Article |
Correspondence to Holly Colognato: colognato{at}pharm.sunysb.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations used in this paper: Csk, COOH-terminal Src kinase; ERK, extracellular signalrelated kinase; FN, fibronectin; GalC, galactocerebroside; Lm2, laminin-2; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; NRG, neuregulin; PDGF
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
However, a key prediction is that each integrin would be able to trigger associated signaling molecules whose effects are specific for one component of cell behavior. Our current understanding of integrin signaling within developing systems has not yet confirmed the identity of signaling molecules with the required specificity. The Src family kinases (SFKs) are nonreceptor tyrosine kinases that integrate external signals received through both integrin and growth factor receptors and, thus, are good candidates to transduce signals that regulate both integrin- and growth factordriven phases of oligodendrocyte development. One of these, Fyn, is expressed throughout the brain, in both neurons and glia, but the peak of its activity during development can be correlated with myelination (Umemori et al., 1992; Osterhout et al., 1999). Fyn may be involved in the oligodendrocyte differentiation process, because transgenic mice lacking Fyn activity are hypomyelinated (Umemori et al., 1994; Sperber et al., 2001) and cultured oligodendrocytes from Fyn / mice or those expressing dominant-negative Fyn show defects in the numbers of newly formed oligodendrocytes, as well as in the formation of complex branches of myelin membrane (Osterhout et al., 1999; Sperber and McMorris, 2001). Furthermore, mice deficient in the laminin 2 chain have a similar region-specific hypomyelination, suggesting that Fyn and laminins may operate in the same signaling pathway, and that integrin receptors may contribute to this pathway (Chun et al., 2003). Another SFK, Lyn, is also expressed in oligodendrocytes, but no function has been described. However, in the hematopoietic lineage, ß1 integrin has been found in a complex with Lyn after fibronectin (FN)-mediated adhesion (Miller et al., 1999), and it was recently shown that ß1 and Src can be directly associated, indicating that different cell types may regulate unique combinations of SFKintegrin associations based on the cell functions required (Arias-Salgado et al., 2003).
Distinct roles for Fyn and Lyn could provide a mechanism that enables each integrin to have distinct functions in oligodendrocyte development. Here, we tested this hypothesis by determining the associations among integrins, growth factor receptors, and the two SFKs, using siRNAs to knock down each SFK and examine the effect in developing oligodendrocyte cultures freshly isolated from the brain, and by examining the activation of each SFK by integrin signaling. We found that Lyn and Fyn were associated with Vß3 and
6ß1, respectively, and that Lyn, but not Fyn, was required for PDGF-stimulated proliferation of oligodendrocyte progenitors. However, at later stages of differentiation Fyn, but not Lyn, was associated with
6ß1 and was required for laminin-mediated amplification of growth factormediated survival and for differentiation with enhancement of myelin membrane formation. These results suggest a model in which integrins determine the consequences of growth factor signaling in oligodendrocytes via an associated SFK, and demonstrate how different SFKs can act within a single cell lineage as effectors that are specific for individual aspects of cell behavior and are able to integrate multiple upstream signaling cues.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Loss of protein expression was also confirmed using immunohistochemistry. Because isolated progenitors expanded in PDGF and FGF respond to growth factors and ECM differently from the way progenitors that are freshly isolated from cortical cultures respond, we avoided expansion in experiments examining cell behavior; instead, we evaluated a mixed population in which the subset of transfected cells was identified by YFP expression. Shown in Fig. 1 D are two cell populations that have been transfected with different vectors: one expressed YFP alone (control, left and right), and one expressed YFP and Fyn siRNA (Fyn(), middle). In controls (Fig. 1 D, left), many cells expressed both Fyn and YFP (arrowheads), whereas in Fyn() cells, the transfected cell population (YFP) and the Fyn-expressing population were mutually exclusive. Control-transfected cells (Fig. 1 D, right) had normal morphology and differentiated identically to nontransfected oligodendrocytes, as illustrated by cells labeled with myelin basic protein (MBP) and GFP antibodies (arrows).
