Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development

Abstract

Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.

Figure 1.

siRNA depletes Src family kinases in primary oligodendrocytes. (A) Src family kinase (SFK) protein expression in oligodendrocytes differentiated for 1, 2, 4, or 6 d after growth factor withdrawal. (B) SFK Western blots of G418-selected progenitors transfected with Fyn and Lyn depletion constructs. (C) Western blots of oligodendrocyte progenitors transfected with Src depletion construct. (A–C) Blots were also probed with actin antibodies as protein loading controls. (D) Immunostaining to visualize Fyn protein in a mixed population containing YFP+-transfected cells. Control (left) and Fyn(−) (middle) micrographs depict newly differentiated oligodendrocytes labeled with antibodies against Fyn (red) and GFP (green) (arrowheads). Double-labeled cells do not appear in the Fyn(−)-transfected population. (right) Control cells expressing late differentiation markers MBP (red) and YFP (green) (arrows).

Figure 2.

Progenitors require Lyn, but not Fyn or Src, for PDGF-mediated proliferation on the αVβ3 ligand. Oligodendrocyte progenitors exposed to 0, 0.1, 1, or 10 ng/ml PDGF for 24 h were evaluated for the percentage of cells that incorporated BrdU. Profiles of control (black squares), Fyn-depleted (gray diamonds), Lyn-depleted (gray triangles), and Src-depleted (gray circles) cells are shown. On poly-d-lysine (PDL), all progenitors showed a similar dose-dependent increase in BrdU incorporation. In contrast, Lyn-depleted progenitors grown on the αVβ3 ligand fibronectin (FN) showed reduced BrdU incorporation (*, P < 0.05). Error bars represent SD.

Figure 3.

Progenitors do not require Lyn for PDGF-mediated migration. Oligodendrocyte progenitors were concentrated in agarose drops and stimulated to migrate using PDGF. (A) Micrograph depicting Lyn-deficient cells (YFP+) migrating on FN in response to 1 ng/ml PDGF. (left) Anti-GFP; (right) phase. (B) Migration of SFK-deficient cells on FN in response to 1 ng/ml PDGF at day 2. Each bar depicts the mean migration distance of all YFP+ cells that have exited drops. Error bars represent SD.

Figure 4.

Newly formed oligodendrocytes require Fyn for laminin-mediated amplification of survival. SFK-depleted progenitors were differentiated on PDL or laminin-2 (Lm2) for 4 d with increasing amounts of the soluble growth factors PDGF or neuregulin (NRG). Survival was evaluated using TUNEL on a YFP/GalC double-positive cell population (transfected, newly formed oligodendrocytes). Low, medium, and high amounts of growth factors are depicted by light gray, dark gray, and black bars, respectively. Error bars represent SD. (A) Fyn depletion caused a significant shift in the PDGF dose–response curve when cells were differentiated on Lm2 (low, *, P < 0.05; medium, **, P < 0.01). (B) NRG-mediated survival was significantly reduced in Fyn-depleted cells differentiated on Lm2 (**, P < 0.01 for low, medium, and high concentrations of NRG), whereas no significant reduction in survival occurred in Lyn-depleted cells.

Figure 5.

Fyn is required for a laminin-mediated switch in NRG signaling and for integrin activation to increase survival. (A) Survival of newly formed SFK-depleted oligodendrocytes in the presence of the PI3K pathway signaling inhibitor wortmannin (hatched bars), the MAPK pathway signaling inhibitor PD098059 (light gray bars), or DMSO control (black bars). Lm2 switches NRG-mediated survival from PI3K-sensitive to PI3K-insensitive, and Fyn depletion, but not Lyn depletion, abolishes this effect. Error bars represent SD. (B) Oligodendrocytes treated for 30 min with NRG. PhosphoERK is enhanced by Lm2 in control cells, but not in Fyn(−) cells. (C) Survival of newly formed SFK-depleted oligodendrocytes in the presence (light gray bars) or absence (dark gray bars) of integrin-activating manganese. Integrin activation using manganese increased NRG-mediated survival, and this increase was lost in the absence of Fyn, but not of other SFKs. Error bars represent SD.

Figure 6.

