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Keywords:

  • CREB phosphorylation;
  • Signal transduction;
  • Oligodendrocyte differentiation

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

Abstract : We have shown previously that the pattern of expression of the transcription factor CREB (cyclic AMP-response element binding protein) in developing oligodendrocytes (OLGs) suggests a role during a period that precedes the peak of myelination in rat brain. We have now investigated the signaling pathways that could be responsible for activating CREB by phosphorylation at different stages along OLG maturation. CREB phosphorylation was studied in short-term cultures of immature OLG precursor cells and young OLGs isolated from 4- and 11-day-old rat cerebrum, respectively. The results indicated that at both developmental stages, CREB phosphorylation could be stimulated by either increased concentrations of cyclic AMP and cyclic AMP-dependent protein kinase activation or increased Ca2+ levels and a protein kinase C activity. The results also showed that CREB phosphorylation in immature OLG precursor cells could be up-regulated by treatment with histamine, carbachol, glutamate, and ATP (neuroligands known to increase Ca2+ levels in these cells), by signaling cascade(s) that involve a protein kinase C activity, as well as the mitogen-activated protein kinase pathway. In contrast, in cells isolated from 11-day-old rats, at a developmental stage that immediately precedes the beginning of the active period of myelin synthesis, CREB phosphorylation was only stimulated by treatment with the β-adrenergic agonist isoproterenol in a process that appears to be mediated by a cyclic AMP/cyclic AMP-dependent protein kinase-dependent pathway. These results support the idea that CREB could be a mediator of neuronal signals that, coupled to specific signal transduction cascades, may play different regulatory roles at specific stages along OLG differentiation.

Oligodendrocytes (OLGs), the cells that produce the myelin membrane in the CNS, undergo several stages of differentiation leading to the formation of myelinating cells. After several rounds of proliferation, the immature OLG precursors, characterized by their reactivity with the A2B5 (Levi et al., 1987) and/or the O4 (Sommer and Schachner, 1981) monoclonal antibodies, develop into committed young OLGs. These multipolar cells, which react with both O4 and O1 antibodies (Sommer and Schachner, 1981), further mature into fully differentiated OLGs that express myelin proteins, including the enzyme 2′, 3′-cyclic nucleotide 3′-phosphohydrolase, myelin basic protein (MBP), and proteolipid protein. Although the sequence of molecular events that control such an orderly series of developmental changes is still poorly understood, results from different laboratories have shown that OLG differentiation is stimulated by cyclic AMP (cAMP) (McMorris, 1983 ; Pleasure et al., 1986 ; Raible and McMorris, 1989, 1990). The mechanism(s) underlying this effect of cAMP are not clear. However, we have shown previously that developing OLGs express elevated levels of the transcription factor cAMP-response element binding protein (CREB) (Sato-Bigbee and Yu, 1993 ; Sato-Bigbee et al., 1994).

CREB belongs to a large family of bZip (basic leucine zipper) transcription factors that bind to a consensus cAMP-response element sequence (TGACGTCA) present in the promoter of cAMP- and/or Ca2+-responsive genes (Montminy et al., 1990 ; Sheng et al., 1991). Our previous results suggested that CREB may be a mediator of the cAMP-dependent stimulation of MBP expression and cell process outgrowth in developing OLGs (Sato-Bigbee and DeVries, 1996a,b).