Lyn regulates integrin-specific proliferation, but not migration
To determine whether SFKs play a role in the responses of oligodendrocyte progenitors, we evaluated two essential functions of progenitors, proliferation and migration, using cells in which individual SFKs were depleted. PDGF is a mitogen for oligodendrocyte progenitors, and the proliferative response seen at physiological PDGF concentrations (0.11.0 ng/ml) can be enhanced by Vß3 integrin engagement (Baron et al., 2002). We evaluated proliferation of SFK-depleted progenitors in the presence of increasing amounts of PDGF and in the presence or absence of the
Vß3 ligand FN (Fig. 2). With increasing PDGF concentrations, proliferation increased equally well in control and Fyn-, Lyn-, and Src-depleted progenitors grown on non-integrin substrate poly-D-lysine (PDL; Fig. 2, left). However, proliferation of Lyn-depleted cells on the
Vß3 ligand FN was reduced in response to physiological levels of PDGF (Fig. 2, right; *, P < 0.05). In contrast, depletion of Fyn or Src did not reduce PDGF-mediated proliferation on either substrate. Proliferation of progenitors grown on the
6ß1 ligand laminin-2 (Lm2) also increased with increasing PDGF; however, SFK depletion had no effect (not depicted).
|
|
|
A laminin-mediated switch in NRG survival signaling requires Fyn
NRG-mediated survival is minimal when freshly isolated cells are grown on PDL or FN; however, Lm2 amplifies NRG's ability to mediate survival (Colognato et al., 2002). We observed a robust amplification of NRG-mediated survival when control-transfected newly formed oligodendrocytes were grown on Lm2 (Fig. 4 B, control). In contrast, Fyn-depleted cells on Lm2 did not amplify NRG-mediated survival, and a significant reduction in survival (Fig. 4 B, **, P < 0.01) was observed at all NRG concentrations. However, Lyn-depleted cells grown on Lm2 retained the ability to amplify NRG-mediated survival, and, as with PDGF, depletion of Src had no effect. In cells grown on PDL, Fyn and Lyn depletion had no significant effect on NRG-mediated survival (not depicted), indicating that, as with PDGF, the SFK plays a role in amplification of survival by the ECM substrate rather than by the growth factor signal alone.
Laminin switches the preferred signaling pathways activated during NRG-mediated survival (Colognato et al., 2002). Thus, on nonlaminin substrates, survival is sensitive to PI3K inhibition but insensitive to MAPK inhibition. This pattern is reversed by Lm2, such that survival is insensitive to PI3K inhibition but sensitive to MAPK inhibition (Fig. 5 A). Here, we observed that the laminin-driven switch in NRG signaling did not occur in Fyn-deficient cells, whereas Lyn-deficient cells remained able to switch. Wortmannin treatment of Fyn() cells grown with NRG on Lm2 significantly reduced survival (**, P < 0.01) compared with control and Lyn-depleted cells grown on Lm2 (Fig. 5 A). Furthermore, Fyn depletion caused the cells to become less sensitive to the MEK inhibitor PD098059 (Fig. 5 A). In addition, cells grown on Lm2 and treated with NRG show enhanced phosphorylation of extracellular signalrelated kinase (ERK), yet do not amplify phosphorylation of Akt. Using a modified electroporation technique to obtain a high percentage of siRNA-positive cells (50%), Western blots of oligodendrocyte lysates revealed that Fyn-depleted cells treated with NRG were unable to amplify ERK phosphorylation (Fig. 5 B).
|
Differentiation on laminin requires Fyn
The same factors that are critical for newly formed oligodendrocytes to survive also regulate entry into the myelin-forming stage of differentiation. To investigate whether SFKs regulate the ability of the ECM to alter oligodendrocyte differentiation, we evaluated MBP expression in the presence of integrin ligands in SFK-depleted cells. The percentage of MBP-expressing cells was determined, and the relative change between SFK-deficient cells and control cells is shown in Fig. 6 A. In cells differentiating on PDL, depletion of Fyn, Lyn, or Src had no effect on the percentage of cells that acquired MBP expression by days 2 or 4 (Fig. 6 A, black bars). In contrast, Fyn-depleted cells differentiated on Lm2 showed a large reduction in the percentage of cells expressing MBP, at both days 2 and 4 (Fig. 6 A, gray bars). However, Lyn- or Src-depleted cells showed no change in differentiation on either substrate.
