. Likewise, EGF receptor down-regulation and the development of saxitoxin and tetanus toxin binding sites are either much reduced or completely absent in src-differentiated compared with NGF-differentiated PC12 cells.     

Variant PC12 cell line that spontaneously differentiates and extends neuritic processes

The rat pheochromocytoma PC12 cells differentiate into neuronal-like cells in response to treatment with neurotrophins. The cells have been extensively used for investigating neuronal differentiation and axonal growth. Here we report the isolation of a variant PC12 cell line, named PC12-N1, which spontaneously differentiates and extends neuritic processes. The PC12-N1 cells expressed many neuronal specific proteins, including the synaptosomal associated protein of 25 kDa (SNAP-25), synaptotagmin, and synaptobrevin (also known as VAMP). The cells also expressed neurofilament protein of 68 kDa, a marker for differentiated neurons. In addition to the spontaneous neurite outgrowth, the PC12-N1 cells showed a marked increase in neurite outgrowth upon treatment with nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cyclic AMP (cAMP). The activation of mitogen-activated protein (MAP) kinases was examined by immunoblot analysis using phospho-specific antibodies. No overactivation was observed with ERK1/2 or p38. However, the c-Jun N-terminal kinase JNK/SAPK was activated approximately 10-fold over the parental PC12 cells. These results suggest that activation of JNK/SAPK may be involved in the spontaneous neurite extension in the PC12-N1 cells. Moreover, the PC12-N1 cells may be used as a model for investigating molecular signaling mechanisms underlying neuronal differentiation and axonal outgrowth.     

Involvement of Gas7 in nerve growth factor-independent and dependent cell processes in PC12 cells

Gas7, a growth arrest-specific gene originally isolated from serum-starved mouse fibroblast cells, is expressed in vivo predominantly in the brain and is required for neurite formation in cultured mouse cerebellar neurons (Ju et al. [1998] Proc. Natl. Acad. Sci. USA 95: 11423-11428). Here we report that Gas7 plays a key role in the morphological differentiation of PC12 preneuronal rat pheochromocytoma cells (PC12 cells). We found that overexpression of murine Gas7 in PC12 cells leads to an expanded cell morphology and promotes spike-like cell processes that resemble the early stages of neurite formation. These processes undergo elongation upon addition of nerve growth factor (NGF). We also found that the addition of NGF induces the production of endogenous rat-Gas7 (rGas7), which is transiently elevated prior to the appearance of NGF-promoted neurite outgrowths. Furthermore, inhibition of endogenous rGas7 production by antisense nucleotides complimentary to the translation initiation region of a rGas7 cDNA (AJ131902) reduces the NGF-promoted neurite outgrowths. Our results demonstrate that Gas7 by itself influences early cell morphological development and likely functions as an early-stage intermediary in NGF-induced neuronal differentiation of PC12 culture cells.     

Neuritin (cpg15) enhances the differentiating effect of NGF on neuronal PC12 cells

Neuritin is a small, highly conserved GPI-anchored protein involved in neurite outgrowth. We have analyzed the involvement of neuritin in NGF-induced differentiation of PC12 cells by investigating the time-course of neuritin expression, the effects of its overexpression or silencing, and the possible mechanisms of its regulation and action. Real-time PCR analysis has shown that neuritin gene is upregulated by NGF in PC12 cells hours before neurite outgrowth becomes appreciable. PC12 cells transfected with a plasmid expressing neuritin display a significant increase in the response to NGF: 1) in the levels of SMI312 positive phosphorylated neurofilament proteins (markers for axonal processes) and tyrosine hydroxylase; 2) in the percentage of cells bearing neurites; as well as 3) in the average length of neurites when compared to control cells. On the contrary, neuritin silencing significantly reduces neurite outgrowth. These data suggest that neuritin is a modulator of NGF-induced neurite extension in PC12 cells. We also showed that neuritin potentiated the NGF-induced differentiation of PC12 cells without affecting TrkA or EGF receptor mRNAs expression. Moreover, the S-methylisothiourea (MIU), a potent inhibitor of inducible nitric oxide synthases, partially counteracts the NGF-mediated neuritin induction. These data suggest that NGF regulates neuritin expression in PC12 cells via the signaling pathway triggered by NO. This study reports the first evidence that neuritin plays a role in modulating neurite outgrowth during the progression of NGF-induced differentiation of PC12 cells. PC12 cells could be considered a valuable model to unravel the mechanism of action of neuritin on neurite outgrowth. (c) 2007 Wiley-Liss, Inc.     