Transcriptional activation by CREB is regulated by phosphorylation at Ser133 (Yamamoto et al., 1988), and this phosphorylation was attributed initially to cAMP-dependent protein kinase (PKA) (Yamamoto et al., 1988 ; Gonzalez et al., 1989). Later studies have shown that this phosphorylation may also be attributed to Ca2+/calmodulin-dependent kinases (CamKs) (Sheng et al., 1991), protein kinase C (PKC) (Xie and Rothstein, 1995), and growth factor-induced kinases, including p90 ribosomal S6 kinase (RSK), a member of the mitogen-activated protein kinase (MAPK) pathway (Ginty et al., 1994 ; Xing et al., 1996). Preliminary results suggested that CREB phosphorylation in OLGs may not only result from increased cAMP levels and PKA activation, but also from elevated Ca2+ levels and PKC activity (Sato-Bigbee and DeVries, 1996b). Both cAMP and Ca2+ levels in OLGs can be regulated by several neuroligands (Kastritsis and McCarthy, 1993 ; Borges et al., 1994 ; Takeda et al., 1995), raising the possibility that CREB could function as a mediator of neuronal signals to the OLGs. Recently, Pende et al. (1997) have studied CREB phosphorylation in cultured glial precursors isolated from embryonic rat cerebral cortex and showed that in this cell population, CREB phosphorylation can be stimulated by growth factors and by glutamate or carbachol, neuroligands that increase Ca2+ levels in these cells. These authors proposed that in these glial precursors, the MAPK/RSK pathway mediates CREB phosphorylation in response to Ca2+, PKC activation, and growth factor stimulation. Their results together with our previous observations suggest that CREB may play different roles along OLG differentiation in response to different extracellular signals. To study this possibility further, we have now investigated the signaling pathways that may regulate CREB activation along the OLG lineage. The results indicated that in both immature precursor cells and committed young OLGs, CREB could be activated by increased cAMP or Ca2+ levels. However, whereas CREB phosphorylation in immature precursor cells was stimulated by neuroligands known to increase Ca2+ levels, CREB phosphorylation in more differentiated cells was increased by β-adrenergic stimulation in a process that most likely involves elevation of cAMP levels and PKA activation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

Materials

Percoll, dibutyryl cAMP (db-cAMP), isoproterenol, carbachol, histamine, glutamate, and ATP were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Matrigel was obtained from Becton Dickinson (Franklin Lakes, NJ, U.S.A.). Thapsigargin (Tg), chelerythrine, KT5720, SQ 22536, KN-93, and PD 98059 were from Calbiochem (San Diego, CA, U.S.A.). Anti-CREB and anti-phosphorylated CREB (anti-P-CREB) antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid, NY, U.S.A.). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). SuperSignal Ultra chemiluminescence detection system was purchased from Pierce (Rockford, IL, U.S.A.). All electrophoresis reagents were obtained from Bio-Rad (Richmond, CA, U.S.A.).

Isolation and culture of OLGs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

OLGs were isolated from Sprague—Dawley rat cerebrum at various postnatal days using a Percoll gradient according to the method of Berti-Mattera et al. (1984) as modified by Sato-Bigbee and DeVries (1996a). In brief, the cerebra were minced and then dissociated in Ca2+/Mg2+-free Hanks' balance salt solution, 25 mM HEPES (pH 7.2), 1 mg/ml glucose, 0.1 mg/ml DNase, and 1 mg/ml acetyltrypsin. After incubation for 45 min at 37°C, the tissue was forced through a 74-μm-pore-size nylon mesh, and the resulting cell suspension was mixed with 1.5 volumes of isosmotic Percoll and centrifuged at 30,000 g for 15 min. The band corresponding to the OLGs and their precursor cells was collected and washed with Hanks' balanced salt solution, and the final cell suspension was incubated for 40 min on tissue culture-treated plastic Petri dishes to allow the attachment of residual microglial and astrocyte contamination (~5—10%). The dishes were then gently swirled for ~10 s, and the nonadherent cells were plated in 24-well-cluster plates (5 × 105 cells/well) that had been coated previously with reduced growth factor Matrigel (15 μl/well). Cells were grown in chemically defined medium [Dulbecco's modified Eagle's medium (DMEM)/Ham F-12 medium (1 : 1, vol/vol) supplemented with 1 mg/ml bovine serum albumin, 50 μg/ml transferrin, 5 μg/ml insulin, 30 nM sodium selenite, 0.11 mg/ml sodium pyruvate, 10 nM biotin, 2 μM hydrocortisone, 15 nM triiodothyronine, 50 units/ml penicillin, and 50 μg/ml streptomycin] at 37°C in 5% CO2. Astrocyte contamination, as judged by staining with anti-glial fibrillary acidic protein antibody, was <5%.

Measurement of intracellular free Ca2+ concentration

Intracellular Ca2+ measurements were performed according to Limbrick et al. (1995). The oligodendroglial cells were loaded with 1 μM indo-1 acetoxymethyl ester dye in a physiological recording solution (145 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, pH 7.3, adjusted to 325 mOsm with sucrose). Cells were rinsed with the recording solution and incubated for an additional 15 min to allow cleavage of the indo-1 acetoxymethyl ester to the free acid by cellular esterases. The intracellular Ca2+ concentration was measured by using the ratio program of the image scan analysis mode of a confocal ACAS Ultima Interactive Laser Cytometer (Meridian Instruments, Okemos, MI, U.S.A.). To excite indo-1, a 100-mW 360-nm line of argon laser was passed through an Olympus D Plan Apo UV 40 × short working distance objective mounted on an Olympus IMT-2 inverted microscope. Emission wavelengths were passed through a 225-μm aperture before reaching the photomultiplier tubes. Photocurrents from each photomultiplier tube were then measured by a computer-driven data acquisition system. The ratio of emitted intensity at 405 nm (indo-1—Ca2+ complex) and 485 nm (free indo-1) was monitored to indicate the relative concentration of free intracellular Ca2+. Sequential images were acquired with a time resolution of 20 s.

