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Differentiation of bipolar CG-4 line oligodendrocytes is associated with regulation of CREB, MAP kinase and PKC signalling pathways
Neuroscience Research 41 (2001) 217– 226
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Differentiation of bipolar CG-4 line oligodendrocytes is associated with regulation of CREB, MAP kinase and PKC signalling pathways Shaun McNulty a,*, Michael Crouch b, Darren Smart a, Martin Rumsby c a
GlaxoSmithKline Pharmaceuticals New Frontiers Science Park, 3rd A6enue, Harlow, CM19 5AW, Essex, UK b Di6ision of Neuroscience, National Uni6ersity of Australia, Acton, Canbera, Australia c Department of Biology, Uni6ersity of York, York, YO105YW, UK Received 22 May 2001; accepted 25 July 2001
Abstract Undifferentiated bipolar CG-4 cell line oligodendrocytes provide a model system for the O-2A progenitor cell from which oligodendrocytes are derived both in vivo and in vitro. The exchange of neuroblastoma conditioned basal media for basal media causes differentiation of undifferentiated bipolar CG-4 cells into multipolar oligodendrocyte-like cells whilst replacement with basal media containing 20% foetal bovine serum favours the formation of type-2 astrocyte-like cells. Here, we demonstrate that activation of these differentiation pathways correlates with distinct changes both in cell metabolism and in signal transduction. Exchange of neuroblastoma conditioned media for basal media correlates with stimulation of basal metabolic activity, reduced phosphorylation of p44/42 MAP kinase and reduced phophorylation of the transcription factor CREB. In contrast, differentiation with basal medium containing 20% foetal bovine serum (FBS), into type 2 astrocyte-like cells, correlates with reduction in basal metabolic activity, increased phosphorylation of p44/42 MAP kinase and increased phophorylation of the transcription factor CREB. Inhibition of protein kinase C blocked both the metabolic and morphological changes associated with differentiation towards mature multipolar oligodendrocyte-like cells. Inhibition of PKA and MEK did not effect metabolic activity. The rapid return of neuroblastoma conditioned basal media to cells treated with basal media, increased phosphorylation of CREB and MAP kinase. These results demonstrate that protein kinase C and p44/42 MAP kinase signalling pathways are modulated during bipolar CG-4 cell differentiation and demonstrate that the transcription factor CREB may play a pivotal role in differentiation along oligodendrocyte-or astrocyte-lineages. © 2001 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. Keywords: CG-4; CG-4 line oligodendrocytes; Type 2-astrocytes; CREB; Signal transduction; MAP kinase; Calcium; Phosphorylation; Kinase; Differentiation; Protein kinase C
1. Introduction
Abbre6iations: BSA, bovine serum albumin; CaM kinase II, calcium/calmodulin dependent kinase II; CRE, calcium/cyclic AMP response-element; CREB, calcium/cyclic AMP response-element binding-protein; cyclic AMP, cyclic adenosine monophosphate; ir-, immunoreactivity; FCS, Foetal calf serum; MAPK, Mitogen activated protein kinase; PBS, Phosphate buffered saline; pCREB, Phospho Ser133 CREB; PKA, Protein kinase A; PKC, Protein kinase C. * Corresponding author. Tel.: + 44-1279-622450; fax: + 44-1279622230. E-mail address: shaun – [email protected] (S. McNulty).
Oligodendrocytes are responsible for the formation and maintenance of myelin around nerve axons in the central nervous system and are derived from bipolar oligodendrocyte-type 2-astrocyte (O-2A) progenitor cells whose division is regulated by growth factors including PDGF and IGF1 (Noble et al., 1988; McMorris and Dubois-Dalcq, 1988). Bipolar O-2A progenitor cells develop at discrete ventral sites in cord and brain and then proliferate and migrate throughout the CNS before undergoing differentiation along a defined lineage to a mature multipolar oligodendrocyte which
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expresses characteristic phenotypic markers and synthesises myelin. Analysis of the morphology and phenotype of oligodendroglia has revealed distinct stages of development ranging from pre-O-2A progenitor cells and pro-oligodendrocytes to fully mature myelin-producing oligodendrocytes (Armstrong, 1998). However, the cellular mechanisms controlling progenitor oligodendrocyte differentiation are not fully understood. Undifferentiated bipolar CG-4 cells retain many of the properties of O-2A progenitor glial cells (Louis et al., 1992). When transplanted in vivo, these undifferentiated bipolar CG-4 cells can migrate to areas of damage and re-myelinate affected neurons (Tontsch et al., 1994; Franklin et al., 1995, 1996). Bipolar CG-4 cells maintain morphology and proliferate when cultured in a defined medium containing growth factors from B104 neuroblastoma cell conditioned medium. Investigations undertaken in vitro have shown that undifferentiated bipolar CG-4 cells are able to undergo differentiation along two distinct pathways leading either into multipolar oligodendrocyte-like, or into type 2-astrocyte-like cells (Louis et al., 1992) depending on the differentiation medium. Undifferentiated bipolar CG-4 cells are maintained in basal media containing 30% B104 neuroblastoma conditioned media (neuroblastoma conditioned basal media, Louis et al., 1992). Exchange of this media for basal media containing low (B 0.5%) serum concentrations causes differentiation into multipolar oligodendrocyte-like cells. Exchange of conditioned media for media containing 20% serum causes differentiation into type-2 astrocyte-like cells (Louis et al., 1992). Oligodendrocyte-like cells derived from bipolar CG-4 cells are sensitive to treatment with hydrogen peroxide, characteristic of mature primary cultures of oligodendrocytes (Bhat and Zhang 1999). A cationic substrate such as poly-L-lysine (Rumsby et al., 1998) or pleiotrophin (Rumsby et al., 1999) enables undifferentiated bipolar CG-4 cells to disperse, migrate and produce morphological features similar to those of the O2-A progenitor (Rumsby et al., 1998). These properties make the CG-4 line an ideal model system for studies of the signalling pathways necessary for differentiation of O-2A oligodendrocyte precursors. Regulation of oligodendrocyte gene expression is essential in order to control effectively the processes of differentiation and myelination. Increased cytosolic levels of second messengers including cyclic AMP and intracellular-free calcium ([Ca2 + ]i) lead to the activation of cellular kinases, including protein kinase A (PKA), protein kinase C (PKC) and Ca2 + /calmodulin dependent kinase II (Cam kinase II). These kinases control transcriptional events through phosphorylation of transcription factors including the calcium/cyclic AMP response-element binding-protein (CREB). Phosphorylation of CREB at Ser133 (pCREB) causes activation and pCREB is able to regulate the transcription of
genes containing the calcium/cyclic AMP response-element (CRE) including c-fos (Gonzalez and Montminy, 1989; Sheng et al., 1990, 1991). Activation of MAP kinase signalling pathways also increases phosphorylation of CREB through activation of a CREB kinase, RSK2 (Ginty et al., 1994; Xing et al., 1996). Recent observations (Sato-Bigbee et al., 1999; Johnson et al., 2000) have demonstrated that CREB is present in oligodendrocytes with peak expression of CREB preceding the peak of myelination. Phosphorylation of CREB was shown to be sensitive both to PKC and to MAP kinase signal transdution pathways (Sato-Bigbee et al., 1999). Separate studies have indicated that phosphorylation of CREB in oligodendrocytes may be mediated by PKCMAP kinase- dependent signal transduction pathways (Pende et al., 1997). Raible and McMorris (1989) have shown that cAMP accelerates the differentiation of precursor cells already committed to becoming oligodendrocytes. Such studies have led to the hypothesis that CREB may play an important role during oligodendrocyte differentiation. Activation of PKC with phorbol ester was shown (Avossa and Pfeiffer, 1993) to induce a transient reversion of O4+ GC- progenitor oligodendrocytes to a vimentiun+ A2B5 + O4- phenotype. In the continued presence of the phorbol ester TPA, these cells redeveloped a mature phenotype expressing GC and MBP markers. Asotra and Macklin (1994) noted that PKC modulates myelin gene expression in enriched oligodendrocyte cultures and is involved in the control of myelin basic protein gene expression by cAMP (Anderson and Miskimins, 1994). Recently, it has been reported that activation of protein kinase C (PKC) by phorbol esters may prevent the differentiation of primary O2-A progenitors into oligodendrocytes (Baron et al., 1998). However, prolonged treatment of cells with phorbol esters causes down regulation of PKC in a variety of cell types complicating the interpretation of results obtained by their use (Murasawa et al., 1998; Gailly, 1998). The Cytosensor microphysiometer (Molecular Devices, UK) measures changes in the extracellular acidification rate of cultures (Owicki et al., 1990). Physiological changes, such as receptor activation, alter rates of energy metabolism causing changes in the levels of acid metabolites, which are secreted from the cell in order to maintain the intracellular pH (Owicki et al., 1990). The microphysiometer is able to detect these minute changes in the excretion of acid metabolites (Owicki et al., 1994). The versatility of the microphysiometer is due to the fact that its output is independent of the signal transduction pathway employed by the receptor (Smart and Wood, 2000). These properties make the microphysiometer an excellent tool for investigating the signalling pathways utilised by CG-4 progenitor cells.
