Go 6983 | Thr Signal

Inhibition of protein kinase C promotes differentiation of neuroblastoma · glioma NG108-15 hybrid cells

Chia-Yu Chang,1,2 Kuo-Hsing Ma,3 Jehng-Kang Wang,4 Ya-Ling Tung4 and Sheau-Huei Chueh4

Abstract

Differentiation of neuroblastoma · glioma NG108-15 hybrid cells can be induced by different means, but the mechanisms involved are unclear. Our aim was to characterize the role of protein kinase C (PKC) in this process. The PKCs present in NG108-15 cells, i.e. PKCa, PKCd, PKCe and PKCf, were inhibited using a cocktail of Go6983 and Ro318220 or were downregulated by treatment with phorbol 12-myristate 13-acetate (PMA). In high-glucose Dulbecco’s modified Eagle medium, neuritogenesis was induced by 24 h treatment with a cocktail of Go6983 and Ro318220 or by 48 h treatment with PMA, the latter process thus requiring a longer treatment. However, when cells treated with PMA for only 24 h were placed in extracellular standard salts solution, e.g. Locke’s buffer, for 3 h, morphological and functional differentiation occurred, with rounding of the cell body, actin polymerization subjacent to the plasma membrane and an increase in voltage-sensitive Ca2+ channel activity in the absence of cell death. This rapid differentiation was not due to autophagy, growth arrest or increased cyclic AMP response element binding protein phosphorylation, but coincided with combined activation of p38 mitogen-activated protein kinase (MAPK) and inhibition of extracellular signal-regulated kinase (ERK) and Akt, as confirmed by the effects of selective inhibitors. Furthermore, PKC activation blocked thapsigargin-induced neuritogenesis, whereas PKC downregulation did not. These results show that PKC downregulation promotes differentiation and this effect is accelerated by exposure to Locke’s buffer. Although this experimental paradigm cannot be related to the in vivo situation and disease, it implies that combined inhibition of Akt and p44 ⁄ p42 ERK and activation of p38 MAPK promotes differentiation.

Keywords: Akt, p38 mitogen-activated protein kinase, p44 ⁄ p42 extracellular signal-regulated kinase, phorbol ester

Introduction

Based on their amino acid sequences and required co-activators, protein kinase C (PKC) isozymes are classified into three groups, the diacylglycerol- and Ca2+-dependent conventional isozymes (PKCa, PKCb and PKCc), the diacylglycerol-dependent novel isozymes (PKCd, PKCe, PKCh, PKCl and PKCg), and the atypical isozymes (PKCk and PKCf) (Mellor & Parker, 1998). The conventional and novel PKCs can be activated by the binding of phorbol esters, the activated PKC then translocates from the cytoplasm to the plasma membrane and this is often followed by its rapid proteolytic degradation (Pontremoli et al., 1990; Pears & Parker, 1991). Thus, chronic exposure to phorbol ester leads to overall downregulation of PKC levels in the cell, whereas acute exposure activates the enzyme.

Neuronal differentiation is associated with changes in the activity, cellular distribution and expression of specific PKC isozymes. Neuritogenesis is an important process during neuronal differentiation. Using the protein kinase inhibitor H7, a correlation has been suggested between PKC inhibition and induction of neuritogenesis (Minana et al., 1990; Ono et al., 1991; Shea et al., 1992; Rocchi et al., 1995;Singleton et al., 2000), whereas neuritogenesis induced by many agonists is dependent on PKC activity (Cambray-Deakin et al., 1990; Lazarovici et al., 1998; Mahoney et al., 2003; Das et al., 2004; Miloso et al., 2004; Canals et al., 2005). Thus, PKCs are involved in neuronal differentiation, but their precise role and the downstream consequences of their activation or inhibition remain unclear (Violin & Newton, 2003).

In an attempt to clarify the role of PKCs in neuronal differentiation and the underlying mechanism, we used NG108-15 cells as a model system (Hamprecht, 1977; Nirenberg et al., 1983). These cells have been widely used to study many aspects of neuronal function and differentiation (Nelson et al., 1976; McGee et al., 1978). In addition, PKC expression has been previously characterized in NG108-15 cells, which express PKCa, PKCd, PKCe and PKCf (Tohda et al., 1991; Chen et al., 1995). Acute treatment of these cells with phorbol ester for up to 15 min results in activation of PKCa, PKCd and PKCe, whereas chronic treatment results in their downregulation (Battaini et al., 1994; Greenland & Mukhopadhyay, 2004). Using more specific inhibitors directed against the PKC isozymes present in NG108-15 using this cocktail or PKC downregulation caused by chronic treatment with phorbol ester induced NG108-15 cell differentiation. We found that neuritogenesis was induced by 24 h treatment with the inhibitor cocktail or by 48 h of phorbol 12-myristate 13-acetate (PMA) treatment. Thus, the latter process required a longer treatment; however, when cells treated with PMA for only 24 h were placed in extracellular standard salts solution, e.g. Locke’s buffer (LB) for only 3 h, morphological and functional differentiation occurred. The mechanism of the differentiation process was examined.

Materials and methods

Materials

All culture materials including high-glucose Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum, hypoxanthine, aminopterin and thymidine were from Invitrogen (Carlsbad, CA, USA). Poly-l-lysine, PMA, Hoechst 33258, amphotericin B, bovine serum albumin, dibutyryl cyclic AMP (dbcAMP), lysis buffer (CelLyticTM M cell lysis reagent for cultured mammalian cells), 3-methyladenine (3-MA), monodansylcadaverine (MDC), rapamycin and PD098059 were from Sigma (St Louis, MO, USA). The protein kinase assay kit, LY294002, Go6983 and Ro318220 were purchased from Merck (Darmstadt, Germany). SB203580 was from Biomol (Plymouth Meeting, PA, USA). The Bradford assay reagent was from Bio-Rad Laboratories (Hercules, CA, USA). Fura-2 and rhodamine phalloidin were purchased from Invitrogen Molecular Probes (Eugene, OR, USA). The enhanced chemiluminescence substrate was provided by PerkinElmer Life and Analytical Sciences (Shelton, CT, USA). The rabbit polyclonal antibodies against p44 ⁄ p42 extracellular signal-regulated kinase (ERK) (Cat. no. 9102), phospho-p44 ⁄ p42 ERK (Thr202 ⁄ Tyr204) (Cat. no. 9101), Akt (Cat. no. 9272), phospho-Akt (Ser473) (Cat. no. 9271), p38 mitogen-activated protein kinase (MAPK) (Cat. no. 9212), phospho-p38MAPK(Thr180 ⁄ Tyr182)(Cat. no.9211),phospho-cyclic AMP response element binding protein (CREB) (Cat. no. 9198) and horseradish peroxidase-conjugated anti-rabbit IgG antibodies (Cat. no. 7074) were from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal IgG panel against the C-terminal region of each rat PKC isoform (Cat. no. PK49) was provided by Oxford Biomedical Research (Oxford, MI, USA). The rabbit polyclonal antibodies against LIM kinase 1 (Cat. no. sc-5576) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were of analytical grade and were obtained from Merck.