Effect of neuroligands on CREB phosphorylation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

OLG cultures were incubated for 15 min in chemically defined medium containing 1—10 μM isoproterenol, 10—100 μM histamine, 50—500 μM carbachol, 10—100 μM glutamate, or 1—10 μM ATP. To assess the role of different protein kinases, the cells were preincubated for 10 min in the presence of the following kinase inhibitors : 10 μM chelerythrine (PKC inhibitor), 10 μM KT5720 (PKA inhibitor), 30 μM KN-93 (CamK inhibitor), or 50 μM PD 98059 [MAPK kinase (MEK) inhibitor] ; this was followed by a 15-min incubation in the presence of both kinase inhibitor and neuroligand. Inhibitor concentrations are higher than IC50 values corresponding to the purified enzymes, but are in agreement with the concentrations used by other investigators to inhibit effectively the kinases in cell culture systems (Campenot et al., 1994 ; Balboa and Insel, 1995 ; Bennett et al., 1995 ; Muthalif et al., 1996 ; Pagh-Roehl et al., 1996). After incubation, the culture plates were placed on ice, and the cells were rinsed with cold DMEM and immediately processed for western blot analysis.

Western blot analysis

OLG cultures containing equal cell numbers per well were placed on ice, rinsed with cold DMEM, and the medium rapidly removed. Cells were immediately lysed in 70 μl of 60 mM Tris-HCl buffer (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate, and 5% 2-mercaptoethanol. The samples were frozen and stored at —70°C until required. For immunoblot analysis, 20-μl samples were subjected to sodium dodecyl sulfate—polyacrylamide gel electrophoresis in 12% acrylamide gels, and proteins were then electrotransferred to nitrocellulose membranes. Nonspecific antibody binding to the blots was blocked by incubation in buffer containing 10 mM Na2HPO4, 2.7 mM KCl, and 137 mM NaCl [phosphate-buffered saline (PBS)], 3% nonfat dry milk, and 0.05% Tween 20 (pH 7.4) (blocking solution) for 1 h at room temperature. Blots were then incubated with either anti-CREB (1 : 500) or anti-P-CREB (1 : 1,000) for 2 h. The anti-P-CREB antibody specifically recognizes CREB when phosphorylated at Ser133 (Ginty et al., 1993). After three 10-min rinses with PBS, blots were incubated for 30 min in blocking solution, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1 : 3,000) for 1 h. All antibodies were diluted in blocking buffer. After two 5-min rinses in PBS containing 0.05% Tween 20 and three 5-min rinses in PBS, immunoreactive bands were detected using the SuperSignal Ultra chemiluminescent substrate. The relative amount of immunoreactivity in each band was determined by scanning densitometric analysis of the x-ray films using a Shimadzu scanner densitometer. To avoid possible differences due to the chemiluminescence detection assay, samples to be compared in each individual experiment were always run in the same gel, and detection of immunoreactive bands by chemiluminescence was carried out simultaneously in the same nitrocellulose filter.

Statistical analysis

Statistical analysis was performed by one-way analysis of variance. Differences were considered statistically significant when p values were <0.05.

CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

As shown in Fig. 1A, incubation of cultured neonatal OLGs for various times in the presence of the cell-permeable cAMP analogue db-cAMP resulted in a dramatic and sustained stimulation of CREB phosphorylation. This effect could be attributed to activation of PKA, because the stimulation of CREB phosphorylation by db-cAMP was abolished by co-incubation with KT5720, a highly specific inhibitor of this kinase (Gadbois et al., 1992) (Fig. 1B). Additional experiments showed that the cAMP-dependent phosphorylation was not affected by treatment with chelerythrine [a highly specific PKC inhibitor (Herbert et al., 1990)] or KN-93 [a CamK inhibitor (Mamiyo et al., 1993)] (data not shown).