Differentiation in response to laminin requires Fyn. (A) Decreased MBP expression in cells grown on laminin in the absence of Fyn. The percentage of SFK-depleted cells expressing the late stage differentiation marker MBP was expressed relative to the percentage of MBP+ in control cells. SFK-depleted cells grown on PDL (black bars) and on Lm2 (gray bars) were compared at days 2 and 4 after growth factor withdrawal. Error bars represent SD. (B) Myelin membrane classification scheme. Examples of MBP-expressing cells are shown. Stages 1, 2, and 3 show increasing levels of process outgrowth and branching, without myelin membrane, whereas stages 4, 5, and 6 show increasing levels of complexity and myelin membrane. (C) Fyn-depleted cells have less myelin membrane acquisition and complexity on Lm2 substrates. The percentage of cells within each category is shown for SFK-depleted YFP/MBP double-positive cells. Oligodendrocytes differentiated on PDL or Lm2 (control), black squares; Fyn(−), light gray squares; Lyn(−), gray triangles; and Src(−), dark gray circles (*, P < 0.050). Error bars represent SD. (D) Typical MBP-expressing (control) and Fyn-depleted oligodendrocytes grown on Lm2.

Figure 7.

SFK associations with integrins and growth factors. (A) Newly formed oligodendrocytes immunostained with antibodies against Fyn (red) and α6β1 integrin (green). Merged panel is shown on the right. (B) Oligodendrocyte lysates from cells differentiated on ECM substrates in the presence or absence of PDGF or NRG. Western blot on α6 integrin antibody immunoprecipitation complexes to detect Fyn. (C) Western blot on Fyn antibody immunoprecipitation complexes to detect ErbB4 NRG receptor subunit. (D) Western blot on PDGFαR antibody immunoprecipitation complexes to detect Lyn. (E) Western blots on Lyn antibody immunoprecipitation complexes to detect the β3 integrin subunit.

Figure 8.

SFK activity is regulated by laminin in differentiating oligodendrocytes, not progenitors. (A) Active Lyn is detected in response to the αVβ3 ligand FN. Immunoprecipitation complexes using antibodies against Fyn, Lyn, or control mouse IgG (−) were evaluated by Western blot for the presence of autophosphorylated SFK phosphoY418. (B) Expression and solubility of Fyn, Lyn, and COOH-terminal Src kinase (Csk) change during oligodendrocyte lineage progression and in response to Lm2. Western blots were performed using antibodies specific for SFKs and SFK regulatory kinase Csk. S, Triton X-100–soluble protein; I, Triton X-100–insoluble protein. Blots were also probed with actin antibodies as protein loading controls. (C) Phosphorylation of the SFK negative regulatory site is reduced by laminin in oligodendrocytes, not progenitors. The same lysates as in B were used, but Western blots were analyzed using antibodies against two SFK sites: phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). (D) FN does not alter phosphorylation of the SFK negative regulatory site. Lysates of progenitors and oligodendrocytes grown on control substrate PDL or on the αVβ3 ligand FN. Western blots were performed using SFK antibodies specific for phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). Blots were also probed with actin antibodies as protein loading controls. (A–D) In the absence of ECM ligands, cells were grown on non-integrin substrate PDL.

Figure 9.

Model for integrin regulation of SFK activity during oligodendrocyte lineage progression. In oligodendrocyte progenitors, Lyn is associated with the PDGFαR–αVβ3 integrin complex and contributes to proliferation signaling. Catalytic Y397 of Lyn (orange) is phosphorylated after αVβ3 integrin ligation. Fyn is maintained in the inactive state by Csk phosphorylation of Fyn-inhibitory COOH-terminal Y531 (yellow). After axonal contact and ligation by α6β1 of the α2 chain laminins expressed in myelinating axon tracts, Lyn dissociates from the integrin–growth factor complex and Csk is downregulated, reducing Fyn phosphorylation at Y531 and promoting Fyn activity. Active Fyn–α6β1 complexes can then trigger PI3K and MAPK signaling, depending on the ligand binding of PDGFαR and ErbB2/4 receptors, respectively, thereby promoting oligodendrocyte survival, differentiation, and myelin formation.