ERK1/2 signaling is essential for the chemoattraction exerted by human FGF2 and human anosmin-1 on newborn rat and mouse OPCs via FGFR1

RESEARCH ARTICLE

ERK1/2 Signaling is Essential for theChemoattraction Exerted by Human

FGF2 and Human Anosmin-1 on NewbornRat and Mouse OPCs via FGFR1

Ver�onica Murcia-Belmonte, Eva M. Medina-Rodr�ıguez, Ana Bribi�an,

Fernando de Castro, and Pedro F. Esteban

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferationand migration, as well as differentiation events such as myelination. Anosmin-1 is an extracellular matrix (ECM) glycoproteinthat interacts with the fibroblast growth factor receptor 1 (FGFR1) to exert its biological actions through this receptor,although the intracellular pathways underlying anosmin-1 signaling remain largely unknown. This protein is defective in the X-linked form of Kallmann syndrome (KS) and has a prominent role in the migration of neuronal and oligodendroglial precur-sors. We have shown that anosmin-1 exerts a chemotactic effect via FGFR1 on neuronal precursors from the subventricularzone (SVZ) and the essential role of the ERK1/2 signaling. We report here the positive chemotactic effect of FGF2 andanosmin-1 on rat and mouse postnatal OPCs via FGFR1. The same effect was observed with the truncated N-terminal regionof anosmin-1 (A1Nt). The introduction in anosmin-1 of the missense mutation F517L found in patients suffering from KSannulled the chemotactic activity; however, the mutant form carrying the disease-causing mutation E514K also found in KSpatients, behaved as the wild-type protein. The chemoattraction exhibited by FGF2 and anosmin-1 on OPCs was blocked bythe mitogen-activated protein kinase (MAPK) inhibitor U0126, suggesting that the activation of the ERK1/2 MAPK signalingpathway following interaction with the FGFR1 is necessary for FGF2 and anosmin-1 to exert their chemotactic effect. In fact,both proteins were able to induce the phosphorylation of the ERK1/2 kinases after the activation of the FGFR1 receptor.

GLIA 2014;62:374–386Key words: migration, Kallmann syndrome, oligodendrocyte precursor, remyelination

Introduction

The progenitors of oligodendrocytes give rise to oligoden-

drocyte precursor cells (OPCs) that are still able to prolif-

erate and have a high migratory capacity. Migration of OPCs

is essential for these cells to colonize the entire CNS before

myelination and seems to be a tightly controlled process

modulated by a variety of signals (de Castro and Bribi�an,

2005; de Castro et al., 2013; Jarjour and Kennedy, 2004).

Among these, FGF2 seems to play an important role. FGF2

was first described to induce the expression of PDGFRa in

rat OPCs increasing their sensitivity to PDGFa (McKinnon

et al., 1990) and contributing to adopt a migratory pheno-

type (McKinnon et al., 1993). A direct effect of FGF2 on

OPC migration was later described (Milner et al., 1997) as

well as the role of FGFR1 signaling in OPC migration invivo, since cells carrying a dominant-negative form of this

receptor are unable to migrate and remain within the ven-

tricles where they are injected (Osterhout et al., 1997). The

implication of the FGF2/FGFR1 system in the motility of

mouse OPCs has been shown in explants from the embryonic

optic nerve (ON) where FGF2 has a motogenic and a posi-

tive chemoattractive effect (Bribi�an et al., 2006, 2008), and a

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22609

Published online December 21, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received Aug 12, 2013, Accepted for publication Nov 12, 2013.

Address correspondence to Dr. Pedro F. Esteban or Dr. Fernando de Castro, Grupo de Neurobiolog�ıa del Desarrollo-GNDe, Hospital Nacional de Parapl�ejicos,

Finca “La Peraleda”, s/n, E-45071-Toledo, Spain. E-mail: [email protected] or [email protected]

From the Grupo de Neurobiolog�ıa del Desarrollo-GNDe, Hospital Nacional de Parapl�ejicos, Finca “La Peraleda, s/n, E-45071-Toledo, Spain.

Additional Supporting Information may be found in the online version of this article.

374 VC 2013 Wiley Periodicals, Inc.

mailto:[email protected]

mailto:[email protected]

chemotactic effect has also been demonstrated on cortical

OPCs from adult mice (Clemente et al., 2011). The down-

stream signaling activated in the migration of OPCs has been

explored but the precise mechanisms involved are not fully

understood (Rajasekharan, 2008). Different extracellular cues

and membrane proteins affecting OPC migration seem to

activate the Rho signaling pathway (Binam�e et al., 2013; Liu

et al., 2012; Novgorodov et al., 2007) and the PDGFa-

promoted migration of OPCs has been described to be associ-

ated with the activation of Fyn kinase, Cdk5, and Wave2

(Miyamoto et al., 2008). Both PDGFa and FGF2-induced

migration require the participation of the ERK1/2 signaling

pathway (Frost et al., 2009; Vora et al., 2011).