image

Figure 1. Effect of db-cAMP on CREB phosphorylation in OLGs. A : OLGs were isolated from 3-day-old rat cerebrum. After 3 days in culture, the cells were incubated for 0 (control), 5, 15, and 45 min in the presence of 1 mM db-cAMP. Levels of phosphorylated CREB were determined by western blot analysis with anti-P-CREB antibody followed by densitometric scanning of immunoreactive bands. Control (0 min) vs. 5, 15, and 45 min : p < 0.001. B : CREB phosphorylation in cells incubated for 15 min in (a) medium alone, (b) medium + 1 mM db-cAMP, and (c) medium + 1 mM db-cAMP + 10 μM KT5720 (PKA inhibitor). a vs. b : p < 0.001 ; a vs. c : NS ; b vs. c : p < 0.001. In both A and B, the immunoreactive bands are the results from representative western blots ; the results represented in the bar graphs are the means ± SEM from three to five independent experiments.

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However, CREB phosphorylation was also highly stimulated following treatment of the cells with Tg, a specific inhibitor of the Ca2+ -ATPase responsible for the accumulation of Ca2+ in the internal stores in the endoplasmic reticulum (Thastrup et al., 1990). The effect of Tg in OLGs was determined by confocal microscopic analysis of Ca2+ levels in individual cells. As shown in Fig. 2, treatment of OLGs with Tg resulted in a transient increase in the cytoplasmic Ca2+ levels of all cells. This increase in Ca2+ levels was followed by a significant time-dependent stimulation of CREB phosphorylation (Fig. 3A). This Ca2+-dependent stimulation of CREB phosphorylation appears to involve a PKC activity, because CREB phosphorylation in the presence of Tg was decreased significantly by co-incubation with chelerythrine (Fig. 3B). Figure 3B also shows that the stimulation of CREB phosphorylation by Tg does not appear to involve activation of a CamK, because phosphorylated CREB levels in cultures incubated with Tg were not reduced by co-incubation with KN-93. The results have also shown that it is unlikely that the increased CREB phosphorylation by Tg is due to a CamK activation of adenylate cyclase and subsequent increase in cAMP and PKA activation, because the stimulation of CREB phosphorylation by Tg was not affected significantly by SQ 22536 [an inhibitor of adenylate cyclase (Goldsmith and Abrams, 1991)].

image

Figure 2. Effect of Tg on Ca2+ levels in OLGs. OLGs were isolated from 2-3-day-old rat cerebrum. After 3 days in culture, cells were incubated for various times in the presence of 1.5 μM Tg (the arrow indicates the time at which Tg was added to the cultures). Each curve represents the cytoplasmic Ca2+ levels in individual cells, as determined after incubation with indo-1 followed by image scan analysis as indicated under Materials and Methods.

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image

Figure 3. Effect of Tg on CREB phosphorylation in OLGs. A : OLGs were isolated from 3-day-old rats. After 3 days in culture, the cells were incubated in the presence of 1.5 μM Tg for 0 (control), 5, 15, 45, and 90 min. At the end of the incubation time, levels of CREB phosphorylation were determined by western blot analysis. 0 min (control) vs. 5, 45, and 90 min : p < 0.05 ; 0 min vs. 15 min : p < 0.01. B : Cell cultures were incubated for 15 min under the following conditions : (a) medium alone ; (b) Tg ; (c) Tg + 10 μM chelerythrine (PKC inhibitor) ; (d) Tg + 30 μM KN-93 (CamK inhibitor) ; (e) Tg + 5 μM SQ 22536 (adenylate cyclase inhibitor). a vs. b, d, and e : p < 0.01 ; b vs. c : p < 0.001 ; b vs. d and e : NS. In both A and B, the immunoreactive bands are the results from representative western blots ; the results in the bar graphs are the means ± SEM from three to five independent experiments.

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Thus, CREB phosphorylation in developing OLGs could be regulated by both cAMP- and Ca2+-dependent signaling pathways. To investigate whether these cascades may be differentially expressed along OLG differentiation, we studied the effect of db-cAMP and Tg on CREB phosphorylation in short-term cultures prepared from cells isolated from 4-, 11-, and 21-day-old rat cerebrum.

