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It is becoming increasingly clear that voltage-operated Ca++ channels (VOCCs) play a fundamental role in the development of oligodendrocyte progenitor cells (OPCs). Since direct phosphorylation by different kinases is one of the most important mechanisms involved in VOCC modulation, the aim of this study was to evaluate the participation of serine-threonine (Ser/Thr) kinases and tyrosine kinases (TK) on Ca++ influx mediated by VOCCs in OPCs. Calcium imaging revealed that OPCs exhibited Ca++ influx following plasma membrane depolarization via L-type VOCCs. Furthermore, VOCC-mediated Ca++ influx declined with OPC differentiation, indicating that VOCCs are developmentally regulated in OPCs. PKC activation significantly increased VOCC activity in OPCs, while PKA activation produced the opposite effect. The results also indicated that OPC morphological changes induced by PKC activation were partially mediated by VOCCs. Our data clearly suggest that TKs exert an activating influence on VOCC function in OPCs. Furthermore, using the PDGF response as a model to probe the role of TK receptors (TKr) on OPCs Ca++ uptake, we found that TKr activation potentiated Ca++ influx after membrane depolarization. Interestingly, this TKr modulation of VOCCs appeared to be essential for the PDGF enhancement of OPC migration rate, since cell motility was completely blocked by TKr antagonists, as well as VOCC inhibitors, in migration assays. The present study strongly demonstrates that PKC and TKrs enhance Ca++ influx induced by depolarization in OPCs, while PKA has an inhibitory effect. These kinases modulate voltage-operated Ca++ uptake in OPCs and participate in the modulation of process extension and migration.
It is becoming increasingly clear that expression of Ca++ channels in the oligodendroglial lineage is highly regulated and their activity may be related to different stages of oligodendrocyte (OL) development. Understanding the mechanisms of voltage-dependent Ca++ influx is important because changes in intracellular Ca++ ([Ca++]int) are central to many cellular activities. For example, in OL progenitor cells (OPCs), voltage-dependent Ca++ influx plays a key role in several important processes such as proliferation, apoptosis and cell migration (Paez et al., 2009a; b). We recently found that increased voltage-dependent Ca++ influx was associated with enhanced OPC motility, and this effect was accompanied by increases in the amplitude of spontaneous somatic Ca++ transients which appeared to be essential for OPC migration (Paez et al., 2009b).
Voltage-operated Ca++ channels (VOCCs), which are common in neurons and muscle, provide transmembrane Ca++ for transmitter release, contraction, the coupling and integration of synaptic inputs to action potentials and other intracellular signaling processes. Six types of VOCCs (P/Q, N, L, R and T) have been classified on the basis of electrophysiological and pharmacological properties (Akopian et al., 1996; MacVicar, 1984; Oh, 1997; Puro et al., 1996; Robitaille et al., 1996). Immunohistochemical studies have reported the expression of L-, N- and R-type VOCCs in OLs in vivo (Butt, 2006).
The pore of a voltage-gated Ca++ channel is formed by an α-subunit, which consists of 4 homologous domains connected by 6 transmembrane helices. Gating of this pore is regulated by phosphorylation at multiple cytoplasmic regions on the α-subunit including the amino- and carboxy-terminals, and the loops between each domain. This structure allows for complex interactions between the α-subunit and many regulatory protein complexes. The Cav1 family of α1 subunits conducts L-type Ca++ currents, and is regulated primarily by second messenger-activated protein phosphorylation pathways. The Cav2 family of α1 subunits conducts N-type, P/Q-type, and R-type Ca++ currents, and is regulated primarily by direct interaction with G proteins and secondarily by protein phosphorylation (Catterall, 2000). The latter regulation is important for electrically active cells, such as neurons. Both L-type channels and T-type channels are regulated through PKC and PKA. Several of the α-subunit isoforms for L-type Ca++ channels contain PKC and PKA phosphorylation sites (Puri et al., 1997).
An emerging body of evidence suggests that VOCCs are also regulated by phosphorylation of tyrosine residues (Wijetunge et al., 2002; Strauss et al., 1997). Several growth factors, such as PDGF and bFGF, activate receptor tyrosine kinases (TKr) and trigger complex intracellular signal transduction pathways finally leading to cell proliferation and migration in OPCs and other cell types (Taniguchi, 1995). Ca++ entry from extracellular sources is known to play a key role in these events. However, the nature of the Ca++ channels involved and a possible regulation through direct channel phosphorylations by TKr remains controversial (Wijetunge et al., 2000; Schroder et al., 2004).
The aim of this study was to evaluate the participation of several kinases on the regulation of voltage-operated Ca++ channels in OPCs. [Ca++]int was measured in real time in cultured OPCs and live brain sections, using a spectrofluorometric technique with Fura-2 as an intracellular Ca++ indicator. High extracellular K+ was used as a depolarization stimulus to activate and open VOCCs, enhancing [Ca++]int in OPCs (Paez et al., 2007; 2008; 2009b).