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The aims of the present study were to investigate how signal transduction pathways and metabolic activity alter when undifferentiated bipolar CG-4 cells are differentiated into multipolar oligodendrocyte- or into type 2 astrocyte-like cells. Phosphorylation of CREB and activation of MAP kinase were examined using specific antisera raised against synthetic phospho-peptides. Changes in [Ca2 + ]i were estimated by imaging cells loaded with the calcium-sensitive dye Fura 2-AM. The Cytosensor microphysiometer was utilised for studies of metabolic changes during differentiation.
37 °C inside the Cytosensor, and maintained by a flow (120 ml/min) of bicarbonate-free neuroblastoma conditioned basal media (pH 7.4). The flow was halted for 22s at the end of each 2 min pump cycle, and the rate of acidification (mV/s) measured for 15 s during that period. After a stable baseline acidification rate was achieved, the cells were perfused with bicarbonate-free basal media containing 0.1% foetal calf serum. In some experiments, the medium used contained either 10 mM chelerythrine chloride, 100 nM calphostin C, 10 mM tryphostin 23, 10 mM Ro 31-8220, 10 mM KN-93, 10 mM LY294002, 10 mM H-89 or 10 mM PD098059.
2. Materials and methods
2.3. Cytosensor analysis
2.1. Cell line preparation and growth conditions
Treatment effects were quantitatively determined as the change in the acidification rate from the baseline to the second data point after switching media and are expressed as mV/s or as a percentage of the paired control response, as appropriate. Conversion to percentage basal is inappropriate for signal transduction studies (Smart and Wood, 2000). Data are presented as mean9S.E. mean unless otherwise stated.
Undifferentiated bipolar CG-4 cells were maintained in neuroblastoma conditioned basal media. This consisted of basal media (DMEM containing insulin 5 mg/ml, transferrin 50 mg/ml, putrescene 100 mM, progesterone 20 nM, selenium 30 nM, biotin 40 nM, 1% penicillin streptomycin and BSA 1.25 mg/ml) supplemented with B104 conditioned media (30% final conditioned media concentration), as described previously (Rumsby et al., 1998). For measurements of intracellular free calcium concentration [Ca2 + ]i, undifferentiated bipolar CG-4 cells were seeded at a density of 1.5×105 onto sterile poly-L-lysine-coated 22 mm diameter coverslips contained in six-well plates and incubated overnight at 37 °C with 5% CO2 and 95% humidity to permit the cells to adhere. Cells were used after 48 h in culture. For Western blot analysis, cells were plated at a density of 2.5× 105 into six-well plates and incubated overnight at 37 °C with 5% CO2 and 95% humidity. Cells were used after 48 h in culture. For analysis of metabolic changes, undifferentiated bipolar CG-4 cells were seeded into poly-L-lysine-coated Cytosensor capsule cups at 0.25×106 cells/cup and cultured for a further 24 h. Differentiation of undifferentiated bipolar CG-4 cells into multipolar oligodendrocyte-like cells was undertaken by exchanging neuroblastoma conditioned basal media for basal media containing either 0 or 0.1% foetal calf serum (FCS). Differentiation of undifferentiated bipolar CG-4 cells into type-2 astrocyte-like cells was undertaken by exchanging neuroblastoma conditioned basal media for basal media supplemented with 20% FCS.
2.2. Cellular acidification measurements using the Cytosensor microphysiometer The Cytosensor microphysiometer was used to measure the extracellular acidification rate, as described previously (McConnell et al., 1992; Jordan et al., 1998). The seeded cups were placed in sensor chambers at
2.4. Western blot analysis of CREB phosphorylation and MAP kinase acti6ation Western blots to identify CREB-ir were performed using a validated, commercially available kit (New England Biolabs, USA) as described previously (McNulty et al., 1997, 1998). Undifferentiated bipolar CG-4 cells were maintained as described above. Neuroblastoma conditioned basal media was exchanged for the required differentiation media and cells maintained for the necessary experimental period at 37 °C/5% CO2. At the end of this period, media was aspirated and cells were washed once in phosphate-buffered saline (PBS) containing 1 mM NaF, prior to extraction in 200 ml of Novex sample buffer. Extracts were then sonicated for 5 s and centrifuged at 4000 rpm to remove debris. Samples (15 ml) were loaded onto the stacking gel lanes of 10% mini polyacrylamide gels (Novex) and proteins were separated using a Novex XCell II Mini-Cell electrophoresis system for 2 h at 100 V. Proteins were then transferred (25 V, for 2 h) to nitrocellulose membranes prior to detection. Membranes were incubated for 1 h at 20 °C in blocking solution (10 ml, TBS containing 5% dried milk, 0.1% Tween 20). After blocking, membranes were incubated overnight at 4 °C in primary antibody solution (10 ml total volume, pCREB 1:1000, total CREB 1:2000, pMAPkinase 1:1000 in TBS containing 5% bovine serum albumin (BSA), 0.1% Tween 20). The next day, antiserum was removed and blots were washed three times in TBS (10 ml) containing 0.1% Tween, prior to incubation with secondary antibody for 30 min at room temperature (10 ml, 1:1000
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HRP linked goat anti rabbit or 10 ml, 1:1000 HRP linked goat anti mouse (New England Biolabs, USA), in blocking solution). Blots were then rinsed three times for 5 min in TBS (10 ml) prior to development using a modified enhanced chemiluminescence detection system (Amersham).
2.5. Analysis of changes in intracellular free calcium concentration Coverslips containing undifferentiated bipolar CG-4 cells were prepared and maintained as described. Cultured cells (grown attached to 22 mm diameter coverslips) were loaded with Fura-2 (Grynkiewicz et al., 1985) by incubation for 1 h at 20 °C with neuroblastoma conditioned basal media containing Fura-2-AM (2 mM, Molecular Probes). This procedure enables cells to load with Fura-2-AM, which becomes hydrolysed to the free acid form inside the intact cell. After loading, coverslips were mounted into imaging chambers and perfused with neuroblastoma conditioned basal media to remove extracellular Fura-2AM. Measurements of changes in the [Ca2 + ]i in individual cells were made from fluorescence ratio values (excitations 340/380 nm, emission \510 nm) using a spectral Wizard monochromator, cooled integrating CCD camera and a dedicated suite of software (Merlin, Life Sciences Resources, Cambridge, UK). Data are expressed as ratio units (340/380).
rosine kinase inhibitor tryphostan 23 (10 mM) and the MAP kinase kinase inhibitor PD098059 (10 mM) were without effect (Fig. 1b). Treatment of undifferentiated bipolar CG-4 cells with chelerythrine chloride caused a dose dependent inhibition of the acidification response (Fig. 2). Treatment of undifferentiated bipolar CG-4 cells with a concentration of serum (20%) which causes differentiation into type 2 astrocyte-like cells caused an apparent biphasic acidification response. This response was typified as an initial-post exchange artefact response peak, followed by a decline in acidification rate below the initial peak and approaching the initial baseline levels of metabolic activity (Fig. 3).
3. Results
3.1. Effect of medium change that induces cell differentiation on metabolic acti6ity The undifferentiated bipolar CG-4 cells achieved a stable baseline acidification rate of 50– 180 mV/s within 60 min of the start of perfusion with neuroblastoma conditioned basal media. Changing to basal medium in order to differentiate cells towards the oligodendrocyte lineage caused an apparently biphasic acidification response, typified as an initial peak followed by a relatively stable plateau of increased metabolic activity (Fig. 1a). Examination of the raw data traces (data not shown) revealed that the initial peak was an artefact caused by the relative buffering capacities of the two media, whilst the plateau phase was due to changes in cellular metabolism. Three PKC inhibitors, chelerythrine chloride (10 mM), calphostin C (100 nM) and Ro31-8220 (10 mM), caused significant inhibition (40– 50%) of the acidification response (Fig. 1b). The PI-3 kinase inhibitor, LY294002 (10 mM), also caused a small ( 25%) but significant inhibition of the acidification response (Fig. 1b). The PKA inhibitor H-89 (10 mM), the ty-
Fig. 1. Representative Cytosensor traces for CG-4 cells undergoing differentiation in the absence ( ) or presence ( ) of 10 mM chelerythrine chloride (a). The arrow indicates the point at which the perfusion media was switched from neuroblastoma conditioned basal media to basal medium containing 0.1% serum to induce differentiation into multipolar oligodendrocyte-like cells. The effects of protein kinase inhibitors (10 mM for 10 min pre-exposure) on the acidification response of undifferentiated bipolar CG-4 cells switched from neuroblastoma conditioned basal media to basal media are shown (b). Data are mean 9S.E. mean, with the n value for each treatment given in parentheses above the relevant bar. * denotes significant (PB 0.05) inhibition compared with controls (mANOVA followed by Student’s t-test). Unless stated, experiments were repeated three times.