Cells isolated from 4-day-old rats (Fig. 4A) are immature OLG precursors that either are bipolar or possess several simple processes, can be labeled with the O4 and/or A2B5 antibodies, and are glial fibrillary acidic protein-negative. In contrast, cells isolated from 11-day-old animals (Fig. 4B) are young OLGs that are multipolar and can be labeled with the O4 and O1 antibodies ; only very few of these cells are MBP-positive. With longer times in culture, the majority of the cells isolated from both 4- and 11-day-old rats develop into mature OLGs that express MBP. Cells obtained from 21-day-old rats (Fig. 4C) are already mature MBP-positive OLGs that elaborate complex branched processes and, with longer times in culture, produce large membrane extensions.

image

Figure 4. Cell cultures prepared from oligodendroglial cells isolated from rat cerebrum at different developmental stages. Oligodendroglial cells were isolated from 4-, 11-, and 21-day-old rat cerebrum as described under Materials and Methods and cultured in chemically defined medium for 2 days. A and B : Cells isolated from 4- and 11-day-old animals, respectively, stained with O4 antibody. C : Cells obtained from 21-day-old rat brain, stained with anti-MBP antibody.

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In these cells, total CREB expression was analyzed after 36 h in culture by using an antibody that detects CREB regardless of whether the protein is phosphorylated or nonphosphorylated (Fig. 5B). As shown before by DNA binding assays (Sato-Bigbee and Yu, 1993) and immunocytochemistry (Sato-Bigbee et al., 1994), CREB expression was higher in the cells from younger animals, but it was down-regulated to background levels of detection in the cells from 21-day-old rats (Fig. 5B). Western immunoblot analysis with the anti-P-CREB antibody (Fig. 5A) indicated that in cells from both 4- and 11-day-old animals, CREB phosphorylation was highly stimulated by incubation with both db-cAMP (lanes b) and Tg (lanes c). Thus, both cAMP-and Ca2+ -dependent signaling cascades could potentially regulate CREB phosphorylation at these two different stages of OLG differentiation.

image

Figure 5. Effect of db-cAMP and Tg on CREB phosphorylation at different stages of OLG differentiation. Oligodendroglial cells were isolated from 4-, 11-, and 21-day-old rat cerebrum. After 36 h in culture, cells were incubated for 15 min in (a) medium alone, (b) 1 mM db-cAMP, or (c) 1.5 μM Tg. P-CREB (phosphorylated CREB) and total CREB (phosphorylated + nonphosphorylated protein) were analyzed by western immunoblot with their respective specific antibodies. All samples correspond to an equivalent number of cells. The results show a representative western blot from three different experiments.

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It is interesting that under the present culture conditions, the very low but still detectable basal levels of CREB phosphorylation appeared to be due solely to a PKA activity. As shown in Fig. 6, in cells from both 4-and 11-day-old rats, basal levels of CREB phosphorylation were only significantly decreased by incubation with the PKA inhibitor. Thus, an active PKA capable of phosphorylating CREB is functional in these cell cultures, even in the absence of exogenously added db-cAMP.

image

Figure 6. Effect of kinase inhibitors upon basal levels of CREB phosphorylation in cultured OLGs. Cells were isolated from 4-and 11-day-old rat cerebrum. After 36 h in culture, the cells were incubated for 15 min in (a) medium alone, (b) PKA inhibitor, (c) PKC inhibitor, or (d) CamK inhibitor. P-CREB was detected by western immunoblot. Autoradiographic films were overexposed to reveal the low basal levels of CREB phosphorylation. The immunoreactive bands are representative of three different experiments.

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Different neuroligands stimulate CREB phosphorylation at specific developmental stages

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

As indicated before, several lines of evidence have clearly shown that both cAMP and Ca2+ levels in OLGs can be increased by receptor-mediated interaction with various neuroligands. Thus, we investigated whether treatment with those ligands could also modulate CREB phosphorylation along OLG differentiation.

For these studies, cells isolated from 4-and 11-day-old rat cerebrum were incubated in the presence of histamine, carbachol, glutamate, and ATP, neuroligands known to increase the intracellular concentration of Ca2+ in neonatal OLGs (Kastritsis and McCarthy, 1993 ; He and McCarthy, 1994 ; Takeda et al., 1995), and isoproterenol, which has been shown to raise the cellular levels of cAMP in these cells (Cohen and Almazan, 1993).

