www.elsevier.com/locate/ymcne

Mol. Cell. Neurosci. 25 (2004) 345–354

Blocked MAP kinase activity selectively enhances neurotrophic

growth responses$

Susanna Althini,a Dmitry Usoskin,a Annika Kylberg,a Paul L. Kaplan,b,1 and Ted Ebendala,*

aDepartment of Neuroscience, Unit for Developmental Neuroscience, Biomedical Centre, Uppsala University, Uppsala, SwedenbCuris Inc., Boston, MA 02138, USA

Received 5 June 2003; revised 3 October 2003; accepted 21 October 2003

Bone morphogenetic proteins (BMPs) 4 and 6 as well as MEK

inhibitors PD98059 and U0126 potentiate neurotrophin 3 (NT3)- and

neurturin (NTN)-induced neurite outgrowth and survival of peripheral

neurons from the E9 chicken embryo. Preexposure to BMP4 or

PD98059 was sufficient to prime the potentiation of subsequently added

NT3. Phosphorylation of Erk2, induced by NT3, was reduced by MEK

inhibition but unaffected by BMP signaling. Real-time PCR showed

that neither BMP stimulation nor MEK inhibition increased Trk

receptor expression and that the BMP-induced genes Smad6 and Id1

were not upregulated by PD98059. In contrast, both MEK inhibition

and BMP signaling suppressed transcription of the serum-response

element (SRE)-driven Egr1 gene. A reporter assay using NGF-

stimulated PC12 cells demonstrated that MEK/Erk/Elk-driven tran-

scriptional activity was inhibited by Smad1/5 and by PD98059. Thus,

suppression of SRE-controlled transcription represents a likely

convergence point for pathways regulating neurotrophic responses.

D 2004 Elsevier Inc. All rights reserved.

Introduction

Development, growth, and maintained functions of neurons are

regulated by neurotrophic proteins (Kaplan and Miller, 2000;

Patapoutian and Reichardt, 2001). One gene family involved

encodes the neurotrophins (NTs) that include nerve growth factor

(NGF), brain-derived neurotrophic factor (BDNF), and neurotro-

phin 3 and 4 (NT3 and NT4, respectively; reviewed by Huang and

Reichardt, 2001). Another gene family encodes GDNF receptor

ligands (GFRL) comprising the glial cell line-derived neurotrophic

factor (GDNF), neurturin (NTN), artemin (ART), and persephin

(PSP; Airaksinen and Saarma, 2002; Baloh et al., 2000). The

neurotrophins and the GFRLs activate receptor tyrosine kinases

1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2003.10.015

$ Supplementary data associated with this article can be found, in the

online version, at doi: 10.1016/j.mcn.2003.10.015.

* Corresponding author. Department of Neuroscience, Unit for

Developmental Neuroscience, Biomedical Centre, Uppsala University,

Box 587, Room A2:203d, Husargatan 3, D1, Plan 4, 5, SE-751 23 Uppsala,

Sweden. Fax: +46-18-559-017.

E-mail address: Ted.Ebendal@neuro.uu.se (T. Ebendal).1 Present address: Genzyme Inc., Cambridge, MA 02139, USA.

Available online on ScienceDirect (www.sciencedirect.com.)

(TrkA, TrkB, TrkC for NTs and Ret for GFRLs) in addition to

binding to accessory receptors (p75 and GFRa1–4, respectively).

A mitogen-activated protein kinase (MAPK) pathway used by

ligand-activated Trk and Ret leads to the phosphorylation and

activation of Erk1/2. Other receptors expressed by neurons include

serine/threonine kinases that are activated by members of the

superfamily of transforming growth factor-hs (TGFhs) includingbone morphogenetic proteins and growth/differentiation factors

(BMPs and GDFs; Ebendal et al., 1998; Massague and Chen,

2000; Mehler et al., 1997; Miyazawa et al., 2002).

We previously showed that BMP7 strongly potentiates neuro-

trophic responses to NT3 and GDNF, in line with the idea that the

tyrosine- and the serine/threonine-kinase receptor pathways coop-

erate to promote neuron survival and process outgrowth in neurons

(Bengtsson et al., 1998). The synergistic effects are now demon-

strated to occur in a range of different nerve cells indicating that the

observed reaction is widespread among neurons. Other observa-

tions also suggest that further members of the TGFh superfamily

have the ability to potentiate the neurotrophic actions of GDNF

(Farkas et al., 1999; Unsicker and Krieglstein, 2000).

The present paper describes an analysis of the pathways

involved in the synergistic actions of neurotrophic factors and

brain morphogenetic proteins (BMPs) using pharmacological

inhibitors of kinases. A general requirement of PI3K for neurite

outgrowth is demonstrated. A striking potentiation of NT3 and

NTN neurotrophic effects by MEK inhibitors is also shown. These

effects are mimicking the synergistic actions of BMP acting in

concert with these neurotrophic factors. Finally, it is shown that

BMP in addition to activating its cognate target genes Smad6 and

Id1 is suppressing transcriptional activation at the serum-response

element (SRE) of the Egr1 gene. This finding identifies a new

convergence point for MEK/Erk/Elk and BMP/ALK/Smad signal-

ing pathways.

Results

BMPs and MEK inhibitors potentiate neurotrophic activities

BMP7 was previously found to potentiate the neurotrophic

effects of NT3 and GDNF (Bengtsson et al., 1998). We now

extend the range of neurotrophic factors tested with BMPs to

S. Althini et al. / Mol. Cell. Neurosci. 25 (2004) 345–354346

include NTN (Creedon et al., 1997; Rosenthal, 1999). Moreover,

BMP6 belonging to the Gbb-60A subfamily of BMPs was added

(Miyazawa et al., 2002; see supplementary data material Fig. 1).

