Lysophosphatidylcholine suppresses apoptosis and induces neurite outgrowth in PC12 cells through activation of phospholipase D2

Lysophosphatidylcholine (LPC) is a bioactive lipid generated by phospholipase A2-mediated hydrolysis of phosphatidylcholine. In the present study, we demonstrate that LPC stimulates phospholipase D2 (PLD2) activity in rat pheochromocytoma PC12 cells. Serum deprivation induced cell death of PC12 cells, as demonstrated by decreased viability, DNA fragmentation, and increased sub-G1 fraction of cell cycle. LPC treatment protected PC12 cells partially from the cell death and induced neurite outgrowth of the cells. Overexpression of PLD2 drastically enhanced the LPC-induced inhibition of apoptosis and neuritogenesis. Pretreatment of the cells with 1-butanol, a PLD inhibitor, completely abrogated the LPC-induced inhibition of apoptosis and neurite outgrowth in PC12 cells overexpressing PLD2. These results indicate that LPC possesses the neurotrophic effects, such as anti-apoptosis and neurite outgrowth, through activation of PLD2.     

p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.     

Dexamethasone differentiates NG108-15 cells through cyclooxygenase 1 induction

Cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid into prostanoids which participate in various cellular functions including apoptosis, mitogenesis, inflammation, immune modulation and differentiation. Moreover, the synthetic glucocorticoid, dexamethasone has immune modulating and anti-inflammatory effects in vivo. Recently, dexamethasone was found to enhance retinoic acid-induced neuronal differentiation. In this study, we investigated the mechanisms of dexamethasone-mediated neuronal differentiation. Immunoblotting and morphological analysis demonstrated that dexamethasone induced neuronal differentiation through COX 1 induction. This phenomenon was inhibited by indomethacin, a COX inhibitor. In addition, the addition of exogenous prostaglandin E2 (PGE2), a substance produced by the COX-mediated pathway, triggered neurite outgrowth of cells treated with COX inhibitor. Taken together, COX 1 appears to play an important role in dexamethasone-mediated neuronal differentiation.     

Changes in neurite outgrowth but not in cell division induced by low EMF exposure: influence of field strength and culture conditions on responses in rat PC12 pheochromocytoma cells

The effects of low electromagnetic field (EMF) exposure (4.5-15.8 microT, 50 Hz AC) on neurite outgrowth and cell division in rat PC12 pheochromocytoma cells were examined. The study involved two separate experimental series in which culture conditions during exposure to the magnetic fields differed. In series 1 (14 experiments in which culture conditions were not strongly conducive to cell differentiation [15% serum]), exposure to 4.5-8.25 microT for 23 h significantly inhibited neurite outgrowth by 21.5 +/- 1.3% (by Manova, p = 0.003). In contrast, in series 2 (12 experiments in which culture conditions promoted cellular differentiation [4% serum]), exposure to 4.35-8.25 microT for 23 h significantly stimulated neurite outgrowth by 16.9 +/- 1.1% (by Manova, p = 0.009). Thus, in both series, exposure to a narrow range of low EMF has significant, but opposite effects on neurite outgrowth. Exposure to higher fields, 8.25-12.5 microT (series 1) and 8.25-15.8 microT (series 2) had no significant effect on neurite outgrowth. These data, when considered with other reports, suggest that neuronal differentiation can be altered by low level EMF exposure. While this may not be detrimental, it merits further research. At present, the reasons for the significant changes in neurite outgrowth being confined to the same narrow field strength are unclear. As stated above, culture conditions in series 2 were more conducive to cell differentiation than those in series 1. This is reflected in the lower number of cells in control samples in series 2, at the end of the 23-h incubation, than in series 1 (- 16.9 +/- 1.7%, p = 0.003). As the same numbers were plated in both series, the medium used in series 1 allows more of the PC12 cells to divide; this is consistent with some cells reverting to a non-neuronal adrenal chromaffin phenotype [L. Greene, A. Tischler. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U. S. A., 73 (1976) 2424-2426]. Exposure to both ranges of magnetic fields (4.35-8.25 and 8.25-15.8 microT) has no effect on cell division. Thus, there is no evidence in this study that there is a mitogenic effect arising from low EMF exposure.     