As shown in Fig. 7, treatment with isoproterenol of cells isolated from 4-day-old rats resulted in only a modest increase of CREB phosphorylation. However, CREB phosphorylation in these immature OLG precursors was stimulated significantly by incubation in the presence of histamine, carbachol, glutamate, and ATP.

image

Figure 7. Effect of neuroligands on CREB phosphorylation in cells isolated from 4-day-old rat cerebrum. P-CREB expression was studied by immunoblot analysis of cells incubated for 15 min in medium alone [control (ctr)] or in the presence of 10 μM isoproterenol (iso), 100 μM histamine (his), 500 μM carbachol (car), 100 μM glutamate (glu), or 10 μM ATP. A representative western blot is shown. The results on the bar graph are the averages ± SEM from three to five independent experiments. Control vs. isoproterenol : p < 0.05 ; control vs. histamine, carbachol, glutamate, and ATP : p < 0.001.

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In contrast to the results observed in cells from 4-day-old animals, CREB phosphorylation in cells from 11-day-old rats was highly stimulated by treatment with isoproterenol (Fig. 8). However, Fig. 8 shows that CREB phosphorylation in these more mature cells was not affected significantly by incubation with histamine, carbachol, glutamate, or ATP.

image

Figure 8. Effect of neuroligands on CREB phosphorylation in OLGs isolated from 11-day-old rats. Cell cultures were incubated in the presence of various neuroligands under the same conditions indicated in the legend to Fig. 7. Relative levels of CREB phosphorylation were determined by densitometric scanning of western immunoblots. The autoradiographic bands correspond to a representative western blot. The results are the means ± SEM from three to five independent experiments. Control vs. isoproterenol : p < 0.001.

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Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

To investigate the signaling pathways that could mediate CREB phosphorylation in the presence of each neuroligand, we have examined the effect of co-incubation with various kinase inhibitors.

As shown in Fig. 9A, the stimulation of CREB phosphorylation by either histamine, carbachol, glutamate, or ATP was not observed if the incubation medium containing each neuroligand was also supplemented with the PKC inhibitor chelerythrine. In addition, the stimulation of CREB phosphorylation by histamine, carbachol, glutamine, or ATP was abolished by incubation with PD 98059, an inhibitor of MEK kinase (the protein kinase that phosphorylates MAPK in the MAPK cascade). In contrast, the stimulatory effect of these neuroligands was not affected by the presence of the CamK inhibitor KN-93 or the PKA inhibitor KT5720 (data not shown). Thus, it appears that the stimulatory effect of histamine, carbachol, glutamine, and ATP on CREB phosphorylation in cells from 4-day-old rats involves a PKC, as well as a MAPK, signaling pathway. Figure 9A also indicates that, as shown before (see Fig. 6), basal levels of CREB phosphorylation in the absence of neuroligands are not decreased by the PKC inhibitor and, in addition, are also not affected by inhibition of MEK kinase.

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Figure 9. Identification of kinases involved in the stimulation of CREB phosphorylation by different neuroligands. A : Identification of kinases that mediate the stimulation of CREB phosphorylation by histamine (his), carbachol (car), glutamate (glu), and ATP. Cultures of cells isolated from 4-day-old rats were preincubated for 10 min in medium alone, or in the presence of either 10 μM chelerythrine (PKC inhibitor) or 50 μM PD 98059 (MEK inhibitor). After this, cells were incubated for an additional 15 min under the following conditions : neuroligand alone (black columns) ; neuroligand + PKC inhibitor (white columns) ; neuroligand + MEK inhibitor (hatched columns). Control cultures (ctr) were incubated under similar conditions, but in media lacking neuroligands. The results are the means ± SEM from three independent experiments. SEM values were <15% of the mean values. Histamine, carbachol, glutamate, and ATP vs. neuroligands + MEK or PKC inhibitors : p < 0.02. B : Identification of kinases that mediate the stimulation of CREB phosphorylation by isoproterenol. Cultures of cells isolated from 11-day-old rats were preincubated for 10 min in (a) medium alone, or in the presence of (b) 10 μM KT5720 (PKA inhibitor), (c) 30 μM KN-93 (CamK inhibitor), (d) 10 μM chelerythrine (PKC inhibitor), or (e) 50 μM PD 98059 (MEK inhibitor). After this, the cells were incubated for an additional 15 min in the same media (control, black columns) or in the same media + isoproterenol (isoproterenol, hatched columns). Results are the means ± SEM from three individual experiments. SEM values were <15% of the mean values. Control, a vs. b : p < 0.01. Isoproterenol, a vs. b : p < 0.001.