NTN gave a stronger outgrowth response than GDNF in both

sympathetic and ciliary ganglia in accordance with results from

Forgie et al. (1999). The NTN-induced neurite outgrowth in

sympathetic cultures was efficiently increased by BMP6. The

synergistic effects were obvious also in ciliary ganglia (supple-

mentary data material Fig. 2). Moreover, BMP4, a more distant

relative belonging to the Dpp-subfamily of BMPs (Miyazawa et

al., 2002), very efficiently increased the fiber outgrowth in com-

bination with NT3 or NTN (Figs. 1A–B) but did not stimulate the

neurons when added alone (supplementary data material Fig. 1D).

Fig. 1. Potentiation of neurotrophic responses. Darkfield micrographs of sympathe

fiber outgrowth. (B) BMP4 has no neurotrophic effect whereas it is in combination

in nerve fiber outgrowth in NT3-stimulated neurons with MEK inhibited by PD980

NT3-stimulated ganglia. (E) Scored fiber outgrowths in sympathetic ganglia confi

the NT3-stimulated cultures, even with a 1-day delay of the MEK inhibitor. Similar

PD98059. (F) Inhibition of Erk phosphorylation by PD98059 demonstrated by Wes

antibodies showed an increased signal with NT3 or with NT3 plus BMP4 90 min

Erk (p-Erk2) without affecting the level of total Erk2 protein (ratios of phosphoryl

graph below). Phospho-Smad1/5 antibodies demonstrate activation of these BMP

We next addressed the issue of signal-transducing pathways

from the Trk and Ret receptor tyrosine kinases being essential for

the BMP potentiation of neurite outgrowth. The role of the Raf/

MEK1/Erk1,2 pathway in potentiation of NT3 was tested by the

use of the pharmacological MEK inhibitors. In cultures with

sympathetic ganglia stimulated by NT3 and BMP4, PD98059 did

not inhibit outgrowth but rather appeared to further enhance the

fiber halo (not shown). This was the impetus to test the MEK

inhibitor with different neurotrophic factors but without the extra

addition of BMPs. Unexpectedly, PD98059 alone, when combined

with NT3, gave rise to drastically increased nerve fiber outgrowths

(Fig. 1C). The neurites were similar to those formed from sympa-

thetic ganglia stimulated by NGF. The increased NT3-induced

tic ganglia explanted to collagen gels for 2 days. (A) NT3 results in sparse

with NT3 strongly potentiates neurite formation. (C) Drastic improvement

59. (D) Inhibition of PI3K with LY294002 blocked the effect of PD98059 in

rmed significant increases in fiber outgrowth when PD98059 was added to

results were obtained testing GDNF and PD98059 as well as with NTN and

tern blotting of embryonic day 9 chicken sympathetic ganglia. Phospho-Erk

after stimulation. In contrast, the MEK inhibitor reduced phosphorylation of

ated over total Erk2 protein as determined by densitometry shown in the bar

Smads (p-Smad1/5) only in ganglia stimulated with BMP4.

S. Althini et al. / Mol. Cell. Neu

outgrowth in sympathetic ganglia with PD98059 was seen already

after 20 h of culture. The substantial reduction in neurite outgrowth

found upon PI3K inhibition by LY294002 was not reverted by the

MEK inhibition (Fig. 1D). In contrast to the positive effects of

PD98059 on NT3-stimulated ganglia, MEK inhibition did not

further enhance NGF-induced outgrowth, even when NGF was

given at suboptimal concentrations (not shown). The selective

inhibition of Erk2 phosphorylation by PD98059 and U0126

(Davies et al., 2000), as well as the reduction of Akt phosphory-

lation upon addition of the PI3 kinase inhibitor LY294002, were

confirmed by Western blot analysis of lysates of ganglia stimulated

by NGF (supplementary data material Fig. 3). In order to test

whether signaling from tyrosine kinase receptors other than Trk

could be potentiated by MEK inhibition, sympathetic ganglia were

stimulated by GDNF (Ebendal et al., 1995) or NTN, signaling by

activation of receptor tyrosine kinase Ret. PD98059 potentiated the

fiber outgrowth responses stimulated by GDNF and to some extent

also NTN (Fig. 1E). Thus, a striking similarity in potentiating

nerve growth between BMPs and a MEK inhibitor was found

among NT3-, GDNF-, or NTN-stimulated neurons.

The effect of PD98059 on NT3-induced neurite outgrowth

resembles the effects of the Trk kinase inhibitor K252b and its

derivative L753000 established previously (Knusel et al., 1992;

Pollack et al., 1999). We presently confirmed that K252b, like

PD98059, strongly potentiates the bioactivity of NT3 under the

current culture conditions (supplementary data material Fig. 4).

Fig. 2. Neuron survival as well as fiber outgrowth stimulated by MEK inhibition in

grown for 2 days in collagen gels in control medium or in medium with added

PD98059, NT3, or both. (B) Ciliary neurons responded to GDNF or NTN by incre

significantly. (C) U0126 increased NT3-induced fiber outgrowth in sympathetic (

explanted nodose ganglia stimulated for 2 days with NT3 (D), NT3 plus BMP6

Phosphorylation of Smad and Erk signal transducers

To examine whether signals from BMP and neurotrophic factor

receptors converge on the downstream Smad and Erk substrata, we

performed Western blot analysis of Erk1/2 (represented by the

single protein species MAP kinase Erk2/p42 in the chicken) and

Smad1/5 phosphorylation in explanted E9 sympathetic ganglia.