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Fig. 2. Pooled data showing the effect of chelerythrine chloride (10 mM for 10 min pre-exposure) on the acidification response of undifferentiated bipolar CG-4 cells switched from neuroblastoma conditioned basal media to basal media are shown. Data are mean 9 S.E. mean, where n =3. * denotes significant (P B0.05) inhibition compared with the acidification rate of control cultures.
3.2. Effect of medium change that induces cell differentiation on phosphorylation of CREB Experiments were undertaken to investigate whether differentiation of undifferentiated bipolar CG-4 cells using standard protocols into either multipolar oligodendrocyte-like cells or type-2 astrocyte-like cells resulted in modulation of the phosphorylation of CREB. Western blot analysis for total CREB-ir or pCREB-ir revealed a single band of immunoreactivity (ir) of a molecular weight of 44 KDa (Fig. 4). Control levels of pCREB-ir were low but treatment with basal media containing serum (20%) causing differentiation along a type-2 astrocyte pathway, strongly increased immunoreactivity (Fig. 4a). In contrast, either the com-
Fig. 3. Representative Cytosensor traces for undifferentiated bipolar CG-4 cells exposed to 0 ( ), 0.5% ( ) or 20% () serum. The arrow indicates the point at which the perfusion was changed from neuroblastoma conditioned basal media into basal medium containing serum at the stated concentration. This trace is typical of n = 3.
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plete removal of growth factors, or including serum at a concentration of 0.1% to induce differentiation to multipolar oligodendrocyte-like cells caused a reduction in pCREB-ir levels below those observed in control non-differentiated cultures. The identity of this band was confirmed in blots probed using antiserum that recognises both phosphorylated and non-phosphorylated forms of CREB (Fig. 4b). However, no systematic changes in immunoreactivity were evident between the experimental groups demonstrating that the observed changes in pCREB-ir can not be accounted for by changes in the total level of CREB protein. Pre-treatment with chelerythrine chloride (10 mM), for a period of 10 min prior to and during differentiation into oligodendrocyte-like cells was without significant effect on pCREB-ir levels (data not shown). The return of neuroblastoma conditioned basal media to CG-4 cultures during the early phase of differentiation into multipolar oligodendrocyte-like cells is known to rescue progenitor like morphology and characteristics. Experiments were undertaken to investigate the effects of the return of neuroblastoma conditioned basal media to cells starved of growth factors for a period of 30 min (Fig. 4c, d). CG-4 cultures undergoing differentiation towards multipolar oligodendrocyte-like cells, demonstrated characteristic low levels of pCREBir (Fig. 4a, c) compared with basal levels. The return of neuroblastoma conditioned basal media increased strongly pCREB-ir over the levels observed either in differentiating cultures or cultures maintained in the presence of neuroblastoma conditioned basal media for the duration of the experiment. These treatments were without effect on levels of CREB-ir obtained from blots of the same experimental samples (Fig. 4d).
3.3. Effect of medium change that induces cell differentiation on phosphorylation of p44 /42 MAP kinase Experiments were undertaken to investigate whether differentiation using standard protocols into either oligodendrocyte- or astrocyte lineages resulted in modulation of the phosphorylation of MAP kinase p44/42 (Fig. 5a, b). Control levels of immunoreactivity were low, however, treatment with basal media containing serum (20%) strongly increased phospho p44/42 MAP kinase immunoreactivity (Fig. 5a). In contrast, the complete removal of growth factors or including serum at a concentration of 0.1% caused a reduction in phospho p44/42 MAP-ir levels below those observed in control non-differentiated cultures. Pre-treatment with chelerythrine chloride (10 mM), for a period of 10 min prior to and during differentiation into oligodendrocyte-like cells was without significant effect on p42/44 MAP kinase levels (data not shown). Experiments were undertaken to investigate the effects of the re-addition
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Fig. 4. Effect of neuroblastoma conditioned basal media (c), basal media containing 0.1% FBS (s1), basal media alone (n1), or basal media with 20% FBS added (s20) on the levels of pCREB-ir (a) and CREB-ir (b) in CG-4 cell cultures. Cells were treated for a period of 15 min prior to extraction. The effects of continual treatment of CG-4 cells with neuroblastoma conditioned basal media alone (C), or either neuroblastoma conditioned basal media (B104) or basal media (n1) following a 30 min treatment with basal media on pCREB-ir (c) and on CREB-ir (d). Following a differentiation period of 30 min, CG-4 cultures were treated for an additional period of 15 min prior to extraction. Each experimental group was run in duplicate and experiments were repeated three times with similar results. Equal concentrations of protein were loaded to each well.
of neuroblastoma conditioned basal media to cells starved of growth factors for a period of 30 min (Fig. 5b). As observed for pCREB-ir, the return of neuroblastoma conditioned basal media strongly increased phospho p42/44 MAP kinase immunoreactivity of cells compared with cultures maintained in basal media or cultures maintained in the presence of neuroblastoma conditioned basal media for the duration of the experiment.
3.4. Effect of medium change that induces cell differentiation on the concentration of intracellular-free calcium Studies were undertaken to investigate if changing neuroblastoma conditioned basal media to basal medium affected the [Ca2 + ]i within undifferentiated bipolar CG-4 cells. Experiments were conducted on four individual batches of undifferentiated bipolar CG-4 cells and undertaken simultaneously with Cytosensor Microphysiometry investigations of metabolic activity in order to confirm the responsive nature of these cells. A representative trace of the average response of 8 cells from a single coverslip is shown in Fig. 6. For all experiments undertaken, the removal of growth factors during differentiation was without effect on basal [Ca2 + ]i demonstrated by an unchanged 340:380 emission ratio. This study was undertaken on four coverslips from each of three separate cell preparations.
Fig. 5. Effect of neuroblastoma conditioned basal media (c), basal media containing 0.1% FBS (S1), basal media alone (n1), or basal media with 20% FBS added (S 20) on the level of phospho MAPK p44/42-ir levels in CG-4 cells treated for a 15 min period. (a). The effects of continual treatment of CG-4 cells with neuroblastoma conditioned basal media alone (C), or neuroblastoma conditioned basal media (B104) or basal media (n1) following a 30 min treatment period with basal media on phospho MAPK p44/42-ir levels (b). Following the differentiation period of 30 min, CG-4 cultures were treated for an additional period of 15 min prior to extraction. Experiments were repeated three times and each experimental treatment run in duplicate within each experiment, equal concentrations of protein (20 mg) were loaded to each well.
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Fig. 6. Effect of treatment with basal media on the 340:380 ratio levels of Fura 2-loaded undifferentiated bipolar CG-4 cells. Cells were loaded with Fura 2 for a 1 h period prior to analysis. The arrow indicates the change in perfusion media from neuroblastoma conditioned basal media to basal medium containing 0.1% serum. The trace shown is from combined data of eight cells on an individual coverslip. At least four coverslips were analysed from each of three separate culture preparations with similar results obtained on each occasion.
3.5. Effect of treatment with the PKC inhibitor chelerythrine chloride on CG-4 cell morphology during differentiation Inhibition of protein kinase C partially inhibited the increase in the acidification rate observed during the exchange of neuroblastoma conditioned basal media for basal media (Figs. 1 and 2). A separate series of experiments were undertaken to investigate the effects of treatment with the PKC inhibitor chelerythrine chloride on the morphology change observed in CG-4 cells during the differentiation process. Cultures were washed twice in basal media and maintained in the presence or absence of chelerythrine chloride in differentiation media at a concentration of 10 mM for 2 h at 37 °C with 5% CO2 and 95% humidity. After this time, coverslips were mounted using Shandon immu-mount and photographed using differential interference optics. Representative photomicrographs obtained from CG-4 cultures are presented in Fig. 7. Control undifferentiated bipolar CG-4 cells possess a characteristic bipolar morphology consistent with undifferentiated bipolar CG-4 cells (Fig. 7a). Treatment with differentiation media induced process formation characteristic of the early phases of differentiation towards multipolar oligodendrocyte-like cells (Fig. 7b). The change in morphology observed during the differentiation process was inhibited by the presence of the PKC inhibitor chelerythrine chloride (Fig. 7c).
4. Discussion In the present study, we investigated the signal transduction and metabolic effects, of changing medium to induce differentiation of undifferentiated bipolar CG-4
Fig. 7. Differential interference contrast photomicrograph of CG-4 cells treated with neuroblastoma conditioned basal media alone (a) with basal media (b) or with basal media with chelerythrine chloride added (10 mM, c). Cultures were treated for a 2 h period prior to mounting in aqueous mounting media and photomicrography. Experiments were repeated three times and representative cells presented.