The glycoprotein anosmin-1 is a 680 aminoacid extrac-

ellular matrix (ECM) protein encoded by the human KAL1gene (Franco et al., 1991; Legouis et al., 1991), whose

sequence is highly conserved across species including rodents,

although to date, no ortholog has been identified in rat or

mouse. This gene is responsible for the X-linked form of

Kallmann syndrome (KS) characterized by anosmia (lack of

the sense of smell) and hypogonadotropic hypogonadism

(Kallmann et al., 1944; Maestre de San Juan, 1856).

Anosmin-1 seems to have a crucial role in the formation of

the neural crest cells by controlling the expression and activ-

ities of FGF8, BMP5, and WNT3a (Endo et al., 2012).

Among other biological effects anosmin-1 participates in cell

migration; promotes the migration of immortalized GnRH

neurons (Cariboni et al., 2004), immortalized GnRH neuro-

blast cells (Hu et al., 2009) and neuronal precursor cells

(NPs) isolated from the forebrain subventricular zone (SVZ)

(Garc�ıa-Gonz�alez et al., 2010). Regarding the migration of

OPCs in vitro, anosmin-1 negatively modulates the moto-

genic effect of FGF-2 mediated by FGFR1 on mouse ON

OPCs without affecting the directionality of migration

(Bribi�an et al., 2006), while acting as a substrate molecule, it

is able to hinder OPC migration probably by increasing their

adhesion (Bribi�an et al., 2008). On the contrary, the blockade

of anosmin-1 or a closely related protein using an antibody

raised against human anosmin-1, hinders the migratory

capacity of ON embryonic OPCs (Bribi�an et al., 2008). In

OPCs isolated from adult mouse cerebral cortex, anosmin-1

also limits the chemotactic effect promoted by FGF2 via

FGFR1 and plays a chemorepellent effect (Clemente et al.,

2011). Outside the nervous system, anosmin-1 facilitates the

migration of colon cancer cells, a process connected with

metastasis (Jian et al., 2009).

Anosmin-1 interacts with different molecules present in

the ECM such us: urokinase plasminogen activator (uPA),

heparan-sulfate (HS), laminin, fibronectin, and anosmin-1

itself (Bribi�an et al., 2008; B€ulow and Hobert, 2004;

Garc�ıa-Gonz�alez et al., 2010; Hu et al., 2004, 2009;

Murcia-Belmonte et al., 2010). The gene coding for FGFR1,

KAL2, is associated with the autosomal inheritance of KS and

the interaction of anosmin-1 with FGFR1, as well as the

effects mediated via this receptor, is the most studied and

best known mechanism of action of this protein (Ayari and

Soussi-Yanicostas, 2007; Bribi�an et al., 2006; Esteban et al.,

2013; Garc�ıa-Gonz�alez et al., 2010; Gonz�alez-Mart�ınez et al.,

2004; Hu et al., 2009; Murcia-Belmonte et al., 2010). Thus,

anosmin-1 would regulate the activity of this receptor provid-

ing a molecular link between two of the genes responsible for

KS (Gonz�alez-Mart�ınez et al., 2004). Different missense

mutations have been identified in the KAL1 locus in KS

patients. Some of these mutations located in the first and

third FnIII domains of anosmin-1 (N267K, E514K, and

F517L) (Georgopoulos et al., 2007; Hardelin et al., 1993;

Maya-Nu~nez et al., 1998) annul or reduce, in vitro, the bind-

ing to the receptor of the full-length protein or of the

domains involved in the interaction (Esteban et al., 2013; Hu

et al., 2009; Murcia-Belmonte et al., 2010); in contrast, at

least one mutation described in the WAP domain, C172R

(Oliveira et al., 2001), has no effect in the binding to

FGFR1 (Esteban et al., 2013). Nevertheless, the presence of

any of these mutations within the protein makes full-length

anosmin-1 inactive (Cariboni et al., 2004; Esteban et al.,

2013; Gonz�alez-Mart�ınez et al., 2004; Hu et al., 2009;

Murcia-Belmonte et al., 2010). The binding to FGFR1 and

the subsequent activation of the receptor entail the triggering

of intracellular signaling pathways, most notably ERK1/2 and

p38 MAPKs, PI3K, and the small GTPases Rac1 and Cdc42,

responsible for the different effects promoted by anosmin-1

(Esteban et al., 2013; Gonz�alez-Mart�ınez et al., 2004; Hu

et al., 2013).