The results show that phosphorylation of Erk2 (at positions T193,

Y195 of chErk2; GenBank accession number AY033635) was

stimulated by NT3 after 90 min, and that MEK inhibitor

PD98059 effectively reduced phosphorylation of Erk2 without

affecting the amount of Erk protein (Fig. 1F). Addition of BMP4

did not reduce the levels of phospho-Erk compared to those seen in

controls or in the NT3-stimulated explants. Phosphorylation of the

C-terminal SSXS motif of Smad1/5 in these ganglia was strongly

increased by the addition of BMP4 but unaffected by the presence

of NT3 or PD98059. Thus, we obtained no evidence for crosstalk

between NT3 and BMP pathways at the level of activating

phosphorylation of Erk or Smad.

Specificity of MEK inhibitor-induced neurite outgrowth

We thereafter asked whether the MEK inhibition, like BMP

stimulation (Bengtsson et al., 1998; Farkas et al., 1999), potentiates

neuron survival supported by neurotrophic factors. This was tested

in a survival assay using dissociated sympathetic and ciliary

rosci. 25 (2004) 345–354 347

different types of neurons. Dissociated sympathetic or ciliary neurons were

neurotrophic factors. (A) Sympathetic neurons incubated for 2 days with

ased survival. PD98059 added together with NTN further enhanced survival

SYMP.) and nodose (NOD.) ganglia. (D–F) Darkfield micrographs of the

(E), or NT3 with PD98059 (F).

S. Althini et al. / Mol. Cell. Neu348

neurons. Increased survival was found when MEK inhibition was

combined with NT3 in sympathetic neurons (Fig. 2A) and with

NTN, but not GDNF, in ciliary neurons (Fig. 2B). Hence, inhibi-

tion of MEK like addition of BMP gave a strong burst to neurite

formation and increased neuron survival in various ganglionic

neurons stimulated by NT3, GDNF, or NTN.

A similar effect was found by applying the chemically unrelated

inhibitor U0126 that has a high degree of specificity for MEK

(Favata et al., 1998). U0126 was tested on sympathetic as well as

nodose ganglia (harbouring large sensory neurons) in the presence of

NT3 (Fig. 2C). At levels blocking MEK activity, U0126 distinctly

potentiated the fiber outgrowth response to NT3 whereas the vehicle

(0.1% DMSO with or without U0124, data not shown) lacked

influence. Thus, Erk inhibition by two different MEK inhibitors

(PD98059, U0126), like BMP stimulation, strongly potentiates

neurotrophic effects also in sensory neurons (Figs. 2D–F).

Fig. 3. (A–D) Sequential stimulation of sympathetic ganglia. (A) A 4-h priming pe

NT3 for 2 days. The resulting outgrowth is indistinguishable from that arising from

BMP4 before transfer to a collagen gel with NT3 resulted in stronger fiber outg

PD98059 also potentiated later fiber outgrowth stimulated by NT3. (D) Preincub

neurotrophic factors was not sufficient to elicit fiber outgrowth. (E) Results from qu

were dissected for immediate RNA preparation (start) or incubated for 4 h in contr

RNA preparation. Total RNA (10 ng of each sample) was used for one-step RT-

standards. TrkC was not upregulated by exposure to BMP4 whereas Smad6 and Id

Egr1 was upregulated in control culture medium whereas addition of either PD98

A brief period of BMP stimulation or MEK inhibition is sufficient

for neurotrophic synergy

Potentiation responses occurred after 1–2 days of simulta-

neous presence of BMP4 and NT3, irrespective of the order in

which they were added. If BMP4 is added to the sympathetic

ganglia when the culture is set up, NT3 can be given to the

culture after 1 day of delay and still give a dense outgrowth

during the next 1–2 days of incubation. Moreover, BMP4 added

1 day after initial NT3 stimulation robustly potentiated neurite

outgrowth during the following days of incubation. Full out-

growth potentiation occurred even when PD98059 was added to

the NT3-stimulated ganglia with up to 24-h delay (Fig. 1E),

indicating that timing of MEK inhibition in relation to onset of

neurotrophic stimulation is not critical to induce the growth

spurt.

rosci. 25 (2004) 345–354

riod in control medium before embedding the explants in collagen gels with

ganglia exposed to NT3 immediately upon dissection. (B) Incubation with

rowth than when preincubation was in control medium. (C) Priming with

ation with BMP4 followed by cultivation in a collagen gel without added

antitative real-time RT-PCR. Chicken embryonic day 9 sympathetic ganglia

ol culture medium (BME), with PD98059 or with BMP4 before subjected to

PCR. Values were normalized using averaged GAPDH and 18S rRNA as

1 transcripts were strongly upregulated by BMP4. The immediate early gene

059 or BMP4 resulted in significantly reduced Egr1 levels after 4 h.

S. Althini et al. / Mol. Cell. Neurosci. 25 (2004) 345–354 349

Considering the possibility that synergistic actions on NT3

depended on a limited priming phase rather than the continuous

presence of BMP or MEK inhibitors, we tested periods of

preincubation of the ganglia in BMP and PD98059 before

stimulation with NT3 (Figs. 3A–D). Four hours of preexposure

to BMP4 (Fig. 3B) or PD98059 (Fig. 3C) was found sufficient

for later enhanced outgrowth. A 1-h exposure period to BMP4 or

PD98059 did not substantially increase the later NT3 response.

Reversing the sequence of exposure so that the sympathetic

ganglion were first exposed to NT3 for 4 h and then stimulated

only by BMP4 for 2 days did not result in neurite outgrowth. It is

thus clear that the neurotrophic signaling must be maintained

active during culture whereas the synergistic effects by activating

BMP receptors and blocking MEK require only a limited priming

period.