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cells into either multipolar oligodendrocyte-like or type-2 astrocyte-like cells. Differentiation along both the oligodendrocyte and the astrocyte pathways alters basal metabolic activity, however, the observed temporal patterns are distinct. Inhibition of PKC and PI3 kinase but not of protein kinase A, tyrosine kinases or MAP kinase kinase reduced the increase in metabolism observed during differentiation into multipolar oligodendrocytelike cells. Treatment with chelerythrine chloride caused a dose-dependent inhibition of metabolic activity, which correlated with a block in the morphological changes, which occur during differentiation. Differentiation into multipolar oligodendrocyte-like cells caused a reduction below basal levels in the phosphorylation state of CREB and of p44/p42 MAP kinase without reducing the total amount of CREB or modulating [Ca2 + ]i. Reintroduction of neuroblastoma conditioned basal media to cells treated with basal media caused a strong increase in phosphorylation of CREB and of MAP kinase. Differentiation into astrocyte-like cells caused an increase in phosphorylation of CREB and MAP kinase. Multiple distinct signalling pathways including those sensitive to cAMP, Ca2 + and MAP kinase have been shown to converge to activate by phosphorylation CREB in vitro. Here we have shown that for undifferentiated bipolar CG-4 cells, the phosphorylation state of CREB, is reduced during the initial stages of differentiation to the multipolar mature oligodendrocyte form. Removal of the growth factors present in neuroblastoma conditioned basal media which maintain the undifferentiated bipolar CG-4 cells in their progenitor-like state did not directly modulate [Ca2 + ]i. Therefore, it is unlikely that reduced activation of Cam kinase transduction pathways cause the observed reduction in phosphorylation of CREB during differentiation towards the oligodendrocyte lineage. It is possible that the observed reduction in MAP kinase p44/42 phosphorylation may account for the observed drop in phosphorylation of CREB since activation of the MAP kinase transduction pathways increases phosphorylation of CREB through a distinct CREB kinase, RSK2 (Ginty et al., 1994; Xing et al., 1996). This would be consistent with the simultaneous increase in both MAP kinase and CREB phosphorylation when neuroblastoma conditioned basal media is added back to CG-4 cells undergoing differentiation. Investigations of metabolic changes utilising the cytosensor microphysiometer demonstrated that differentiation into oligodendrocyte-like cells or into astrocyte like cells occur with distinct metabolic profiles, providing evidence for the modulation of distinct signalling pathways. These differences were confirmed by analysis of the phosphorylation state of CREB and of MAP kinase proteins indicative of their activation state during differentiation. Importantly increased intracellular levels of cAMP are thought to increase the rate of appearance of a differentiated multipolar oligodendrocyte phenotype
without necessarily changing the lineage commitment of O-2A progenitors cells (Mirsky et al., 1980; Raible and McMorris, 1989). Although this indicates that increased cAMP production may not be central to lineage commitment during differentiation, this does not exclude a role for the transcription factor CREB. Several reports have indicated that CREB is present in oligodendrocytes and plays an important role in oligodendrocyte biology (Sato-Bigbee and Yu, 1993; Sato-Bigbee et al., 1994). These include the presence of a response element related to the CREB-responsive element in the myelin-oligodendrocyte glycoprotein (MOG) gene (Jaquet et al., 1999) and the ability to influence myelin basic protein levels (Sato-Bigbee and DeVries, 1996). Phosphorylation of CREB at ser133 which activates this transcription factor, can occur in both mature (Sato-Bigbee et al., 1999) and in progenitor oligodendrocytes (Sato-Bigbee et al., 1999; Pende et al., 1997). Our data support the possibility that phosphorylation of CREB plays an important role in differentiation of the bipolar CG-4 cell line. The preliminary stages of differentiation of CG-4 cells into multipolar oligodendrocyte-like cells are associated with reduced CREB phosphorylation and do not require increased phosphorylation and activation of this transcription factor. However, differentiation into type 2-astrocytelike cells increases phosphorylation of CREB and MAP kinase. Therefore, phosphorylation and activation of these proteins may be important for differentiation to take place along the astrocyte pathway. The precise roles of PKC and PKA transduction pathways during the differentiation of undifferentiated bipolar CG-4 cells are unknown. However their roles during differentiation of the O-2A lineage oligodendroglial progenitor into oligodendrocyte or type-2 astrocytes have been partially addressed (Raible, 1989, Baron, 1998, Yong et al., 1988). These studies have provided alternate views of the role of PKC during differentiation, reporting either that activation of PKC prevents (Baron et al., 1998), or enhances (Yong et al., 1988) the differentiation of O-2A progenitor cells into oligodendrocytes in culture. Our data demonstrates that inhibition of PKC blocks both the early changes in metabolism and also the gross morphological re-modeling which occur during the first few minutes of the differentiation process. These data are in agreement with that presented for O-2A cells in the studies of Yong et al., 1988. The discrepancy between reports of an inhibitory role for PKC, as presented recently by Baron (Baron et al., 1998) and a stimulatory role (Yong et al., 1988) may be a consequence of down regulation of PKC associated with the prolonged phorbol ester-induced activation of PKC. Alternatively this may represent a dual effect of PKC on differentiation pathways with early processes being enhanced by PKC activation and later processes being inhibited. We observed no change in the basal [Ca2 + ]i levels during the differentiation of O-2A progenitor cells into oligodendrocyte-like cells. This indicates that iso-
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forms of PKC whose activation state is insensitive to calcium levels may be involved in the differentiation process. In the present study we have shown that inhibition of PKA is without apparent effect on the early metabolic changes which occur during undifferentiated bipolar CG-4 cell differentiation into multipolar oligodendrocyte-like cells. This is consistent with the study of Raible and McMorris who demonstrated that cyclic AMP regulates the relative rate of differentiation of oligodendrocyte progentitors but not the choice of oligodendrocyte or astrocyte lineage (Raible and McMorris, 1989). Inhibition of PKC blocks only part of the changes in metabolic activity associated with differentiation towards oligodendrocyte-like cells, indicating that additional signalling pathways may also play a significant part in this process. In support of this, we have presented preliminary data indicating that inhibition of PI3 kinase using the inhibitor LY294002 can partially block the changes in metabolic activity which occur during the first few minutes of the differentiation process. These studies support the roles of PKC, MAP kinase and CREB in the process of cell differentiation and provide evidence for the control of transcriptional processes in a model system of O-2A progenitor oligodendrocyte differentiation. In addition, changes in the MAP kinase phosphorylation state indicate that the transcription factor Elk1 may also play a part in the control of transcription during differentiation into multipolar oligodendrocyte-like cells. Indeed, co-operative or synergistic effects of p42/p44 MAP kinase signalling (through activation of the transcription factor Elk1) and PKA- dependent signalling (through activation of CREB), can occur at the level of the control of gene expression. For example, control of transcription of the immediate early gene c-fos can, under certain circumstances, can be modulated both by Elk1 acting through the serum response element (SRE) and CREB acting through the cyclic AMP response element (CRE), (Xing et al., 1996). These studies reveal that both CREB and MAP kinase activation states are modulated during CG-4 cell differentiation and also during the reversal of differentiation and that different pathways are activated depending on the differentiation route initiated. It is now necessary to dissect these pathways further to determine how CREB and MAP kinase pathways function in detail.
Acknowledgements We acknowledge financial support from the Wellcome trust (funding to MG Rumsby) and Jim Murray and Mark Hepworth for assistance in the preparation of this manuscript.