In the present report, we show that human FGF2 and

surprisingly human anosmin-1, both acting through FGFR1,

have a clear positive chemotactic effect on newborn rat and

mouse cortical OPCs. The introduction of disease-causing

missense mutations within anosmin-1 that annul the chemo-

attractive effect on rat SVZ NPs (Murcia-Belmonte et al.,

2010), yields opposite effects: while the F517L substitution

renders a nonfunctional protein, the A1E514K mutated pro-

tein behaves as wild-type anosmin-1 and displays a positive

chemotactic effect on OPCs via FGFR1. Consistent with pre-

vious reports that show that the N-terminal region of

anosmin-1 comprising the CR, WAP, and FnIII.1 domains

(A1Nt) retains some of the biological effects of full-length

anosmin-1 (B€ulow et al., 2002; Esteban et al., 2013;

Gonz�alez-Mart�ınez et al., 2004; Hu et al., 2004; Murcia-

Belmonte et al., 2010), this truncated protein shows a posi-

tive chemotropic effect on OPCs that is also mediated via

FGFR1. We also describe the activation of the ERK1/2 path-

way by FGF2, anosmin-1, A1Nt, and A1E514K via FGFR1

Murcia-Belmonte et al.: Chemotactic Effect of Anosmin-1 on OPCs

March 2014 375

and show its essential role in the chemoattractive effect of

FGF2 and anosmin-1 on rat and mouse OPCs.

Materials and Methods

AnimalsNewborn P0 Wistar rats and newborn P0 C57BL/6 and CD1 mice

were used in all this work. All the experiments using animals were

performed in accordance with Spanish (RD223/88) and European

Community Council Directive of November 24, 1986 (86/609/

ECC) regulation and they were approved by the animal review board

at the Hospital Nacional de Parapl�ejicos (registred as SAPA001).

Cell CultureCHO cells were grown in Dulbecco’s Modified Eagle’s Medium

(DMEM, GIBCO) supplemented with 8% fetal Bovine Serum

(FBS, GIBCO), 100 U/mL of penicillin and 100 mg/mL of strepto-

mycin (GIBCO) at 37�C and 5% CO2. Cells were transfected using

X-tremeGENE DNA Transfection Reagent (Roche) according to the

protocol provided by the manufacturer.

Plasmids Used and Extracellular Matrix ProteinExtract PreparationA human KAL1 cDNA was used in the construction of the expres-

sion plasmids carrying C-terminal HA-tagged versions of full-length

anosmin-1 (A1), the N-terminal region of anosmin-1 comprising the

CR, WAP, and FnIII.1 domains (A1Nt, M1-A289) and full-length

anosmin-1 with the E514K or F517L substitutions (A1E514K and

A1F517L). These plasmids have been used before in our laboratory

and a detailed description of their construction can be found else-

where (Esteban et al., 2013; Murcia-Belmonte et al., 2010).

Untransfected CHO cells and transiently transfected CHO

cells with the different expression plasmids were cultured for 24–36

h and ECM protein extracts were prepared as described previously

(Garc�ıa-Gonz�alez et al., 2010; Murcia-Belmonte et al., 2010).

Briefly, the cells were washed with calcium/magnesium-free Hank’s

Balanced Salt Solution and then incubated with gentle rocking for

30–45 min at 4�C in 1.5 mL/culture dish (10 cm in diameter) of

20 mM phosphate buffer (PB) pH 7.4, containing 500 mM NaCl

and complete EDTA free protease inhibitor (Roche). The ECM pro-

teins released into the buffer were concentrated 10–15 times and

dialyzed to eliminate the excess of NaCl against 20 mM PB pH 7.4

with complete EDTA free protease inhibitor (Roche) using an Ami-

con Ultra-4 Ultracel-30k (Millipore Corporation, Billerica, MA).

Equal amounts of total protein from the CT and the different

anosmin-1 concentrated ECM extracts were used in the chemotaxis

and signaling experiments (0.1 lg/mL). The presence of the proteins

in concentrated ECM extracts was confirmed by western blot, using

and anti-HA rat monoclonal peroxidase-conjugated antibody (High

Affinity 3F10; Roche).

Chemotaxis Assays and Cell ImmunostainingOPCs were obtained from the cortex of P0 postnatal Wistar rats and

C57BL/6 and CD1 mice, following an adapted protocol for OPC

isolation by shaking (McCarthy and de Vellis, 1980; Molina-

Holgado et al., 2002). OPC migration studies were performed in

chemotaxis chambers with polycarbonate membranes (pore size 8

mm; Corning Costar). The membranes were coated with poly-L-

lysine and laminin as described previously (Merch�an et al., 2007;

Murcia-Belmonte et al., 2010). OPCs from rat or mouse were

seeded (40,000 cells/transwell) in the upper chamber in Bottenstein-

Sato (BS) medium supplemented with 1% fetal bovine serum

(Bribi�an et al., 2006; Spassky et al., 2002) while in the lower com-

partment the same culture medium was supplemented for the differ-

ent experimental groups as follows: (I) CT; (II) FGF2 (20 ng/mL;

recombinant human FGF2, RD Systems 233-FB); (III) A1; (IV)