Cell culture

For expansion, neuroblastoma · glioma NG108-15 hybrid cells (passage 25–35) were cultured in 100-mm dishes in high-glucose DMEM supplemented with 5% fetal bovine serum, 100 lm hypoxanthine, 1 lm aminopterin and 16 lm thymidine at 37 C in an atmosphere of 95% air and 5% CO2 as described previously (Chueh et al., 1995). For experiments, the cells were first plated on 100-mm dishes, 35-mm dishes, 12-well plates or 24-mm coverslips depending on the experiment and cultured for another 24 h under the same conditions as for expansion, then were transferred to serum-free highglucose DMEM and treated as described.

Treatment of cells

To compare the effect of PKC inhibition or downregulation on neuritogenesis, at 24 h after plating, the cells were placed in serumfree high-glucose DMEM supplemented with 0.015% dimethyl sulfoxide (DMSO) (control) or 1 lm Go6983 plus 1 lm Ro318220 to inhibit PKCs or with 300 nm PMA to downregulate PKCs, and morphological changes were examined after 24 or 48 h. As a positive control, cells were treated with 1 mm dbcAMP, as NG108-15 cells differentiate when the intracellular concentration of cyclic AMP is increased (Nelson et al., 1976; McGee et al., 1978).

We have previously shown that, after 3 h treatment of NG108-15 cells in LB with thapsigargin, a sarco(endo)plasmic reticulum Ca2+ ATPase inhibitor, not only is the intracellular Ca2+ pool depleted, but neurite outgrowth is induced via arachidonic acid generation (Chin et al., 2002). In experiments examining the effect of PKC activation or downregulation on thapsigargin-induced neuritogenesis, at 24 h after plating, the cells were placed in serum-free high-glucose DMEM and were left untreated (control) or treated with 300 nm PMA for 1 min (PKC activation) or 24 h (PKC downregulation); all three groups of cells were then placed in LB [(in millimolar)150 NaCl, 5 KCl, 1 MgCl2, 2.2 CaCl2, 5 glucose and 10 HEPES, pH 7.4] with or without 1 lm thapsigargin, incubated for another 3 h and morphological changes examined.

As cells placed in LB for 3 h after PMA treatment for 24 h showed morphological and functional differentiation, we characterized the underlying mechanism; at 24 h after plating, cells were incubated for 24 h with 300 nm PMA in serum-free high-glucose DMEM, the bathing solution was replaced with serum-free high-glucose DMEM or LB without PMA and incubation was continued for a further 3 h. Control cells underwent the same treatment except for the addition of 0.015% DMSO instead of PMA.

Morphological changes, neurite outgrowth and varicosity assay

Cells wereplated on poly-l-lysine-coated 24-mm coverslipsat a density of 2.5 · 104 cells per coverslip. After the indicated treatment, the cells were fixed by incubation with 3.7% formaldehyde in phosphatebuffered saline (PBS) for 10 min at room temperature, then morphological changes, the percentage of cells showing neurite outgrowth and the number of varicosities were analysed by phase-contrast microscopy (Nikon TE200). Processes longer than the cell body were scored as neurites. Neurite-positive cells and the number of varicosities were quantified in 10 randomly chosen fields for each coverslip, with 40–60 cells per field, and then the percentage of cells with neurites and the number of varicosities per 100 cells were calculated.

Measurement of cell numbers and apoptotic cells

Cells plated in 35-mm dishes at a density of 1 · 105 cells per dish and treated as indicated were harvested from both the attached and floating fractions and the cells combined and thoroughly washed with PBS. Total viable cell number was determined by Trypan Blue exclusion using a haemocytometer. For measurement of apoptotic cells, the cells were fixed by incubation with 3.7% formaldehyde in PBS at room temperature for 10 min, spread on slides, allowed to dry and treated with 10 lm Hoechst 33258 for 20 min at room temperature. Nuclear morphology was then examined on an Olympus IX-70 fluorescence microscope, the number of apoptotic and non-apoptotic nuclei counted in a population of 300 cells, and the percentage of apoptotic cell per 300 cells calculated as described previously (Chin et al., 2002).

Measurement of the intracellular Ca2+ concentration

Changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured using the fluorescent dye fura-2, as described previously (Grynkiewicz et al., 1985; Chin et al., 2010). Briefly, the cells were plated on coverslips at a density of 2.5 · 105 cells per coverslip. After the indicated treatment, the cells were incubated for 30 min at 37 C with 5 lm fura-2 AM in LB, the coverslip was mounted in a modified Cunningham chamber attached to the stage of a fluorescence microscope (model DMIRB; Leica, Germany), and [Ca2+]i changes were measured on-line using a computer-controlled dual-excitation fluorometric Ca2+ imaging system equipped with a high-speed scanning polychromatic light source (model C7773; Hamamatsu Photonics, Hamamatsu, Japan) and a CCD camera (HISCA; model C6790; Hamamatsu Photonics) controlled by aquacosmos 2.5 software (Hamamatsu Photonics) to acquire quantitative fluorescence intensity data using excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. The sampling rate was 1 Hz, and seven cells per coverslip were examined. The average 340 ⁄ 380-nm fluorescence ratio (F340 ⁄ F380) for these seven cells was used to reflect the [Ca2+]i change.