Regulation of neurite outgrowth by intermittent irradiation of visible light

The effect of neurite outgrowth of PC12 cells on collagen-coated glass plates under intermittent light irradiation at 525 nm and 0.4 mW/cm2 of intensity was investigated. Neurite outgrowth of PC12 cells was significantly suppressed when PC12 cells were cultivated under intermittent light irradiation with a total irradiation time of more than 2 min/h. No temperature increase was observed in the culture medium under either continuous or intermittent light irradiation. Therefore, suppression of neurite outgrowth under light irradiation was not due to the increase of temperature in the culture medium, but rather the effect of light on the PC12 cells, especially the signal transmittance of light to PC12 cells. The light irradiation interval also affected the neurite outgrowth of PC12 cells when the total irradiation time was constant. A high extension ratio of neurite outgrowth was observed under a long time interval of nonirradiation between light irradiations (1 min of irradiation every hour) as compared with frequent light irradiation intervals (5 s of irradiation every 5 min) with the same total irradiation period per hour. The neurite outgrowth ratio was thought to be dependent on the light intensity, the total time of light irradiation in the intermittent light irradiation, and the interval of light irradiation in the intermittent light irradiation.     

Cadmium(II) specifically interacts with cellular signaling to induce proto-oncogenes c-fos and c-jun in rat PC12 cells

Cadmium(II) compounds are carcinogens but only weakly mutagens. Because Cd(II) at low micromolar concentrations stimulates cell growth and induces some proto-oncogenes with several mammalian cell lines, the stimulation of cell proliferation is discussed as a major mechanism of the carcinogenicity of this metal. In the present work, the induction of the cellular proto-oncogenes c-fos and c-jun by Cd(II) was studied in rat PC12 cells. Several cellular signal transduction elements were tested as mediators of the proto-oncogene induction by cadmium. Cd(II) does not evoke mobilization of free intracellular Ca²⁺ in PC12 cells, and there was no impairment of the effect of Cd(II) by inhibitors of protein kinase A or of MAPK kinase. However, bisindolylmaleimide I, a specific inhibitor of protein kinase C abolished the proto-oncogene induction by Cd(II). Hence, a critical role for protein kinase C in the mitogenic effect of Cd(II) is inferred, and a substitution of structural Zn²⁺ ions by Cd²⁺ ions in this enzyme is discussed as the putative mechanism. Received: 30 July 1997 / Revised: 9 October 1997 / Accepted: 11 October 1997     

SELECTIVE NEURITE OUTGROWTH ON SILVER NEGATIVE ION (Ag~- )-IMPLANTED POLYSTYRENE SURFACES

INTRODUCTIONA negative ion-implantation technique[1,2] developed by us for use in semiconductors, integrated circuits (IC),and large scale-integrations (LSI) has been studied in its application on biotechnology. The implantation of highion energy beams causes the impairment of insulators such as common synthetic polymers. Our negative ion-implantation, however, is considered to result in less charge-up problems compared with positive ion-implantation. That is, negative ionic species can…     

Cadmium(II) specifically interacts with cellular signaling to induce proto-oncogenes c-fos and c-jun in rat PC12 cells

Cadmium(II) compounds are carcinogens but only weakly mutagens. Because Cd(II) at low micromolar concentrations stimulates cell growth and induces some proto-oncogenes with several mammalian cell lines, the stimulation of cell proliferation is discussed as a major mechanism of the carcinogenicity of this metal. In the present work, the induction of the cellular proto-oncogenes c-fos and c-jun by Cd(II) was studied in rat PC12 cells. Several cellular signal transduction elements were tested as mediators of the proto-oncogene induction by cadmium. Cd(II) does not evoke mobilization of free intracellular Ca²⁺ in PC12 cells, and there was no impairment of the effect of Cd(II) by inhibitors of protein kinase A or of MAPK kinase. However, bisindolylmaleimide I, a specific inhibitor of protein kinase C abolished the proto-oncogene induction by Cd(II). Hence, a critical role for protein kinase C in the mitogenic effect of Cd(II) is inferred, and a substitution of structural Zn²⁺ ions by Cd²⁺ ions in this enzyme is discussed as the putative mechanism. Received: 30 July 1997 / Revised: 9 October 1997 / Accepted: 11 October 1997     

Tailored chondroitin sulfate glycomimetics via a tunable multivalent scaffold for potentiating NGF/TrkA-induced neurogenesis

The challenges inherent in the synthesis of large glycosaminoglycan (GAG) polysaccharides have made chemically accessible multivalent glycoligands a valuable tool in the field of GAG mimetics. However, the difficulty of positioning sulfated sugar motifs at desired sites has hindered efforts to precisely tailor their biofunctions. Here, we achieved precise orientation of sulfated disaccharide motifs by taking advantage of a structurally well-defined polyproline scaffold, and describe systematic explorations into the importance of the spatial arrangement of sulfated sugars along the scaffold backbone in designing multivalent glycoligands. Our protein binding studies demonstrate that the specific conformational display of pendant sugars is central to direct their multivalent interactions with NGF. By employing computational modeling and cellular studies, we have further applied this approach to engineer NGF-mediated signaling by regulating the NGF/TrkA complexation process, leading to enhanced neuronal differentiation and neurite outgrowth of PC12 cells. Our findings offer a promising strategy for the pinpoint engineering of GAG-mediated biological processes and a novel method of designing new therapeutic agents that are highly specific to GAG-associated disease.     