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Rumsby, M.G., Afsari, F., Stark, M., Hughson, E., 1998. Process extension by rat CG-4 line progenitor oligodendrocytes: a positively-charged surface promotes a bipolar but not a differentiated multipolar morphology and is not necessary for proliferation. Neurosci. Res. Commun. 23, 101 – 109. Rumsby, M., Suggitt, F., Haynes, L., Hughson, E., Kidd, D., McNulty, S., 1999. Substratum of Pleiotrophin (HB-GAM) stimulates rat CG-4 line oligodendrocytes to adopt a bipolar morphology and disperse: Primary O-2A progenitor glial cells disperse similarly on pleiotrophin. Glia 26, 361 – 367. Sato-Bigbee, C., Yu, R.K., 1993. Presence of cyclic AMP response element-binding protein in oligodendrocytes. J. Neurochem. 60 (6), 2106 – 2110. Sato-Bigbee, C., Chan, E.L., Yu, R.K., 1994. Oligodendroglial cyclic AMP response element-binding protein: a member of the CREB family of transcription factors. J. Neurosci. Res. 38 (6), 621 –628. Sato-Bigbee, C., DeVries, G.H., 1996. Treatment of oligodendrocytes with antisense deoxyoligonucleotide directed against CREB mRNA: effect on the cyclic AMP-dependent induction of myelin basic protein expression. J. Neurosci. Res. 46 (1), 98 – 107. Sato-Bigbee, C., Pal, S., Chu, A.K., 1999. Different neuroligands and signal transduction pathways stimulate CREB phosphorylation at specific developmental stages along oligodendrocyte differentiation. J. Neurochem. 72 (1), 139 – 147. Sheng, M., Dougan, S.T., Greenberg, M.E., 1990. Membrane depolarisation and calcium induce c-fos transcription via phosphorylation of CREB. Neuron. 4, 571 – 582. Sheng, M., Thompson, M.A., Greenberg, M.E., 1991. CREB: A Ca2 + -regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252, 1427 – 1430. Smart, D., Wood, M.D., 2000. Cytosensor techniques for examining signal transduction of neurohormones. Biochem. Cell. Biol. 78, 281 – 288. Tontsch, U., Archer, D.R., Dubois-Dalcq, M., Duncan, I.D., 1994. Transplantation of an oligodendrocyte cell line leading to extensive myelination. Proc. Natl. Acad. Sci. 91, 11616 – 11620. Xing, J., Ginty, D.D., Greenberg, M.E., 1996. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273, 959 – 963. Yong, V.W., Sekiguchi, S., Kim, M.W., Kim, S.U., 1988. Phorbol ester enhances morphological differentiation of oligodendrocytes in culture. J. Neurosci. Res. 19 (2), 187 – 194.
www.elsevier.com/locate/neures
Differentiation of bipolar CG-4 line oligodendrocytes is associated with regulation of CREB, MAP kinase and PKC signalling pathways Shaun McNulty a,*, Michael Crouch b, Darren Smart a, Martin Rumsby c a
GlaxoSmithKline Pharmaceuticals New Frontiers Science Park, 3rd A6enue, Harlow, CM19 5AW, Essex, UK b Di6ision of Neuroscience, National Uni6ersity of Australia, Acton, Canbera, Australia c Department of Biology, Uni6ersity of York, York, YO105YW, UK Received 22 May 2001; accepted 25 July 2001
Abstract Undifferentiated bipolar CG-4 cell line oligodendrocytes provide a model system for the O-2A progenitor cell from which oligodendrocytes are derived both in vivo and in vitro. The exchange of neuroblastoma conditioned basal media for basal media causes differentiation of undifferentiated bipolar CG-4 cells into multipolar oligodendrocyte-like cells whilst replacement with basal media containing 20% foetal bovine serum favours the formation of type-2 astrocyte-like cells. Here, we demonstrate that activation of these differentiation pathways correlates with distinct changes both in cell metabolism and in signal transduction. Exchange of neuroblastoma conditioned media for basal media correlates with stimulation of basal metabolic activity, reduced phosphorylation of p44/42 MAP kinase and reduced phophorylation of the transcription factor CREB. In contrast, differentiation with basal medium containing 20% foetal bovine serum (FBS), into type 2 astrocyte-like cells, correlates with reduction in basal metabolic activity, increased phosphorylation of p44/42 MAP kinase and increased phophorylation of the transcription factor CREB. Inhibition of protein kinase C blocked both the metabolic and morphological changes associated with differentiation towards mature multipolar oligodendrocyte-like cells. Inhibition of PKA and MEK did not effect metabolic activity. The rapid return of neuroblastoma conditioned basal media to cells treated with basal media, increased phosphorylation of CREB and MAP kinase. These results demonstrate that protein kinase C and p44/42 MAP kinase signalling pathways are modulated during bipolar CG-4 cell differentiation and demonstrate that the transcription factor CREB may play a pivotal role in differentiation along oligodendrocyte-or astrocyte-lineages. © 2001 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. Keywords: CG-4; CG-4 line oligodendrocytes; Type 2-astrocytes; CREB; Signal transduction; MAP kinase; Calcium; Phosphorylation; Kinase; Differentiation; Protein kinase C
1. Introduction
Abbre6iations: BSA, bovine serum albumin; CaM kinase II, calcium/calmodulin dependent kinase II; CRE, calcium/cyclic AMP response-element; CREB, calcium/cyclic AMP response-element binding-protein; cyclic AMP, cyclic adenosine monophosphate; ir-, immunoreactivity; FCS, Foetal calf serum; MAPK, Mitogen activated protein kinase; PBS, Phosphate buffered saline; pCREB, Phospho Ser133 CREB; PKA, Protein kinase A; PKC, Protein kinase C. * Corresponding author. Tel.: + 44-1279-622450; fax: + 44-1279622230. E-mail address: shaun – [email protected] (S. McNulty).
Oligodendrocytes are responsible for the formation and maintenance of myelin around nerve axons in the central nervous system and are derived from bipolar oligodendrocyte-type 2-astrocyte (O-2A) progenitor cells whose division is regulated by growth factors including PDGF and IGF1 (Noble et al., 1988; McMorris and Dubois-Dalcq, 1988). Bipolar O-2A progenitor cells develop at discrete ventral sites in cord and brain and then proliferate and migrate throughout the CNS before undergoing differentiation along a defined lineage to a mature multipolar oligodendrocyte which
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expresses characteristic phenotypic markers and synthesises myelin. Analysis of the morphology and phenotype of oligodendroglia has revealed distinct stages of development ranging from pre-O-2A progenitor cells and pro-oligodendrocytes to fully mature myelin-producing oligodendrocytes (Armstrong, 1998). However, the cellular mechanisms controlling progenitor oligodendrocyte differentiation are not fully understood. Undifferentiated bipolar CG-4 cells retain many of the properties of O-2A progenitor glial cells (Louis et al., 1992). When transplanted in vivo, these undifferentiated bipolar CG-4 cells can migrate to areas of damage and re-myelinate affected neurons (Tontsch et al., 1994; Franklin et al., 1995, 1996). Bipolar CG-4 cells maintain morphology and proliferate when cultured in a defined medium containing growth factors from B104 neuroblastoma cell conditioned medium. Investigations undertaken in vitro have shown that undifferentiated bipolar CG-4 cells are able to undergo differentiation along two distinct pathways leading either into multipolar oligodendrocyte-like, or into type 2-astrocyte-like cells (Louis et al., 1992) depending on the differentiation medium. Undifferentiated bipolar CG-4 cells are maintained in basal media containing 30% B104 neuroblastoma conditioned media (neuroblastoma conditioned basal media, Louis et al., 1992). Exchange of this media for basal media containing low (B 0.5%) serum concentrations causes differentiation into multipolar oligodendrocyte-like cells. Exchange of conditioned media for media containing 20% serum causes differentiation into type-2 astrocyte-like cells (Louis et al., 1992). Oligodendrocyte-like cells derived from bipolar CG-4 cells are sensitive to treatment with hydrogen peroxide, characteristic of mature primary cultures of oligodendrocytes (Bhat and Zhang 1999). A cationic substrate such as poly-L-lysine (Rumsby et al., 1998) or pleiotrophin (Rumsby et al., 1999) enables undifferentiated bipolar CG-4 cells to disperse, migrate and produce morphological features similar to those of the O2-A progenitor (Rumsby et al., 1998). These properties make the CG-4 line an ideal model system for studies of the signalling pathways necessary for differentiation of O-2A oligodendrocyte precursors. Regulation of oligodendrocyte gene expression is essential in order to control effectively the processes of differentiation and myelination. Increased cytosolic levels of second messengers including cyclic AMP and intracellular-free calcium ([Ca2 + ]i) lead to the activation of cellular kinases, including protein kinase A (PKA), protein kinase C (PKC) and Ca2 + /calmodulin dependent kinase II (Cam kinase II). These kinases control transcriptional events through phosphorylation of transcription factors including the calcium/cyclic AMP response-element binding-protein (CREB). Phosphorylation of CREB at Ser133 (pCREB) causes activation and pCREB is able to regulate the transcription of
genes containing the calcium/cyclic AMP response-element (CRE) including c-fos (Gonzalez and Montminy, 1989; Sheng et al., 1990, 1991). Activation of MAP kinase signalling pathways also increases phosphorylation of CREB through activation of a CREB kinase, RSK2 (Ginty et al., 1994; Xing et al., 1996). Recent observations (Sato-Bigbee et al., 1999; Johnson et al., 2000) have demonstrated that CREB is present in oligodendrocytes with peak expression of CREB preceding the peak of myelination. Phosphorylation of CREB was shown to be sensitive both to PKC and to MAP kinase signal transdution pathways (Sato-Bigbee et al., 1999). Separate studies have indicated that phosphorylation of CREB in oligodendrocytes may be mediated by PKCMAP kinase- dependent signal transduction pathways (Pende et al., 1997). Raible and McMorris (1989) have shown that cAMP accelerates the differentiation of precursor cells already committed to becoming oligodendrocytes. Such studies have led to the hypothesis that CREB may play an important role during oligodendrocyte differentiation. Activation of PKC with phorbol ester was shown (Avossa and Pfeiffer, 1993) to induce a transient reversion of O4+ GC- progenitor oligodendrocytes to a vimentiun+ A2B5 + O4- phenotype. In the continued presence of the phorbol ester TPA, these cells redeveloped a mature phenotype expressing GC and MBP markers. Asotra and Macklin (1994) noted that PKC modulates myelin gene expression in enriched oligodendrocyte cultures and is involved in the control of myelin basic protein gene expression by cAMP (Anderson and Miskimins, 1994). Recently, it has been reported that activation of protein kinase C (PKC) by phorbol esters may prevent the differentiation of primary O2-A progenitors into oligodendrocytes (Baron et al., 1998). However, prolonged treatment of cells with phorbol esters causes down regulation of PKC in a variety of cell types complicating the interpretation of results obtained by their use (Murasawa et al., 1998; Gailly, 1998). The Cytosensor microphysiometer (Molecular Devices, UK) measures changes in the extracellular acidification rate of cultures (Owicki et al., 1990). Physiological changes, such as receptor activation, alter rates of energy metabolism causing changes in the levels of acid metabolites, which are secreted from the cell in order to maintain the intracellular pH (Owicki et al., 1990). The microphysiometer is able to detect these minute changes in the excretion of acid metabolites (Owicki et al., 1994). The versatility of the microphysiometer is due to the fact that its output is independent of the signal transduction pathway employed by the receptor (Smart and Wood, 2000). These properties make the microphysiometer an excellent tool for investigating the signalling pathways utilised by CG-4 progenitor cells.