Electrophysiological recording

The Ca2+ current was measured by the whole-cell voltage clamp technique using a patch clamp amplifier (Axopatch I-D; Axon Instruments, Foster City, CA, USA), as described previously (Lo et al., 2002). The cells were plated on coverslips at a density of 2.5 · 105 cells per coverslip, then, after the indicated treatment, were mounted in a recording chamber superfused with bathing buffer consisting of (in millimolar) 137 tetraethylammonium chloride, 4 CsCl, 0.5 MgCl2, 1.8 CaCl2, 10 glucose and 10 HEPES, pH 7.4, while the pipette buffer consisted of (in millimolar) 130 CsCl, 1 MgCl2, 10 EGTA, 5 Mg2ATP, 0.1 NaGTP, 5 Na2 phosphocreatine and 10 HEPES, titrated to a pH of 7.2 with CsOH. The Ca2+ current was measured as an inward current during depolarization from a holding potential of )70 mV to a test potential of up to +50 mV in 10-mV steps, each for 150 ms, at a frequency of 0.1 Hz using a perforated patch clamp and amphotericin B.

F-actin determination

All steps were at room temperature. After the indicated treatment, NG108-15 cells plated on coverslips at a density of 2.5 · 104 cells per coverslip were fixed in 3.7% formaldehyde for 10 min, rinsed twice in LB and permeabilized in 0.1% Triton X-100 for 5 min. After several washes, they were incubated for 30 min with 2 U ⁄ mL rhodamine phalloidin in PBS containing 1% bovine serum albumin, then examined by fluorescence microscopy using a Zeiss model LSM510 confocal laser scanning microscope (Oberkochen, Germany).

Immunoblotting

For p44 ⁄ p42 ERK, Akt, p38 MAPK, LIM kinase 1 and CREB immunoblots, cells plated on 100-mm dishes at a density of 2.5 · 106 cells per dish were treated as indicated, detached and thoroughly washed, and the cell pellets were suspended in 150 lL ice-cold lysis buffer (CelLyticTM M cell lysis reagent for cultured mammalian cells) and incubated for 30 min at 4 C to lyse the cells. After centrifugation at 16 000 g for 30 min at 4 C, the supernatant was collected. For PKC blots, the cell pellets were suspended in sonication buffer containing (in millimolar) 25 Tris–HCl, 250 sucrose, 2.5 magnesium acetate, 1 dithiothreitol and 2.5 EGTA, pH 7.4, supplemented with 20 lg ⁄ mL leupeptin and 200 lm phenylmethylsulfonyl fluoride, and sonicated. After centrifugation at 100 000 g for 40 min at 4 C, the supernatant was collected and designated as the cytosolic soluble fraction. The pellet was resuspended in sonication buffer supplemented with 0.5% Triton X-100 and the suspension incubated for 20 min on ice, then centrifuged at 16 000 g for 30 min at 4 C. The final supernatant was collected and designated as the particulate fraction. Protein content was determined using the Bradford assay. Aliquots of each fraction were mixed with an equal volume of twofold concentrated reducing SDS-PAGE sample buffer and boiled for 10 min. After electrophoresis, the proteins were electrophoretically transferred to nitrocellulose paper. After blocking overnight at 4 C with blocking buffer (5% non-fat milk in PBS), the blots were incubated for 1 h at room temperature with primary antibodies in blocking buffer plus 0.1% Tween 20 and bound antibodies detected by incubation for 1 h at room temperature with appropriate horseradish peroxidase-conjugated secondary antibodies in the same buffer, followed by incubation with enhanced chemiluminescence substrate. All antibodies were diluted 1 : 1000.

Autophagy detection

Cells plated on coverslips at a density of 2.5 · 104 cells per coverslip were treated as indicated, then were incubated for 15 min at room temperature with 50 lm monodansylcadaverine (MDC), fixed in 3.7% formaldehyde for 10 min at room temperature and rinsed twice in LB. Fluorescence images were taken immediately using a computercontrolled fluorescence microscope (model DMIRB; Leica) equipped with a HISCA CCD camera (model C6790; Hamamatsu Photonics) controlled by aquacosmos 2.5 software (Hamamatsu Photonics) with excitation and emission wavelengths of 340 and 505 nm, respectively.

PKC activity assay

After treatment of cells with PMA or the Go6983 ⁄ Ro318220 cocktail for the indicated time, PKC activity was measured using a nonradioactive protein kinase assay kit (Calbiochem Merck Biosciences, Darmstadt, Germany) according to the manufacturer’s instructions. Briefly, after the indicated treatment, cells plated on 100-mm dishes at a density of 2.5 · 106 cells per dish were detached and thoroughly washed and the cell pellet suspended in ice-cold sample preparation buffer. After sonication on ice, the sonicate was centrifuged at 100 000 g for 60 min at 4 C, then aliquots of the supernatant (about 5–10 lg protein in 12 lL) were used for the PKC assay. The basal PKC activity of control cells was determined in each experiment and the PKC activity of the PMA- or Go6983 ⁄ Ro318220 cocktail-treated cells expressed as a percentage of this.

Statistical analysis

All numerical data are expressed as means ± standard deviation (SD). Data from each experiment were analysed by one-way analysis of variance (anova) followed by Newman–Keuls post hoc test when needed to analyse data between two or more groups. Differences were considered to be significant at P < 0.05. The software package graphpad prism (version 5.03; GraphPad Software, San Diego, CA, USA) was used.

Results

Correlation between reduction in PKC activity and induction of differentiation

To analyse the potential role of PKC in neuronal differentiation, we examined whether PKC inhibition or PKC downregulation in high-glucose DMEM induced differentiation of NG108-15 cells. As shown in Fig. 1A, at 24 and 48 h, the control group consisted of undifferentiated NG108-15 cells (panels a and b) with flat, angularshaped cell bodies, with only a few bearing short neurites. As a positive control, treatment of the cells with 1 mm dbcAMP for 24 or 48 h resulted in typical differentiated NG108-15 cells (panels c and d). Not only did the morphology change from a polygonal flattened to an oval rounded cell body, but increased neurite outgrowth and varicosity formation were also seen (Braas et al., 1983; Krystosek, 1985). When cells were incubated for 24 or 48 h with a cocktail of Go6983 and Ro318220 (1 lm each), morphological changes were induced (compare panels e and f with a), similar to those seen with dbcAMP treatment. In contrast, no morphological differentiation was induced by PKC downregulation after 24 h treatment with 300 nm PMA, the cells appearing identical to the control cells (compare panel g with a), whereas differentiation was seen after 48 h of PMA treatment (compare panel h with a). These results suggest that inhibition and downregulation of PKC both induced differentiation of NG108-15 cells, but that the latter process required longer treatment. Activated PKC translocates from the cytoplasm to the plasma membrane and this is often followed by its rapid degradation (Pontremoli et al., 1990; Pears & Parker, 1991). We therefore measured PKC levels in both the cytosolic and the particulate fractions before and after 24 h treatment with 300 nm PMA or the Go6983 ⁄ Ro318220 cocktail. As shown in Fig. 1B and C, Western blots showed that undifferentiated NG108-15 cells expressed PKCa, PKCd, PKCe and PKCf in both the cytosolic (S) and particulate (P) fractions, with PKCb, PKCc and PKCg being undetectable. In the PMA-treated cells (Fig. 1B), levels of PKCa, PKCd and PKCe were profoundly reduced in both fractions, with PKCa and PKCd being barely detectable (5 and 4% of the original PKCa in the cytosolic and particulate fraction, respectively, the corresponding values for PKCd being 1 and 2%), while PKCf levels were unchanged. However, PKCe was still detectable in the particulate fraction at higher levels than in the cytosolic fraction (22 and 7% of the control values, respectively). In Go6983 ⁄ Ro318220-treated cells (Fig. 1C), levels of PKCa, PKCd, PKCe and PKCf were slightly reduced in the cytosolic fraction by 20, 44, 15 or 3%, respectively, compared with corresponding control cells, while, in the particulate fraction, the reduction was greater, 58, 63, 53 and 44%, respectively. Go6983 ⁄ Ro318220-treated cells relative to that in control cells after different lengths of treatment. PKC activity decreased to about 30% of control levels in cells treated with PMA for between 12 and 24 h, while, in Go6983 ⁄ Ro318220-treated cells, a similar decrease was seen after 1 h. These data show a correlation between a reduction in PKC activity and induction of differentiation.