Insulin-like growth factor-I receptor and estrogen receptor crosstalk mediates hormone-induced neurite outgrowth in PC12 cells

Estradiol (E(2)) and insulin-like growth factor-I (IGF-I) can act independently or in concert to promote neurite outgrowth in vivo and in cultured neurons. This study examined the role of crosstalk between estrogen receptor (ER)alpha and the IGF-I receptor as a critical mediator of hormone- and growth factor-dependent neurite outgrowth in a homogenous cell system. We used control PC12 cells and PC12 cells stably transfected with ER alpha, both of which express IGF-I receptor. Cells were treated for 1 week with vehicle, 1 nM E(2) or 100 ng/ml IGF-I alone or with E(2) or IGF-I in the presence of either the IGF-I receptor antagonist JB1 or the ER antagonist ICI 182,780. IGF-I significantly increased neurite outgrowth, as measured by the percentage of process-bearing cells, and absolute neurite length per cell in both control and ER alpha-transfected PC12 cells. In contrast, E(2) increased process formation and extension only in PC12 cells that were stably transfected with ER alpha. ICI 182,780 and JB1 blocked the IGF-I-induced increases in neurite length in both cell types. The efficacy of ICI 182,780 in control PC12 cells may have been due to the upregulation of ER alpha in these cells by the 7-day treatment with IGF-I. The ER and IGF-I receptor antagonists similarly blocked the E(2)-induced increase in neurite lengths in ER alpha-transfected cells. Immunofluorescent analysis of the cellular distribution of an axonal marker, phospho-neurofilament, verified that the processes extended by PC12 cells were neurites. These data suggest that receptor crosstalk between IGF-I receptors and ER alpha has an important role in neurite formation and extension even in a single-cell system.     

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.     

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells (DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium (DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor (NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2 (MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction (qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors (NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCs-CM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls; however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Chinese Library Classification No. R459.9; R364; R622     

5'-Nucleotidase Activates and an Inhibitory Antibody Prevents Neuritic Differentiation of PC12 Cells

Ecto-5'-nucleotidase catalyses the hydrolysis of AMP at the surface of a variety of cells whereas it is absent from others. In addition to its catalytic activity, a function in neural development and also its interaction with extracellular matrix proteins has been reported. In order to further elucidate the biological function of ecto-5'-nucleotidase we have investigated the effect of 5'-nucleotidase on nerve growth factor-induced differentiation of PC12 cells. Furthermore, we compared the effect of an inhibitory versus a non-inhibitory monospecific antibody against the enzyme on neuritic differentiation and survival of PC12 cells that constitutively express the enzyme. When coverslips are coated with the soluble form of ecto-5'-nucleotidase in addition to collagen, there is a considerable increase in nerve growth factor-induced neurite length during the first 24 h of culture. Addition of an antibody to a culture medium that inhibits 5'-nucleotidase activity to 33% of control values dramatically reduces the number of neurites per cell within 3 days of culture. The cells round up, cluster and eventually die. On the contrary, another antibody that had no significant effect on enzyme activity affected neither nerve growth factor-induced neurite formation nor survival of PC12 cells. Addition of adenosine (200 nM, 10 or 20 μM) to the culture medium did not influence PC12 cell differentiation. The effects induced by the inhibitory antibody could be only partially prevented by simultaneous application of adenosine. Our results suggest that 5'-nucleotidase is essential for nerve growth factor-induced neurite outgrowth and survival of PC12 cells. The results obtained cannot be simply explained on the basis of the availability and extracellular production of adenosine.     