|
Fyn associates with 6ß1, whereas Lyn associates with PDGF
R and
Vß3 integrin
Having shown that Lyn and Fyn regulate proliferation and survival/differentiation, respectively, we asked whether each SFK was associated with the integringrowth factor receptor complexes responsible for these different stages of oligodendroglial development. Using immunofluorescence microscopy, we detected 6ß1 and Fyn in newly formed oligodendrocytes in an overlapping distribution (Fig. 7 A). Next, detergent lysates of newly formed oligodendrocytes grown on PDL, Lm2, and FN in the presence or absence of growth factors were evaluated by immunoprecipitation for the formation of protein complexes (Fig. 7, BE). Antibodies specific for the
6 integrin subunit isolated complexes containing Fyn, but not Lyn, and the
6ß1Fyn association was independent of substrate or growth factor stimuli (Fig. 7 B). Immunoprecipitations using Fyn antibodies also revealed a potential association between Fyn and the ErbB4 NRG receptor subunit (Fig. 7 C). This association was difficult to detect but, interestingly, was only observed in cells treated with NRG on non-integrin substrates. PDGF
R immunoprecipitations revealed an association between PDGF
R and Lyn that, in the absence of PDGF, was most robust on FN but, in the presence of PDGF, was also observed in cells grown on PDL or Lm2 (Fig. 7 D). No association between Fyn and PDGF
R was observed (unpublished data). Lyn immunoprecipitations revealed an association between Lyn, but not Fyn, and the
Vß3 integrin that was also enhanced by FN but, in contrast to the LynPDGF
R association, was not detected after PDGF treatment (Fig. 7 E).
|
|
We found that oligodendrocytes express Csk, the kinase that phosphorylates the COOH-terminal SFK negative regulatory site, and that levels of Csk within the SFK-containing insoluble fractions were decreased in cells grown on Lm2, but only in oligodendrocytes, not in progenitors (Fig. 8 B). Therefore, we conclude that the 6ß1 ligand Lm2 alters Fyn activity by regulating the dephosphorylation of the inhibitory COOH-terminal Y531, at least in part by down-regulation of Csk, with the catalytic tyrosine (Y420) being unaffected by Lm2 engagement. We also evaluated SFK phosphorylation in cells grown on FN (Fig. 8 D). In contrast with our observations in cells grown on Lm2, we detected no change in phosphoY527 immunoreactivity.
Two findings make it unlikely that a significant level of the immunoreactivity detected by the antibody against phosphoY527 can be attributed to phosphorylation of the homologous inhibitory Y508 in Lyn. First, comparison of the soluble/insoluble fractions in the precursors shows Lyn to be enriched in the insoluble fraction, whereas phosphoY527 immunoreactivity was detected entirely in the soluble fraction, where Fyn and Csk are highly expressed. Second, we did not detect immunoreactivity using the antibody against Src phosphoY527 after Lyn-specific pulldowns (unpublished data). We conclude that, in contrast with Fyn, Lyn is activated by Vß3 ligands, resulting in the autophosphorylation of the catalytic Y397, whereas the inhibitory Y508 remains unphosphorylated and does not contribute to the regulation of Lyn.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We found that, in addition to having distinct integrin associations, Fyn and Lyn are activated by different molecular mechanisms. We observed that oligodendrocytes express Csk, a kinase that negatively regulates the function of SFKs (Schmedt et al., 1998). Csk expression was highest in progenitors, and its down-regulation in oligodendrocytes grown on Lm2 correlated with reduced phosphorylation of the negative regulatory tyrosine in Fyn. Laminin may influence the activity of Csk directly, such as by reducing Csk levels or Csk accessibility, or indirectly by accelerating the differentiation process during which Csk activity is modulated. Preliminary data suggest that oligodendrocytes express Csk binding protein, a transmembrane molecule that has been shown in T cells to target Csk to the plasma membrane and direct a negative feedback loop for SFK signaling (Kawabuchi et al., 2000). Developmentally regulated changes in Csk binding protein levels could, therefore, alter Csk activity. Another possible mechanism for an integrin-dependent change in SFK activity would be regulation of the availability or activity of phosphatases such as receptor protein tyrosine phosphatase, which has been shown to regulate SFKs in an integrin-dependent manner in fibroblasts (von Wichert et al., 2003).
The loss of phosphorylation of Fyn tyrosine 531 triggered by laminin was associated with a switch from PI3K to MAPK NRG survival signaling. This switch in signaling has significant consequences for the development of the oligodendrocyte lineage, in which NRG signaling alone has been shown to stimulate PI3K signaling and keep oligodendrocytes in a differentiation-arrested state (Canoll et al., 1999). We have shown previously that integrin-mediated contact with axons is an important regulatory mechanism for triggering oligodendrocytes to survive and to complete the differentiation program (Colognato et al., 2002). Csk may play an instructive role in this switch, because Csk null fibroblasts grown on laminin-10 show increased MAPK signaling, whereas PI3K signaling is decreased under these circumstances (Gu et al., 2003). Preliminary data using Csk siRNA indicate that when Csk is decreased, Fyn Y531 phosphorylation is reduced and newly formed oligodendrocytes survive better than they do in control cells. Therefore, a prediction from the current study is that reducing Csk expression or activity may also help trigger myelination in the stalled preoligodendrocytes that have been observed in demyelinated multiple sclerosis lesions (Chang et al., 2002).