A1Nt; (V) A1E514K; (VI) A1F517L. The cells were treated during

the experiment with the FGFR blocker SU5402 (10 mM; Calbio-

chem-Merck), the MEK1/2 inhibitor U0126 (10 lM; Sigma-

Aldrich), where indicated, and the rest of the cultures were exposed

to an equal volume of the vehicle DMSO (Sigma-Aldrich) during

the course of the experiment which was carried out at 37�C, 5%

CO2, and at 95% relative humidity. After 20 h, cells were fixed with

4% paraformaldehyde (PFA; for 15 min at RT), washed 3 times

with phosphate buffer saline (PBS, pH 7.4) and the nonmigratory

cells on the upper membrane surface were removed with a cotton

swab. The presence of transmigrated OPCs in the lower chamber

was evaluated by immunostaining with A2B5 antibody (anti-Gangli-

oside GT3 (Eisenbarth et al., 1979); henceforth we will use A2B5 to

make reference to the antibody and the antigen; 1:10, ATCC; CRL-

1520, Hybridoma Bank) and anti-Olig2 (1:200, AB9610 Millipore)

antibodies and their corresponding fluorescent secondary antibodies

(Bribi�an et al., 2006, 2008; Merch�an et al., 2007). After immuno-

staining, the Boyden filters were examined with an In Cell Analyzer

1000 (GE-HealthCare) and 16 microphotographs from each mem-

brane were taken randomly. To quantify chemoattraction, the num-

ber of transmigrated OPCs per field was counted using the software

In Cell Analyzer 1000 Workstation (GE-HealthCare). The data were

expressed as percentage of migrating OPCs relative to control condi-

tions 6 SEM, considered as 100% (Esteban et al., 2013; Garc�ıa-

Gonz�alez et al., 2010; Murcia-Belmonte et al., 2010) and they were

analyzed with the Sigmastat software package (SPSS). For each one

of the conditions assayed, representative images captured with a

Leica TCS-SP5 Confocal Laser-Scanning microscope are shown.

Some of the purified cells were seeded onto coverslips of 14 mm in

diameter coated with poly-L-lysine and laminin and cultured in BS

medium for 2 days before fixation with 4% PFA and immunostain-

ing against A2B5, Olig2, and FGFR1 (1:100, Santa Cruz Biotech-

nologies sc-121-G). Images were taken with a SP5 Leica confocal

microscope and representative images are shown.

SignalingPurified P0 rat OPCs (1 3 106 cells/well) were seeded in poly-L-

lysine coated P12-well plates and purified P0 C57BL/6 mouse

OPCs (80,000 cells/well) were plated in poly-L-lysine coated 96-well

tissue culture plates and incubated for 24 h in culture medium:

DMEM supplemented with 10% Fetal Bovine Serum (FBS; Bio-

Whittaker, Lonza DE14–801F), antibiotic and antimycotic solution

(Sigma-Aldrich A5955) at 37�C, 5% CO2, and at 95% humidity.

After 24 h, cells were serum-starved for 6 h in DMEM and treated

with the FGFR blocker SU5402 (10 mM; Calbiochem-Merck) where

376 Volume 62, No. 3

indicated for 30 min previous to stimulation, while the rest of the

wells were exposed to an equal volume of the vehicle DMSO

(Sigma-Aldrich). Stimulation was carried out for 30 min with FGF2

(20 ng/mL; recombinant human FGF2, RD Systems 233-FB) and

ECM concentrated extracts from CHO control cells (CT) and from

CHO cells transfected with the expression plasmids carrying the dif-

ferent C-terminal HA-tagged versions of anosmin-1. The FGFR

blocker SU5402 (10 mM) or an equal volume of the vehicle DMSO

were used where indicated during stimulation.

After 30 min, rat OPCs were washed with cold PBS and lysed

in lysis buffer (PBS pH 7.4, 1 mM EDTA, 1% NP-40, complete

EDTA free protease inhibitor and PhosSTOP Phosphatase Inhibitor

Cocktail Tablets, Roche). An equivalent amount of lysate was boiled

for 5 min in 23 Laemmli’s sample buffer (Sigma-Aldrich). Mouse

OPCs were fixed with PFA 4% during 15 min and washed with PBS

containing 0.1% Triton X-100. Rat OPC lysates were resolved by

SDS–PAGE, transferred onto nitrocelullose membranes (Santa Cruz

Biotechnology) and immunoblotted for detection with anti-ERK

(Santa Cruz Biotechnology, sc-93) and anti-phospho-ERK (Santa

Cruz Biotechnology, sc-7383) antibodies. Fixed mouse OPCs in 96-

well tissue culture plates were analyzed by In-Cell western using the

Odyssey Infrared Imaged System. OPCs were immunostained with

anti-ERK (Santa Cruz Biotechnology, sc-93) and anti-phospho-ERK

(Santa Cruz Biotechnology, sc-7383) antibodies following the instruc-

tions from the manufacturer. In both cases, secondary antibodies con-

jugated with 680 or 800 infrared fluorescent dyes (IRDye) were used

for simultaneous analysis of ERK and pERK in separate fluorescent

channels using the Odyssey Infrared Imaging System (LICOR). The

quantitative analysis was carried out following the Odyssey LICOR

instruction manual. The values 6 SEM of the relative amount of

pERK relative to the total amount of ERK, normalized against the

value of the relative pERK/ERK amount of the cells exposed to the

ECM extracts of CHO CT cells considered as 100%, are represented.