EGF in combination with depolarization or cAMP produces morphological but not physiological differentiation in PC12 cells

In response to nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) receptor activated Ras/extracellular signal-regulated kinase (ERK) signaling, PC12 cells undergo a prototypical neuronal differentiation program, characterized by neurite extension and upregulation of voltage-gated ion channels. The epidermal growth factor (EGF) receptor also activates Ras/ERK signaling, but produces proliferation instead of differentiation. In the presence of depolarizing concentrations of KCI, however, EGF elicits neurite outgrowth through the synergistic actions of the Ras/ERK and cAMP signaling pathways. To assess if EGF and KCI/cAMP elicit the same suite of differentiation events as does NGF and bFGF, we used patch clamp recording to determine if EGF in the presence of KCI or a cAMP agonist also induced physiological differentiation as defined by upregulation of ion channels. Chronic NGF treatment of PC12 cell cultures elicited robust morphological differentiation, a threefold increase in mean calcium channel current density, and an eightfold increase in mean sodium channel current density. Sibling cultures chronically treated with EGF in the presence of high KCI or a cAMP agonist also displayed morphological differentiation, but had calcium channel current densities which were no larger than untreated, undifferentiated cells. Additionally, the increase in mean sodium channel current density induced by EGF in the presence of KCI or cAMP was no greater than the increase observed with EGF alone. Thus, although EGF in the presence of KCI or cAMP is sufficient to induce morphological differentiation as defined by neurite outgrowth, synergism of the Ras/ERK and cAMP/PKA signaling pathways is not sufficient to promote the fully physiologically differentiated PC12 phenotype. J. Neurosci. Res. 47:16–26, 1997. © 1997 Wiley-Liss, Inc.     

Protein kinase C isozymes that mediate enhancement of neurite outgrowth by ethanol and phorbol esters in PC12 cells

Using PC12 cells to study ethanol's effects on growth of neural processes, we found that ethanol enhances NGF- and basic FGF-induced neurite outgrowth. Chronic ethanol exposure selectively up-regulates δ and ε protein kinase C (PKC) and increases PKC-mediated phosphorylation in PC12 cells. Since PKC regulates differentiation, we investigated the role of PKC in enhancement of neurite outgrowth by ethanol. Like ethanol, 0.3–10 nM phorbol 12-myristate, 13-acetate (PMA) increased NGF-induced neurite outgrowth. However, higher concentrations did not, and immunoblot analysis demonstrated that 100 nM PMA markedly depleted cells of β, δ and ε PKC. PMA (100 nM) also down-regulated β, δ and ε PKC in ethanol-treated cells and completely prevented enhancement of neurite outgrowth by ethanol. In contrast, the cAMP analogue 8-bromoadenosine cAMP did not completely mimic the effectsof ethanol on neurite outgrowth, and ethanol was able to enhance neurite formation in mutant PC12 cells deficient in protein kinase A (PKA). These findings implicate β, δ or εPKC, but not PKA, in the neurite-promoting effects of ethanol and PMA. Since chronic ethanol exposure up-regulates δ and ε, but not βPKC, these findings suggest that δ or εPKC regulate neurite outgrowth.     

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.     

Differentiation of PC12 cells with K-ras: comparison with nerve growth factor.

The cell line PC12, derived from a rat pheochromocytoma, has served as a model for studies on the mechanism of action of nerve growth factor, as well as for the exploration of neuronal differentiation in general. When treated with nanomolar concentrations of nerve growth factor, these neoplastic chromaffin-like cells stop dividing and acquire, for all intents and purposes, the phenotype of mature sympathetic neurons. This phenotype is characterized by the extensive outgrowth of electrically excitable neurites, the ability to form functional synapses, and the acquisition of a number of biochemical markers. Treatment of PC12 cells with retroviral vectors encoding the K-ras, the N-ras, or the v-src oncogenes also produces a marked morphological differentiation very similar to that seen upon treatment with nerve growth factor. Treated cells stop dividing and develop an extensive network of neurites. It has recently been shown that PC12 cells differentiated with v-src, while resembling, morphologically, those treated with nerve growth factor, differ substantially in the biochemical characteristics normally associated with nerve growth factor-induced differentiation. Cells infected with K-ras also develop a neurite network similar to that seen after treatment with nerve growth factor. In addition, such cells develop tetanus toxin-binding sites and saxitoxin-binding sites, as do cells treated with nerve growth factor. Decreases in the binding of epidermal growth factor and in the activity of calpain also occur and these, as well, are characteristic of nerve growth factor-treated cells. But the adhesive properties of cells infected with K-ras are different than those of nerve growth factor-treated cells, and the former do not show an increase in the NILE glycoprotein. Finally, K-252a, an inhibitor of the actions of nerve growth factor on PC12 cells, has no effect on the neurite outgrowth produced by infection with K-ras. Thus, many of the key markers of nerve growth factor-induced differentiation of PC12 cells also appear upon differentiation with K-ras, but there are, nevertheless, some crucial differences in the properties of these two sets of cells.     