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In contrast to the results shown above, the stimulation of CREB phosphorylation by isoproterenol observed in cultures prepared from cells isolated from 11-day-old rats was abolished by co-incubation with the specific PKA inhibitor KT5720 (Fig. 9B). On the other hand, the isoproterenol effect on CREB phosphorylation was not affected significantly by co-incubation with either PKC, CamK, or MEK inhibitors. Therefore, the β-adrenergic stimulation of CREB phosphorylation appears to depend on the activation of a cAMP/PKA signaling cascade.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

The pattern of CREB expression in OLGs suggests a role in a developmental period that precedes the peak of myelination in rat brain (Sato-Bigbee and Yu, 1993 ; Sato-Bigbee et al., 1994). Moreover, CREB synthesis inhibition studies supported the idea that this transcription factor is one of the mediators of the cAMP-dependent stimulation of OLG differentiation (Sato-Bigbee and DeVries, 1996a). We have now studied the signal transduction systems that could regulate CREB phosphorylation along OLG maturation. The present results indicated that in OLG precursor cells as well as in young OLGs, CREB phosphorylation could be stimulated by elevating the intracellular levels of cAMP or Ca2+. Although the stimulation of CREB phosphorylation by increased cAMP levels depends on PKA activation, the increased CREB phosphorylation that follows a rise in Ca2+ levels appears to involve a PKC activity. Results from several laboratories (Cohen and Almazan, 1993, 1994) have shown that Ca2+ and cAMP levels in cultured OLGs can be increased by receptor-mediated interaction with several neuroligands, raising the possibility that neuronal signals mediated by neurotransmitters could regulate CREB phosphorylation in OLGs. The expression of some of those receptor—neurotransmitter systems is regulated developmentally, showing lower levels in the more mature cells (He and McCarthy, 1994 ; Takeda et al., 1995), further suggesting the role of neuronal signals in modulating OLG maturation.

Although most studies on OLG development use cells isolated from neonatal brain or spinal cord, which are then cultured for various days to allow their differentiation to occur in vitro, recent results from He et al. (1996) indicated that neonatal OLGs become unresponsive to neurotransmitters after several days in culture in the absence of neurons. These authors have shown that neuronal contact and activity may contribute to maintaining neurotransmitter-activated signaling pathways coupled to mobilization of Ca2+ in OLGs. Thus, we decided to study CREB phosphorylation and the effect of neuroligands in cells that were isolated directly from rat cerebrum at different postnatal ages and were then allowed to grow in vitro for only 36 h. One might expect that these cells would still express the signaling systems that they possessed in the brain before their isolation. Using these cell cultures, we have shown that different signaling cascades appear to regulate CREB phosphorylation at different stages of OLG development. We have found that in the immature OLG precursors, CREB phosphorylation was stimulated by histamine, carbachol, glutamate, or ATP, all neuroligands that have been shown to increase Ca2+ levels in OLGs (Kastritsis and McCarthy, 1993 ; Borges et al., 1994 ; Takeda et al., 1995). Although CREB is known to be a substrate of CamK (Sheng et al., 1991), our results indicated that in the oligodendroglial cells, the signaling pathway(s) responsible for this stimulation appear to involve a PKC activity, as well as a MAPK pathway. Similar results were shown recently by Pende et al. (1997), who proposed that CREB phosphorylation in glial cell progenitors in response to glutamate, carbachol, and basic fibroblast growth factor, is mediated by the MAPK/RSK pathway with involvement of PKC. In relation to these observations, Larocca and Almazan (1997) and Pende et al. (1997) have shown that treatment of OLG precursor cells with carbachol results in activation of MAPK in a process that involves PKC. At this moment, it is difficult to determine the possible function of a MAPK/CREB pathway in the OLG lineage. However, several indirect lines of evidence suggest a role in regulating cell proliferation. Carbachol treatment of OLG progenitors not only results in MAPK activation (Larocca and Almazan, 1997 ; Pende et al., 1997) and CREB phosphorylation (present results ; Pende et al., 1997), but also elevates c-fos gene expression accompanied by increased cell proliferation (Cohen et al., 1996). In addition, results from Bhat and Zhang (1996) and Pende et al. (1997) have demonstrated the activation of MAPK by treatment of OLG precursors with platelet-derived growth factor and basic fibroblast growth factor, two factors known to promote OLG proliferation (Bogler et al., 1990 ; McKinnon et al., 1990).

