Protein kinase C isoforms in human glioblastoma cells.

Protein kinase C (PKC), an enzyme involved in signal transduction, responds to diacyl glycerol and also to phorbol ester, a ligand analogous to diacyl glycerol. We have studied the expression of the major isoforms (alpha, beta I, beta II, and gamma) in eight human glioblastoma cell lines. In all eight lines, PKC-alpha mRNA and protein were expressed. In none of the eight did a probe for PKC-beta I and -beta II mRNA give positive results nor were Western blots for PKC-beta II positive. The half-life for PKC alpha mRNA was approximately 16 h and levels of the mRNA were increased slightly following addition of phorbol myristate acetate (PMA) or transforming growth factor-beta (TGF beta). PKC-gamma was present in most of the glioblastomas. In cell line A172, 82% of the PKC-alpha was present in the cytosol with the remainder evenly divided between plasma membrane and nucleus. Thirty minutes after addition of PMA, 33% of the total original protein was in the plasma membrane and 48% in the nuclear fraction. By 21 h, no PKC-alpha was recovered from any fraction. PKC-gamma was also down-regulated in the presence of PMA, but there was no evidence for translocation to the plasma membrane or nuclear fraction. In a more detailed study, translocation of PKC-alpha in the presence of PMA was complete by 10 min, and a major decrease in the PKC translocated to the plasma-membrane fraction occurred some time between 2 and 4 h after PMA addition, while a major decrease in the translocated nuclear fraction occurred some time after 6 h. cAMP alone had no effect on the PKC alpha protein level or distribution, nor did it alter the translocation and down-regulation due to PMA exposure. In these studies the level of PKC-alpha mRNA in tumors was similar to that in normal glial 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.     

Protein kinase C mediates potentiation of synaptic transmission by phorbol ester at parallel fibers in the dorsal cochlear nucleus

Many cells in the outer two layers of the dorsal cochlear nucleus (DCN) express high levels of the phospholipid-activated, calcium dependent kinase, protein kinase C (PKC), an enzyme that can phosphorylate numerous proteins involved in neurotransmission and postsynaptic signaling. We investigated the effects of stimulating PKC with phorbol esters (phorbol 12-13 diacetate; PDAc) on parallel fiber synaptic transmission in brain slices of the guinea pig DCN. Phorbol esters increased the amplitude of the postsynaptic components of the field potential, including the excitatory post-synaptic field potential (fEPSP) and the population spike following electric stimulation of parallel fibers. Phorbol esters simultaneously decreased paired-pulse facilitation, suggesting that transmitter release mechanisms were affected. Potentiation of synaptic transmission and diminished paired-pulse potentiation were also observed in intracellular recordings of DCN neurons. The effects of phorbol esters were antagonized by the specific PKC blockers bisindolylmaleimide and calphostin C. Although modulation of the synaptic potentials appears to be mediated by presynaptic PKC, the differential effects of PDAc on the fEPSP and the population spike also suggest the involvement of postsynaptic PKC and postsynaptic targets. These experiments demonstrate that protein kinase C is capable of profoundly modulating synaptic transmission at parallel fiber synapses in the DCN.     

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.     

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.     

Dose-dependent phorbol ester facilitation or blockade of hippocampal long-term potentiation: relation to membrane/cytosol distribution of protein kinase C activity

We have proposed that the translocation/activation of protein kinase C (PKC) in synergism with a Ca²⁺-mediated event plays an essential role in hippocampal long-term potentiation (LTP). In a previous study, we saw no effect of PKC-activating phorbol esters alone on baseline responses, although it has been reported by others to enhance synaptic transmission. To resolve this discrepancy, we investigated the dose-response to phorbol esters of both baseline and potentiated granule cell responses elicited with perforant path stimulation. It was confirmed that iontophoretic ejection of phorbol ester to the dentate hilus, which alone had no effect on baseline responses, prolonged the persistence of potentiation produced by 2 trains of 400 Hz stimulation. These data support the proposed synergistic model in which the effects of phorbol ester and high frequency stimulation together produce a long-lasting potentiation of synaptic activation. A similar synergism was observed with ejection of a lower dose of phorbol ester into the perforant path synaptic zone in the molecular layer. Higher doses delivered to the synaptic zone without 400 Hz stimulation were sufficient to enhance baseline synaptic responses, but these doses inhibited the initial potentiation induced with 2 trains of 400 Hz stimulation delivered immediately after ejection. There was at times a slowly developing enhancement observed after the initial blockade. Thus, induction of a persistent synaptic enhancement was observed without initial potentiation. Measurement of PKC activity in membrane and cytosol indicated that PKC activation is only associated with the persistence phase of LTP. In contrast, there was no change in PKC subcellular distribution associated with the blockade of initial potentiation by higher doses of PDBu.     