Quantitative and Statistic AnalysisThe data were compared using by one-way ANOVA or the corre-

sponding tests on ranks and were presented as the mean 6 SEM.

Statistical analysis of the quantitative results was conducted using the

Sigmastat software package (SPSS). Minimal statistical significance

was fixed at P <0.05 (*).

Results

Chemoattractive Effect of Anosmin-1 and DifferentMutant Forms of This Protein on Cortical OPCsWe have previously described the chemoattractive effect of

anosmin-1 on rat SVZ neuronal precursors (NPs) via FGFR1

and that this effect is lost in full-length anosmin-1 harboring

different missense mutations found in KS patients (Esteban

et al., 2013; Garc�ıa-Gonz�alez et al., 2010; Murcia-Belmonte

et al., 2010). On the other hand, our group has reported a

negative modulatoy role of anosmin-1 on the motogenic and

chemotactic effect exerted by FGF2/FGFR1 on embryonic

and adult OPCs (Bribi�an et al., 2006; Clemente et al., 2011).

To broaden the knowledge of the effects of anosmin-1 on

OPC migration, we used rat and C57BL/6 mouse P0 cortical

OPCs in chemotaxis assays. We first confirmed the presence

of FGFR1 by immunostaining in rat and mouse OPCs posi-

tive for Olig2 and A2B5 used in our experiments (Fig. 1).

Our results indicated that both human FGF2 and human

anosmin-1 had a clear chemoattractive effect on rat and

mouse OPCs (Fig. 2A,B; Supporting Information Fig. 1A,C)

(Henceforth, we will refer to human FGF2 and human

anosmin-1 used in all the experiments as FGF2 and anosmin-

1). This chemotactic effect is suppressed by the FGFR inhibi-

tor SU5402, suggesting that it is mediated by FGFR1 (Fig.

2A,B; Supporting Information Fig. 1A,C). Since, the results

obtained with anosmin-1 were not in agreement with previ-

ous results reported by our own group in embryonic ON

OPCs and adult cortical OPCs from CD1 mice (Bribi�an

et al., 2006; Clemente et al., 2011), we performed a series of

chemotaxis assays with P0 cortical OPCs from CD1 mice.

Also in this case, both FGF2 and anosmin-1 showed a clear

chemotactic effect on these cells (Fig. 3).

We asked next if, as in the case of GN11 cells and rat

SVZ NPs, the presence of the E514K and F517L substitu-

tions would annul this chemotactic effect (Cariboni et al.,

2004; Murcia-Belmonte et al., 2010). Surprisingly, while the

F517L mutation reduced the migration of rat and mouse

OPCs to CT levels, the E514K substitution did not (Fig.

2A,B; Supporting Information Fig. 1A,C). The addition of

the FGFR inhibitor SU5402 annulled the effect of the

A1E514K protein, indicating that this effect is also exerted

via FGFR1 (Fig. 2A,B; Supporting Information Fig. 1A,C).

The presence of the different versions of anosmin-1 used in

these experiments and in all the subsequent chemotaxis and

signaling experiments was detected by western blot in CHO

ECM concentrated extracts (Fig. 2C).

The activation of the ERK1/2 MAPK pathway by FGF2

and anosmin-1, via FGFR1, is necessary to promote their che-

motropic effect on rat SVZ neuroblasts (Esteban et al., 2013),

but the intracellular signaling controlling OPC migration is

poorly understood (Rajasekharan, 2008). To investigate the

FGFR1 downstream signaling involved in the chemotropic

effect elicited by FGF2 and anosmin-1 on rat and mouse corti-

cal OPCs, we performed chemotaxis assays using U0126, a

specific inhibitor of the ERK1/2 MAPK pathway. In the che-

motaxis assays performed the blockade of ERK activation

inhibits the chemotactic effect exerted by FGF2, anosmin-1,

and the mutant form A1E514K on both rat and mouse OPCs

(Fig. 4A,B; Supporting Information Fig. 1B,D).

The Truncated Form of Anosmin-1 (A1Nt) Retainsthe Chemotropic Effect on Cortical OPCsThe N-terminal region of anosmin-1 (A1Nt) comprising the

CR, WAP, and FnIII.1 domains, is able to bind to FGFR1


March 2014 377

(Hu et al., 2009; Murcia-Belmonte et al., 2010). This trun-

cated form of anosmin-1 retains some of the biological func-

tions attributed to full-length anosmin-1 (B€ulow et al.,

2002; Esteban et al., 2013; Gonz�alez-Mart�ınez et al., 2004;

Hu et al., 2004; Murcia-Belmonte et al., 2010). When

tested in chemotaxis assays, A1Nt showed a chemotactic

effect on both rat and mouse newborn cortical OPCs (Fig.

