Protein deacetylase SIRT1 in the cytoplasm promotes nerve growth factor-induced neurite outgrowth in PC12 cells

SIRT1, a NAD⁺-dependent protein deacetylase, is known to have neural functions. However, despite its cytoplasmic expression in some neural cells, its cytoplasmic function, if any, is unknown. Here we found that PC12 (pheochromocytoma) cells expressed SIRT1 in the cytoplasm. Nerve growth factor (NGF)-induced neurite outgrowth of these cells was promoted by activators of SIRT1, while inhibitors of SIRT1 or SIRT1-siRNA significantly inhibited it. The overexpression of a mutant SIRT1 that localised to the cytoplasm but not the nucleus enhanced the NGF-dependent neurite outgrowth, and a cytoplasmic dominant-negative SIRT1 suppressed it. Thus, cytoplasmic SIRT1 increases the NGF-induced neurite outgrowth of PC12 cells.     

Neurotrophic effect of Semaphorin 4D in PC12 cells

Semaphorins provide crucial attractive and repulsive cues involved in axon guidance during neural development. Out of them, Semaphorin 4D (Sema4D) is enriched in the nervous and immune tissues, and acts as proliferative and survival factors of peripheral lymphocytes in the immune system, but is poorly understood in the nervous system. By using PC12 cells which are well known to differentiate into neural cells in response to nerve growth factor (NGF), we found that soluble forms of Sema4D had neurotrophic effects which were inhibited by neutralizing antibodies to Sema4D. Sema4D strikingly potentiated neurite outgrowth in the presence of 50 ng/ml NGF and increased sensitivity to NGF. Cells responded to very low concentrations of NGF in the presence of 1 nM Sema4D. Activation of following signal proteins, protein kinase C (PKC), L-type of voltage-dependent Ca(2+) channel, and phosphatidylinositol (PI) 3-kinase mediated neurotrophic neurite-outgrowth action of Sema4D. These findings suggest a new function of Sema4D as a neurotrophic signal in PC12 cells.     

Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells

The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.     

Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.     

ALK activation induces Shc and FRS2 recruitment: Signaling and phenotypic outcomes in PC12 cells differentiation

Activation of the neuronal receptor tyrosine kinase ALK (anaplastic lymphoma kinase) promoted the neuron-like differentiation of PC12 cells through specific activation of the ERK MAP-kinase pathway. However, the nature of primary signaling events initiated is still poorly documented. Here, we established that Shc and FRS2 adaptors were recruited and phosphorylated following antibody-based ALK activation. We further demonstrated that Shc was recruited to the consensus phosphotyrosine site NPTpY(1507) and FRS2 was likely recruited to a novel non-orthodox phosphotyrosine site within ALK. Finally, we characterized a functional role for Shc and likely FRS2 in ALK-dependant MAP-kinase activation and neuronal differentiation of PC12 cells. These findings hence open attractive perspectives concerning specific characteristics of ALK in the control of the mechanisms driving neuronal differentiation.     

N-Propionylmannosamine-induced over-expression and secretion of thioredoxin leads to neurite outgrowth of PC12 cells

The function of the central nervous system largely depends on growth and differentiation (neurite outgrowth) of neural cells and it is well established that growth factors, especially nerve growth factor NGF stimulate neurite outgrowth. However, additional factors are implicated in this process notably the redox state of the cells. For the first time we could demonstrate that the application of recombinant thioredoxin stimulates neurite outgrowth of PC12 cells to the same extend as NGF. Thioredoxin, a small redox protein is a major player in the cellular protein reduction system. An increased expression and secretion of thioredoxin is achieved by the application of the novel sialic acid precursor N-propionylmannosamine (ManNProp). From earlier studies it is known that this N-acylmannosamine analog stimulates significantly the neurite outgrowth in cell cultures. This finding would give new insights into the mechanism of the nerve-stimulatory action of ManNProp and demonstrates the novel role of thioredoxin during the regulation of nerve growth, encouraging further studies.     

Interleukin-6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells

Abstract: In contrast to the intensively studied nerve growth factor (NGF)-related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin-6 (IL-6). We have examined the mechanisms of IL-6-induced neuronal differentiation of the pheochromocytoma cell line PC12. IL-6 independently induced the expression of peripherin , identifying this gene as the first neuronal-specific target of IL-6. However, IL-6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL-6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL-6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk-1 mitogen-activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL-6 receptor, providing another potential mechanism of cooperativity between NGF and IL-6 signaling. We propose that IL-6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor-specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL-6 receptor system.     

Clusterin interacts with SCLIP (SCG10-like protein) and promotes neurite outgrowth of PC12 cells

Clusterin has been known as a chaperone-like molecule capable of interacting with various proteins. In this study, we show that clusterin interacts with the microtubule-destabilizing stathmin family protein SCLIP by GST pull-down and co-immunoprecipitation assays. Interestingly, SCLIP interacts with 80 kDa mature form of clusterin in the cytosolic fraction of PC12 cells permeabilized by low concentration of a weak nonionic detergent digitonin, but not with intracellular variants of clusterin known as binding isoforms of Ku70 or TGF-beta receptors. Both clusterin and SCLIP are co-localized at the perinuclear region and growth cone of PC12 cells. In addition, we show that the minimal domains for the interaction are mapped to the C-terminal valine-rich region (367-447) of clusterin and the N-terminal palmitoylation and membrane attachment site (1-34) of SCLIP. Finally, we demonstrate that ectopic expression of clusterin in PC12 cells elongates neurite-formation triggered by NGF and induces spontaneous neurite outgrowth even in the absence of NGF. Taken together, these results suggest that the clusterin interacts with SCLIP and the interaction may act as an important modulator during neuronal differentiation.     