Enriched oligodendrocytes were prepared as described by Amur-Umarjee et al. (1993). First, cerebral hemispheres from 1 day old mice were mechanically dissociated and were plated on poly-D-lysine-coated flasks in Dulbecco's modified Eagle's medium and Ham's F12 (1:1 vol/vol) (Invitrogen Life Technologies, Carlsbad, CA), containing 100μg/ml gentamycin and supplemented with 4mg/ml anhydrous dextrose, 3.75mg/ml HEPES buffer, pH=7.4, 2.4mg/ml sodium bicarbonate and 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, CA). After 24 hours the medium was changed and the cells were grown in DMEM/F-12 supplemented with insulin (5μg/ml), transferrin (50μg/ml), sodium selenite (30nM), d-biotin (10mM), 0.1% BSA (Sigma Aldrich, St. Louis, MO), 1% horse serum and 1% FBS (Omega Scientific, Tarzana, CA). After 9 days, OPCs were purified from the mixed glial culture by the differential shaking and adhesion procedure of Suzumura et al. (1984) and allowed to grow on polylysine-coated coverslips in defined culture media (Agresti et al., 1996) including PDGF-AA (10ng/ml) and bFGF (10ng/ml) (Peprotech, Rocky Hill, NJ). OPCs were kept in mitogens (PDGF and bFGF) for 2 days and then induced to differentiate by switching the cells to a mitogen-free medium (mN2) (Oh et al., 2003). mN2: DMEM/F-12 supplemented with d-glucose (4.5gm/l), insulin (5μg/ml), human transferrin (50μg/ml), sodium selenite (30nM), T3 (15nM), d-biotin (10mM), hydrocortisone (10nM), 0.1% BSA, 1% horse serum and 1% FBS.
Calcium imaging acquisitions of GFP-labeled living OPCs were performed on coronal slices at postnatal day four (P4) and eight (P8), as described elsewhere (Kakita and Goldman, 1999). Briefly, mice were anesthetized with isofurane, after which brains were rapidly removed and stored in ice cold bicarbonate buffered solution (pH=7.4) gassed with 95% O2 and 5% CO2. Coronal slices (300μm) were cut on a vibratome. Brain tissue was kept in ice-cold bicarbonate solution during these procedures. The slices were then cultured with Eagle's Basal Medium with Earle's salts (BME; Invitrogen) supplemented with 18.6mM NaHCO3, 1% BSA (fraction 5; Sigma, St. Louis, MO), 5μg/ml insulin, 5μg/ml transferrin, 5μg/ml sodium selenite (Sigma), 20U/ml penicillin–streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen) and 27mM glucose. After that, brain slices were ready for calcium imaging studies.
At the completion of the calcium-imaging experiment, the cells were stained with antibodies against NG2, O4, O1, PDGFrα and MBP and examined by confocal microscopy. For MBP immunostaining, the cells were rinsed briefly in PBS and fixed in 4% buffered paraformaldehyde for 30 min at room temperature. After rinsing in PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature and then processed for immunocytochemistry following the protocol as outlined by Reyes and Campagnoni (2002). Essentially, fixed cells were incubated in a blocking solution (5% goat serum in PBS) followed by an overnight incubation at 4°C with a polyclonal antibody for MBP (1:700). Staining with NG2 (1:50), PDGFαR (1:100), O4 (1:20) and anti-galactocerebroside antibody O1 (1:20) was performed on live cells without permeabilization for 1h at room temperature before fixation. Cells were then incubated with the appropriate secondary antibodies (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA), mounted onto slides with Aquamount (Lerner Laboratories, Pittsburgh, PA), and fluorescent images were obtained using an Olympus (Melville, NY) spinning disc confocal microscope.
To examine differences in morphology, OPCs cultured for 2 days in defined culture media (Agresti et al., 1996) including PDGF-AA (10ng/ml) and bFGF (10ng/ml) were stained with the O4 antibody analyzed as follows: 1) the number of primary processes per OL, i.e., processes which directly bud from the cell; 2) the number of secondary processes per primary process. A process was considered as secondary when it branched directly from a primary process. Thin, short processes, which were occasionally found along the entire length of primary process, were not considered as secondary; and 3) the length of primary processes. The percentage of primary processes with lengths corresponding to at least 4 cell soma diameters was determined. This method has been used previously for monitoring morphological changes in OL as well as other cell types (Yong et al., 1988; 1991; Šišková et al., 2009). The morphological parameters were obtained from 50 cells from at least 4 independent cultures. The total number of primary and secondary processes, and the length of primary processes, was measured using the image analysis software SlideBook™ 4.1 (Intelligent Imaging Innovations, San Diego, CA).
Cultured OPCs were incubated in a stage top chamber with 5%CO2 at 37°C (Live-Cell Control Unit), which was placed over the stage of an Olympus (Melville, NY) Spinning Disc Confocal Inverted Microscope equipped with a motorized z-stage. A 20× objective was used for acquiring images. Bright-field images were taken every 6 min over a period of 24 hr using a CCD camera (Hamamatsu ORCA-ER) and analyzed with Image analysis software (SlideBook™ 4.1, Intelligent Imaging Innovations, San Diego, CA). Cell migration speed and distances were analyzed off-line by tracing individual cells using the motion tracking function of SlideBook software. The brightest part of each cell body was used as the tracking target. Subsequently, migratory values were statistically analyzed under different experimental conditions. Data are presented as mean ± S.E.M. Statistical significance was assessed by using the Student paired t test, in which p<0.05 was defined as statistically significant.