The 4-h priming period necessary for later potentiation of

neurotrophic activity may be involved synthesis of new transcripts

in the ganglionic neurons. Actinomycin D was added to cultures

with NT3, NT3 with BMP4, or NT3 with PD98059 and consis-

tently resulted in total inhibition of fiber outgrowth (supplementary

data material Figs. 5A–B). In contrast, NGF with actinomycin D

gave a short but fairly dense outgrowth as noted before (Partlow

and Larrabee, 1971), indicating that suppression of RNA synthesis

does not totally inhibit neurite outgrowth in sympathetic ganglia

stimulated with NGF (supplementary data material Figs. 5C–D). It

took only a 4-h preincubation period with actinomycin D to impair

NGF outgrowth in this manner and to block BMP4 potentiation

(simultaneous incubation) of later NT3 potentiation. PD98059

priming for 4 h in the presence of actinomycin D resulted in lack

Fig. 4. Transcriptional transactivation by Elk1-Gal4 fusion protein measured by a

BMP4 reduced the NGF-induced ability of Elk1 to activate reporter transcription. (

activity of Elk1. This effect was blocked by U0126 at the concentration (1 AM) u

Like BMP4, expression of a constitutively activated BMP-receptor ALK2 reduce

alleviated when inhibitory Smad7 was added. Smad1 expression also reduced the

of later NT3 responsiveness. These observations support the idea

that new RNA need to be synthesized during the priming period

allowing for later increases in neurotrophic responses.

To examine the possibility that BMP4 or PD98059 directly

affects the expression of select genes in the primed sympathetic

ganglia, we turned to quantitative real-time RT-PCR (Fig. 3E and

supplementary data material Table 1). No differences in the levels

of TrkA (not shown) or TrkC transcripts were detected in

sympathetic ganglia incubated during 4 h in control medium

and in medium with BMP4 or PD98059 (Fig. 3E). Likewise,

RNA samples from sympathetic ganglia grown for 22 h with

NT3, NT3 plus BMP4, or with NT3 plus PD98059 did not shift

TrkA or TrkC mRNA levels (not shown). Smad6 mRNA was

increased significantly after 4 h with BMP4 but not when the

ganglia were incubated only with PD98059. Another BMP-

regulated gene, inhibitor of DNA-binding 1 (Id1), was found

strongly upregulated by BMP4, whereas the 4-h incubation in

culture medium (with or without MEK inhibition) lowered the

level significantly. The neurotrophin-regulated immediate early

gene Egr1 was upregulated after 4 h in plain culture medium.

Addition of either PD98059 or BMP4 both resulted in signifi-

cantly reduced Egr1 levels after 4 h.

BMP signals and MEK inhibition independently reduces

transcriptional activity of Elk1

Since mRNA levels for the neurotrophin-regulated early

growth response gene 1 (Egr1) were reduced in the ganglia after

4-h treatment either with PD98059 or with BMP4, it was consid-

µ µ

Gal4-luciferase reporter in PC12 cells stimulated by NGF. (A) Addition of

B) Constitutively active MEK plasmid strongly increases the transcriptional

sed here to stimulate neurite outgrowth and survival in ganglionic neurons.

d the Elk1-controlled reporter activity. The effect of ALK2 signaling was

transactivation, an effect that was inhibited by co-expressed Smad7.

S. Althini et al. / Mol. Cell. Neurosci. 25 (2004) 345–354350

ered to be of interest to examine events upstream of Egr1 but

downstream of Erk1/2. The Egr1 promoter is under strong control

of the MEK/Erk/Elk1 pathway (for a review, see Thiel and Cibelli,

2002) and contains several serum-response elements (SRE). In

order to examine the transcriptional activation by phosphorylated

Elk1, we turned to NGF-stimulated PC12 cells as a neuronal

model system and measured transcriptional activity of Elk1-Gal4

fusion protein by a transactivated Gal4-luciferase reporter in

transfected PC12 cells. Addition of BMP4 significantly reduced

the ability of Elk1 to activate transcription of the reporter (Fig.

4A). Transfection with a constitutively active MEK plasmid

strongly increased the transcriptional activity of Elk1 (Fig. 4B).

This effect was blocked almost completely by U0126 at the

concentration (1 AM) used here to stimulate neurite outgrowth

and survival in ganglionic neurons. Like BMP4, constitutively

activated BMP-receptor ALK2 reduced the Elk1-controlled report-

er. This effect was not seen when the inhibitory Smad7 was added

to the system. Transfection with a Smad1 expression plasmid also

reduced the transactivation, an effect reversed by co-expressed

Smad7 (Fig. 4B).

Discussion

The present results demonstrate a striking similarity among

BMPs on one hand and MEK inhibitors on the other hand in the

potentiation of neurotrophic activities of NT3, GDNF, and NTN.

The potentiation was found in embryonic autonomic and sensory

neurons using two structurally unrelated MEK inhibitors PD98059

and U0126 and raises the question of how signaling pathways and

downstream effector molecules interact when these neurons are

stimulated with neurotrophic factors in combination with BMPs or

MEK inhibitors.