In contrast, we did not observe phosphorylation of the negative regulatory tyrosine in Lyn, indicating that Csk may not regulate Lyn activity in oligodendrocytes. Instead, we observed that phosphorylation of a catalytic tyrosine was increased when cells were grown on Vß3 substrates. The ability of Csk to regulate Fyn but not Lyn may result from the distribution of Csk and from the SFKs within lipid raft membrane microdomains. Both Fyn and Lyn are associated with lipid rafts in oligodendrocytes (Kramer et al., 1999). Lipid microdomains are more ordered regions of the plasma membrane that are highly enriched in cholesterol and glycosphingolipids and, thus, are insoluble in many detergents. These microdomains are thought to act as signaling platforms that can sequester or segregate signaling molecules, including integrin receptors. Indeed, laminin causes a redistribution of integrin
6ß1 to rafts in newly formed oligodendrocytes, in which PDGF-mediated survival signaling becomes dependent on the integrity of lipid raft domains (Baron et al., 2003). We confirmed that Fyn and Lyn are detergent insoluble in differentiated oligodendrocytes, but found that Fyn was predominantly detergent soluble in oligodendrocyte progenitors stimulated with PDGF. This indicates that Fyn may be excluded from rafts in proliferating cells. A differential lipid raft association for Fyn and Lyn would provide a mechanism for ensuring that Csk, which is also found in the soluble, nonraft pool, is available to inactivate Fyn, but not Lyn, in proliferating progenitors.
Previous studies have shown that mice lacking Fyn or Fyn kinase activity are hypomyelinated in the brain (Umemori et al., 1994; Sperber et al., 2001). This is believed to be an oligodendrocyte-intrinsic defect because oligodendrocytes with altered or absent Fyn activity differentiate defectively in culture (Osterhout et al., 1999; Umemori et al., 1999; Sperber and McMorris, 2001; Liang et al., 2004). Expression of dominant-negative Fyn resulted in morphological defects such as reduced process outgrowth, whereas Fyn null cells had a reduced propensity to differentiate but exhibited normal morphology. Fyn has been shown to activate p190RhoGAP, and expression of dominant-negative or constitutively active forms of the RhoGTPases Rho, Rac1, and cdc42 perturbs oligodendrocyte process formation, suggesting that these GTPases are one set of downstream targets for Fyn in the oligodendrocyte lineage (Wolf et al., 2001; Liang et al., 2004). Mice deficient in the laminin 2 subunit also have a hypomyelination defect, similar in regional specificity to that in Fyn null mice (Chun et al., 2003). Laminin- and Fyn-deficient mice are both hypomyelinated in the forebrain and optic nerve, but have normal-appearing myelin in the spinal cord. Furthermore, the expression of laminin associated with axon tracts correlates with and peaks with myelination, as does Fyn activity (Umemori et al., 1994; Powell et al., 1998; Farwell and Dubord-Tomasetti, 1999; Osterhout et al., 1999; Colognato et al., 2002). These similarities in phenotype and expression pattern can be explained by our current findings showing that the laminin receptor
6ß1 integrin is constitutively associated with Fyn, and that Fyn is required for laminin to amplify differentiation and survival in response to PDGF and NRG.
Several molecules have been proposed to regulate Fyn in oligodendrocytes. Antibody-mediated clustering of the cell surface molecule myelin-associated glycoprotein (MAG) was found to increase Fyn kinase activity (Umemori et al., 1994). However, MAG null mice do not have gross myelination defects but instead are characterized by subtle defects in myelin structure and periaxonal organization (Montag et al., 1994; Li et al., 1998). MAG/Fyn double knockout mice are more severely hypomyelinated than Fyn knockout mice, suggesting that the relationship between MAG and Fyn is complex and that the two molecules may operate in different signaling pathways (Biffiger et al., 2000). The GPI-anchored IgG superfamily molecule F3/contactin has also been proposed to regulate Fyn, because antibody-mediated clustering of F3/contactin in an oligodendrocyte cell line, Oli-neu, increased Fyn kinase activity (Kramer et al., 1999). However, like MAG null mice, F3 null mice do not have a hypomyelination phenotype similar to that of the Fyn null mice (Berglund et al., 1999). Therefore, Fyn may be able to activate multiple signaling pathways within the oligodendrocyte. An important question for further studies is to identify the mechanism by which adaptor and other molecules regulate the specific patterns of SFK association with the different receptors.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Survival assay
8-well chamber slides (Nunc) were coated for 4 h at 37°C with PDL (Sigma-Aldrich), FN, or Lm2. Each well received 20,000 progenitors suspended in SATO's medium. 1 h after attachment, the indicated growth factors were added and the cells were differentiated for 4 d. For PDGF assays, 0.1, 1.0, or 10 ng/ml PDGF was included, and for NRG assays, 1, 10, or 100 ng/ml NRG was included. Immunostaining using mouse anti-GFP and rabbit anti-GalC antibodies was used to identify transfected, newly formed oligodendrocytes. TUNEL using indirect immunofluorescence was used to visualize nicked DNA according to the manufacturer's instructions (Apoptag). In each well, a minimum of 100 GFP/GalC double-positive cells were scored for TUNEL. Cell survival was determined by the percentage of TUNEL-negative cells in the GFP/GalC double-positive population. To compare different experiments, the percent change in cell survival above or below the internal control (survival on PDL with no treatment or growth factors) was calculated. Experiments were performed a minimum of three times and the mean percent changes and SDs were calculated. Statistical significance was determined using the paired t test.