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The aims of the present study were to investigate how signal transduction pathways and metabolic activity alter when undifferentiated bipolar CG-4 cells are differentiated into multipolar oligodendrocyte- or into type 2 astrocyte-like cells. Phosphorylation of CREB and activation of MAP kinase were examined using specific antisera raised against synthetic phospho-peptides. Changes in [Ca2 + ]i were estimated by imaging cells loaded with the calcium-sensitive dye Fura 2-AM. The Cytosensor microphysiometer was utilised for studies of metabolic changes during differentiation.
37 °C inside the Cytosensor, and maintained by a flow (120 ml/min) of bicarbonate-free neuroblastoma conditioned basal media (pH 7.4). The flow was halted for 22s at the end of each 2 min pump cycle, and the rate of acidification (mV/s) measured for 15 s during that period. After a stable baseline acidification rate was achieved, the cells were perfused with bicarbonate-free basal media containing 0.1% foetal calf serum. In some experiments, the medium used contained either 10 mM chelerythrine chloride, 100 nM calphostin C, 10 mM tryphostin 23, 10 mM Ro 31-8220, 10 mM KN-93, 10 mM LY294002, 10 mM H-89 or 10 mM PD098059.
2. Materials and methods
2.3. Cytosensor analysis
2.1. Cell line preparation and growth conditions
Treatment effects were quantitatively determined as the change in the acidification rate from the baseline to the second data point after switching media and are expressed as mV/s or as a percentage of the paired control response, as appropriate. Conversion to percentage basal is inappropriate for signal transduction studies (Smart and Wood, 2000). Data are presented as mean9S.E. mean unless otherwise stated.
Undifferentiated bipolar CG-4 cells were maintained in neuroblastoma conditioned basal media. This consisted of basal media (DMEM containing insulin 5 mg/ml, transferrin 50 mg/ml, putrescene 100 mM, progesterone 20 nM, selenium 30 nM, biotin 40 nM, 1% penicillin streptomycin and BSA 1.25 mg/ml) supplemented with B104 conditioned media (30% final conditioned media concentration), as described previously (Rumsby et al., 1998). For measurements of intracellular free calcium concentration [Ca2 + ]i, undifferentiated bipolar CG-4 cells were seeded at a density of 1.5×105 onto sterile poly-L-lysine-coated 22 mm diameter coverslips contained in six-well plates and incubated overnight at 37 °C with 5% CO2 and 95% humidity to permit the cells to adhere. Cells were used after 48 h in culture. For Western blot analysis, cells were plated at a density of 2.5× 105 into six-well plates and incubated overnight at 37 °C with 5% CO2 and 95% humidity. Cells were used after 48 h in culture. For analysis of metabolic changes, undifferentiated bipolar CG-4 cells were seeded into poly-L-lysine-coated Cytosensor capsule cups at 0.25×106 cells/cup and cultured for a further 24 h. Differentiation of undifferentiated bipolar CG-4 cells into multipolar oligodendrocyte-like cells was undertaken by exchanging neuroblastoma conditioned basal media for basal media containing either 0 or 0.1% foetal calf serum (FCS). Differentiation of undifferentiated bipolar CG-4 cells into type-2 astrocyte-like cells was undertaken by exchanging neuroblastoma conditioned basal media for basal media supplemented with 20% FCS.
2.2. Cellular acidification measurements using the Cytosensor microphysiometer The Cytosensor microphysiometer was used to measure the extracellular acidification rate, as described previously (McConnell et al., 1992; Jordan et al., 1998). The seeded cups were placed in sensor chambers at
2.4. Western blot analysis of CREB phosphorylation and MAP kinase acti6ation Western blots to identify CREB-ir were performed using a validated, commercially available kit (New England Biolabs, USA) as described previously (McNulty et al., 1997, 1998). Undifferentiated bipolar CG-4 cells were maintained as described above. Neuroblastoma conditioned basal media was exchanged for the required differentiation media and cells maintained for the necessary experimental period at 37 °C/5% CO2. At the end of this period, media was aspirated and cells were washed once in phosphate-buffered saline (PBS) containing 1 mM NaF, prior to extraction in 200 ml of Novex sample buffer. Extracts were then sonicated for 5 s and centrifuged at 4000 rpm to remove debris. Samples (15 ml) were loaded onto the stacking gel lanes of 10% mini polyacrylamide gels (Novex) and proteins were separated using a Novex XCell II Mini-Cell electrophoresis system for 2 h at 100 V. Proteins were then transferred (25 V, for 2 h) to nitrocellulose membranes prior to detection. Membranes were incubated for 1 h at 20 °C in blocking solution (10 ml, TBS containing 5% dried milk, 0.1% Tween 20). After blocking, membranes were incubated overnight at 4 °C in primary antibody solution (10 ml total volume, pCREB 1:1000, total CREB 1:2000, pMAPkinase 1:1000 in TBS containing 5% bovine serum albumin (BSA), 0.1% Tween 20). The next day, antiserum was removed and blots were washed three times in TBS (10 ml) containing 0.1% Tween, prior to incubation with secondary antibody for 30 min at room temperature (10 ml, 1:1000
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HRP linked goat anti rabbit or 10 ml, 1:1000 HRP linked goat anti mouse (New England Biolabs, USA), in blocking solution). Blots were then rinsed three times for 5 min in TBS (10 ml) prior to development using a modified enhanced chemiluminescence detection system (Amersham).
2.5. Analysis of changes in intracellular free calcium concentration Coverslips containing undifferentiated bipolar CG-4 cells were prepared and maintained as described. Cultured cells (grown attached to 22 mm diameter coverslips) were loaded with Fura-2 (Grynkiewicz et al., 1985) by incubation for 1 h at 20 °C with neuroblastoma conditioned basal media containing Fura-2-AM (2 mM, Molecular Probes). This procedure enables cells to load with Fura-2-AM, which becomes hydrolysed to the free acid form inside the intact cell. After loading, coverslips were mounted into imaging chambers and perfused with neuroblastoma conditioned basal media to remove extracellular Fura-2AM. Measurements of changes in the [Ca2 + ]i in individual cells were made from fluorescence ratio values (excitations 340/380 nm, emission \510 nm) using a spectral Wizard monochromator, cooled integrating CCD camera and a dedicated suite of software (Merlin, Life Sciences Resources, Cambridge, UK). Data are expressed as ratio units (340/380).
rosine kinase inhibitor tryphostan 23 (10 mM) and the MAP kinase kinase inhibitor PD098059 (10 mM) were without effect (Fig. 1b). Treatment of undifferentiated bipolar CG-4 cells with chelerythrine chloride caused a dose dependent inhibition of the acidification response (Fig. 2). Treatment of undifferentiated bipolar CG-4 cells with a concentration of serum (20%) which causes differentiation into type 2 astrocyte-like cells caused an apparent biphasic acidification response. This response was typified as an initial-post exchange artefact response peak, followed by a decline in acidification rate below the initial peak and approaching the initial baseline levels of metabolic activity (Fig. 3).