It is interesting that CREB phosphorylation in young OLGs isolated from rat cerebrum at 11 days of age, a developmental period that immediately precedes the beginning of the active period of myelination, showed a different response to neuroligands from the one observed in the immature precursors. We have now shown that in these more differentiated cells, CREB phosphorylation was not affected by the neuroligands that are known to increase Ca2+, but was instead highly stimulated by treatment with isoproterenol, a β-adrenergic agonist that raises cAMP levels in the OLGs (Cohen and Almazan, 1993). Our results also indicated that this stimulatory effect may be mediated by a PKA activity.

In this regard, Raible and McMorris (1990) have clearly shown that cAMP stimulates OLG differentiation without changing the lineage commitment or the proliferation rate of the progenitor cells. Although the mechanism(s) underlying this action of cAMP are not well understood, our previous results suggested that the stimulatory effect of the cyclic nucleotide may be mediated at least partially by CREB. We have shown before that inhibition of CREB synthesis in developing OLGs abolished the cAMP-dependent stimulation of MBP expression and complex process outgrowth (Sato-Bigbee and DeVries, 1996a). The present results indicating that CREB phosphorylation in young OLGs is stimulated by treatment with db-cAMP or by isoproterenol, in a process that requires an active PKA, further support a role for CREB as a mediator of the action of cAMP on OLG maturation. Raible and McMorris (1989) have shown that db-cAMP only stimulates OLG differentiation at a specific developmental period. These authors have demonstrated that db-cAMP treatment does not increase the number of cells expressing MBP until just before OLGs positive for this antigen appear in the control cultures, but cells are no longer responsive to db-cAMP thereafter, when cultures show more mature OLGs. According to our present results, this “cAMP-responsive” period appears to overlap with the time at which CREB phosphorylation in young OLGs is highly stimulated by β-adrenergic treatment. Although still more experimental evidence is required to draw a direct correlation between all of these observations, it is tempting to suggest that β-adrenergic stimulation followed by PKA activation and CREB phosphorylation could be at least one of the signals triggering the final stage of OLG maturation that finally results in active myelin synthesis.

In summary, we have shown that CREB phosphorylation along the oligodendroglial lineage can be stimulated by raising the intracellular levels of either cAMP or Ca2+ and that different neuroligands and signal transduction systems may regulate CREB activation at specific developmental stages. These observations raise the possibility that CREB may play different roles at different stages of OLG differentiation and myelination.

Experiments are currently in progress to explain the bases for the different response to neuroligands observed in immature precursors versus young OLGs. The experiments reported in this study were carried out using each neuroligand at a wide range of concentrations, which include the maximal amounts used by other investigators, to study the effect of these same neuroligands on either Ca2+ or cAMP levels. However, several important questions should be addressed. Do neuroligands that are known to elevate Ca2+ levels in neonatal cells have a different effect on CREB phosphorylation in cells from 4- and 11-day-old rats due to a different capacity to regulate Ca2+ levels at different stages of OLG differentiation ? This possibility could explain the contradictory results indicating that elevation of Ca2+ levels by Tg highly stimulates CREB phosphorylation in cells from both age groups. Similarly, the dramatic stimulation of CREB phosphorylation by isoproterenol observed in cells from 11-day-old rats may be due to an increased capacity of these cells to generate cAMP in response to β-adrenergic stimulation. In support of this idea is the observation that if cAMP levels are raised by supplementing the incubation medium with db-cAMP, CREB phosphorylation is highly stimulated in cells from both 4- and 11-day-old rats. Moreover, preliminary studies suggest that the expression of β-adrenergic receptor in OLGs may be developmentally regulated (unpublished observations). The answers to these questions should provide important clues to understand further the signaling pathways that control OLG proliferation and differentiation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation and culture of OLGs
  5. Effect of neuroligands on CREB phosphorylation
  6. RESULTS
  7. CREB phosphorylation along OLG differentiation can be stimulated by elevation of cAMP or Ca2+ levels
  8. Different neuroligands stimulate CREB phosphorylation at specific developmental stages
  9. Specific kinase systems are involved in mediating the stimulation of CREB phosphorylation by different neuroligands
  10. DISCUSSION
  11. Acknowledgements

The authors thank Drs. John W. Bigbee, George H. DeVries, and Karun Sharma for helpful discussions. This work was supported by grant NS35097 from the National Institutes of Health.

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