Methods were similar to those described previously (Colwell, 2000; Michel et al., 2002; Paz Soldan et al., 2003). Briefly, a cooled CCD camera (Hamamatsu ORCA-ER) was added to the Olympus (Melville, NY) spinning disc confocal microscope to measure fluorescence. To load the dye into cells, primary cultures of oligodendrocytes and brain slices were washed in serum and phenol red-free DMEM and incubated for 45 min at 37°C, 5% CO2 in the same media containing a final concentration of 4μM Fura-2 (AM) (TefLabs, Austin, TX) plus 0.08% Pluronic F-127 (Molecular Probes, Eugene, OR), then washed four times in DMEM and stored in DMEM for 0-1 hours before being imaged (Paz Soldan et al., 2003). Resting calcium levels were made in serum-free HBSS containing 2mM Ca++ but no Mg++. Other measurements were made in HBSS. Calcium influx and resting Ca++ levels were measured on individual cells, and the results were pooled from five separate cell preparations for each condition. The fluorescence of Fura-2 was excited alternatively at wavelengths of 340 and 380 nm by means of a high-speed wavelength-switching device (Lambda DG-4; Sutter Instruments, Novato, CA). Image analysis software (SlideBook™ 4.1, Intelligent Imaging Innovations, San Diego, CA) allowed the selection of several “regions of interest” within the field from which measurements were taken. To minimize bleaching, the intensity of excitation light and sampling frequency was kept as low as possible. In these experiments, measurements were normally made once every 2s.
Free [Ca++] was estimated from the ratio (R) of fluorescence at 340 and 380 nm, using the following equation: [Ca++] = Kd × slope factor × (R - Rmin)/(Rmax - R) (Grynkiewicz et al., 1985). The Kd was assumed to be 140nM, whereas values for Rmin and Rmax were all determined using a calibration kit (Fura-2 Ca++ imaging calibration, Molecular Probes, Eugene, OR) to estimate values. With this method, glass coverslips were filled with a high-Ca++ (Fura-2 plus 10mM Ca++), a low-Ca++ (Fura-2 plus 10mM EGTA), and a control solution without Fura-2. Each solution also contains a dilute suspension of 15μm polystyrene microspheres to ensure uniform coverslip/slide separation and facilitate microscope focusing. The fluorescence (F) at 380 nm excitation of the low Ca++ solution was imaged, and the exposure of the camera adjusted to maximize the signal. These camera settings were then fixed, and measurements were made with 380 and 340 nm excitation of the three solutions. Rmin = F340nm in low Ca++/F380 in low Ca++; Rmax = F340 in high Ca++/F380 in high Ca++; Sf = F380 in low Ca++/F380 in high Ca++.
A non-radioactive PKC activity assay designed for quantifying the activity of PKC in crude enzyme preparations was used in accordance with the manufacturer's recommendations (Assay Designs, Ann Arbor, MI). This PKC activity assay is based on a solid phase enzyme-linked immuno-absorbent assay (ELISA) that utilizes a specific synthetic peptide as a substrate for PKC and a polyclonal antibody that recognizes the phosphorylated form of the substrate. Briefly, cytosolic proteins from untreated and PMA-treated OPCs were added to the appropriate wells, followed by the addition of ATP to initiate the reaction. The kinase reaction was terminated and a substrate phosphospecific antibody was added to the wells. The phosphospecific antibody was subsequently bound by a peroxidase-conjugated secondary antibody. The assay was developed with tetramethylbenzidine substrate (TMB), stopped with acid solution and finally the intensity of the color was measured in a microplate reader at 450nm.
Control and PMA-treated Petri dishes containing ~1.5×106 cells/dish were washed twice with ice-cold PBS. The cells were collected by centrifugation (600×g for 10min at 4°C) and extracted with lysis buffer [20mM MOPS, pH=7.4, 50mM β-glycerolphosphate, 50mM sodium fluoride, 1mM sodium vanadate, 5mM EGTA, 2mM EDTA, 1% NP40, 1mM dithiothreitol (DTT), 1mM benzamidine, 1mM phenylmethanesulphonylfluoride (PMSF) and 10μg/ml leupeptin and aprotinin] for 10min at 4°C. The insoluble material was cleared by centrifugation (13000rpm for 15min at 4°C) and the resulting protein extracts (cytosolic fraction) were stored at -70°C until use. Protein concentration was determined using the BCA method.
Data are presented as mean ± S.E.M. unless otherwise noted. For Fura-2 experiments, the statistical comparisons among different experimental groups were performed by analysis of covariance ANCOVA.
Primary cultures of OPCs were first loaded with a membrane-permeable form of the Ca++ indicator dye Fura-2, before treatment with 20mm K+ to activate voltage-operated Ca++ channels by plasma membrane depolarization. Our data showed enhanced Ca++ influx in OPCs occurred when high K+ was introduced into the medium (Figure 1A). (Note: each line represents an analysis of a single cell). High K+ induced a biphasic increase in OPC intracellular Ca++ ([Ca++]int). The first phase consisted of a sharp peak characteristic of a transient, large increase in [Ca++]int (Peak) (Figure 1A), which was followed by a second phase of slowly declining internal Ca++ concentrations (Plateau phase) (Figure 1A). Importantly, increases in Fura-2 signal in these cells were abolished in the presence of zero Ca++, and were blocked by Cd++, verapamil and nifedipine, confirming that this rise in [Ca++]int resulted from Ca++ influx via L-type VOCCs (Figure 1B). In support of this, the amplitude of Ca++ uptake was enhanced by Bay K 8644, an L-type Ca++ channel agonist which prolongs single channel open time without affecting the close time (Figure 1B).