LB exposure accelerated PKC downregulation-induced morphological differentiation

We have previously shown that 3 h of treatment with thapsigargin induces neurite outgrowth and rounding up of cell bodies in NG10815 cells (Chin et al., 2002). To determine the role of PKC in the differentiation process, we measured the effect of pretreatment with 300 nm PMA for 1 min (PKC activation) or 24 h (PKC downregulation) on thapsigargin-induced differentiation. As shown in Fig. 2, compared with untreated control cells (Fig. 2A), treatment with thapsigargin for 3 h in LB induced morphological differentiation (Fig. 2D) and this effect was blocked by PMA pretreatment for 1 min (Fig. 2E), but not for 24 h (Fig. 2F), suggesting that activation of PKC blocks thapsigargin-induced morphological differentiation. In this experiment, the cells were bathed in high-glucose DMEM during the 1 min or 24 h of PMA pretreatment, then were moved to LB with or without thapsigargin for a further 3 h. Interestingly, cells pretreated with PMA for 24 h, then placed in LB without thapsigargin for 3 h also underwent morphological differentiation (Fig. 2C), an effect not seen in cells pretreated with PMA for 1 min (Fig. 2B). Apart from the lack of amino acids and vitamins in LB, the main difference in the composition of high-glucose DMEM and LB is the glucose concentration, which is 25 mm in the high-glucose DMEM and 5 mm in the LB. To determine whether low glucose or lack of amino acids and vitamins in LB was the crucial factor accelerating the morphological changes, we performed the same experiment using normal DMEM (5.5 mm glucose) or high-glucose LB (25 mm glucose) as the bathing solution after PMA treatment. After PMA treatment, cells placed in high-glucose LB for 3 h showed the same morphological differentiation as those placed in LB, whereas cells placed in normal DMEM did not (data not shown). Thus, the lack of amino acids and vitamins, rather than low glucose, in LB was the crucial factor causing the morphological changes.

Enhancement of functional differentiated characteristics in NG108-15 cells after PMA pretreatment and subsequent LB exposure

We then addressed whether these morphologically changed cells induced by incubating NG108-15 cells for 24 h with PMA, then in LB for 3 h, exhibited characteristics of functional differentiated cells and the underlying mechanism. First, we examined whether voltagesensitive Ca2+ channel activity was increased, as higher voltagesensitive ion channel activity is detected after dbcAMP-induced differentiation of NG108-15 cells (Kasai & Neher, 1992; Hu & Shi, 1997; Kawaguchi et al., 2007). We monitored the increase in [Ca2+]i, indicated by the fura-2 340 ⁄ 380-nm fluorescence ratio, in response to challenge with 75 mm KCl, which causes membrane depolarization; sample traces are shown in Fig. 3A. In cells treated for 24 h with DMSO (trace a) (DMSO 24 h) or PMA (trace b) (PMA 24 h) in highglucose DMEM, the [Ca2+]i increased from a basal level of 1.09 ± 0.11 (n = 178 cells) to respective peak levels of 1.41 ± 0.12 (n = 96 cells) or 1.40 ± 0.09 (n = 82 cells). In the DMSO-pretreated cells, the amplitude of the [Ca2+]i increase was not altered after placing the cells in either high-glucose DMEM (DMSO 24 h + H Glu DMEM 3 h) or LB for 3 h (DMSO 24 h + LB 3 h) (traces c and e, respectively), whereas, in the PMA-pretreated cells, no change was seen on placing the cells in high-glucose DMEM for 3 h (trace d) (PMA 24 h + H Glu DMEM 3 h), but a significant increase was seen when they were placed in LB for 3 h (trace f) (PMA 24 h + LB 3 h), the peak level being about 2.08 ± 0.17 (n = 86 cells). Statistical data for the increased [Ca2+]i induced by 75 mm KCl, subtracting the basal level from the peak level, in cells with different treatments from five experiments is shown in Fig. 3C (left panel) (one-way anova, F5,24 = 162.2, P < 0.0001, followed by Newman–Keuls post hoc test, P < 0.001, PMA 24 h + LB 3 h vs. other all groups). The activity of the voltage-sensitive Ca2+ channels was also measured electrophysiologically by measuring the Ca2+ current as an inward current during depolarization. The current–voltage plot, shown in Fig. 3B, shows that the voltage-sensitive Ca2+ current measured in cells placed for 3 h in LB was greater after PMA pretreatment than after DMSO pretreatment. The maximal current in both DMSO- and PMA-pretreated cells was seen when the membrane potential was depolarized from )70 to 0 mV; representative current traces are shown in the inset. Thus, the activity of the voltage-sensitive Ca2+ channel measured by either the [Ca2+]i change or electrophysiologically showed a marked increase following PMA pretreatment and subsequent LB exposure. We next measured the number of neurites (centre panel in Fig. 3C) and varicosities (right panel in Fig. 3C) seen 3 h after placing the cells in LB or high-glucose DMEM following DMSO or PMA pretreatment. After PMA treatment alone, the number of neurites increased slightly compared with that in control cells and was unaltered after placing the cells in high-glucose DMEM for 3 h, but was increased in cells placed in LB for 3 h. In contrast, no change in the number of neurites was seen when DMSO-pretreated cells were placed in either high-glucose DMEM or LB for 3 h (centre panel in Fig. 3C, one-way anova, F5,54 = 394.4, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, PMA 24 h vs. DMSO 24 h, DMSO 24 h + H Glu DMEM 3 h and DMSO 24 h + LB 3 h; P < 0.001, PMA 24 h + H Glu DMEM 3 h vs. DMSO 24 h, DMSO 24 h + H Glu DMEM 3 h and DMSO 24 h + LB 3 h; P < 0.001, PMA 24 h + LB 3 h vs. other all groups, n = 10 for all groups). Similar results were observed when the number of varicosities was measured (right panel in Fig. 3C, oneway anova, F5,54 = 89.78, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, PMA 24 h vs. DMSO 24 h, DMSO 24 h + H Glu DMEM 3 h and DMSO 24 h + LB 3 h; P < 0.01, PMA 24 h + H Glu DMEM 3 h vs. DMSO 24 h, DMSO 24 h + H Glu DMEM 3 h and DMSO 24 h + LB 3 h; P < 0.001, PMA 24 h + LB 3 h vs. other all groups, n = 10 for all groups). Thus, exposure to LB accelerates PKC depletion-induced functional differentiation, as voltage-sensitive Ca2+ channel activity, sprouting and elongation of neurites, and formation of varicosities were significantly increased.