Dual Contradictory Effect of H-89 on Neuronal Retraction, Death and Inflammation in Differentiated PC12 Cells Subjected to Oxidative Stress

Interrelation between oxidative stress and neuro-inflammation has been discussed extensively to contribute to neuronal dysfunction in neurodegenerative disorders. In this manner, it seems that there is an intriguing link between protein kinase A (PKA), neuronal apoptosis and inflammation. Rat PC12 pheochromocytoma cell can be induced to differentiate into neuron-like cells possessing elongated neurites by nerve growth factor. In this study, we investigated the effect of H-89, a selective inhibitor of PKA, on the neurite retraction along with evaluation of cell death and inflammatory markers in the differentiated PC12 cells, exposed to H₂O₂. We found that dose-dependent inhibition of PKA by low and medium concentrations of H-89 (5, 7 and 10 μM) enhanced the parameters of neurite outgrowth and complexity in the cells co-treated with H₂O₂ as an oxidative stress. Similar concentrations of H-89 significantly inhibited cell death and neurite retraction induced by oxidative stress. Components of TNF-α–NFκB–COX-2 axis, a discussed pathway in neuroinflammation, downregulated dose-dependently by administration of H-89 in H₂O₂-induced PC12 cells. In this condition, PKA inhibition by the high concentrations of H-89 (15 and 20 μM) led to enhanced cell death and inflammation with decreased neurite outgrowth. These findings indicate that H-89 has a dual contradictory effect on oxidative stress and inflammation that affect neurite outgrowth and complexity in differentiated PC12 cells.     

PC12 cell activation by epidermal growth factor receptor: role of autophosphorylation sites

PC12 cells have been used as a model system for neuronal differentiation due to their ability to alter their phenotype to a sympathetic neuron-like cell in response to nerve growth factor or fibroblast growth factor. Under some conditions, epidermal growth factor (EGF) can also induce PC12 cells to differentiate. To study signaling from the EGF receptor without the confounding effects of endogenous EGF receptors we generated a chimeric receptor comprised of the ectodomain of platelet-derived growth factor (PDGF) receptor in-frame with the transmembrane and cytoplasmic domains of EGF receptor, termed PER. Expression of PER in PC12 cells confers the ability of PDGF to induce differentiation whereas PDGF has no effect on untransfected PC12 cells. This response is kinase activity-dependent since a kinase-deficient mutant (K721M) fails to induce differentiation in response to PDGF. Mutation of five tyrosine residues that are autophosphorylated in response to EGF either individually or in combination had minimal effects on the ability of these receptors to induce morphological PC12 cell differentiation. The PER mutant with all five autophosphorylation sites mutated to phenylalanine (5YF) was equivalently capable of interacting with several important signaling molecules, including Shc, Grb2, Gab1, phospholipase Cgamma, and Cbl. Furthermore, both the phosphatidylinositol 3-kinase (PI3K)/Akt and Ras/Erk pathways were activated in a sustained manner when PER or 5YF-expressing cells were stimulated with PDGF. Our results show that the five autophosphorylation sites in the extra-kinase C-terminal domain of EGFR are not required for the ability of EGFR to induce morphological differentiation of PC12 cells.     