Migration assay
Transfected progenitors were suspended in 10 µl SATO defined medium plus PDGF and FGF. The volume of the cell suspension was measured and one third of the volume of 1% low melting temperature agarose (prepared in sterile PBS and equilibrated to 37°C) was added. 1.5-µl drops of cellagarose suspension were added to the center of PDL-coated 8-well chamber slides. The drops were incubated at 4°C for 15 min to solidify agarose, and then flooded with 0.2 ml SATO medium with 0, 1, or 10 ng/ml PDGF and 10 ng/ml FN or Lm2. At day 2, cells were fixed with methanol and immunostained with GFP antibodies to detect transfected cells. The migration distance from the agarose drop boundary was measured on captured images using OpenLab image software. For each experiment, all transfected cells within four drops were measured and averaged for each growth factor/ECM condition. Each experiment was performed at least three times and a representative experiment is shown.
Proliferation assay
8-well Permanox chamber slides were coated for 4 h at 37°C with PDL, FN, or Lm2. Slides were blocked in PBS containing 0.5% heat-inactivated BSA for 30 min and washed with PBS. Progenitors (20,000 per well) in DME were allowed to attach for 1 h, and then an equal volume of DME containing BrdU and PDGF was added. Final concentrations were 0, 0.1, 1, and 10 ng/ml for PDGF and 10 µM for BrdU. At 24 h, cells were washed and fixed in methanol, immunofluorescence was performed with antibodies to GFP to visualize YFP+-transfected cells and to BrdU to visualize cells that have entered S phase, and cells were stained with Hoechst to visualize nuclei. BrdU incorporation was defined as the percentage of healthy GFP+ cells that were positive for BrdU. Each experiment was repeated at least three times, with individual conditions performed in duplicate.
Cell culture
Disassociated rat neonatal cortices were cultured (at 37°C in 7.5% CO2) in DME with 10% FCS on PDL-coated flasks. By day 10, mixed glial cultures were obtained, consisting of oligodendrocyte precursor cells and microglia growing on an astrocyte monolayer. Purified oligodendrocyte precursor cells were isolated using a modification of the mechanical dissociation and differential adhesion method described by McCarthy and de Vellis (1980).
Transfection
Overnight incubation with lipid carrier FUGENE (Roche) was used to introduce 5 µg plasmid DNA per 75-cm2 flask of mixed glial cultures. Next, oligodendrocyte progenitors were purified by mechanical dissociation, and then they were selected in 400 µg/ml G418 with 10 ng/ml PDGF and FGF to prevent differentiation and maintain proliferation. In functional assays (survival, proliferation, migration, and differentiation), progenitors were not selected and were plated directly in 8-well chamber slides for analysis with no previous growth factor treatment. Transfected cells (typically 10%) were visualized by YFP fluorescence. Cells prepared for biochemistry were transfected using Nucleofector electroporation system according to the manufacturer's instructions with efficiencies of
50% (Amaxa).