5A–C). The treatment of the cells with the FGFR blocker

SU5402 and the MEK1/2 inhibitor U0126 abolished the

chemoattraction exerted by A1Nt (Fig. 5A–C), indicating

the participation of FGFR1 and the ERK1/2 pathway in

this effect.

FGF2, Anosmin-1, A1E514K, and A1Nt Activatethe ERK MAPK Pathway in OPCsTo confirm that FGF2 and anosmin-1 activated the ERK

MAPK pathway via FGFR1, purified rat and mouse cortical

OPCs were plated on poly-L-lysine-coated P12-well plates or

poly-L-lysine-coated P96-well plates, respectively. The cells

were treated with FGF2 and the different forms of anosmin-1

in the presence or not of the FGFR blocker SU5402. Both

FGF2 and anosmin-1 triggered the activation of the ERK

pathway in rat and mouse cortical OPC cultures (Fig. 6A–C).

While the F517L substitution was not able to induce the acti-

vation of the ERK pathway, the mutant form A1E514K and

FIGURE 1: Expression of FGFR1 in rat and mouse OPCs. Representative images of oligodendrocyte precursor cells purified from P0 new-born rat and C57BL/6 mouse cerebral cortices immunostained for Olig2, A2B5, and FGFR1. The scale bar represents 25 mm.


the truncated N-terminal protein A1Nt triggered ERK phos-

phorylation (Fig. 6A–C). In all the conditions assayed, the

activation is mediated via FGFR1, since the treatment of the

cells with the FGFR blocker SU5402 impeded the activation

of the ERK1/2 pathway (Fig. 6A–C).

Discussion

The role of FGF2/FGFR1 in OPC motility has been reported

before (Armstrong et al., 1990; Bansal et al., 1996; Bribi�an

et al., 2006; Clemente et al., 2011; McKinnon et al., 1993).

This would be corroborated by our present results in

FIGURE 2: FGF2 and anosmin-1 have a chemotactic effect on rat OPCs. In chemotaxis assays using Boyden chambers, FGF2, andanosmin-1 showed a clear chemoattractive effect on newborn rat cortical OPCs (A, B). This effect is mediated through the activation ofFGFR1 since the blocker SU5402 canceled the effect. The F517L substitution annulled the chemoattraction but, surprisingly, the E514Ksubstitution did not seem to affect the chemotactic effect of the protein via FGFR1 (A, B). The presence of transmigrated OPCs on theopposite side of the membrane was evaluated by immunocytochemistry against A2B5/Olig2. A representative image is shown for all theconditions analyzed (A) and the scale bar represents 100 mm. The data are shown as percentage of migrating OPCs 6 SEM relative tocontrol conditions (CT), considered as 100% (B). ANOVA test (Bonferroni correction) or their corresponding tests on ranks were usedand the minimal statistical significance was fixed at P < 0.05 and represented as *. The presence of the different forms of anosmin-1 inthe ECM extracts of CHO cells used in this and in the rest of the experiments was confirmed by western blotting using an anti HA anti-body (A1: full-length anosmin-1; A1E514K full-length mutant anosmin-1 E514K; A1F517L: full-length mutant anosmin-1 F517L; A1Nt:truncated N-terminal anosmin-1) (C).


March 2014 379

newborn rat and mouse cortical OPCs. Anosmin-1 has been

shown to act as a negative modulator of the FGF2/FGFR1-

promoted migratory ability of embryonic ON and adult cort-

ical OPCs from CD1 mice (Bribi�an et al., 2006; Clemente

et al., 2011). As a substrate molecule anosmin-1 reduces

OPC migration most likely by increasing their adhesion, but

the use of an antibody against human anosmin-1, hampers

the correct adhesion and subsequent migration of OPCs

(Bribi�an et al., 2008). Our data strongly support a positive

chemotactic effect of anosmin-1 on cortical OPCs from new-

born animals (P0) mediated through FGFR1, in agreement

with previous results that show that anosmin-1 acts as a che-

moattractive factor for different cell types (Cariboni et al.,

2004; Esteban et al., 2013; Garc�ıa-Gonz�alez et al., 2010; Hu

et al., 2009, 2013; Jian et al., 2009; Murcia-Belmonte et al.,

2010). It has been hypothesized that depending on the bind-

ing dynamics of anosmin-1 to preformed FGF2-FGFR1 pairs

or to FGFR1 directly, it could facilitate FGFR1 signaling and

FIGURE 3: FGF2 and anosmin-1 have a chemotactic effect on newborn cortical OPCs from CD1 mice. In chemotaxis assays using Boydenchambers, FGF2 and anosmin-1 also increased the number of transmigrated newborn cortical OPCs from CD1 mice, as evaluated byimmunocytochemistry against A2B5/Olig2. A representative image is shown for the three conditions analyzed and the scale bar repre-sents 100 mm. The data are shown as percentage of migrating OPCs 6 SEM relative to control conditions (CT), considered as 100%.ANOVA test (Bonferroni correction) or their corresponding tests on ranks were used and the minimal statistical significance was fixed atP < 0.05 and represented as *.


cell migration or hamper both (Hu and Bouloux, 2011; Hu

et al., 2009). Particularly, the glucidic composition and struc-

ture of heparan-sulfate proteoglycans (that can vary with spe-

cies, mouse strain, cell type, or age), could determine the

binding and function of the different anosmin-1 domains

(Andrenacci et al., 2006; B€ulow et al., 2002; B€ulow and

Hobert, 2004; Kim et al., 2008; Tornberg et al., 2011). Differ-

ent lines of evidence support the notion that OPCs and oligo-

dendrocytes are heterogeneous populations of cells that could

express different ECM proteins and membrane receptors and

behave differently depending on origin and age (Kessaris et al.,

2006; Richardson et al., 2006). Therefore, anosmin-1 could

exert different responses in embryonic, newborn, or adult

OPCs, as well as in OPCs from different CNS regions.

Mutant forms of anosmin-1 behave as wild-type or

mutant proteins depending on the cellular type and effect

FIGURE 4: The chemotactic effect of FGF2 and anosmin-1 on rat OPCs mediated via FGFR1 requires the activation of the ERK1/2 MAPKpathway. The blockade of ERK signaling by the MEK inhibitor U0126 (U0) annulled the migration promoted by these proteins in ratOPCs. The chemoattraction promoted by the mutant form A1E514K is also blocked when the ERK1/2 activation is inhibited (A, B).Microphotographs of A2B5/Olig2 positive OPCs are showed for all the conditions analyzed (A). The scale bar represents 100 mm. Thedata are presented as percentage of migrating OPCs 6 SEM relative to control conditions (CT), considered as 100% (B). ANOVA test(Bonferroni correction) or their corresponding tests on ranks were used and the minimal statistical significance was fixed at P < 0.05 andrepresented as *.


March 2014 381

studied (Andrenacci et al., 2006; B€ulow et al., 2002; Hu

et al., 2004). The E514K and F517L substitutions in FnIII.3

produce a 2-fold and a 6-fold reduced binding affinity to

FGFR1 in vitro (Hu et al., 2009), or impede the binding of

FnIII.3 (Murcia-Belmonte et al., 2010), canceling the chemo-

tropic effect of anosmin-1 on GN11 cells and NPs (Cariboni

et al., 2004; Murcia-Belmonte et al., 2010). Together with

the distinctive composition of the ECM of rat and mouse

newborn cortical OPCs, could explain a more efficient bind-

ing to FGFR1 of the A1E514K protein that would behave as

a wild-type protein in these cells. The N-terminal region of

anosmin-1 binds to FGFR1 (Hu et al., 2009; Murcia-

FIGURE 5: The truncated N-terminal region of anosmin-1 (A1Nt) retained the chemotactic activity on rat and mouse OPCs through theactivation of FGFR1 and the ERK1/2 MAPK pathway. Transmigrated OPCs on the opposite side of the membrane were immunostainedagainst A2B5/Olig2 and a representative image is shown for all the conditions analyzed (A). The scale bar represents 100 mm. The dataare shown as percentage of migrating OPCs 6 SEM relative to control conditions (CT), considered as 100% (B: rat OPCs; C: mouseOPCs). ANOVA test (Bonferroni correction) or their corresponding tests on ranks were used and the minimal statistical significance wasfixed at P < 0.05 and represented as *.


FIGURE 6: FGF2 and anosmin-1 activated ERK1/2 in rat and mouse OPCs. The missense mutation E514K did not interfere with thecapacity of the protein to activate this pathway in rat OPCs, contrasting with the effect of the F517L substitution that convertedanosmin-1 in a nonfunctional protein that was not able to activate this pathway. The treatment with SU5402 impeded the activation,indicating that FGF2 and anosmin-1 activated the ERK1/2 pathway through the activation of FGFR1 (A). The truncated form of anosmin-1, A1Nt, is able to induce the activation of ERK1/2 via FGFR1 (B). Approximately 1 3 106 cells were incubated for 24 h in poly-L-lysine-coated P12-well plates in DMEM supplemented with 10% FBS, were serum starved for 6 h in DMEM and treated with the FGFR blockerSU5402 where indicated. The cells were then stimulated for 30 min with FGF2 and ECM extracts from CHO control cells and from CHOcells expressing the C-terminal HA-tagged full-length anosmin-1 (A1) and the mutant forms A1E514K, A1F517L, and A1Nt. Lysates wereresolved by SDS–PAGE and using the Odyssey Infrared Imaging System (LI-COR), a quantitative analysis was carried out following theOdyssey LI-COR instruction manual. In the case of mouse OPCs 80,000 cells were plated in poly-L-lysine-coated P96-well plates andtreated with the different proteins. An in-cell western assay was performed using the Odyssey Infrared Imaging System (LI-COR) againstERK and pERK (C). In all the panels values 6 SEM are represented as the relative amount of pERK/ERK, normalized against the value ofthe relative pERK/ERK amount of the cells exposed to the ECM extracts of CHO CT cells considered as 100%.