Chromatographic Resolution and Characterization of a Nerve Growth Factor-Dependent Kinase That Phosphorylates Microtubule-Associated Proteins 1 and 2 in PC12 Cells

When the supernatant fractions from extracts of control and nerve growth factor (NGF)- or dibutyryl cyclic AMP-treated PC12D cells were applied to DEAE-Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule-associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP-treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF-treated cells. This activity was designated previously as NGF-dependent MAP kinase. The apparent molecular mass of the active kinase was 45-50 kDa. The apparent Km value was 35 microM for ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE-Sepharose column was assayed in the presence of Mn2+ instead of Mg2+, another NGF-stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg(2+)-activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF-dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF-induced activation of NGF-dependent MAP kinase was blocked by the presence of K-252a. In contrast, the activation of NGF-dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K-252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the activation of NGF-dependent MAP kinase.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms

Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.     

Activation of stress-activated protein kinases correlates with neurite outgrowth induced by protease inhibition in PC12 cells

PC12 cells are well characterized for their ability to differentiate into neuronal-like cells when challenged with nerve growth factor. It has been reported that the calpain and proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (CI) is also able to induce neurite outgrowth in PC12 cells. In this study, we report that the inhibitor of proteasomal chymotrypsin-like activity, carbobenzoxy-Ile-Glu-(O-tert-butyl)-Ala-Leu-aldehyde (PSI), can also induce differentiation of PC12 cells. Induction of neurite outgrowth with PSI, CI, or its close analogue, carbobenzoxy-Leu-Leu-leucinal (MG132), was associated with stress-activated protein kinase (SAPK) activation. Neurite formation induced by protease inhibition was independent of mitogen-activated protein kinase/extracellular signal-regulated kinase, p38/reactivating kinase, or phosphatidylinositol 3-kinase activities. The exact mechanism by which protease inhibition activates SAPKs remains to be elucidated; however, our results suggest that the SAPK signal transduction cascade may be an alternative and/or parallel pathway in the regulation of neuronal differentiation.     

Salvia macilenta exhibits antiglycating activity and protects PC12 cells against H2O2-induced apoptosis

Salvia macilenta is a member of the genus Salvia (Laminaceae) whose antioxidant activity and neuroprotective effect has been shown previously. The present study aimed to examine the antiglycating and antiapoptotic abilities of methanolic extract of this plant. Moreover, the effect of S. macilenta on neurite outgrowth and complexity after exposure to H₂O₂ has been studied. Base on our results, S. macilenta has antiglycating activity and protects PC12 cells against oxidative stress-induced apoptotic cell death, as examined by Hoechst staining and Western blot analysis of caspase-3, Bax, Bcl-2 and PARP. We further showed that S. macilenta decreased neurite growth and complexity impairment in differentiated PC12 cells exposed to oxidative stress. It caused a decrease in cell body area, neurite width, and the proportion of bipolar cells, while significantly increasing neurite length, the number of primary neurites per cell and the ratio of nodes to primary neuritis. All around, the mentioned results open a new horizon for future works to use this plant as a potential neuroprotective agent.     

低频脉冲电场对PC12细胞突起生长和NGF受体分布的影响

以PC12细胞为实验材料,研究低频脉冲电场(f=50Hz,τ=20ms,Epp=1V／m)对神经细胞突起生长及膜受体聚簇的影响。结果显示,该电场能促进NGF受体的聚簇。电场处理15min使PC12细胞表面的NGF受体发生明显的聚簇,30min组次之,5min组聚簇效果较弱。这表明细胞膜受体可能是电磁场与细胞相互作用的位点之一。运用细胞突起图形处理软件,追踪测定经电场处理后PC12细胞的突起数量和长度,发现该电场能显著促进细胞突起的生长,但对突起数量没有明显的影响。     

Bisphenol-A suppresses neurite extension due to inhibition of phosphorylation of mitogen-activated protein kinase in PC12 cells

An endocrine disrupter, bisphenol-A is widely used in the production of plastics and coatings. Recently, it was reported that bisphenol-A affected neurotransmitters in the mammalian brain. On the basis of these reports, it was considered that bisphenol-A affected neuronal differentiation. In this study, the morphological changes in nerve growth factor (NFG)-induced differentiation caused by bisphenol-A were confirmed using a PC12 cell system. When a low concentration of bisphenol-A was added to medium containing NGF, it inhibited neurite extension. In addition, to clarify whether bisphenol-A affects the early and late stages of the NGF-signaling pathway in cell differentiation, changes of phosphorylation of MAP kinases and cAMP-response element binding protein (CREB) in PC12 cells treated with and without BPA in medium containing NGF were investigated using western blot analysis. As results, bisphenol-A significantly inhibited phosphorylation of CREB and ERK1/2 MAPK.     

Blockage of Nerve Growth Factor Action in PC12h Cells by Staurosporine, a Potent Protein Kinase Inhibitor

Staurosporine, which has a structure similar to that of K-252a, a potent protein kinase inhibitor that blocks nerve growth factor (NGF) action in PC12 and PC12h cells, is also known as a potent inhibitor of several protein kinases. This study shows that in PC12h cells staurosporine has a dual action: at lower concentrations than that required by K-252a, it is an inhibitor of NGF induction of neurite formation and of changes in the phosphorylation of specific proteins, whereas at concentrations higher than that required to inhibit NGF-induced neurite outgrowth, it rapidly enhances outgrowth by itself.