PC12 neurite regeneration and long-term maintenance in the absence of exogenous nerve growth factor in response to contact with Schwann cells

We have investigated mouse and rat ganglionic Schwann cells as possible sources of neurite outgrowth-promoting factors by co-culturing Schwann cells with nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells primed by pretreatment with NGF. NGF-primed PC12 cells are capable of neurite regeneration when provided with an appropriate neurite promoting factor such as NGF. When primed PC12 cells were co-cultured with Schwann cells in the absence of exogenous NGF, PC12 cells that directly contacted Schwann cells became enlarged and flattened, attaining a neuron-like morphology within one day. When contact with Schwann cells was established, PC12 cells regenerated neurites by the first day of co-culture and these were maintained throughout the experiments (7 weeks). Most PC12 cells cultured in the same collagen-coated dishes with Schwann cells, but not directly in contact with them, failed to regenerate neurites. Instead, they began to proliferate, forming cell clusters. Neurite regeneration by PC12 cells in contact with Schwann cells was not blocked by antibody to NGF. These results demonstrate the presence of a neurite-promoting activity localized to the vicinity of the Schwann cell surface which is capable of eliciting regeneration and long-term maintenance of PC12 neurites in the absence of exogenous NGF. This activity does not appear to be due to NGF.     

Pheophytin a, a low molecular weight compound found in the marine brown alga Sargassum fulvellum, promotes the differentiation of PC12 cells

We identified and characterized a neurodifferentiation compound from the marine brown alga Sargassum fulvellum collected from the Japanese coastline. Several instrumental analyses revealed the compound to be pheophytin a. Pheophytin a did not itself promote neurite outgrowth of PC12 cells. However, when PC12 cells were treated with a low concentration of pheophytin a (3.9 microg/ml) in the presence of a low level of nerve growth factor (10 ng/ml), the compound produced neurite outgrowth similar to that produced by a high level of nerve growth factor (50 ng/ml). Pheophytin a also enhanced signal transduction in the mitogen-activated protein kinase signaling pathway, which is also induced by nerve growth factor. The effect of pheophytin a on neurite outgrowth of PC12 cells was completely blocked by U0126, a representative mitogen-activated protein kinase kinase inhibitor. These results suggest that pheophytin a enhances the neurodifferentiation of PC12 cells in the presence of a low level of nerve growth factor and that this effect is mediated by activation of a mitogen-activated protein kinase signaling pathway.     

Metabolites of sesamin,a major lignan in sesame seeds,induce neuronal differentiation in PC12 cells through activation of ERK1/2 signaling pathway

Sesamin, a major lignan in sesame seeds, exhibits various health benefits. Here, we investigated effects of sesamin, its stereoisomer episesamin, and their metabolites on neuronal differentiation in rat pheochromocytoma PC12 cells. Among all compounds tested, primary metabolites of sesamin and episesamin, SC-1 and EC-1 {S- and R-epimer of 2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo [3.3.0]octane}, were the most potent to induce neuronal differentiation. SC-1 alone induced neuronal differentiation through extracellular signal-regulated kinase (ERK) 1/2 activation that is essential for nerve growth factor (NGF)-induced neuronal differentiation, as shown by the suppression with MEK1/2 inhibitors, PD98059 and U0126. However, SC-1 did not increase phosphorylation of TrkA, a high-affinity NGF receptor, and a TrkA inhibitor, K252a, did not affect SC-1-induced neuronal differentiation. Furthermore, SC-1 potentiated neuronal differentiation in cells co-treated with NGF, which was associated with enhanced ERK1/2 activation and increased expression of neuronal differentiation markers. Interestingly, when treated with SC-1 and a high dose of NGF, formation of synaptic connections and synaptophysin accumulation at the neurite terminals were markedly enhanced. These results indicate that (1) SC-1 alone induces neuronal differentiation, (2) SC-1 potentiates neuronal differentiation in NGF-treated cells, (3) SC-1 enhances formation of synaptic connections in cells treated with a high dose of NGF, all of which are associated with ERK1/2 activation. It is therefore concluded that SC-1 may promote neuronal differentiation by tapping into the ERK1/2-MAPK (mitogen-activated protein kinase) signaling pathway downstream from the TrkA receptor in PC12 cells.