MEK inhibition enhances neuron survival and neurite outgrowth

The synergies seen with BMPs and neurotrophins (NTs) or

BMPs and members of the GDNF-family-ligands are likely to

represent interactions at some level of signal transduction pathways

or transcriptional regulation. The major BMP signaling pathway

engages type II and type I serine/threonine kinase receptors

(BMPRII, ALK2, ALK3, and ALK6) to activate the cytoplasmic

receptor-regulated proteins Smad1, 5, and 8 (Massague and Chen,

2000; Miyazawa et al., 2002; Moustakas et al., 2001; ten Dijke et

al., 2000; von Bubnoff and Cho, 2001). In contrast, the neuro-

trophic factors examined here are signaling via the tyrosine kinase

receptors TrkA, TrkC, and Ret (Airaksinen and Saarma, 2002;

Baloh et al., 2000; Creedon et al., 1997; Kaplan and Miller, 2000;

Patapoutian and Reichardt, 2001). Binding of NTs to Trk activates

several different pathways including the PI3K/Akt kinase pathway.

The PI3K pathway is activating the downstream Akt kinase,

demonstrated to have a major function to enhance neuronal

survival (Brunet et al., 2001; Crowder and Freeman, 1998; Kaplan

and Miller, 2000).

Another tyrosine-kinase receptor pathway is utilizing the Ras/

MEK/extracellular signal-regulated kinase (Erk) cassette. Down-

stream substrates of the MAP kinase Erk2 (p42) include ternary

complex factor Elk and the Rsk kinase that might activate

transcription factors including CREB (Kaplan and Miller, 2000).

Application of MEK inhibitors utilized in the present study has

unexpectedly demonstrated that this pathway in many cases is

dispensable for neurotrophic factor-induced neuron survival and

fiber outgrowth. The synthetic, reversible inhibitors PD98059 and

U0126 readily pass the cell membrane and exhibit a high degree of

specificity for inhibition of MEK whereas some other tested

kinases are not blocked (Alessi et al., 1995; Davies et al., 2000;

Favata et al., 1998).

As noted above, the effects of the MEK inhibitors are similar to

the synergistic effects of neurotrophic activities exerted by BMPs.

NT3 by itself rescued about 50% of seeded sympathetic neurons

after 2 days in culture. We now find that adding PD98059 to the

cultures significantly increased the survival rate to over 80%,

which matches the previously reported 75% survival with NT3

combined with BMP7 (Bengtsson et al., 1998). Inhibition of MEK/

Erk signaling in response to NGF has previously been shown to be

without negative effects on sympathetic neuron survival (Creedon

et al., 1996; Virdee and Tolkovsky, 1996). Moreover, NGF

stimulated nerve fiber outgrowth in embryonic chicken sympathet-

ic and dorsal root ganglia were found to be unaffected by the

addition of PD98059 at doses inhibiting the chicken Erk2 (MAP

kinase p42; Klinz et al., 1996). The effect of inhibiting MEK seen

here is strikingly similar to the potentiation of NT3 bioactivity

earlier described for K252b (Knusel et al., 1992; Maroney et al.,

1997) or its analogue L753000 (Pollack et al., 1999) and ascribed

to the inhibition of TrkA kinase activity. Moreover, a previous

report noted potentiation by PD98059 and U0126 on NT3-stimu-

lated neurite outgrowth from explanted dorsal root ganglia from the

adult mouse (Wiklund et al., 2002).

Mechanisms involved in neurotrophic synergy

There are several levels of possible crosstalk between signals

arising from activated BMP and NT3/GDNF/NTN. Inhibition of

MEK may mimic BMP actions by influencing these signaling

cascades at one or several levels. A first level of possible

convergence points comprises of inhibited phosphorylation at

activating sites in Erk2 by receptor-activated Smads 1 and 5. This

would account for the similarity in potentiation of neurotrophic

activity found between added BMP and MEK inhibitors. Some

reports show that BMP may inhibit growth factor-induced Erk

activation (Ghosh Choudhury et al., 1999) and that TGFh has

inhibitory effects on Erk2 in epithelial cells that may be mediated

by activation of serine/threonine phosphatases (Giehl et al., 2000).

However, such a model is not supported by the present results from

Western blot analysis of phospho-Erk, showing that PD90859

effectively blocks activating phosphorylation of Erk but that

BMP does not affect the Erk phosphorylation.

Despite the rapid phosphorylation events upon receptor stim-

ulation, a priming period of 4 h with BMP4 or PD98059 was

required for later potentiation of responses to NT3. This argues

for the requirement of physiological changes in the cells, possibly

involving de novo synthesis of transcripts, for example, encoding

transcription factors, signal mediators, or receptors. This is also

supported by our observations on cultures treated with actinomy-

cin D.

We have previously given reasons why it is likely that the

activity of NT3 under the present conditions is mainly mediated via

TrkA rather than via TrkC (Bengtsson et al., 1998). Nevertheless,

potentiation of NT3 activity seen when BMP is added would be

easily explained by upregulation of the cognate NT3 receptors

TrkC and TrkA (and, in consequence, also upregulation of GDNF

and NTN receptors by BMPs to account for the potentiation of

Fig. 5. Model of the signaling crosstalk between neurotrophic factors

(NTFs) and bone morphogenetic proteins (BMPs) related to the effects of

MEK inhibition. Direct stimulatory modifications are indicated by straight

arrows while inhibitory actions are shown with an end bar. Transcriptional

regulations are shown by knicked arrows. The dotted line from Erk1/2 to

Smad1/5 indicate a putative inhibitory modification shown to have

marginal if any effects in the present system.

S. Althini et al. / Mol. Cell. Neurosci. 25 (2004) 345–354 351

these trophic factors). We now excluded this possibility by real-

time PCR, again failing to show any shifts in TrkA or TrkC mRNA

levels in the ganglia treated with BMP4.