The role of chalcones: helichrysetin,xanthohumol, and flavokawin-C in promoting neurite outgrowth in PC12 Adh cells

Chalcones are a group of compounds widely distributed in plant kingdom. The aim of this study was to assess the neurite outgrowth stimulatory activity of selected chalcones, namely helichrysetin, xanthohumol and flavokawin-C. Using adherent rat pheochromocytoma (PC12 Adh) cells, the chalcones were subjected to neurite outgrowth assay and the extracellular nerve growth factor (NGF) levels were determined. Xanthohumol (10 μg/mL) displayed the highest (p < 0.05) percentage of neurite-bearing PC12 Adh cells and the highest (p < 0.05) NGF level in the culture medium of xanthohumol-treated cells. While, helichrysetin induced a moderately high numbers of neurite-bearing cells, flavokawin-C did not stimulate neurite outgrowth. This work supports the potential use of xanthohumol as a potential neuroactive compound to stimulate neurite outgrowth.     

ATP-induced focal adhesion kinase activity is negatively modulated by phospholipase D2 in PC12 cells

Extracellular ATP has been known to modulate various cellular responses including mitogenesis, secretion and morphogenic activity in neuronal cells. In the ATP-induced morphogenic activity, focal adhesion kinase(s) such as Fak have been suggested to play a critical role. Binding of ATP to its specific cell surface receptor in PC12 cells induces phospholipase D (PLD) activity. However, the role of PLD on ATP-induced Fak activation in PC12 cells remains unclear. In this study, we investigated the role of PLD on the ATP-induced Fak activation and paxillin phosphorylation using two established cell lines: wild type PLD2- and lipase-inactive mutant PLD2-inducible PC12 cells. Stimulation of cells with ATP caused PLD2 activation via classical protein kinase C activation. ATP also induced Fak activation, and paxillin phosphorylation, and were dramatically reduced by wild type PLD2 overexpression but not by lipase-inactive mutant PLD2 overexpression. When the PC12 cells were pretreated with propranolol, a specific inhibitor for phosphatidic acid phosphohydrolase resulting in the accumulation of PA, ATP-induced Fak activation and paxillin phosphorylation were also reduced. We found that inhibition of tyrosine phosphatases by pervanadate completely blocked PLD2-dependent Fak and paxillin dephosphorylation. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced Fak and paxillin phosphorylation possibly through tyrosine phosphatases.     

Differentiation of human gingival mesenchymal stem cells into neuronal lineages in 3D bioconjugated injectable protein hydrogel construct for the management of neuronal disorder

The success of regeneration attempt is based on an ideal combination of stem cells, scaffolding and growth factors. Tissue constructs help to maintain stem cells in a required area for a desired time. There is a need for easily obtainable cells, potentially autologous stem cells and a biologically acceptable scaffold for use in humans in different difficult situations. This study aims to address these issues utilizing a unique combination of stem cells from gingiva and a hydrogel scaffold, based on a natural product for regenerative application. Human gingival mesenchymal stem cells (HGMSCs) were, with due induction, differentiated to neuronal lineages to overcome the problems associated with birth tissue-related stem cells. The differentiation potential of neuronal lineages was confirmed with suitable specific markers. The properties of mesenchymal stem cells in encapsulated form were observed to be similar to free cells. The encapsulated cells (3D) were then subjected to differentiation into neuronal lineages with suitable inducers, and the morphology and gene expression of transient cells were analyzed. HGMSCs was differentiated into neuronal lineages as both free and encapsulated forms without any significant differences. The presence of Nissl bodies and the neurite outgrowth confirm the differentiation. The advantages of this new combination appear to make it a promising tissue construct for translational application.     

Basic fibroblast growth factor-induced translocation of p21-activated kinase to the membrane is independent of phospholipase C-gamma1 in the differentiation of PC12 cells

p21-activated kinase (PAK) targeting to the plasma membrane is essential for PC12 cell neurite outgrowth. Phospholipase C-gamma1 (PLC-gamma1) can mediate the PAK translocation in response to growth factors, since PLC-gamma1 binds to both tyrosine-phosphorylated receptor tyrosine kinases and PAK through its SH2 and SH3 domain, respectively. In the present study, we examined a potential role for PLC-gamma1 in the basic fibroblast growth factor (bFGF)-induced PAK translocation using stable PC12 cell lines that overexpress in a tetracycline-inducible manner either the wild-type FGFR-1 or the Y766F FGFR-1 mutant. Phosphatidylinositol hydrolysis was increased 6.5-fold in response to bFGF in the wild type cells but negligible in the mutant cells. The recombinant GST-PLC-gamma1 SH3 was able to bind to PAK1 but not GST alone. However, examination of PLC-gamma1 as an adaptor for translocation of PAK1 in cells showed that both cells transfected with pEGFP-PAK1 was able to differentiate for 24 h, as visualized by laser confocal microscopy. Translocation of PAK1 to growth cones occurs at similar levels in both wild and mutant cells. These results suggest that a protein(s) other than PLC-gamma1 is functionally relevant for PAK targeting.     