Modification of actin polymerization was also detected in parallel with the morphological differentiation and enhanced Ca2+ channel activity. As shown in Fig. 4A, in cells treated with DMSO (panel a) or PMA (panel b) alone, F-actin (stained with rhodamine phalloidin) was readily detectable in lamellipodia and filopodia around the periphery of the cells, while typical prominent actin stress fibres were not visible. When PMA-pretreated cells were placed in LB for 3 h, F-actin was condensed into a clearly discernible dense cortex ring close to the plasma membrane (panel f), an effect not seen when the same cells were placed in high-glucose DMEM (panel d). In contrast, when DMSO-pretreated cells were placed in either high-glucose DMEM or LB, the F-actin distribution within the cell was unaltered, being undistinguishable from that of control cells (compare panels c and e with panel a). In dbcAMP-induced differentiated NG108-15 cells, polymerization of actin is increased and stabilized by phosphorylation of cofilin, which is catalysed by LIM kinase 1 via the CREB phosphorylation pathway (Tojima et al., 2003). However, in our study, no significant change in phospho-CREB or LIM kinase 1 expression was seen in DMSO- or PMA-pretreated cells placed in LB or high-glucose DMEM for 3 h (Fig. 4B).

PMA pretreatment and subsequent LB exposure does not result in apoptosis or autophagy

As differentiation is normally associated with cessation of cell growth, we determined whether LB exposure inhibited cell growth and caused cell death in PMA-pretreated cells. PMA treatment alone or followed by 3 h incubation in LB did not affect cell growth, as cell numbers were indistinguishable in DMSO- or PMA-pretreated cells placed in F5,24 = 0.7462, P = 0.5968, n = 5 for all groups). We then measured apoptotic cells using DNA condensation. As shown in Fig. 5B, 5 or 6% of the cells were apoptotic after 24 h treatment with DMSO or PMA, respectively, and the percentage of apoptotic cells was almost unchanged when the cells were subsequently placed in high-glucose DMEM for 3 h (4 or 7% for DMSO- or PMA-pretreated cells, respectively) and was increased, but to about the same level (10 or 12%), when DMSO- or PMA-pretreated cells were placed in LB for 3 h (Fig. 5B, one-way anova, F5,24 = 124.0, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, DMSO 24 h + LB 3 h vs. DMSO 24 h, DMSO 24 h + H Glu DMEM 3 h, PMA 24 h and PMA 24 h + H Glu DMEM 3 h; P < 0.001, PMA 24 h + LB 3 h vs. DMSO 24 h, DMSO 24 h + H Glu DMEM 3 h, PMA 24 h and PMA 24 h + H Glu DMEM 3 h, n = 5 for all groups). Thus, the level of apoptosis in DMSO- or PMA-treated cells was indistinguishable. DMSO 24 h + H Glu DMEM 3 h, PMA 24 h and PMA 24 h + H Glu DMEM 3 h. (C) Cells were either incubated for 24 h with 300 nm PMA in highglucose DMEM, then placed in high-glucose DMEM (H Glu DMEM) or LB for 3 h (a and b, respectively) or were incubated for 24 h with 1 lm rapamycin (Rapa) or 10 mm 3-MA in high-glucose DMEM (c and d, respectively) before being stained with MDC as described in the Materials and methods. The fluorescence images shown are representative of those obtained in five independent experiments. Scale bar = 20 lm.

Deprivation of extracellular nutrients causes degradation of organelles in the cytoplasm by autophagy to provide the amino acids needed (Klionsky & Emr, 2000). Because morphological differentiation did not occur until the cells had been bathed in LB, we then evaluated the relationship between autophagy and the development of differentiation by comparing the autophagic activity of PMA-pretreated cells placed in high-glucose DMEM or LB for 3 h. Autophagosome formation was measured by staining using MDC, which accumulates in acidic organelles enriched in lipids (Biederbick et al., 1995) and leads to a punctate staining pattern when autophagy is stimulated. As shown in Fig. 5C, a similar number of punctate structures was seen in cells placed in high-glucose DMEM or LB for 3 h after PMA pretreatment (panel a and b, respectively), suggesting that the morphological change and functional differentiation were not attributable to autophagosome accumulation. In the positive and negative controls, NG108-15 cells treated with 200 nm rapamycin for 24 h to stimulate autophagy by inhibiting the mammalian target of rapamycin (mTOR) showed enhanced autophagosome accumulation (panel c), while cells treated with 10 mm 3-MA, an inhibitor of autophagy, displayed decreased numbers of autophagosomes (panel d).