We were interested to see whether BMP-regulated genes were

induced after addition of PD98059. Smad6 (Ishida et al., 2000) and

the inhibitor of DNA-binding genes Id1-3 are rapidly induced by

BMP (Hollnagel et al., 1999; Miyazawa et al., 2002; Miyazono and

Miyazawa, 2002). Although our real-time PCR data show that the

presence of BMP4 for 4 h induces the expression of Smad6 and

Id1, both documented as immediate early BMP-responsive genes,

these responses were not found upon addition of PD98059. This

finding argues against the possibility of an inhibitory action of

MEK/Erk on Smads 1 and 5. Precedence for such a mechanism is

found in reports showing that Erk phosphorylation of residues in

the linker region of Smad1 prevents nuclear localization upon

activation by receptor-mediated phosphorylation of C-terminal

serine residues (Kretzschmar and Massague, 1998; Kretzschmar

et al., 1997). Nevertheless, we did not see any major influence by

Erk on the activity of Smad1 and Smad5 in the studied ganglia.

Similar findings were previously made in NGF-stimulated PC12

cells where we demonstrated that U0126 did not increase Smad1 or

Smad5 transcriptional activity as measured in a reporter assay

using a Smad-binding element (Althini et al., 2003), nor was

increased transcriptional activity seen with U0126 in these cells

when Smad5 was fused with Gal4 in a transactivating reporter

system.

We now show that both BMP4 and PD98059 downregulated

the immediate early gene Egr1, encoding the zinc finger tran-

scription factor early growth response-1 (Egr1, also known as

NGFI-A, zif268, and Krox24; Milbrandt, 1987; Sukhatme et al.,

1988), which is induced upon Erk1/2 activation of Elk1. The

promoter region of Egr1 has several serum-response factor

elements (SRE; Thiel and Cibelli, 2002) that bind serum response

factor (SRF) and Elk1 (or other related Ets proteins) in a ternary

complex. It is thus expected that inhibition of the MEK/Erk/Elk1

pathway will reduce transcription of Egr1 whereas the mecha-

nism for a similar effect by BMP signals appear less obvious.

While on and off DNA interactions between Smad2/3 proteins

and the AP-1 family proteins c-Jun, JunB, JunD, and c-Fos have

been demonstrated to influence transactivation of various pro-

moter elements (Liberati et al., 1999; Verrecchia et al., 2001;

Zhang et al., 1998), no similar findings seem to concern the

BMP-activated Smads 1 and 5 and ternary complex factors.

However, the expression of several immediate early genes,

including Egr1, that are controlled by serum-response elements

are repressed by Wnt signaling (likely mediated by h-catenin)without reduction of Erk/Elk phosphorylation and without inter-

ference in formation of the ternary complex at the SRE (Tice et

al., 2002). By analogy, activated Smad1 and Smad5 may possibly

function in the sympathetic neurons to repress co-activators (e.g.,

CBP/p300) and thus reduce SRE-driven gene expression. Thus,

Egr1 expression represents one convergence point for BMP

signaling and MEK inhibition. Whether this is a critical step in

the increased neurite outgrowth and neuronal survival, demon-

strated here when BMP or MEK inhibitors are added to neurons,

remains to be proven. It may well be the case because Egr1,

among its several functions (O’Donovan et al., 1999), includes

pro-apoptotic properties (Thiel and Cibelli, 2002). Egr1 is rapidly

induced in brain neurons following electrostimulation (O’Dono-

van et al., 1998; Sgambato et al., 1998), and a dominant negative

inhibitor of Egr1-mediated transcription will reduce neuronal

apoptosis (Levkovitz and Baraban, 2001). Several downstream

events controlled by activated Egr1, including activation of the

p53 promoter, binding to c-Jun or transactivation of the lipid

phosphatase PTEN gene opposing the PI3K-Akt pathway, could

account for its apoptotic effects in neurons (Thiel and Cibelli,

2002).

A proposed model

Based on our findings, we suggest a model presented in Fig. 5

for the interplay between neurotrophic factor signaling and signals

from the BMP pathway. Inhibition of MEK activity by PD98059

and U0126 will result in reduced phosphorylation of Erk1/2. This

leads to reduced activation of Elk1 or other ternary complex

factors regulating transcription from genes containing serum-

response elements such as Egr1. Reduction of Erk activation by

MEK inhibitors may also alleviate inhibitory phosphorylation in

the linker region of BMP-receptor-activated Smad proteins, but

this was shown here to be of little effect to activate BMP-

regulated genes such as Id1. In contrast, BMPs by activating the

serine/threonine type I receptor ALK2 causes C-terminal phos-

phorylation and activation of Smads 1 and 5 that upregulates BMP

target genes like Smad6 and Id1. A further effect is an inhibition

of Elk1 transcriptional activation possibly by interference with

common co-activators like the histone acetylase p300 (Li et al.,

2003). Like upstream MEK inhibition, this will result in reduced

levels of Egr1 transcript that may subsequently alter gene activity

further downstream.

l. Neurosci. 25 (2004) 345–354

Significance for in vivo events

It is of note that both MEK inhibitors described here to strongly

support neurotrophic factors also have been shown to robustly

protect brain neurons from ischemic insults in animal models

(Alessandrini et al., 1999; Namura et al., 2001). In a traumatic

brain injury (TBI) model, Mori et al. (2002a) recently found that

phospho-Erk was upregulated while no changes were found in

JNK phosphorylation. Blocking Erk activity with PD98059

resulted in significant reduction of cortical lesion volumes whereas

the application of p38 and JNK inhibitor SB203580 did not

improve outcome of brain trauma. The specific effects of inhibiting

MEK/Erk are also supported by findings in this TBI model using

MEK inhibitor U0126 (Mori et al., 2002b). The possibility exists

that such protection is mediated by endogenous neurotrophic

factors potentiated by MEK inhibition. Such signaling pathways

could be future pharmacological targets and find therapeutic use

(English and Cobb, 2002; Swain et al., 1999).