Neural cell adhesion molecule (NCAM) induces neuronal phenotype acquisition in dominant negative MEK1-expressing hippocampal neural progenitor cells

It has been shown that neural cell adhesion molecule (NCAM)-induced neuronal differentiation is extracellular signal-regulated kinase (ERK)-dependent. However, an involvement of the mitogen activated protein kinase (MAPK) kinase (MEK), an upstream kinase of ERK, has not been directly demonstrated in this process. Therefore, we investigated whether the MEK1 plays a critical role in the NCAM-induced neuronal differentiation of hippocampal neural progenitor cells (NPCs). NPCs were transiently transfected with expression plasmids encoding activated or dominant negative (DN) forms of MEK1. The expression of DN MEK1 inhibited neuronal phenotype acquisition and soluble NCAM rescued the defect in the neuronal phenotype acquisition in DN-MEK1-transfected cells, suggesting that NCAM might contribute to the neuronal differentiation via distinct, parallel pathways including the MEK pathway. In cells expressing wild type MEK1 or constitutively active MEK1 on the other hand, the percentage of cells positive for beta-tubulin type III (Tuj1), a marker for early postmitotic neurons, was higher than seen in vector-transfected cells. These results suggest that the activation of MEK1 is required for obtaining neuronal phenotype in NPCs.     

Involvement of MAPK pathway in hypoxia-induced up-regulation of urokinase plasminogen activator receptor in a human prostatic cancer cell line, PC3MLN4

Clinical studies have shown that tumor hypoxia is associated with invasive growth and metastasis and may be an important prognostic factor adversely influencing survival in patients with tumors. To investigate the mechanisms involved in hypoxia-induced invasive growth and metastasis, hypoxia-mediated urokinase plasmalogen activator receptor (uPAR) expression, cellular invasiveness, and mitogen activated protein kinase (MAPK) activation were measured in a prostate cancer cell line, PC3MLN4. The levels of uPAR expression and cellular invasiveness were increased in hypoxic cells. Hypoxia-induced cellular invasiveness was blocked by an anti-uPAR monoclonal antibody. Phosphorylations of ERK and p38 kinases were also more extensive in hypoxic cells than in normoxic cells. Hypoxia-induced uPAR up-regulation was inhibited by pre-treatments with a specific inhibitor of MEK, PD98059 and a specific inhibitor of p38 MAP kinase, SB203580. Cell growth also increased in hypoxic cells. From these results, hypoxia increased tumor cell invasion by up-regulating uPAR expression, which might be mediated through ERK and p38 kinase signaling pathways in PC3MLN4 prostate cancer cell line.     

Rational design of small molecule inhibitors targeting RhoA subfamily Rho GTPases

Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets. By virtual screening, we have identified a Rho-specific inhibitor, Rhosin. Rhosin contains two aromatic rings tethered by a linker, and it binds to the surface area sandwiching Trp58 of RhoA with a submicromolar Kd and effectively inhibits GEF-catalyzed RhoA activation. In cells, Rhosin specifically inhibited RhoA activity and RhoA-mediated cellular function without affecting Cdc42 or Rac1 signaling activities. By suppressing RhoA or RhoC activity, Rhosin could inhibit mammary sphere formation by breast cancer cells, suppress invasion of mammary epithelial cells, and induce neurite outgrowth of PC12 cells in synergy with NGF. Thus, the rational designed RhoA subfamily-specific small molecule inhibitor is useful for studying the physiological and pathologic roles of Rho GTPase.     

PKCalpha induces differentiation through ERK1/2 phosphorylation in mouse keratinocytes

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKCa isoform. When PKCalpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKCalpha kinase active mutant (PKCalpha- CAT) in the undifferentiated keratinocyte, but not PKCbeta-CAT, also increased differentiation marker expressions. On the other hand, PKCalpha dominant negative mutant (PKCbeta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKCbeta-KR did not, suggesting that PKCalpha is responsible for keratinocyte differentiation. When downstream pathway of PKCalpha in Ca2+ -mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+ -mediated differentiation, and that only ERK1/2 pathway is specific for PKCalpha-mediated differentiation in mouse keratinocytes.     

Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells

Background: Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities.Results: Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aidehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called ‘branched chain amino acid preferring’ (BrAAP) and ‘small neutral amino acid preferring’ (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity.Conclusions: The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate,