Distinct fibroblast growth factor (FGF)/FGF receptor signaling pairs initiate diverse cellular responses in the oligodendrocyte lineage

Abstract

Fibroblast growth factors (FGFs) have been implicated in numerous cellular processes, including proliferation, migration, differentiation, and survival. Whereas FGF-2, the prototypic ligand in a family of 22 members, activates all four tyrosine kinase FGF receptors (FGFR1-FGFR4), other members demonstrate a higher degree of selectivity. Oligodendrocytes (OLs), the myelin-producing cells of the CNS, are highly influenced by FGF-2 at all stages of their development. However, how other FGFs and their cognate receptors orchestrate the development of OLs is essentially undefined. Using a combination of specific FGF ligands and receptor blocking antibodies, we now show that FGF-8 and FGF-17 target OL progenitors, inhibiting their terminal differentiation via the activation of FGFR3, whereas FGF-9 specifically targets differentiated OLs, triggering increases in process growth via FGFR2 signaling; FGF-18 targets both OL progenitors and OLs via activation of both FGFR2 and FGFR3. These events are highly correlated with changes in FGF receptor expression from FGFR3 to FGFR2 as OL progenitors differentiate into mature OLs. In addition, we demonstrate that, although activation of FGFR1 by FGF-2 leads to proliferation of OL progenitors, it produces deleterious effects on differentiated OLs (i.e., aberrant reentry into cell cycle and down-regulation of myelin proteins with a loss of myelin membrane). These data suggest that ligand availability, coupled with changes in FGF receptor expression, yield a changing repertoire of ligand-receptor signaling complexes that contribute critically to the regulation of both normal OL development and potential OL/myelin pathogenesis.

Figure 1.

Diverse effects of different FGFs on proliferation and differentiation of oligodendrocyte progenitors. OL progenitors were grown in the absence (control) or presence of FGF-2, FGF-8, FGF-9, FGF-17, or FGF-18 for 2 d (A, B) or 4 d (D, E) and analyzed by immunofluorescence microscopy for their effects on OL progenitor proliferation (BrdU⁺ OL progenitors; A, B) or differentiation into OLs (MBP⁺ OLs; D, E). The data are expressed as percentage of total OL lineage cells (O4⁺). C, Immunoblot analysis with antibodies to phospho-MAPK of OL progenitors stimulated with FGF-2 or FGF-8 as a function of time from 0-24 h. Error bars represent SEM; n = 3-6 independent experiments each performed in triplicate. Scale bars, 25 μm. Note that, compared with controls, FGF-2 stimulates proliferation and inhibits differentiation of OL progenitors, FGF-8, FGF-17, and FGF-18 inhibit differentiation without significantly affecting proliferation, and FGF-9 has no affect on either proliferation or differentiation of OL progenitors.

Figure 2.

The effect of FGF family members on cell cycle reentry in mature oligodendrocytes. Mature OLs were grown in the absence (control) or presence of FGF-2, FGF-8, FGF-9, FGF-17, or FGF-18 for 2 d (plus BrdU for the last 3 h). The number of OLs incorporating BrdU and the level of expression of Cdk2 and cyclin E were determined to identify cell cycle reentry. A, Example of a mature OL double labeled with O4 and anti-BrdU after FGF-2 treatment. B, Quantification of the percentage of mature OLs incorporating BrdU (expressed as a percentage of total OLs) after treatment with different FGF family members. Inset, Immunoblot analysis for the expression of the cell cycle proteins cyclin E and Cdk2 after exposure to FGF-2, FGF-8, or FGF-9 compared with untreated controls. The blots were reprobed for actin to demonstrate equal protein loading. Error bars represent SEM; n = 3-5 independent experiments, each performed in triplicate.

Figure 3.

Selective induction of process elongation in mature oligodendrocytes by specific FGFs. A, Mature OLs were grown in the absence (control) or presence of FGF-2, FGF-8, FGF-9, FGF-17, or FGF-18 for 2 d, after which they were double immunolabeled with the OL markers O4 and MBP (O4 is shown). B, Quantification of OL areas illustrating that FGF-2, FGF-9, and FGF-18, but not FGF-8 or FGF-17, induced process elongation as shown by increases in cell areas. Scale bars, 25 μm. Error bars represent SEM; n = 3-9 independent experiments, each performed in triplicate.

Figure 4.

Downregulation of myelin proteins is specific to FGF-2. A, Mature OLs were grown in the absence (control) or presence of FGF-2, FGF-8, FGF-9, FGF-17, or FGF-18 for 2 d, and the expressions of MBP, MOG, and FGFR2 were determined by immunoblotanalyses. Equal amounts of protein were loaded in each lane as confirmed by reprobing blots with antibodies to actin. Quantification of the levels of MBP (B) and FGFR2 (C) demonstrated that only FGF-2 induced a statistically significant downregulation of myelin proteins compared with controls. D, OLs were stimulated with FGF-8 or FGF-2 for 15 min and analyzed by immunoprecipitation (IP) with antibodies for FGFR2 and immunoblotting (IB) of the precipitates with phospho-tyrosine-specific antibodies (α-pTyr) as a measure of FGFR2 activation. The blots were then reprobed for FGFR2 to assess total FGFR2 protein, showing that FGFR2 is activated by FGF-2, but not FGF-8, before being subsequently downregulated. Error bars represent SEM; n = 3 independent experiments performed in triplicate. ^*p < 0.01. ^** in A, Nonspecific band.

Figure 5.

Developmental expression patterns of FGFR1, FGFR2, and FGFR3 protein. A, Protein lysates from transfected cell lines overexpressing FGFR1, FGFR2, or FGFR3 were loaded in adjacent lanes and analyzed by immunoblotting to test the specificity of anti-receptor antibodies for each FGFR. B, Equal amounts of total proteins analyzed by immunoblotting shows the developmental protein expression patterns of FGFR1, FGFR2, and FGFR3 in early progenitors (EP), late progenitors (Pro-OLs), terminally differentiated oligodendrocytes (OLs), and myelin (My). A representative experiment of three to five independent experiments is shown. Note that there is a developmental switch from FGFR3 to FGFR2 as late progenitors differentiate into mature OLs.

Figure 6.

Oligodendrocyte progenitors require FGFR1 for FGF-2-induced proliferation, but FGFR3 for FGF-8, FGF-17, and FGF-18 induced arrest of terminal differentiation. A, OL progenitors grown for 3 d in the presence of FGF-2, with or without blocking antibodies to FGFR1 (αR1) or FGFR3 (αR3), were analyzed for proliferation by BrdU incorporation. FGF-2-mediated increase in BrdU⁺ OL progenitors was significantly attenuated by αR1 but not by αR3. An inhibitor of all FGFRs, PD also abolished the increase in BrdU incorporation. B, OL progenitors were grown in the presence of FGF-2, FGF-8, FGF-17, or FGF-18, with or without blocking antibodies to FGFR1 or FGFR3. After 3 d, cells were analyzed for GalC expression indicative of differentiating OLs. The arrest of differentiation mediated by FGF-8, FGF-17, and FGF-18 was prevented by αR3 but not by αR1. The FGF-8-induced block of terminal differentiation was also overcome by PD. Both αR1 and αR3 were required to prevent the FGF-2-induced block of differentiation. Error bars represent SEM; n = 3-12 independent experiments, each performed in triplicate.

Figure 7.

Reentry of mature oligodendrocytes into the cell cycle and downregulation of myelin proteins is mediated by FGFR1; stimulation of process elongation is mediated by FGFR2. Mature OLs were exposed for 2 d to FGF-2, FGF-9, or FGF-18 with or without blocking antibodies to FGFR1 (αR1) or FGFR2 (αR2). A, The stimulation by FGF-2 of BrdU incorporation in mature OLs compared with untreated controls (Cont) was attenuated by αR1 or PD but not by αR2. B, Sizes (areas) of OLs exposed to FGF-2, FGF-9, or FGF-18 are plotted as percentage of untreated OLs. Increases in OL area induced by FGF-2, FGF-9, and FGF-18 were prevented by αR2 or PD but not αR1. DMSO vehicle (Veh) was similar to control. C, Immunoblot analyses and quantification of OLs exposed to FGF-2 demonstrates that the downregulation of FGFR2 (Ca) and MOG (Cb) protein expression was prevented by αR1 and PD but not by αR2 or αR3. Insets, Representative immunoblots show the expression of FGFR2 (Ca) and MOG (Cb). Error bars represent SEM; n = 3-5 independent experiments, each performed in triplicate.

Figure 8.

FGF/FGFR signaling pairs are associated with specific responses during oligodendrocyte development. Left, OL progenitors predominantly express FGFR1 and FGFR3. Activation of FGFR1 (R1) by FGF-2 induces their proliferation and concurrent suppression of differentiation. The selective activation of FGFR3 (R3) by FGF-8 (and related subfamily members FGF-17 and FGF-18) inhibits differentiation. Right, Differentiated OLs no longer express FGFR3 but upregulate the expression of FGFR2 and continue to express FGFR1. Specific activation of FGFR2 (R2) by FGF-9 stimulates process elongation. However, exposure of differentiated OLs to FGF-2, in addition to stimulating process elongation via FGFR2, also leads to the downregulation of myelin proteins and reentry into the cell cycle via FGFR1. These conclusions are based on a combination of specific FGF ligand presentation, blocking antibodies (dotted lines), and analysis of regulated expression of FGFRs.