S. Althini et al. / Mol. Cel352

Experimental methods

Bioassays

Sympathetic ganglia dissected from the E9 chicken embryo,

and in addition, ciliary, nodose, and dorsal root ganglia were

included in the assays according to established methods (Bengts-

son et al., 1998; Ebendal, 1989; Ebendal et al., 1980; Kullander et

al., 1997). The ganglia were placed in a small drop of culture

medium and subsequently embedded in a collagen gel having a

volume of 100 Al. Another 100 Al of culture medium (Eagle’s

Basal Medium, BME, supported with 0.5% fetal calf serum, FCS)

with additives such as double-strength neurotrophic factors or

kinase inhibitors were added over the gel matrix for incubation

in a humidified carbon dioxide incubator for 2 days. Scores were

set on a standardized scale from 0 (no outgrowth) to 5 (very dense

circular fiber halos around the explanted ganglia by two indepen-

dent observers using coded cultures. Experiments were repeated at

least twice with 4–20 ganglia scored in each group. For examina-

tion of dissociated neurons, ganglia were incubated with 0.5%

trypsin for 30 min and then suspended in culture medium for

inclusion in collagen gels (Bengtsson et al., 1998).

Growth factors and kinase inhibitors

The following growth factors were used at indicated final

concentrations: recombinant human BMP4 (100–200 ng/ml) from

R&D Systems, Minneapolis, MN, USA (cat. # 314-BP). BMP6

(tested at 200 ng/ml) was from Creative Biomolecules, Ltd,

Hopkinton, MA, USA. NTN and GDNF (both used at 50 ng/ml)

were from PeproTech Inc. (Rocky Hill, NJ, USA). Mouse hNGFwas prepared in our laboratory (Kullander et al., 1997). NT3 (used

at 10 ng/ml) was from Austral Biologicals, San Ramon, CA, USA.

Kinase inhibitors were dissolved in dimethylsulfoxide (DMSO)

and tested on ganglionic neurons. We used two substances inhibit-

ing MEK signaling: PD98059 at 50 AM (Calbiochem EMD

Biosciences Inc., La Jolla, CA, USA, #513000; Alessi et al.,

1995) and U0126 at 1 AM (from Promega Biosceinces Inc., San

Luis Obispo, CA, USA, Cat. #V1121 or Calbiochem #662005;

Favata et al., 1998). MAP kinase p38 inhibitor SB203580 at 10 AM(Calbiochem #559395) was also tested. For blocking PI3 kinase

signaling, LY294002 at 50 AM (obtained from Calbiochem

#440202) was applied. Kinase inhibitor K252b (Calbiochem

#420301) was tested at 0.1 AM considering its ability to inhibit

TrkA. Control cultures received either 0.1% DMSO in the culture

medium or the inactive derivatives U0124 (5 AM; Calbiochem,

#662006) or SB202474 (50 AM; Calbiochem, #559387) dissolved

in DMSO. To inhibit RNA synthesis, actinomycin D-mannitol

(Sigma Aldrich corporation, St Louis, MO, USA, cat. #A 5156)

was added at an effective concentration of 1 Ag/ml.

Immunoblotting and kinase assay

E9 chicken sympathetic ganglia were treated for 90 min with

control medium, NT3, BMP4, NT3 plus BMP4 or NT3 and

PD98059. After incubation, ganglia were washed in PBS and

lysed in 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA,

1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1

mM h-glycerolphosphate, 1 mM Na3VO4, 1 Ag/ml leupeptin, and

1 mM phenylmethylsulfonyl fluoride (PMSF). SDS-boiled samples

were run on a 10% polyacrylamide gel before being transferred to a

PVDF membrane and subsequently incubated with primary anti-

bodies overnight at 4jC. For detection of phosphorylated versus

total Erk2 and Akt, we used the Phospho-Plus Antibody Kits from

Cell Signalling Technology (Beverly, MA, USA; cat. #9110 and

9270, respectively). A rabbit polyclonal antibody was used to

detect phospho-Smad1/5/8 (pS1, diluted 1:1000, Peter ten Dijke,

the Netherlands Cancer Institute, Amsterdam, the Netherlands).

Secondary antibodies (anti-rabbit or anti-mouse IgG) conjugated

with horseradish peroxidase were used for enhanced chemilumi-

nescence detection (LumiGlo substratum, Cell Signalling). To

quantitate band densities, films were digitalized and analyzed for

average density using the Jandel Video Analysis system (JAVA,

SPSS, Chicago, IL, USA).

Real-time RT-PCR

In order to examine levels of specific transcripts, E9 chicken

sympathetic ganglia were subjected to RNA isolation using Qiagen

Rneasy (Qiagen Inc., Valencia, CA, USA). The Qiagen QuantiTect

SYBR Green RT-PCR kit for a one-step reaction (reverse tran-

scription at 50jC for 30 min) yielding amplified fragments of

about 100 base pairs was used with primers for chicken TrkC,

TrkA, Smad6, Id1, and Egr1 (seesupplementary data material Table

1 for primer pairs and GeneBank accession numbers). For normal-

ization of data, averaged expression of GAPDH and 18S rRNA

was determined. Quantitative real-time RT-PCR was run for 50

cycles (94jC for 15 s, 60jC for 30 s, and 72jC for an additional 30

s) using an iCycler thermal cycler (Bio-Rad Laboratories, Inc.,

Hercules, CA, USA) following a hot start (95jC for 15 min). After

completion of the cycles, melting curves obtained by increasing the

temperature from 55 to 96jC in increments of 0.5jC were

examined to ascertain specificity of the PCR-products.