Protein analysis
Cells were washed with ice-cold PBS and lysed in 1% Triton X-100, 10 mM Tris, pH 7.4, 5 mM EDTA, and 150 mM NaCl on ice. Cells were scraped and transferred to microfuge tubes and placed on ice for 15 min. Lysates were centrifuged at 14,000 rpm to separate detergent-insoluble and -soluble material. The Triton-insoluble pellet was solubilized in 10 mM Tris and 1% SDS by trituration through a 21-gauge needle. Before immunoprecipitations, lysates were prepared as above but incubated at 37°C to solubilize lipid raft-associated proteins. Protein concentration was determined by protein assay (Bio-Rad Laboratories) and lysates were boiled for 5 min in Laemmli solubilizing buffer and 3% ß-mercaptoethanol. Proteins were separated by SDS-PAGE using 7.5% or 10% acrylamide minigels (Bio-Rad Laboratories) and blotted onto 0.45 µm nitrocellulose. Membranes were blocked for 1 h in 0.1% Tween 20 and TBS (TBS-T) containing either 5% milk or 5% BSA, and then in primary antibodies in blocking buffer overnight at 4°C. Membranes were washed in TBS-T, incubated for 1 h in HRP-conjugated secondary antibodies (Amersham Biosciences), washed again in TBS-T, and developed using chemiluminescence. For immunoprecipitation of protein complexes, lysates were incubated with antibodies and protein A/G beads (Santa Cruz Biotechnology, Inc.) at 4°C overnight on a rotating wheel. Bead immune complexes were washed four times and prepared for electrophoresis and Western blotting.
Immunocytochemistry and image acquisition
To detect YFP in fixed cells, we incubated the cells for 10 min in PBS containing 3% PFA and 2% sucrose, and then performed immunocytochemistry with GFP antibodies (Molecular Probes) in PBS containing 0.4% BSA and 0.1% Triton X-100. For double immunofluorescence, additional primary antibodies were included to detect MBP, GalC, CNPase, or Fyn, followed by FITC- and TRITC-labeled donkey secondary antibodies. To visualize Fyn and the 6 integrin subunit, we fixed cells with methanol for 5 min at 20°C. Slides were mounted in Immunofluor (ICN Biomedicals) and evaluated at room temperature using an Axioplan fluorescence microscope (Carl Zeiss MicroImaging, Inc.) fitted with 10x eyepiece magnification using 20x (0.5 NA) and 40x (0.75 NA) objectives. Images were acquired using a digital camera (model C4742-95; Hamamatsu) and imaging software (OpenLab) and were exported as TIFF files to Adobe Photoshop.
Analysis of myelin membrane morphology
Oligodendrocytes transfected with siRNA constructs were differentiated for 2 or 4 d in Sato's medium with 0.5% FCS (differentiation medium). YFP-positive cells were evaluated for the expression of MBP using immunocytochemistry and graded according to morphological characteristics and degree of myelin membrane formation (Results).
Reagents
Reagents were obtained from Sigma-Aldrich unless otherwise indicated.
Antibodies.
The following rabbit polyclonal antibodies were used: GalC, Fyn, PDGFR, and ErbB4 NRG receptor subunit (Santa Cruz Biotechnology, Inc.); SFK phosphoY527 and phosphoY418 (Biosource International); GFP (Molecular Probes); total ERK (New England Biolabs, Inc.); and ß3 integrin subunit (CHEMICON International). The following mouse monoclonals were used: GFP; Fyn, and Csk (Transduction); pp60src (Oncogene Research Products); a rat mAb against MBP (Serotec); a hamster IgM against ß1 integrin (BD Biosciences); and FITC or TRITC donkey secondary antibodies (Jackson ImmunoResearch Laboratories). For triple immunofluorescence, aminomethylcoumarin-streptavidin (Vector Laboratories) was used to detect biotin-conjugated secondary antibodies.
Proteins.
Human recombinant PDGF-A and FGF-2 (PeproTech) were used at 10 ng/ml, except where otherwise indicated. The active component of NRG-1 was a recombinant protein comprising the EGF-like domain (Neomarkers). PDL, FN, and human placental laminin, a mixture of laminins that is primarily Lm2, were used at 10 µg/ml.
Other chemicals.
Inhibitors (Calbiochem) were suspended in DMSO and used at 50 nM (wortmannin) and at 25 µM (PD098059). In control wells, the equivalent volume of DMSO was added. Manganese was used at 50 µM.
![]() |
Acknowledgments |
---|
This work was funded by a National Multiple Sclerosis Society Career Transition Fellowship and a National Institutes of Health Ruth L. Kirschstein National Research Service Award postdoctoral fellowship (NS11035; both to H. Colognato) and a Wellcome Trust Research Leave Fellowship (to C. ffrench-Constant).
Submitted: 12 April 2004
Accepted: 7 September 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arias-Salgado, E.G., S. Lizano, S. Sarkar, J.S. Brugge, M.H. Ginsberg, and S.J. Shattil. 2003. Src kinase activation by direct interaction with the integrin beta cytoplasmic domain. Proc. Natl. Acad. Sci. USA. 100:1329813302.