Imaging experiments were performed at 1, 2, 3 or 4 days in vitro (DIV) allowing analysis of the developmental regulation of voltage-gated Ca++ influx. Immediately after shake-off from mixed glial cultures, OPCs were grown in the presence of mitogens [PDGF (10ng/ml) and bFGF (10ng/ml)] for 48 hours (1 and 2DIV) and then induced to exit the cell cycle and differentiate by transferring the cells to a mitogen-free medium (mN2). After shifting to the differentiation medium there is a sharp decrease in cell division, a decline in early immunocytochemical markers, such as NG2 and A2B5, and an increase in intermediate (e.g. O4) and mature (e.g. O1, MBP) markers consistent with differentiation of the OPCs (Paez et al., 2009a). During the first two DIV in the presence of growth factors 75% of the cells were NG2+ and 95% of the cells were A2B5+, 25% of the cells were O4+ and 8% were O1+. After switching to a growth factor-free medium (mN2) for two days, immunocytochemical staining of the cells demonstrated that less than 25% of the cells remained NG2+, 70% of the cells became O4+ and 35% of the cells started to express O1. Under these conditions, Ca++ uptake in OPCs after depolarization occurred at 1 and 2 DIV, but not at 3 and 4 DIV (Figure 2H). Similar results were found in OPCs cultured in PDGF alone (Figure 2I). In the last experiment, the cells were allowed to divide and spontaneously differentiate (i.e. progress through the lineage under the same culture conditions). This experiment demonstrates that high levels of VOCC expression is a property of immature OPCs and does not solely depend on the growth factors used in the culture medium.
Since no culture is perfectly synchronous, we performed Ca++ imaging on individual cells, saved the data, removed the slide, and immunostained for stage-specific markers to determine the phenotype of the cells from which we had just obtained Ca++ entry data. This involved re-locating the cells with the computer driven stage after storing the coordinates of the cells. Such an experiment is shown in Figure 2A-C where we performed immunocytochemical staining for the NG2 antigen after confocal Ca++ imaging of the field of OPCs that had responded to high K+. Examination of the same field after NG2 staining indicated that NG2+ OPCs responded to depolarization with large increases in intracellular Ca++ (Figure 2G). In contrast, the more mature O1+ cell population responded with a significantly smaller increase in the Fura-2 signal under the same high K+ treatment (Figure 2D-F). Additional experiments measuring [Ca++]int levels after high K+ depolarization with other differentiation markers (e.g. PDGFrα, O4 and MBP) provided further evidence for enhanced Ca++ influx in immature cells (PDGFrα+, NG2+, O4+), which then declined as the OPCs matured (O1+, MBP+) (Figure 2J). Taken together these data suggest that VOCC influx was greatly reduced with OPC differentiation.
We performed “in situ” experiments in live tissue sections to examine Ca++ influx in GFP labeled OPCs isolated from transgenic mice in which the expression of GFP is driven by the PLP promoter (Mallon et al., 2002). In these mice GFP expression provides a convenient marker for cells in the oligodendroglial lineage, thus facilitating experiments to identify OPCs and OLs in live tissue slices. We focused our in situ measurements of OPC Ca++ influx in slice preparations containing the lateral ventricle subventricular zone (SVZ) and corpus callosum (CC) since these regions have been well studied as sources of OPCs. Our goal in these experiments was to confirm the existence of VOCC activity in OPCs in situ. Recordings were made at postnatal day 4 and 8 (P4, P8). OPCs in the SVZ represent precursor and migrating cells, and OPC/OLs in the CC include more mature cells. Slice preparations containing the SVZ and CC were first loaded with the Ca++ indicator dye Fura-2, before treating with high K+ to activate Ca++ entry via VOCCs. Our in situ data from the SVZ showed a significant Ca++ influx in OPCs (Figure 3A and C). Importantly, increases in Fura-2 signal in these SVZ cells were abolished in the presence of zero Ca++, and were blocked by verapamil and nifedipine, confirming that this rise in [Ca++]int results from Ca++ influx via VOCCs (Figure 3E). In agreement with our previous results, Ca++ influx after high K+ depolarization was significantly greater in immature OPCs from the SVZ vs CC OLs suggesting that VOCCs may play a role during the early stages of OPC maturation (Figure 3B, D and F).