3. Results
3.1. Effect of medium change that induces cell differentiation on metabolic acti6ity The undifferentiated bipolar CG-4 cells achieved a stable baseline acidification rate of 50– 180 mV/s within 60 min of the start of perfusion with neuroblastoma conditioned basal media. Changing to basal medium in order to differentiate cells towards the oligodendrocyte lineage caused an apparently biphasic acidification response, typified as an initial peak followed by a relatively stable plateau of increased metabolic activity (Fig. 1a). Examination of the raw data traces (data not shown) revealed that the initial peak was an artefact caused by the relative buffering capacities of the two media, whilst the plateau phase was due to changes in cellular metabolism. Three PKC inhibitors, chelerythrine chloride (10 mM), calphostin C (100 nM) and Ro31-8220 (10 mM), caused significant inhibition (40– 50%) of the acidification response (Fig. 1b). The PI-3 kinase inhibitor, LY294002 (10 mM), also caused a small ( 25%) but significant inhibition of the acidification response (Fig. 1b). The PKA inhibitor H-89 (10 mM), the ty-
Fig. 1. Representative Cytosensor traces for CG-4 cells undergoing differentiation in the absence ( ) or presence ( ) of 10 mM chelerythrine chloride (a). The arrow indicates the point at which the perfusion media was switched from neuroblastoma conditioned basal media to basal medium containing 0.1% serum to induce differentiation into multipolar oligodendrocyte-like cells. The effects of protein kinase inhibitors (10 mM for 10 min pre-exposure) on the acidification response of undifferentiated bipolar CG-4 cells switched from neuroblastoma conditioned basal media to basal media are shown (b). Data are mean 9S.E. mean, with the n value for each treatment given in parentheses above the relevant bar. * denotes significant (PB 0.05) inhibition compared with controls (mANOVA followed by Student’s t-test). Unless stated, experiments were repeated three times.
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Fig. 2. Pooled data showing the effect of chelerythrine chloride (10 mM for 10 min pre-exposure) on the acidification response of undifferentiated bipolar CG-4 cells switched from neuroblastoma conditioned basal media to basal media are shown. Data are mean 9 S.E. mean, where n =3. * denotes significant (P B0.05) inhibition compared with the acidification rate of control cultures.
3.2. Effect of medium change that induces cell differentiation on phosphorylation of CREB Experiments were undertaken to investigate whether differentiation of undifferentiated bipolar CG-4 cells using standard protocols into either multipolar oligodendrocyte-like cells or type-2 astrocyte-like cells resulted in modulation of the phosphorylation of CREB. Western blot analysis for total CREB-ir or pCREB-ir revealed a single band of immunoreactivity (ir) of a molecular weight of 44 KDa (Fig. 4). Control levels of pCREB-ir were low but treatment with basal media containing serum (20%) causing differentiation along a type-2 astrocyte pathway, strongly increased immunoreactivity (Fig. 4a). In contrast, either the com-
Fig. 3. Representative Cytosensor traces for undifferentiated bipolar CG-4 cells exposed to 0 ( ), 0.5% ( ) or 20% () serum. The arrow indicates the point at which the perfusion was changed from neuroblastoma conditioned basal media into basal medium containing serum at the stated concentration. This trace is typical of n = 3.
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plete removal of growth factors, or including serum at a concentration of 0.1% to induce differentiation to multipolar oligodendrocyte-like cells caused a reduction in pCREB-ir levels below those observed in control non-differentiated cultures. The identity of this band was confirmed in blots probed using antiserum that recognises both phosphorylated and non-phosphorylated forms of CREB (Fig. 4b). However, no systematic changes in immunoreactivity were evident between the experimental groups demonstrating that the observed changes in pCREB-ir can not be accounted for by changes in the total level of CREB protein. Pre-treatment with chelerythrine chloride (10 mM), for a period of 10 min prior to and during differentiation into oligodendrocyte-like cells was without significant effect on pCREB-ir levels (data not shown). The return of neuroblastoma conditioned basal media to CG-4 cultures during the early phase of differentiation into multipolar oligodendrocyte-like cells is known to rescue progenitor like morphology and characteristics. Experiments were undertaken to investigate the effects of the return of neuroblastoma conditioned basal media to cells starved of growth factors for a period of 30 min (Fig. 4c, d). CG-4 cultures undergoing differentiation towards multipolar oligodendrocyte-like cells, demonstrated characteristic low levels of pCREBir (Fig. 4a, c) compared with basal levels. The return of neuroblastoma conditioned basal media increased strongly pCREB-ir over the levels observed either in differentiating cultures or cultures maintained in the presence of neuroblastoma conditioned basal media for the duration of the experiment. These treatments were without effect on levels of CREB-ir obtained from blots of the same experimental samples (Fig. 4d).
3.3. Effect of medium change that induces cell differentiation on phosphorylation of p44 /42 MAP kinase Experiments were undertaken to investigate whether differentiation using standard protocols into either oligodendrocyte- or astrocyte lineages resulted in modulation of the phosphorylation of MAP kinase p44/42 (Fig. 5a, b). Control levels of immunoreactivity were low, however, treatment with basal media containing serum (20%) strongly increased phospho p44/42 MAP kinase immunoreactivity (Fig. 5a). In contrast, the complete removal of growth factors or including serum at a concentration of 0.1% caused a reduction in phospho p44/42 MAP-ir levels below those observed in control non-differentiated cultures. Pre-treatment with chelerythrine chloride (10 mM), for a period of 10 min prior to and during differentiation into oligodendrocyte-like cells was without significant effect on p42/44 MAP kinase levels (data not shown). Experiments were undertaken to investigate the effects of the re-addition
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Fig. 4. Effect of neuroblastoma conditioned basal media (c), basal media containing 0.1% FBS (s1), basal media alone (n1), or basal media with 20% FBS added (s20) on the levels of pCREB-ir (a) and CREB-ir (b) in CG-4 cell cultures. Cells were treated for a period of 15 min prior to extraction. The effects of continual treatment of CG-4 cells with neuroblastoma conditioned basal media alone (C), or either neuroblastoma conditioned basal media (B104) or basal media (n1) following a 30 min treatment with basal media on pCREB-ir (c) and on CREB-ir (d). Following a differentiation period of 30 min, CG-4 cultures were treated for an additional period of 15 min prior to extraction. Each experimental group was run in duplicate and experiments were repeated three times with similar results. Equal concentrations of protein were loaded to each well.
of neuroblastoma conditioned basal media to cells starved of growth factors for a period of 30 min (Fig. 5b). As observed for pCREB-ir, the return of neuroblastoma conditioned basal media strongly increased phospho p42/44 MAP kinase immunoreactivity of cells compared with cultures maintained in basal media or cultures maintained in the presence of neuroblastoma conditioned basal media for the duration of the experiment.
3.4. Effect of medium change that induces cell differentiation on the concentration of intracellular-free calcium Studies were undertaken to investigate if changing neuroblastoma conditioned basal media to basal medium affected the [Ca2 + ]i within undifferentiated bipolar CG-4 cells. Experiments were conducted on four individual batches of undifferentiated bipolar CG-4 cells and undertaken simultaneously with Cytosensor Microphysiometry investigations of metabolic activity in order to confirm the responsive nature of these cells. A representative trace of the average response of 8 cells from a single coverslip is shown in Fig. 6. For all experiments undertaken, the removal of growth factors during differentiation was without effect on basal [Ca2 + ]i demonstrated by an unchanged 340:380 emission ratio. This study was undertaken on four coverslips from each of three separate cell preparations.
Fig. 5. Effect of neuroblastoma conditioned basal media (c), basal media containing 0.1% FBS (S1), basal media alone (n1), or basal media with 20% FBS added (S 20) on the level of phospho MAPK p44/42-ir levels in CG-4 cells treated for a 15 min period. (a). The effects of continual treatment of CG-4 cells with neuroblastoma conditioned basal media alone (C), or neuroblastoma conditioned basal media (B104) or basal media (n1) following a 30 min treatment period with basal media on phospho MAPK p44/42-ir levels (b). Following the differentiation period of 30 min, CG-4 cultures were treated for an additional period of 15 min prior to extraction. Experiments were repeated three times and each experimental treatment run in duplicate within each experiment, equal concentrations of protein (20 mg) were loaded to each well.
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Fig. 6. Effect of treatment with basal media on the 340:380 ratio levels of Fura 2-loaded undifferentiated bipolar CG-4 cells. Cells were loaded with Fura 2 for a 1 h period prior to analysis. The arrow indicates the change in perfusion media from neuroblastoma conditioned basal media to basal medium containing 0.1% serum. The trace shown is from combined data of eight cells on an individual coverslip. At least four coverslips were analysed from each of three separate culture preparations with similar results obtained on each occasion.