PMA pretreatment and subsequent LB exposure result in inhibition of Akt and p44 ⁄ p42 ERK and activation of p38 MAPK

MAPK and Akt are downstream effectors of many growth factors and play a key role in cellular differentiation and proliferation. Enhanced phosphorylation of p44 ⁄ p42 ERK is seen in dbcAMP-treated NG10815 cells (Chin et al., 2010). To examine the cellular components involved in the accelerated differentiation seen in PMA-pretreated NG108-15 cells placed in LB, we measured the degree of activation of two MAPK members (p44 ⁄ p42 ERK and p38 MAPK) and Akt using antibodies against phospho-p44 ⁄ p42 ERK, phospho-p38 MAPK or phospho-Akt. As shown in Fig. 6A, phosphorylation of p44 ⁄ p42 ERK was decreased after 24 h treatment of the cells with PMA (compare lane 2 with 1) and the ratio of phosphorylated p44 ⁄ p42 ERK in DMSO-pretreated cells to that in PMA-treated cells was unchanged after placing the cells in high-glucose DMEM or LB for 3 h (compare lanes 3 and 4 and lanes 5 and 6 with lanes 1 and 2). As for Akt, phosphorylation increased to 150% of DMSO-treated control levels following PMA treatment for 24 h (compare lane 2 with 1) and this increased phosphorylation was maintained when the cells were placed in high-glucose DMEM for 3 h (lane 4), but decreased to 50% of DMSO-treated control levels in LB (lane 6). In DMSO-treated control cells, Akt phosphorylation was unchanged after placing the cells in high-glucose DMEM (lane 3), but decreased to the same level as in PMA-treated cells when the cells were placed in LB (lane 5). Phosphorylation of p38 MAPK was barely detectable in cells treated with DMSO or PMA alone (lanes 1 and 2, respectively) or when these cells were placed in high-glucose DMEM for 3 h (lanes 3 and 4, respectively), but was markedly increased when DMSO- or PMApretreated cells were placed in LB (lanes 5 and 6). Figure 6B shows the densitometric data for levels of phospho-p44 ⁄ p42 ERK, phosphoAkt and phospho-p38 MAPK relative to those in control cells treated with DMSO for 24 h from seven independent experiments. For ERK phosphorylation, one-way anova, F5,36 = 52.74, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, DMSO 24 h vs. PMA 24 h; DMSO 24 h + H Glu DMEM 3 h vs. PMA 24 h + H Glu DMEM 3 h; DMSO 24 h + LB 3 h vs. PMA 24 h + LB 3 h, n = 7 for all groups. For Akt phosphorylation, one-way anova, F5,36 = 189.8, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, DMSO 24 h vs. PMA 24 h; DMSO 24 h + H Glu DMEM 3 h vs. PMA 24 h + H Glu DMEM 3 h; DMSO 24 h + LB 3 h vs. DMSO 24 h and DMSO 24 h + H Glu DMEM 3 h; PMA 24 h + LB 3 h vs. PMA 24 h and PMA 24 h + H Glu DMEM 3 h, n = 7 for all groups. For p38 MAPK phosphorylation, one-way anova, F5,36 = 71.26, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, DMSO 24 h + LB 3 h vs. DMSO 24 h, PMA 24 h, DMSO 24 h + H Glu DMEM 3 h and PMA 24 h + H Glu DMEM 3 h; PMA 24 h + LB 3 h vs. DMSO 24 h, PMA 24 h, DMSO 24 h + H Glu DMEM 3 h and PMA 24 h + H Glu DMEM 3 h, n = 7 for all groups. These results show that decreased phosphorylation of p44 ⁄ p42 ERK and Akt and increased phosphorylation of p38 MAPK coincided with differentiation of NG108-15 cells.

Pharmacological inhibition of p44 ⁄ p42 ERK and Akt facilitates differentiation of NG108-15 cells, while inhibition of p38 MAPK antagonizes it

We then addressed the question of whether pharmacological inhibition of p44 ⁄ p42 ERK by PD098059 or of Akt by LY294002 during the LB incubation step facilitated the differentiation of NG108-15 cells and whether inhibition of p38 MAPK by SB203580 inhibited it. The effect of these three inhibitors on cell morphology is shown in Fig. 7A. After 24 h of PMA treatment, inclusion of PD098059 in the LB (panel d) had no effect on cell morphology compared with cells in the absence of inhibitor (panel b), i.e. cell rounding and neurite outgrowth were still seen, whereas incorporation of SB203580 into the LB inhibited the morphological differentiation (compare panel f with panel b). With DMSO-pretreated cells, incorporation of either PD098059 or SB203580 into LB had no effect on cell morphology (compare panels c and e with a). When LY294002 was incorporated into the LB after DMSO or PMA pretreatment, morphological differentiation was still seen in cells treated with PMA (compare panel h with b), but was also seen in cells treated with DMSO (panel g).