Since direct phosphorylation by different kinases is one of the most important mechanisms involved in VOCC modulation, we examined the effect of serine-threonine (Ser/Thr) kinases on Ca++ influx mediated by VOCCs in OPCs. Using Fura-2 Ca++ imaging and high K+ depolarization we tested PKC activation with phorbol 12-myristate 13-acetate (PMA), a biologically active phorbol ester; and PKC inhibition by chelerythrine. These studies were performed on isolated OPCs in culture. The data indicated that 10μM PMA applied two minutes before high K+ stimulation markedly increased the peak of Ca++ influx evoked by depolarization of OPCs (Figure 4B). In contrast, Ca++ influx induced by high K+ was markedly reduced when PKC activity was inhibited by 50μM chelerythrine (Figure 4C). These results clearly suggest that, in OPCs, PKC activity enhances VOCC function. Since this experiment did not determine the effect of activating PKC after K+ stimulation we also examined Ca++ influx by adding PMA 2min after the K+ stimulus had begun. As shown in Figure 4D the Fura-2 ratio of OPCs responding to 20mM K+ was not significantly different compared to control OPCs when PMA was added to the culture medium during K+ depolarization (Figure 4A, E). These data clearly indicate that PKC modulates VOCC activation without affecting channel inactivation.
The influence of PKA on OPC Ca++ channel function is unknown; several studies in neural cells suggest that PKA acts to depress whole-cell Ca++ currents by affecting a hyperpolarizing shift in the voltage dependence of inactivation for the current. If under basal conditions PKA acts to suppress VOCC function, then an inhibitory influence over PKA would be expected to result in increased VOCC activity. We examined these ideas in the following experiments. First, we examined the effect of H89, a specific PKA inhibitor, on Ca++ influx in OPCs. Exposure to high K+ triggered a significantly larger increase in the [Ca++]int levels in OPCs pretreated with 25μM H89 compared to responses in control cells (Figure 5A). We also examined the effects of PKA activators such as 8-PIP-cAMP and 8-AHA-cAMP on Ca++ responses in OPCs. The results showed that the activation of PKA by either of these compounds significantly decreased the voltage-operated Ca++ entry compared to OPCs that were not exposed to these drugs (Figure 5A). This decrease in Ca++ entry indicates that under basal conditions PKA acts to suppress VOCC function in OPCs.
In the experiments described above Ca++ imaging measurements (high K+ depolarization) were performed after 2min of drug exposure. Treating the cells with these PKA modulators before and during depolarization with high K+ allowed us to analyze the effect of this kinase on both VOCC activation and inactivation. To study the role of PKA in VOCC inactivation, OPCs were treated with 8-PIP-cAMP and 8-AHA-cAMP during the depolarization stimulus. As shown in Figure 5A and B the activation of PKA after K+ stimulation caused a significant decrease in the [Ca++]int levels during the peak as well as during the plateau phase indicating that PKA promotes VOCCs inactivation.
Taken together these data suggest that in primary OPC cultures Ser/Thr kinases play a significant role in modulating voltage mediated Ca++ influx in OPCs. The results indicate that PKC and PKA activities influence VOCC-dependent Ca++ influx and that they have opposite effects on the modulation of Ca++ influx evoked by depolarization in OPCs.
Several studies in the literature indicate that Ca++ is important in OL and OPC process extension (Pende et al., 1997; Stariha et al., 1997; Yoo et al., 1999). Previous studies in our lab indicated that VOCC activation induced OPCs to extend sheets and processes (Paez et al., 2007). This was an important finding not only because it established a link between process extension and intracellular Ca++ levels but also because little attention has been given to the role of VOCCs in OPC or OL function.
Yoo et al. (1999) reported that activation of PKC in OLs caused an increase in process extension as well as an increase in intracellular Ca++ levels. Since increased levels of both PKC activity and intracellular Ca++ lead to process outgrowth in OPCs, we investigated the possibility that PKC was influencing Ca++ levels leading to process extension by VOCC modulation. OPCs were exposed to varying levels of the PKC activator PMA (0.1, 1 and 5μM) for two days. Process extension was evaluated by determining the percentage of OPCs with processes that had a length equal to or greater than four times the mean cell body diameter of the OPC population. Figure 6A shows that there was a positive correlation between the concentration of PMA in the media and the percentage of OPCs with processes longer than 4 times the cell body diameter. In control media, OPCs with long processes comprised approximately 30% of the population. As the concentration of PMA was increased, there was a parallel increase in the percentage of these cells. For example, at a PMA concentration of 1μM, the percentage of OPCs with long processes was about 57% (Figure 6A). Furthermore, the total number of primary and secondary processes was determined in control and PMA-treated OPCs after 48h. As shown in Figure 6B the total number of primary processes per cell (PP/Cell) as well as the number of secondary processes per primary process (SP/PP) was significantly higher compared to control OPCs when PMA was added to the culture medium.
Once the effects of PMA on OPC process formation were determined, we examined the activity of PKC in OPC primary cultures under our experimental conditions. Cytosolic proteins from untreated and PMA-treated OPCs were assayed for PKC activity using a non-radioactive assay (see Materials and Methods). PMA increased PKC activity at least 4-fold during the first hour of treatment, whereas the presence of the PKC inhibitor chelerythrine inhibited this activation by ~70% (Figure 6C). The level of inhibition was determined by a comparison of the areas under the activity peaks in Figure 6C. These data indicate that PKC activation for 1 hour is enough to induce OPC process extension 24-48h later, suggesting that OPCs do not need continuous PKC activation to sustain process extension.