Belmonte et al., 2010) and some mutations impede the bind-

ing to the receptor (Hu et al. 2009; Esteban et al., 2013) com-

promising the function of the protein and (B€ulow et al., 2002;

Cariboni et al., 2004; Esteban et al., 2013; Gonz�alez-Mart�ınez

et al., 2004). This truncated protein, A1Nt, exerts some of the

effects of the full-length protein in different cellular environ-

ments (B€ulow et al., 2002; Gonz�alez-Mart�ınez et al., 2004; Hu

et al., 2004), acting via FGFR1 to promote rat NP migration

(Esteban et al., 2013; Murcia-Belmonte et al., 2010). Accord-

ingly, our results showed a chemotactic effect of A1Nt through

FGFR1 on rat and mouse OPCs. We have demonstrated the

expression and secretion of this truncated protein into the cul-

ture medium (Esteban et al., 2013; Murcia-Belmonte et al.,

2010). The extraction of this truncated protein in salt extracts

from the ECM demonstrated that it is retained in the cell sur-

face of transfected CHO cells, consistent with reports that sug-

gest that the first FnIII domain is the main responsible for the

binding of anosmin-1 to the ECM (Hu et al., 2004).

Despite the increasing knowledge of the extracellular

cues governing OPC migration (de Castro and Bribi�an, 2005;

de Castro et al., 2013; de Castro and Zalc, 2013; Jarjour and

Kennedy, 2004) not much is known about the intracellular

pathways involved in the regulation of OPC migration (Raja-

sekharan, 2008). The effect promoted by different ECM and

membrane molecules on OPC migration seems to rely on the

activation of the Rho GTPases, crucial in the rearrangement

of the cytoskeleton involved in cell motility (Binam�e et al.,

2013; Liu et al., 2012; Novgorodov et al., 2007). Similarly,

the activation of different kinases including Fyn kinase and

the ERK1/2 signaling pathway, has been reported as necessary

for the migration of OPCs induced by PDGFa and FGF2

(Frost et al., 2009; Miyamoto et al., 2008; Vora et al., 2011).

In agreement with some of these previous observations our

results clearly showed the implication of the ERK1/2 signal-

ing in the chemotropic effect promoted by FGF2 and the dif-

ferent forms of anosmin-1 in OPCs mediated by FGFR1. We

have previously shown that anosmin-1 via FGFR1 induces rat

SVZ NP migration through the activation of ERK1/2 (Este-

ban et al., 2013). By contrast, the activation of this pathway

does not seem necessary for the chemotactic effect exerted by

anosmin-1 on FNC-B4hTert neurons, response that requires

the activation of PI3K signaling (Hu et al., 2013).

The identification of factors such as FGF2 and

anosmin-1 that participate in OPC migration and the intra-

cellular signaling involved, could be relevant for the develop-

ment of cellular and pharmacological therapies aimed to

facilitate endogenous OPC migration towards the lesions and

to restore the damage in demyelinating diseases and after spi-

nal cord injury (Crawford et al., 2013; Kremer et al., 2011).

Anosmin-1 positive oligodendroglial cells have been described

in the corpus callosum and the ON during development

(Bribi�an et al., 2006, 2008; Clemente et al., 2008). These

observations and the new data showing the participation of

this protein in OPC migration should be taken into consider-

ation in the evaluation of KS satellite symptoms, their expla-

nation and treatment (Garc�ıa-Gonz�alez et al., in press).

Acknowledgment

Grant sponsor: Spanish Ministerio de Econom�ıa y Com-

petitividad MINECO; Grant numbers: SAF2009-07842;

ADE10-0010; and RD07-0060-2007; Grant sponsor:

Fundaci�on Para la Investigaci�on Socio-Sanitaria de Castilla-La

Mancha FISCAM.; Grant numbers: PI2009/29 to PFE; and

MOV2007-JI/19.

The authors are grateful to Dr. Jos�e �Angel Rodr�ıguez

Alfaro and Dr. Javier Mazar�ıo for their help with the confocal

imaging and Isabel Mach�ın, Rafael Lebr�on, Iris S�anchez, and

Jacinto Sarmentero for their technical assistance. PFE was a

researcher hired by SESCAM, currently hired under ADE10-

0010.

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ERK1/2 signaling is essential for the chemoattraction exerted by human FGF2 and human anosmin-1 on newborn rat and mouse OPCs via FGFR1