The effect of incorporation of PD098059, SB203580 or LY294002 into the LB after DMSO and PMA pretreatment on p44 ⁄ p42 ERK, Akt and p38 MAPK phosphorylation is shown in Fig. 7B. Using SB203580 to inhibit p38 MAPK phosphorylation, not only was p38 MAPK phosphorylation inhibited, but p44 ⁄ p42 ERK phosphorylation was increased, while Akt phosphorylation was unaffected (compare lanes 2 and 1 in DMSO-pretreated cells and lanes 6 and 5 in PMApretreated cells). When LY294002 was incorporated into the LB, Akt phosphorylation was inhibited in both DMSO-pretreated cells (compare lanes 3 and 1) and PMA-pretreated cells (compare lanes 7 and 5), as was p44 ⁄ p42 ERK phosphorylation, while p38 MAPK phosphorylation was significantly increased in both sets of pretreated cells (lane 3 or 7 for DMSO- or PMA-treated cells, respectively). Incorporation of PD098059 into the LB inhibited p44 ⁄ p42 ERK phosphorylation in both DMSO-pretreated cells (compare lanes 4 and 1) and PMApretreated cells (compare lanes 8 and 5); however, p38 MAPK phosphorylation in both sets of cells (compare lanes 4 and 1 and lanes 8 and 5) was enhanced, while Akt phosphorylation was enhanced in DMSO-pretreated cells (compare lanes 4 and 1), but unchanged in PMA-pretreated cells (compare lanes 8 and 5). The statistical quantitative data from six experiments for the phosphorylation of p44 ⁄ p42 ERK, Akt, and p38 MAPK analyzed by densitometry are shown in Fig. 7C. In DMSO-pretreated cells, for ERK phosphorylation, one-way anova, F3,20 = 239.1, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, DMSO 24 h + LB 3 h (open bar) vs. DMSO 24 h + LB plus LY294002 3 h and DMSO 24 h + LB plus PD98059 3 h; P < 0.01, DMSO 24 h + LB 3 h vs. DMSO 24 h + LB plus SB203580. For Akt phosphorylation, one-way anova, F3,20 = 79.10, P < 0.0001, followed by Newman–Keuls posthoc test, P < 0.001, DMSO 24 h + LB 3 h (open bar) vs. DMSO 24 h + LB plus LY294002 3 h and DMSO 24 h + LB plus PD98059 3 h. For p38 MAPK phosphorylation, one-way anova, F3,20 = 171.1, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, DMSO 24 h + LB 3 h (open bar) vs. DMSO 24 h + LB plus SB203580, DMSO 24 h + LB plus LY294002 3 h and DMSO 24 h + LB plus PD98059 3 h, n = 6 for all groups. In PMA-pretreated cells, for ERK phosphorylation, one-way anova, F3,20 = 264.5, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, PMA 24 h + LB 3 h (open bar) vs. PMA 24 h + LB plus SB203580, PMA 24 h + LB plus LY294002 3 h and PMA 24 h + LB plus PD98059 3 h. For Akt phosphorylation, one-way anova, F3,20 = 10.75, P = 0002, followed by Newman–Keuls post-hoc test, P < 0.01, PMA 24 h + LB 3 h (open bar) vs. PMA 24 h + LB plus LY294002. For p38 MAPK phosphorylation, one-way anova, F3,20 = 324.6, P < 0.0001, followed by Newman–Keuls post-hoc test, P < 0.001, PMA 24 h + LB 3 h (open bar) vs. PMA 24 h + LB plus SB203580, PMA 24 h + LB plus LY294002 3 h and PMA 24 h + LB plus PD98059 3 h, n = 6 for all groups. Together, these results confirmed that a combined decrease in phosphorylation of both p44 ⁄ p42 ERK and Akt and an increase in p38 MAPK phosphorylation, observed in the presence of LY294002 in both DMSO- and PMA-treated cells and in the presence of PD098059 in PMA-treated cells, facilitates the morphological differentiation of NG108-15 cells.

Discussion

We have demonstrated a negative correlation between PKC activity and induction of differentiation. Both PKC inhibition for 24 h (cocktail of Go6983 and Ro318220) and PKC downregulation for 48 h (chronic PMA treatment) induced differentiation. This means that PKC was almost immediately inhibited by the inhibitor cocktail, while the action of PMA was more complex, consisting of PKC activation during the first few minutes of treatment, followed by its gradual downregulation. Furthermore, the expression of the atypical isozyme

PKCf is not affected by PMA, but is sensitive to the Go6983 ⁄ Ro318220 cocktail, while the expression of the novel PMA receptor PKCl (also known as PKD) and chimaerin is sensitive to PMA, but not affected by the inhibitor cocktail (Gschwendt et al., 1996; Ron & Kazanietz, 1999). These proteins may participate in NG108-15 cell differentiation and this may explain why 24 h incubation with PMA did not induce differentiation.

Cytosolic levels of all PKC isoforms were markedly reduced after 24 h treatment with PMA, but membrane PKCe was still detectable (Fig. 1B). PKCe is involved in neuronal differentiation and neurite elongation (Shirai et al., 2008). Following dbcAMP-induced differentiation, an increase in cytosolic PKCe immunoreactivity is observed, while, in non-differentiated cells, PKCe is present at a higher level in the particulate compartment (Battaini et al., 1994). This may explain why the downregulation of PKC by treatment with PMA for 24 h was not sufficient to induce morphological differentiation, as PKCe levels in the particulate fraction were higher than in the cytosol (Fig. 1B), as in non-differentiated cells (Battaini et al., 1994), but did provide an environment suitable for maintenance of differentiated cells, as thapsigargin-induced neuritogenesis was still seen after 24 h of PMA treatment (Fig. 2). In Go6983 ⁄ Ro318220-treated cells, PKCe was present at a higher level in the cytosolic fraction at 24 h, when differentiation was seen (Fig. 1A and C). Consistent with the notion of a negative correlation between PKC activity and induction of differentiation, inhibition of differentiation would be expected if PKC was activated and, indeed, PKC activation by treatment with PMA for 1 min was found to block thapsigargin-induced neuritogenesis (Fig. 2).

Simultaneous application of dbcAMP and phorbol ester synergistically increases the differentiation of NG108-15 cells, but at least 2 days of exposure is required for neurite extension (Tohda et al., 1991). In this previous study, the concentration of 12-tetradecanoylphorbol 13-acetate (TPA) used was 100 nm and, after 24 h of TPA exposure, PKC activity was not reduced and no neurite elongation or outgrowth was induced. It has also been shown that, after 17 h exposure of NG108-15 cells to 1 lm TPA, cytosolic and membrane PKCa, PKCd and PKCe are downregulated, while PKCf is unaltered (Chen et al., 1995). In the present study, the PMA concentration used was 300 nm. After 24 h exposure, the PKC activity was still about 30% of the original levels. Consistent with the above results, in our PMA-treated cells, levels of PKCa, PKCd and PKCe were significantly reduced, while PKCf levels were unchanged (Fig. 1B). Thus, the phorbol ester concentration and treatment time are critical for downregulation of PKCs.

The fact that Akt inhibition and p38 MAPK activation occurred simultaneously when both DMSO- or PMA-pretreated cells were placed in LB for 3 h (Fig. 6B) might suggest that a mutual antagonistic relationship may exist between p38 MAPK and Akt activity. However, this possibility was ruled out by the fact that inhibition of p38 MAPK by SB203580 increased phosphorylation of p44 ⁄ p42 ERK, but not that of Akt, suggesting a reciprocal relationship between p38 MAPK and p44 ⁄ p42 ERK. This was confirmed by the use of PD098059, which inhibited the phosphorylation of p44 ⁄ p42 ERK and increased that of p38 MAPK (Fig. 7B). Our data also suggest that there is an interaction between the ERK and Akt pathways, as p44 ⁄ p42 ERK phosphorylation was also inhibited by LY294002, while inhibition of p44 ⁄ p42 ERK phosphorylation by PD098059 increased Akt phosphorylation (Fig. 7B). This may explain why LY294002, but not PD098059, induced morphological differentiation in cells placed in LB for 3 h without PMA pretreatment (Fig. 7A, panel g vs. panel c). In both cases, p44 ⁄ p42 ERK phosphorylation was inhibited and p38 MAPK phosphorylation enhanced due to inhibition of p44 ⁄ p42 ERK, but LY294002, which inhibits Akt, induced differentiation, while PD098059, which activates Akt, did not (Fig. 7B, lanes 3 and 4), further indicating that increased phosphorylation of p38 MAPK is required, but not sufficient, to induce differentiation. Our finding that PD98059 enhanced Akt phosphorylation in DMSO-treated cells is consistent with a recent report showing that, in DU145 and PC3 prostate cancer cells, inhibition of the ERK pathway by PD98059 augments epidermal growth factor-induced Akt phosphorylation, as does U0126 (Gan et al., 2010).