Calcium-channel blockers were used to test whether voltage-operated Ca++ entry was associated with the morphological transformation of OPCs promoted by PKC activation. OPCs treated with 1μM PMA were incubated for 48 h either in the presence or absence of the VOCC blockers, verapamil and nifedipine, and process elongation was evaluated as described above. In the presence of verapamil and nifedipine, there was strong inhibition of the elaboration of processes and membrane sheets induced by PMA in OPCs (Figure 6D). Figure 6E illustrates fluorescent images of OPCs treated with PMA. After two days the OPCs, which were generally bipolar in control media, elaborated large processes in the presence of 1μM PMA (Figure 6Eb). This morphological change was significantly reversed in the presence of 25μM or 50μM verapamil (Figure 6Ec and d). The results of these experiments clearly indicate that VOCCs play a key role in mediating the morphological changes through PKC activation in OPCs.
To assess the contributions of TK activation on voltage-operated Ca++ uptake we used genistein, a specific inhibitor of tyrosine protein kinases that acts by binding to the ATP site of the TK, and we also use an inhibitor of tyrosine phosphatases, sodium orthovanadate. Pretreatment of OPC cultures with genistein resulted in a dose-dependent reduction in VOCC Ca++ influx compared to control cells (Figure 7A-B, all data not shown). In contrast, OPCs which were treated with sodium orthovanadate showed a dose-dependent activation of Ca++ influx across VOCCs (Figure 7C all data not shown). Additionally, orthovanadate increased not only Ca++ influx during high K+ stimulation but also caused a sustained increase in [Ca++]int during the plateau phase (Figure 7C-D). This means that the [Ca++]int remained high after the initial transient influx of Ca++ into the cell. These results clearly suggest that TKs exert an activating influence on VOCC function in OPCs.
PDGF is a potent mitogen that induces early OPCs to proliferate in vitro and can prevent premature differentiation in vivo (Noble et al., 1988; Raff et al., 1988; Calver et al., 1998). PDGF exerts its effect by interaction with intrinsic tyrosine kinase receptors (TKr) and Ca++ signals activated after membrane receptor recruitment. This is one of the most conserved immediate responses triggered in the target cells in which PDGF exerts mitogenic activity. Furthermore, the tyrosine kinase activity of the receptors has been found to be essential for transmission of the mitogenic signal into the cell (Escobedo et al., 1988). Accordingly, we used the PDGF response in OPCs as a model to probe the role of TKr on OPC Ca++ uptake after membrane depolarization. OPCs were incubated with different concentrations of PDGF for 1 hour prior to exposure of the cells to medium containing high K+. Exposure to high K+ triggered a significantly larger increase in the Fura-2 signal in OPCs pre-treated with 20ng/ml PDGF compared to responses in non-treated cells (Figure 8A). As the concentration of PDGF was increased, there was a parallel increase in the Ca++ influx across VOCCs. For example, at a PDGF concentration of 100ng/ml, the average amplitude of the Fura-2 signal was ~45% higher compared to non-treated cells (Figure 8A). Additionally, in as little as 20ng/ml PDGF, OPCs incubated with the selective PDGF TKr inhibitor, AG-1296, showed a ~50% decrease in the depolarization-induced Ca++ entry (Figure 8B). The peak [Ca++]int mobilization decreased to 125 ± 5.2 nM in OPCs treated with 1μM AG-1296 vs. 221 ± 3.2 nM in buffer treated cells (P ≤0.01) (Figure 8B and C). Importantly, differences in Ca++ entry between OPCs treated with 20ng/ml and 40ng/ml PDGF were blocked by genistein and were abolished in the presence of AG-1296 (Figure 8C), confirming that TKs are directly involved in PDGF-mediated modulation of Ca++ influx via VOCCs. These results are consistent with the notion that TKr, such as PDGFr, enhance Ca++ influx induced by depolarization in OPCs.
While the effect of PDGF on OPC migration has been well established (McKinnon et al. 2005; Milner et al., 1997; Frost et al., 2009) little has been known about its mechanism. We recently found increased Ca++ uptake via VOCCs associated with enhanced OPC motility, and this effect was accompanied by increases in the amplitude of spontaneous somatic Ca++ transients (Paez et al., 2009b).
For this reason, we examined the role played by VOCCs on PDGF-induced OPC migration by performing cell migration experiments in the presence of PDGF and pharmacological agents to stimulate or inhibit voltage-gated Ca++ influx. First, we examined the effect of PDGF on OPC migration by means of time-lapse video microscopy performed over a period of 24 hours in medium containing different concentrations of PDGF. In this time-lapse cell migration assay, cell movement was assessed by calculating the average cell migration velocity and the total distance traveled by the cell. For this analysis, only OPCs moving more than 50μm in 6 hours were scored (Figure 9A). Tracking of cells was performed using the SlideBook™ 4.1 data analysis program as previously described (Paez et al., 2009b). Under basal experimental conditions (10ng/ml PDGF) the mean rate of OPC migration was 33 ± 2.2 μm/h (Figure 9B). At higher PDGF concentrations (80ng/ml), the average OPCs velocity was almost 70% higher than in basal conditions (Figure 9B). We found a corresponding increase in the total OPC migration distance after 8 hours (Figure 9C). Second, we assessed the effect of specific VOCC blockers such as nifedipine and verapamil on PDGF-dependent modulation of OPC migration. These treatments resulted in a significant slowdown of OPC movement (Figure 10A), indicating that VOCCs, known to contribute to homeostatic Ca++ balance in OPCs and other cells, are important components in the mechanism of PDGF on OPC migration. Furthermore, stimulation of Ca++ influx through VOCCs (through high K+ treatment) significantly increased the PDGF effect on cell movement (Figure 10A), as did treatment of the cells with Bay K 8644, an L-type VOCC agonist (Figure 10A). These data show that changes in [Ca++]int resulting from the modulation of voltage-gated Ca++ influx provide a powerful means by which OPC migration may be regulated by PDGF. Additionally, they are the first to demonstrate that extracellular Ca++ influx through VOCCs is an important component in the mechanism of PDGF on OPC motility.