Translocation of protein kinase C in rat hippocampal slices

Tumor-promoting phorbol esters specifically activate protein kinase C and mimic the effects of neurotransmitters in certain systems. Treatment of hippocampal slices with phorbol dibutyrate caused translocation of protein kinase C activity from cytoplasm to membranes. Experiments with carbachol, norepinephrine, glutamate, KCl, and LiCl failed to demonstrate a similar translocation. Translocation more readily provides an index of protein kinase C involvement for phorbol esters than for other agents in hippocampus.     

蛋白激酶C调节脊髓神经元突起的生长(英文)

目的关于蛋白激酶C(PKC)在神经元突起生长和神经再生中的作用,目前仍存有争议。本研究主要观察PKC对离体培养的脊髓神经元生长的调节作用,旨在阐明PKC对突起生长的调节作用。方法分离纯化胎龄14天(E14)的SD胎鼠的脊髓前角神经元,进行原代培养,并检测不同时相点膜/浆PKC活性(m/c-PKCactivity)的比值。结果神经元培养3-11d期间,神经元内m/c-PKC比值以及PKC-βII在突起中的表达水平均与突起生长呈显著相关关系(r=0.95,P<0.01;r=0.73,P<0.01)。此外,PKC激动剂PMA能显著提高m/c-PKC比值,且与神经突起的生长一致(r=0.99,P<0.01)。而PKC抑制剂GF109203X则能显著抑制突起生长,且不被PMA作用所逆转。结论PKC的活性在脊髓神经元突起生长调节中具有重要作用,其中βII亚型可能扮演重要角色。     

Opposing influences of protein kinase activities on neurite outgrowth in human neuroblastoma cells: initiation by kinase A and restriction by kinase C.

The respective roles of cAMP-dependent protein kinase (protein kinase A [PKA]) and protein kinase C (PKC) in the early stages of neurite outgrowth were examined in SH-SY-5Y human neuroblastoma cells. Forskolin or dbcAMP, agents that increase intracellular cAMP levels, and intracellular delivery of PKA catalytic subunit induced neurite outgrowth. The PKA inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), prevented the increases, and decreased further the percentage of cells possessing short, filopodia-like neurites in the absence of inducers. In contrast to effects on PKA activation, PKC activation by 12-0-tetradecanoylphorbol-13-acetate (TPA) reduced the percentage of filopodia-like neurites elaborated by otherwise untreated cells, and prevented neurite outgrowth induced by PKA activators. PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), staurosporine, and sphingosine induced neurite outgrowth. Neurites induced by PKA activation contained higher levels of tubulin immunoreactivity than those induced by PKC inhibition. Furthermore, PKA-induced neurites rapidly retracted in the presence of colchicine, while those elaborated following PKC inhibition were more resistant. These data suggest that neurites elaborated in response to PKA activation are dependent upon microtubule polymerization, and that neurite induction following PKC inhibition is mediated by a different mechanism. PKA activators and PKC inhibitors exerted additive effects on neurite outgrowth, suggesting that the distinct pathways regulated by these two kinases function cooperatively during neuritogenesis.     

Protein kinase C in astrocytes: a determinant of cell morphology

Protein kinase C-like activity was found to be present in astrocytes prepared from rat neocortex and maintained in culture. Exposure to phorbol 12-myristate 13-acetate (PMA) caused a redistribution of this kinase from the cytosol to the membrane fraction of these cells. Also PMA was found to cause a profound change in astrocyte morphology; cells were converted from flat, polygonal, undifferentiated cells to process-bearing cells.     

Sodium-dependent glutamate transport in Müller glial cells: regulation by phorbol esters

The regulation of the Na⁺-dependent high affinity glutamate/aspartate transporter system expressed in cultured Müller glia cells from chick retina was studied. Treatment of the cells with the Ca²⁺/diacylglycerol dependent protein kinase C (PKC) activator, phorbol 12-tetradecanoil-13-acetate (TPA) produced a decrease in [³H]-aspartate uptake which was reversed by staurosporine and partially by H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochoride], two PKC inhibitors. Long-term treatment with TPA resulted in a drastic decrease in the uptake activity, correlated with a substantial fall in the expression of the transporter protein. These findings suggest that PKC is involved in transport modulation at two different levels: phosphorylation and transporter expression in retinal Müller glial cells.     

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.     