3.5. Effect of treatment with the PKC inhibitor chelerythrine chloride on CG-4 cell morphology during differentiation Inhibition of protein kinase C partially inhibited the increase in the acidification rate observed during the exchange of neuroblastoma conditioned basal media for basal media (Figs. 1 and 2). A separate series of experiments were undertaken to investigate the effects of treatment with the PKC inhibitor chelerythrine chloride on the morphology change observed in CG-4 cells during the differentiation process. Cultures were washed twice in basal media and maintained in the presence or absence of chelerythrine chloride in differentiation media at a concentration of 10 mM for 2 h at 37 °C with 5% CO2 and 95% humidity. After this time, coverslips were mounted using Shandon immu-mount and photographed using differential interference optics. Representative photomicrographs obtained from CG-4 cultures are presented in Fig. 7. Control undifferentiated bipolar CG-4 cells possess a characteristic bipolar morphology consistent with undifferentiated bipolar CG-4 cells (Fig. 7a). Treatment with differentiation media induced process formation characteristic of the early phases of differentiation towards multipolar oligodendrocyte-like cells (Fig. 7b). The change in morphology observed during the differentiation process was inhibited by the presence of the PKC inhibitor chelerythrine chloride (Fig. 7c).
4. Discussion In the present study, we investigated the signal transduction and metabolic effects, of changing medium to induce differentiation of undifferentiated bipolar CG-4
Fig. 7. Differential interference contrast photomicrograph of CG-4 cells treated with neuroblastoma conditioned basal media alone (a) with basal media (b) or with basal media with chelerythrine chloride added (10 mM, c). Cultures were treated for a 2 h period prior to mounting in aqueous mounting media and photomicrography. Experiments were repeated three times and representative cells presented.
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cells into either multipolar oligodendrocyte-like or type-2 astrocyte-like cells. Differentiation along both the oligodendrocyte and the astrocyte pathways alters basal metabolic activity, however, the observed temporal patterns are distinct. Inhibition of PKC and PI3 kinase but not of protein kinase A, tyrosine kinases or MAP kinase kinase reduced the increase in metabolism observed during differentiation into multipolar oligodendrocytelike cells. Treatment with chelerythrine chloride caused a dose-dependent inhibition of metabolic activity, which correlated with a block in the morphological changes, which occur during differentiation. Differentiation into multipolar oligodendrocyte-like cells caused a reduction below basal levels in the phosphorylation state of CREB and of p44/p42 MAP kinase without reducing the total amount of CREB or modulating [Ca2 + ]i. Reintroduction of neuroblastoma conditioned basal media to cells treated with basal media caused a strong increase in phosphorylation of CREB and of MAP kinase. Differentiation into astrocyte-like cells caused an increase in phosphorylation of CREB and MAP kinase. Multiple distinct signalling pathways including those sensitive to cAMP, Ca2 + and MAP kinase have been shown to converge to activate by phosphorylation CREB in vitro. Here we have shown that for undifferentiated bipolar CG-4 cells, the phosphorylation state of CREB, is reduced during the initial stages of differentiation to the multipolar mature oligodendrocyte form. Removal of the growth factors present in neuroblastoma conditioned basal media which maintain the undifferentiated bipolar CG-4 cells in their progenitor-like state did not directly modulate [Ca2 + ]i. Therefore, it is unlikely that reduced activation of Cam kinase transduction pathways cause the observed reduction in phosphorylation of CREB during differentiation towards the oligodendrocyte lineage. It is possible that the observed reduction in MAP kinase p44/42 phosphorylation may account for the observed drop in phosphorylation of CREB since activation of the MAP kinase transduction pathways increases phosphorylation of CREB through a distinct CREB kinase, RSK2 (Ginty et al., 1994; Xing et al., 1996). This would be consistent with the simultaneous increase in both MAP kinase and CREB phosphorylation when neuroblastoma conditioned basal media is added back to CG-4 cells undergoing differentiation. Investigations of metabolic changes utilising the cytosensor microphysiometer demonstrated that differentiation into oligodendrocyte-like cells or into astrocyte like cells occur with distinct metabolic profiles, providing evidence for the modulation of distinct signalling pathways. These differences were confirmed by analysis of the phosphorylation state of CREB and of MAP kinase proteins indicative of their activation state during differentiation. Importantly increased intracellular levels of cAMP are thought to increase the rate of appearance of a differentiated multipolar oligodendrocyte phenotype
without necessarily changing the lineage commitment of O-2A progenitors cells (Mirsky et al., 1980; Raible and McMorris, 1989). Although this indicates that increased cAMP production may not be central to lineage commitment during differentiation, this does not exclude a role for the transcription factor CREB. Several reports have indicated that CREB is present in oligodendrocytes and plays an important role in oligodendrocyte biology (Sato-Bigbee and Yu, 1993; Sato-Bigbee et al., 1994). These include the presence of a response element related to the CREB-responsive element in the myelin-oligodendrocyte glycoprotein (MOG) gene (Jaquet et al., 1999) and the ability to influence myelin basic protein levels (Sato-Bigbee and DeVries, 1996). Phosphorylation of CREB at ser133 which activates this transcription factor, can occur in both mature (Sato-Bigbee et al., 1999) and in progenitor oligodendrocytes (Sato-Bigbee et al., 1999; Pende et al., 1997). Our data support the possibility that phosphorylation of CREB plays an important role in differentiation of the bipolar CG-4 cell line. The preliminary stages of differentiation of CG-4 cells into multipolar oligodendrocyte-like cells are associated with reduced CREB phosphorylation and do not require increased phosphorylation and activation of this transcription factor. However, differentiation into type 2-astrocytelike cells increases phosphorylation of CREB and MAP kinase. Therefore, phosphorylation and activation of these proteins may be important for differentiation to take place along the astrocyte pathway. The precise roles of PKC and PKA transduction pathways during the differentiation of undifferentiated bipolar CG-4 cells are unknown. However their roles during differentiation of the O-2A lineage oligodendroglial progenitor into oligodendrocyte or type-2 astrocytes have been partially addressed (Raible, 1989, Baron, 1998, Yong et al., 1988). These studies have provided alternate views of the role of PKC during differentiation, reporting either that activation of PKC prevents (Baron et al., 1998), or enhances (Yong et al., 1988) the differentiation of O-2A progenitor cells into oligodendrocytes in culture. Our data demonstrates that inhibition of PKC blocks both the early changes in metabolism and also the gross morphological re-modeling which occur during the first few minutes of the differentiation process. These data are in agreement with that presented for O-2A cells in the studies of Yong et al., 1988. The discrepancy between reports of an inhibitory role for PKC, as presented recently by Baron (Baron et al., 1998) and a stimulatory role (Yong et al., 1988) may be a consequence of down regulation of PKC associated with the prolonged phorbol ester-induced activation of PKC. Alternatively this may represent a dual effect of PKC on differentiation pathways with early processes being enhanced by PKC activation and later processes being inhibited. We observed no change in the basal [Ca2 + ]i levels during the differentiation of O-2A progenitor cells into oligodendrocyte-like cells. This indicates that iso-
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forms of PKC whose activation state is insensitive to calcium levels may be involved in the differentiation process. In the present study we have shown that inhibition of PKA is without apparent effect on the early metabolic changes which occur during undifferentiated bipolar CG-4 cell differentiation into multipolar oligodendrocyte-like cells. This is consistent with the study of Raible and McMorris who demonstrated that cyclic AMP regulates the relative rate of differentiation of oligodendrocyte progentitors but not the choice of oligodendrocyte or astrocyte lineage (Raible and McMorris, 1989). Inhibition of PKC blocks only part of the changes in metabolic activity associated with differentiation towards oligodendrocyte-like cells, indicating that additional signalling pathways may also play a significant part in this process. In support of this, we have presented preliminary data indicating that inhibition of PI3 kinase using the inhibitor LY294002 can partially block the changes in metabolic activity which occur during the first few minutes of the differentiation process. These studies support the roles of PKC, MAP kinase and CREB in the process of cell differentiation and provide evidence for the control of transcriptional processes in a model system of O-2A progenitor oligodendrocyte differentiation. In addition, changes in the MAP kinase phosphorylation state indicate that the transcription factor Elk1 may also play a part in the control of transcription during differentiation into multipolar oligodendrocyte-like cells. Indeed, co-operative or synergistic effects of p42/p44 MAP kinase signalling (through activation of the transcription factor Elk1) and PKA- dependent signalling (through activation of CREB), can occur at the level of the control of gene expression. For example, control of transcription of the immediate early gene c-fos can, under certain circumstances, can be modulated both by Elk1 acting through the serum response element (SRE) and CREB acting through the cyclic AMP response element (CRE), (Xing et al., 1996). These studies reveal that both CREB and MAP kinase activation states are modulated during CG-4 cell differentiation and also during the reversal of differentiation and that different pathways are activated depending on the differentiation route initiated. It is now necessary to dissect these pathways further to determine how CREB and MAP kinase pathways function in detail.
Acknowledgements We acknowledge financial support from the Wellcome trust (funding to MG Rumsby) and Jim Murray and Mark Hepworth for assistance in the preparation of this manuscript.
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