Since the PDGFr is directly involved, through its tyrosine kinase activity, in modulating Ca++ influx via VOCCs, we investigated the role of tyrosine protein kinases in PDGF-modulation of OPC migration. We tracked OPCs in medium containing 40ng/ml of PDGF and the general TK antagonist genistein. Figure 10B shows that the average speed of OPC migration was lower when genistein was present in the media. For example, in 40ng/ml PDGF the average migration speed of OPCs was 52 ± 5.1 μm/h (n=20), but as the concentration of genistein was increased, it fell to an average speed of 14 ± 3.8 μm/h (n=20) in the presence of 10μM genistein (Figure 10B). Furthermore, PDGF modulation of OPC velocity essentially disappeared when the selective PDGF receptor TK inhibitor, AG-1296, was added to the external medium (Figure 10B). In contrast, PDGF-induced migration was unaffected by PD173074, a potent and selective inhibitor of bFGF TKr signaling in OPCs (Bansal et al., 2003), and was increased by the tyrosine phosphatase inhibitor, sodium orthovanadate (Figure 10B). These results indicate that the tyrosine kinase activity of the PDGFr is a key component of the mechanism of action of PDGF on OPC movement.
Voltage-activated Ca++ currents have been examined in OPC culture preparations from a range of different tissues, providing somewhat differing results. Perinatal progenitor cells taken from rat optic nerve were found to lack Ca++ currents (Barres et al., 1990), although their presence was detected in progenitor cells obtained from adult optic nerve (Borges et al., 1995). Perinatal OPCs from the mouse cortex (Verkhratsky et al., 1990; von Blankenfeld et al., 1992) exhibit both low-voltage and high-voltage activated (LVA and HVA) Ca++ currents, although the expression of these currents varies from cell to cell (Williamson et al. 1997). Oligodendrocyte Ca++ currents have also been examined in situ; recordings from early postnatal slices containing the corpus callosum, revealed the presence of both HVA and LVA Ca++ currents in OLs located in this white matter region (Berger et al., 1992). Supporting this finding is the evidence presented here from primary OPC cultures and tissue slices depolarized with high K+. Depolarization consistently leads to plasma membrane Ca++ entry, which is blocked in the presence of VOCC antagonists. Thus, the available evidence indicates the presence of VOCCs in OPCs and immature OLs, and that a considerable component of this current is carried by L-type channels.
Several earlier studies have reported that Ca++ responses through VOCCs appear to diminish with maturation of OLs from progenitors to mature cells in culture (von Blankenfeld et al., 1992; Berger et al., 1992; Takeda et al., 1995). At the same time other studies have described no difference in the functional expression of VOCCs in immature and mature cultured OLs (von Blankenfeld et al., 1992; Paez et al., 2008). It is possible that voltage-operated Ca++ influx plays a role during the first steps of OL maturation (e.g. migration and proliferation) because expression of VOCCs decreases during development. However, OL cultures are mixtures of cells at different stages of maturation. The earlier studies were hampered by an inability to positively identify the phenotypes of individual cultured cells from which the Ca++ influx measurements were made. In the present study we examined Ca++ influx in individual OPCs. Following acquisition of the Ca++ imaging data, the cells were immunocytochemically stained for stage-specific markers. These experiments clearly revealed developmentally regulated activity of VOCCs, e.g. mature cells expressing MBP and O1 showed a small increase in [Ca++]int after high K+ stimulation under culture conditions. In situ, Ca++ influx after K+ stimulation was significantly greater in immature OPCs from the SVZ compared to more mature OLs found in the CC. Thus, the in vitro and in situ data indicate that voltage-operated Ca++ influx, present in immature OPCs, disappeared as the cells matured indicating that VOCCs play a role during the early stages of OPCs maturation.
In this study, the effect of several kinases and phosphatases on depolarization-induced VOCC activity was investigated in OPCs. PKA modulation is a physiologically important mechanism influencing VOCC function in diverse excitable tissues such as heart and muscle. PKA modulates Ca++ influx through phosphorylation of a serine residue on the carboxy terminus distal to the calmodulin interaction sites of VOCC α1 subunit (Catterall, 2000). The influence of PKA on OPC Ca++ channel function is unknown, but several studies in neuronal cells suggest that PKA acts to depress whole-cell Ca++ currents causing a hyperpolarizing shift in the voltage dependence of inactivation for the current (Johnson et al., 2005; Kamp and Hell, 2000; Keef et al., 2001). In our experiments, PKA activation promoted two major changes in OPC Ca++ currents: a drastic reduction in the Ca++ current amplitude (peak) and the acceleration of the inactivation kinetics (i.e., significant decrease in the [Ca++]int during the plateau phase). These results clearly indicate that PKA exerts an inhibitory influence on VOCC function in OPCs.