Physiological levels of β-amyloid peptide stimulate protein kinase C in PC12 cells

Alzheimer's β-amyloid peptide (Aβ) is normally present at nanomolar concentrations in body fluids and in the medium of cultured cells. In vitro experiments have shown that Aβ has neurotrophic effects and can promote neuronal adhesion and elongation of axon-like processes. In an attempt to understand the molecular mechanisms underlying such effects, we have recently reported that nanomolar doses of Aβ can stimulate protein tyrosine phosphorylation and activate phosphatidylinositol-3-kinase in neuronal cells. Here we show evidence that Aβ can also activate protein kinase C, a serine/threonine kinase, in PC12 cells. First, using a serine-containing S6 peptide as an exogenous substrate, we found that nanomolar levels of Aβ peptides 1–40 or 1–42 significantly stimulated an S6 phosphorylating kinase activity, whereas the Aβ_40–1 reverse sequence peptide had no effect. Down-regulation of PKC by prolonged (18 h) treatment with 1 μM PMA prevented the Aβ-induced S6 phosphorylation. Using a more specific PKC substrate, N-terminal acetylated peptide (4–14) from myelin basic protein, we then demonstrated that Aβ indeed increased PKC activity and that this activity could be blocked by the PKC inhibitor, staurosporine. Finally, immunoblotting experiments showed that Aβ induced translocation of PKCγ from cytosol to membrane and also significantly reduced cytosolic PKCα levels. Taken together, these data suggest that physiological levels of Aβ can regulate PKC activity.     

Repeated,intermittent treatment with amphetamine induces neurite outgrowth in rat pheochromocytoma cells (PC12 cells)

Repeated, intermittent treatment with amphetamine (AMPH) leads to long-term neurobiological adaptations in rat brain including an increased number and branching of dendritic spines. This effect depends upon several different cell types in the intact brain. Here we demonstrate that repeated, intermittent AMPH treatment induces neurite outgrowth in cultured PC12 cells without the requirement for integrated synaptic pathways. PC12 cells were treated with 1 micro M AMPH for 5 min a day, for 5 days. After 10 days of withdrawal, there was an increase in the percentage of cells with neurites ( approximately 30%) and the length of neurites as well as an increase in the level of GAP-43 and neurofilament-M. Neurite outgrowth was enhanced as withdrawal time was increased. Neurite outgrowth was much greater following repeated, intermittent treatment with AMPH compared to continuous or single treatment with AMPH. Pretreatment with cocaine, a monoamine transporter blocker, inhibited the AMPH-mediated increase in neurite outgrowth. Neither NGF antibody nor DA receptor antagonists blocked AMPH-induced neurite outgrowth, demonstrating that AMPH-induced neurite outgrowth is not dependent on endogenous NGF release or DA receptors. Thus we have demonstrated that repeated, intermittent treatment with AMPH has a neurotrophic effect in PC12 cells. The effect requires the action of AMPH on the norepinephrine transporter, and shares characteristics in its development with other forms of sensitization but does not require an intact neuroanatomy.     

Pretreatment with the protein kinase C activator phorbol 12,13-dibutyrate attenuates the ethanol-induced loss of the righting reflex in mice: modification by diabetes

The effects of the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu) on the ethanol-induced loss of the righting reflex were studied in diabetic and non-diabetic mice. The ethanol-induced loss of the righting reflex was significantly less in diabetic mice than in non-diabetic mice. Intracerebroventricular (i.c.v.) pretreatment with PDBu dose- and time-dependently reduced the ethanol-induced loss of the righting reflex in non-diabetic mice. The reduction of the ethanol-induced loss of the righting reflex caused by PDBu was reversed by concomitant i.c.v. pretreatment with calphostin C, a selective PKC inhibitor. On the other hand, PDBu had no effect on the ethanol-induced loss of the righting reflex in diabetic mice. I.c.v. pretreatment with calphostin C (10 pmol) increased the ethanol-induced loss of the righting reflex in diabetic mice but not in non-diabetic mice. These results suggest that the activation of PKC reduces the ethanol-induced loss of the righting reflex in mice. Furthermore, it is possible that this attenuation of the ethanol-induced loss of the righting reflex in diabetic mice may be due in part to increased PKC activity.     

PKC activators (phorbol ester or bryostatin) stimulate outgrowth of NGF-dependent neurites in a subline of PC12 cells

Nerve growth factor (NGF) stimulation of PC12 cells activates signaling pathways leading to new protein expression and growth of neurites. In wild type PC12 cells, incubation with phorbol ester (PMA) will activate protein kinase C (PKC) leading to the expression of many proteins necessary for neurite outgrowth, but this activation of PKC alone will not stimulate growth of long neurites. Here, we show in the subline of PC12-N09, which lacks NGF-stimulated growth of long neurites, that a brief incubation with PKC activators, PMA or bryostatin 1 (bryostatin), before NGF incubation, stimulates the growth of long neurites. However, incubation in the reverse order is ineffective. A short incubation with PMA or bryostatin followed by NGF induced tyrosine phosphorylation of MAP kinase (MAPK), which is of the same duration as that induced by NGF alone. Thus, PMA preincubation did not increase the length NGF activation of MAPK. Twenty-four hr after incubation with PMA or bryostatin, PKC isoforms were downregulated but PKC isoforms δ-, and ϵ- were still present. In these cells chronically treated with either PMA or bryostatin to downregulate PKC, NGF incubation preceded by PMA preincubation still led to long neurite outgrowth. These results suggest that a PMA or bryostatin incubation followed by NGF activates PKC isoforms δ-, and ϵ-leading to outgrowth of long neurites, and that the PMA signaling is independent of the MAPK pathway. J. Neurosci. Res. 53:214–222, 1998. © 1998 Wiley-Liss, Inc.     