NG108-15 cells do not possess well-defined ‘stress fibres’, and, in these cells, cytochalasin B disrupts actin microfilament assembly and blocks thrombin-induced cell rounding (Jalink & Moolenaar, 1992), suggesting that the shape change is mediated by the cortical actin cytoskeleton. In support of this, we observed that, after placing the cells in LB following PMA pretreatment, no typical prominent actin stress fibres were visible, F-actin condensed into a dense cortical ring close to the plasma membrane and the cells were rounded (Fig. 4). NG108-15 cell differentiation caused by dbcAMP leads to increased LIM kinase 1 expression via the CREB phosphorylation pathway (Tojima et al., 2003; Tojima & Ito, 2004). However, in our study, levels of phospho-CREB and LIM kinase 1 were not significantly increased after differentiation (Fig. 4). Furthermore, in NG108-15 cells, increased autophagy caused by rapamycin via inhibition of mTOR promotes dbcAMP-induced differentiation, while phosphorylation of the downstream substrate of mTOR, p70S6K, is hardly detectable (Chin et al., 2010). In the current study, the accelerated differentiation was not attributable to autophagy (Fig. 5C). However, Ro318220 is also known to inhibit p70S6K (Alessi, 1997) and may conceivably affect other protein kinases that favour differentiation.

The initial cell density can influence the time required for cell death or differentiation. In the present study, for a better resolution and more accurate measurement, the seeding cell number was different in different types of experiments, ranging from 2.5 · 104 cells per coverslip for morphology and neurite outgrowth studies to 2.5 · 106 cells per 100-mm dish for immunoblotting, the actual density being 0.55 · 104 and 3.18 · 104 ⁄ cm2, respectively. However, in a given type of experiment, the plating density was the same for the Go6983 ⁄ Ro318220- or PMA-treated cells and serum was withdrawn to halt cell growth. Thus, the influence of different cell densities on Go6983 ⁄ Ro318220- or PMA-treated cells should be negligible.

In conclusion, PKC downregulation by PMA promotes differentiation and this effect is accelerated by LB exposure. The accelerated differentiation is not due to autophagy or growth arrest or to increased CREB phosphorylation, but coincides with combined activation of p38 MAPK and inhibition of ERK and Akt. Thus, differentiation of NG108-15 cells can be induced by different means and the corresponding mechanisms are different.

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Inhibition of protein kinase C promotes differentiation of neuroblastoma · glioma NG108-15 hybrid cells

Chia-Yu Chang,1,2 Kuo-Hsing Ma,3 Jehng-Kang Wang,4 Ya-Ling Tung4 and Sheau-Huei Chueh4

Abstract

Keywords: Akt, p38 mitogen-activated protein kinase, p44 ⁄ p42 extracellular signal-regulated kinase, phorbol ester

Introduction

Neuronal differentiation is associated with changes in the activity, cellular distribution and expression of specific PKC isozymes. Neuritogenesis is an important process during neuronal differentiation. Using the protein kinase inhibitor H7, a correlation has been suggested between PKC inhibition and induction of neuritogenesis (Minana et al., 1990; Ono et al., 1991; Shea et al., 1992; Rocchi et al., 1995; Singleton et al., 2000), whereas neuritogenesis induced by many agonists is dependent on PKC activity (Cambray-Deakin et al., 1990; Lazarovici et al., 1998; Mahoney et al., 2003; Das et al., 2004; Miloso et al., 2004; Canals et al., 2005). Thus, PKCs are involved in neuronal differentiation, but their precise role and the downstream consequences of their activation or inhibition remain unclear (Violin & Newton, 2003).

Materials and methods

Materials

Cell culture

Treatment of cells

Morphological changes, neurite outgrowth and varicosity assay

Measurement of cell numbers and apoptotic cells

Measurement of the intracellular Ca2+ concentration

Electrophysiological recording

F-actin determination

Immunoblotting

Autophagy detection

PKC activity assay

Statistical analysis

Results

Correlation between reduction in PKC activity and induction of differentiation

LB exposure accelerated PKC downregulation-induced morphological differentiation

Enhancement of functional differentiated characteristics in NG108-15 cells after PMA pretreatment and subsequent LB exposure

PMA pretreatment and subsequent LB exposure does not result in apoptosis or autophagy

PMA pretreatment and subsequent LB exposure result in inhibition of Akt and p44 ⁄ p42 ERK and activation of p38 MAPK

Pharmacological inhibition of p44 ⁄ p42 ERK and Akt facilitates differentiation of NG108-15 cells, while inhibition of p38 MAPK antagonizes it

Discussion

References

Alessi, D.R. (1997) The protein kinase C inhibitors Ro 318220 and GF 109203X are equally potent inhibitors of MAPKAP kinase – 1b (Rsk-2) and p70 S6 kinase. FEBS Lett., 402, 121–123.

Biederbick, A., Kern, H.F. & Elsasser, H.P. (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur. J. Cell Biol., 66, 3–14.

Cambray-Deakin, M.A., Adu, J. & Burgoyne, R.D. (1990) Neuritogenesis in cerebellar granule cells in vitro: a role for protein kinase C. Brain Res. Dev. Brain Res., 53, 40–46.

Chin, T.-Y., Hwang, H.-M. & Chueh, S.-H. (2002) Distinct effects of different calcium-mobilizing agents on cell death in NG108-15 neuroblastoma · glioma cells. Mol. Pharmacol., 61, 486–494.

Chueh, S.-H., Song, S.-L. & Liu, T.-Y. (1995) Heterologous desensitization of opioid-stimulated Ca2+ increase by bradykinin or ATP in NG108-15 cells. J. Biol. Chem., 270, 16630–16637.

Greenland, K.J. & Mukhopadhyay, A.K. (2004) Selective activation of protein kinase C isoforms by angiotensin II in neuroblastoma · glioma cells. Mol. Cell. Endocrinol., 213, 181–191.