Baron, W., S.J. Shattil, and C. ffrench-Constant. 2002. The oligodendrocyte precursor mitogen PDGF stimulates proliferation by activation of alpha(v)beta3 integrins. EMBO J. 21:19571966.
Baron, W., L. Decker, H. Colognato, and C. ffrench-Constant. 2003. Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains. Curr. Biol. 13:151155.[CrossRef][Medline]
Barres, B.A., R. Schmid, M. Sendnter, and M.C. Raff. 1993. Multiple extracellular signals are required for long-term oligodendrocyte survival. Development. 118:283295.
Berglund, E.O., K.K. Murai, B. Fredette, G. Sekerkova, B. Marturano, L. Weber, E. Mugnaini, and B. Ranscht. 1999. Ataxia and abnormal cerebellar microorganization in mice with ablated contactin gene expression. Neuron. 24:739750.[Medline]
Biffiger, K., S. Bartsch, D. Montag, A. Aguzzi, M. Schachner, and U. Bartsch. 2000. Severe hypomyelination of the murine CNS in the absence of myelin-associated glycoprotein and fyn tyrosine kinase. J. Neurosci. 20:74307437.
Brummelkamp, T.R., R. Bernards, and R. Agami. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science. 296:550553.
Calver, A.R., A.C. Hall, W.P. Yu, F.S. Walsh, J.K. Heath, C. Betsholtz, and W.D. Richardson. 1998. Oligodendrocyte population dynamics and the role of PDGF in vivo. Neuron. 20:869882.[Medline]
Canoll, P.D., J.M. Musacchio, R. Hardy, R. Reynolds, M.A. Marchionni, and J.L. Salzer. 1996. GGF/neuregulin is a neuronal signal that promotes the proliferation and survival and inhibits the differentiation of oligodendrocyte progenitors. Neuron. 17:229243.[Medline]
Canoll, P.D., R. Kraemer, K.K. Teng, M.A. Marchionni, and J.L. Salzer. 1999. GGF/neuregulin induces a phenotypic reversion of oligodendrocytes. Mol. Cell. Neurosci. 13:7994.[CrossRef][Medline]
Chang, A., W.W. Tourtellotte, R. Rudick, and B.D. Trapp. 2002. Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis. N. Engl. J. Med. 346:165173.
Chun, S.J., M.N. Rasband, R.L. Sidman, A.A. Habib, and T. Vartanian. 2003. Integrin-linked kinase is required for laminin-2induced oligodendrocyte cell spreading and CNS myelination. J. Cell Biol. 163:397408.
Colognato, H., W. Baron, V. Avellana-Adalid, J.B. Relvas, A. Baron-Van Evercooren, E. Georges-Labouesse, and C. ffrench-Constant. 2002. CNS integrins switch growth factor signalling to promote target-dependent survival. Nat. Cell Biol. 4:833841.[CrossRef][Medline]
Decker, L., and C. ffrench-Constant. 2004. Lipid rafts and integrin activation regulate oligodendrocyte survival. J. Neurosci. 24:38163825.
Farwell, A.P., and S.A. Dubord-Tomasetti. 1999. Thyroid hormone regulates the expression of laminin in the developing rat cerebellum. Endocrinology. 140:42214227.
Fernandez, P.A., D.G. Tang, L. Cheng, A. Prochiantz, A.W. Mudge, and M.C. Raff. 2000. Evidence that axon-derived neuregulin promotes oligodendrocyte survival in the developing rat optic nerve. Neuron. 28:8190.[Medline]
Frost, E.E., P.C. Buttery, R. Milner, and C. ffrench-Constant. 1999. Integrins mediate a neuronal survival signal for oligodendrocytes. Curr. Biol. 9:12511254.[CrossRef][Medline]
Fruttiger, M., L. Karlsson, A.C. Hall, A. Abramsson, A.R. Calver, H. Bostrom, K. Willetts, C.H. Bertold, J.K. Heath, C. Betsholtz, and W.D. Richardson. 1999. Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice. Development. 126:457467.
Gu, J., S. Nada, M. Okada, and K. Sekiguchi. 2003. Csk regulates integrin-mediated signals: involvement of differential activation of ERK and Akt. Biochem. Biophys. Res. Commun. 303:973977.[CrossRef][Medline]
Kawabuchi, M., Y. Satomi, T. Takao, Y. Shimonishi, S. Nada, K. Nagai, A. Tarakhovsky, and M. Okada. 2000. Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases. Nature. 404:9991003.[CrossRef][Medline]
Klinghoffer, R.A., C. Sachsenmaier, J.A. Cooper, and P. Soriano. 1999. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 18:24592471.