PKC is a multigene family of 10 phospholipid-dependent, serine-threonine kinases central to many signal transduction pathways (Nishizuka, 1992). Several PKCs have been shown to regulate voltage-gated ion channels through the direct phosphorylation of the α1 subunit (Stea et al., 1995; Zhu and Ikeda, 1994). More recently the formation of a functional PKC-VOCC complex that is critical for rapid and efficient stimulation of Ca++ channel activity by PKC has been described in neurons (Chen et al., 2006). In our studies reported here we used the PKC activator PMA to demonstrate that PKC has an excitatory influence on VOCC function in OPCs participating in the depolarization-induced activation of the channels. These results were confirmed using the selective PKC inhibitor chelerythrine, which produced a clear decrease in depolarization induced Ca++ influx in OPCs. Thus, our hypothesis is that PKC and VOCC are part of a signaling cascade implicated in OPC development, of which process remodeling is only one regulated property.
While PKC activation in OPCs leads to a decrease in the number of cells acquiring a mature phenotype (Bhat et al., 1992; Radhakrishna and Almazan, 1994; Baron et al., 1998; Heinrich et al., 1999; Avossa and Pfeiffer, 1993), PKC activation in immature OLs has been reported to enhance elaborate process extensions (Althaus et al., 1991; Yong et al., 1988; 1991). Yoo et al. (1999) have shown that activation of PKC in OL not only causes an increase in process extension, but also an increase in intracellular Ca++ levels, suggesting that the PKC pathway for induction of processes is at least partially dependent upon increases in [Ca++]int (Yoo et al., 1999).
We showed that the activation of VOCCs in OPC cultures can induce process extension (Paez et al., 2007). The data presented here show that process growth after PKC activation in OPCs is influenced strongly by L-type Ca++ channels because it was significantly reduced by the L-type channel blockers nifedipine and verapamil. These experiments served to define specific channels within the VOCC family that are involved in the morphological remodeling induced by PKC in OPCs. In this regard, astrocyte morphological changes induced by PKC are accompanied by the upregulation and activation of voltage-gated Ca++ channels and are abrogated in the presence of L-type channel blockers (Burgos et al., 2007). Thus, PKC can regulate Ca++ influx in OPCs under depolarizing conditions and the evidence supports the involvement of VOCCs in OPC process remodeling induced by PKC. This represents a novel regulatory pathway involving VOCC that participates in PKC-dependent oligodendrocyte morphological differentiation.
Even though an active and direct participation of TKs in VOCC function in different tissues has been demonstrated (Keef et al., 2001; Schroder et al., 2004; Wang and Lipsius, 1998), nothing is known about VOCC regulation through tyrosine phosphorylation in OPCs. Using pharmacological approaches, we found that TKs exert an excitatory influence on VOCC function in OPCs. Inhibition of TKs with genistein resulted in a dose-dependent reduction in VOCCs Ca++ influx whereas inhibition of tyrosine phosphatases (with sodium orthovanadate) showed a dose-dependent activation of Ca++ influx across VOCCs. These data are consistent with those of Cataldi et al. (1996) and Vela et al. (2007) who showed that TK inhibition reduced L-type Ca++ channel activity evoked by high K+ in GH3 cells.
The effect of TK receptor (TKr) activity on OPC voltage-operated Ca++ influx was examined using PDGF as a model TKr system active in OPCs. Our present findings show that PDGF produces an increase in the activity of VOCC channels in OPCs, suggesting a novel and relevant physiological role of PDGF in the control of the OL lineage progression.
PDGF binds to the PDGFα receptor, which belongs to a family of TKrs that has its own cytoplasmic associated TK activity (Taniguchi, 1995). PDGF promotes OPC proliferation, migration and survival via PDGFrα, the only PDGF receptor isoform in these cells detected by ligand binding (Pringle et al., 1989) and molecular expression (McKinnon et al., 1990). The TK activity of this receptor has been found to be essential for the transmission of the mitogenic and chemotactic signaling in the cell (Escobedo et al., 1988). Our results are consistent with the notion that TKr, such as PDGFr, enhance Ca++ influx induced by depolarization in OPCs. The PDGF effect on OPC Ca++ entry was abolished in the presence of AG-1296, a selective PDGF receptor TK inhibitor, confirming that the TK activity of PDGFr is essential for VOCC modulation. Furthermore, we have shown that extracellular Ca++ uptake through VOCCs, resulting from PDGFr modulation, is an important component in the mechanism of OPC migration. In this paper, we propose that VOCCs modulation by PDGFr is a key component of the migratory mechanism activated by PDGF in OPCs. Our conclusion is supported by experiments in which PDGF promotion of cell migration was efficiently obliterated by specific VOCC inhibitors as well as TKr antagonists.
In summary, the present work demonstrates that PKC and TKr enhance Ca++ influx induced by depolarization in OPCs, while PKA has an inhibitory effect. These kinases modulate voltage-operated Ca++ uptake in OPCs and therefore participate in the regulation of essential early OPC developmental functions such as processes extension and migration.
Grant sponsors: This investigation was supported (in part) by NIH grant NS33091 (ATC) and a Postdoctoral Fellowship FG1723A1/1 (PMP) from the NMSS.