Activation of protein kinase C by phorbol dibutyrate potentiates [

Intracellularly injected phorbol 12,13-dibutyrate (PdiB), a phorbol ester that activates protein kinase C (PKC), altered the postsynaptic responses of neurons of the motor cortex of cats. PdiB increased the amplitudes and durations of EPSPs and decreased the amplitudes and durations of IPSPs elicited by stimulation of the ventrolateral (VL) thalamus or the pyramidal tract (PT). The changes lasted for 50 min or longer. Corresponding changes in peak excitatory and inhibitory postsynaptic currents (EPSCs, IPSCs) were measured directly with the single electrode voltage clamp technique. Quantitative analysis of EPSCs in response to VL thalamic stimulation and IPSCs in response to PT stimulation made in a subgroup of fast PT cells suggested that PdiB acted within the injected neuron rather than presynaptically to alter the synaptic currents. No consistent changes in resting membrane parameters that would account for these modifications were found. Control injections of a phorbol ester that did not activate PKC failed to induce changes in synaptic responses or resting membrane properties. These observations suggest that activation of PKC, in vivo, can induce long-lasting changes in synaptic responses of neocortical neurons by direct modification of postsynaptic ion channel conductivities.     

Activation of protein kinase C induces long-term changes of postsynaptic currents in neocortical neurons

Intracellularly injected phorbol 12,13-dibutyrate (PdiB), a phorbol ester that activates protein kinase C (PKC), altered the postsynaptic responses of neurons of the motor cortex of cats. PdiB increased the amplitudes and durations of EPSPs and decreased the amplitudes and durations of IPSPs elicited by stimulation of the ventrolateral (VL) thalamus or the pyramidal tract (PT). The changes lasted for 50 min or longer. Corresponding changes in peak excitatory and inhibitory postsynaptic currents (EPSCs, IPSCs) were measured directly with the single electrode voltage clamp technique. Quantitative analysis of EPSCs in response to VL thalamic stimulation and IPSCs in response to PT stimulation made in a subgroup of fast PT cells suggested that PdiB acted within the injected neuron rather than presynaptically to alter the synaptic currents. No consistent changes in resting membrane parameters that would account for these modifications were found. Control injections of a phorbol ester that did not activate PKC failed to induce changes in synaptic responses or resting membrane properties. These observations suggest that activation of PKC, in vivo, can induce long-lasting changes in synaptic responses of neocortical neurons by direct modification of postsynaptic ion channel conductivities.     

Changes in levels of microtubule-associated proteins in relation to the outgrowth of neurites from PC12D cells, a forskolin- and nerve growth factor-responsive subline of PC12 pheochromocytoma cells

Immunoblotting analysis and immunofluorescence studies of proteins that react with MAP₁- and MAP₂-specific antibodies in PC12 rat pheochromocytoma cells were carried out. When cells of the PC12D subline of PC12 cells, which rapidly extend neurites in response to NGF or drugs that elevate intracellular levels of cyclic AMP, were examined, they were found to contain a relatively higher level of MAP₁ or of a MAP₁-like protein than conventional PC12 cells. Immunoblotting study showed that levels of MAP₁ and MAP₂ or of MAP₁ or MAP₂-like proteins increased in PC12D cells, but not in conventional PC12 cells, in response to forskolin. Immunofluorescence studies also revealed increases in levels of MAP₁ and MAP₂ or of MAP₁ or MAP₂-like proteins in conjunction with the outgrowth of neurites from the cells. These results support the hypothesis that the induction of MAPs may be one of the first steps required for outgrowth of neurites from PC12 cells. Furthermore, PC12D cells may contain a sufficiently high level of MAP₁ or MAP₁-like protein to permit the extension of neurites in the absence of the lag period normally required by PC12 cells. The MAP₁ or a MAP₁-like protein was localized in the cell soma and neurites. An increase of MAP₂-specific immunoreactivity in perikarya was observed in the differentiated cells. After immunostaining with a monoclonal antibody that reacted with phosphorylated MAP₁, intense fluorescence was seen in the growth cones of neurites. This observation supports the hypothesis that the phosphorylation of MAP₁ or of a MAP₁-like protein may play a regulatory role in the formation of neurites in growth cones.