Kramer, E.M., C. Klein, T. Koch, M. Boytinck, and J. Trotter. 1999. Compartmentation of Fyn kinase with glycosylphosphatidylinositol-anchored molecules in oligodendrocytes facilitates kinase activation during myelination. J. Biol. Chem. 274:2904229049.
Li, C., B. Trapp, S. Ludwin, A. Peterson, and J. Roder. 1998. Myelin associated glycoprotein modulates glia-axon contact in vivo. J. Neurosci. Res. 51:210217.[CrossRef][Medline]
Liang, X., N.A. Draghi, and M.D. Resh. 2004. Signaling from integrins to Fyn to Rho family GTPases regulates morphologic differentiation of oligodendrocytes. J. Neurosci. 24:71407149.
McCarthy, K.D., and J. de Vellis. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85:890902.[Abstract]
Miller, L.A., J.J. Hong, M.S. Kinch, M.L. Harrison, and R.L. Geahlen. 1999. The engagement of beta1 integrins on promonocytic cells promotes phosphorylation of Syk and formation of a protein complex containing Lyn and beta1 integrin. Eur. J. Immunol. 29:14261434.[CrossRef][Medline]
Montag, D., K.P. Giese, U. Bartsch, R. Martini, Y. Lang, H. Bluthmann, J. Karthigasan, D.A. Kirschner, E.S. Wintergerst, K.A. Nave, et al. 1994. Mice deficient for the myelin-associated glycoprotein show subtle abnormalities in myelin. Neuron. 13:229246.[Medline]
Osterhout, D.J., A. Wolven, R.M. Wolf, M.D. Resh, and M.V. Chao. 1999. Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase. J. Cell Biol. 145:12091218.
Powell, S.K., C.C. Williams, M. Nomizu, Y. Yamada, and H.K. Kleinman. 1998. Laminin-like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro. J. Neurosci. Res. 54:233247.[CrossRef][Medline]
Schmedt, C., K. Saijo, T. Niidome, R. Kuhn, S. Aizawa, and A. Tarakhovsky. 1998. Csk controls antigen receptor-mediated development and selection of T-lineage cells. Nature. 394:901904.[CrossRef][Medline]
Sperber, B.R., and F.A. McMorris. 2001. Fyn tyrosine kinase regulates oligodendroglial cell development but is not required for morphological differentiation of oligodendrocytes. J. Neurosci. Res. 63:303312.[CrossRef][Medline]
Sperber, B.R., E.A. Boyle-Walsh, M.J. Engleka, P. Gadue, A.C. Peterson, P.L. Stein, S.S. Scherer, and F.A. McMorris. 2001. A unique role for Fyn in CNS myelination. J. Neurosci. 21:20392047.
Travis, M.A., J.D. Humphries, and M.J. Humphries. 2003. An unraveling tale of how integrins are activated from within. Trends Pharmacol. Sci. 24:192197.[CrossRef][Medline]
Tuschl, T. 2002. Expanding small RNA interference. Nat. Biotechnol. 20:446448.[CrossRef][Medline]
Umemori, H., A. Wanaka, H. Kato, M. Takeuchi, M. Tohyama, and T. Yamamoto. 1992. Specific expressions of Fyn and Lyn, lymphocyte antigen receptor-associated tyrosine kinases, in the central nervous system. Brain Res. Mol. Brain Res. 16:303310.[Medline]
Umemori, H., S. Sato, T. Yagi, S. Aizawa, and T. Yamamoto. 1994. Initial events of myelination involve Fyn tyrosine kinase signalling. Nature. 367:572576.[CrossRef][Medline]
Umemori, H., Y. Kadowaki, K. Hirosawa, Y. Yoshida, K. Hironaka, H. Okano, and T. Yamamoto. 1999. Stimulation of myelin basic protein gene transcription by Fyn tyrosine kinase for myelination. J. Neurosci. 19:13931397.
von Wichert, G., G. Jiang, A. Kostic, K. De Vos, J. Sap, and M.P. Sheetz. 2003. RPTP- acts as a transducer of mechanical force on
v/ß3-integrincytoskeleton linkages. J. Cell Biol. 161:143153.
Wolf, R.M., J.J. Wilkes, M.V. Chao, and M.D. Resh. 2001. Tyrosine phosphorylation of p190 RhoGAP by Fyn regulates oligodendrocyte differentiation. J. Neurobiol. 49:6278.[CrossRef][Medline]