PC12 cell transfection and luciferase assay

In order to evaluate effects of BMPs and MEK inhibitors on

substrata downstream of Erk2, we analyzed transcriptional activity

of an Elk1-Gal4 fusion protein by a luciferase reporter system

(PathDetect Elk1 trans-Reporting system, cat. #219005. Strata-

gene, La Jolla, CA, USA). Briefly, rat pheochromocytoma PC12

cells were seeded on a thin collagen film and grown under low-

S. Althini et al. / Mol. Cell. Neurosci. 25 (2004) 345–354 353

serum conditions (0.5%) for 1 day in the presence of NGF at 50 ng/

ml. The differentiating cells were then transfected using Lipofec-

tamin 2000 (Gibco, InVitrogen Life Technologies, San Diego, CA,

USA). The cells also received appropriate control plasmids (mock

transfections) or plasmids encoding Smad1, Smad7, constitutively

active ALK2, or constitutively active MEK1 (pFC-MEK1) as

detailed previously (Althini et al., 2003). After further incubation

for 2 days, the PC12 cells were harvested for determination of

luciferase activity (reporter lysis buffer and luciferase assay sub-

strate from Promega, Madison, WI, USA) in a Wallac Victor

luminometer (model 1420 Multilabel Counter) and the data nor-

malized to h-galactosidase encoded by a co-transfected pCMV-

hgal plasmid.

Statistical analysis

The SigmaStat version 2.0 software (SPSS, Inc) was used for

statistical analysis. Error bars indicate standard error of the mean

throughout the figures, *P < 0.1, **P < 0.05, and ***P < 0.001.

Acknowledgments

We are grateful for valuable discussions and help given by Peter

ten Dijke and Carl-Henrik Heldin. The Swedish Research Council,

The Swedish Foundation for Strategic Research, and Curis, Inc.

supported this work.

References

Airaksinen, M.S., Saarma, M., 2002. The GDNF family: signalling,

biological functions and therapeutic value. Nat. Rev., Neurosci. 3,

383–394.

Alessandrini, A., Namura, S., Moskowitz, M.A., Bonventre, J.V., 1999.

MEK1 protein kinase inhibition protects against damage resulting

from focal cerebral ischemia. Proc. Natl. Acad. Sci. U. S. A. 96,

12866–12869.

Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T., Saltiel, A.R., 1995.

PD098059 is a specific inhibitor of the activation of mitogen-acti-

vated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270,

27489–27494.

Althini, S., Usoskin, D., Kylberg, A., ten Dijke, P., Ebendal, T., 2003. Bone

morphogenetic protein signalling in NGF-stimulated PC12 cells. Bio-

chem. Biophys. Res. Commun. 307, 632–639.

Baloh, R.H., Enomoto, H., Johnson, E.M., Milbrandt, J., 2000. The GDNF

family ligands and receptors-implications for neural development. Curr.

Opin. Neurobiol. 10, 103–110.

Bengtsson, H., Soderstrom, S., Kylberg, A., Charette, M.F., Ebendal, T.,

1998. Potentiating interactions between morphogenetic protein and neu-

rotrophic factors in developing neurons. J. Neurosci. Res. 53, 559–568.

Brunet, A., Datta, S.R., Greenberg, M.E., 2001. Transcription-dependent

and -independent control of neuronal survival by the PI3K-Akt signal-

ing pathway. Curr. Opin. Neurobiol. 11, 297–305.

Creedon, D.J., Johnson, E.M., Lawrence, J.C., 1996. Mitogen-activated

protein kinase-independent pathways mediate the effects of nerve

growth factor and cAMP on neuronal survival. J. Biol. Chem. 271,

20713–20718.

Creedon, D.J., Tansey, M.G., Baloh, R.H., Osborne, P.A., Lampe, P.A.,

Fahrner, T.J., Heuckeroth, R.O., Milbrandt, J., Johnson, E.M., 1997.

Neurturin shares receptors and signal transduction pathways with glial

cell line-derived neurotrophic factor in sympathetic neurons. Proc. Natl.

Acad. Sci. U. S. A. 94, 7018–7023.

Crowder, R.J., Freeman, R.S., 1998. Phosphatidylinositol 3-kinase and

Akt protein kinase are necessary and sufficient for the survival of

nerve growth factor-dependent sympathetic neurons. J. Neurosci. 18,

2933–2943.

Davies, S.P., Reddy, H., Caivano, M., Cohen, P., 2000. Specificity and

mechanisms of action of some commonly used protein kinase inhibi-

tors. Biochem. J. 351, 95–105.

Ebendal, T., 1989. Use of collagen gels to bioassay nerve growth factor

activity. In: Rush, R.A. (Ed.), Nerve Growth Factors. IBRO Handbook

Series: Methods in the Neurosciences, vol. 12. Wiley, Chichester, pp.

81–93.

Ebendal, T., Olson, L., Seiger, A., Hedlund, K.O., 1980. Nerve growth

factor in the rat iris. Nature 286, 25–28.

Ebendal, T., Tomac, A., Hoffer, B.J., Olson, L., 1995. Glial cell line-de-

rived neurotrophic factor stimulates fiber formation and survival in

cultured neurons from peripheral autonomic ganglia. J. Neurosci. Res.

40, 276–284.

Ebendal, T., Bengtsson, H., Soderstrom, S., 1998. Bone morphogenetic

proteins and their receptors: potential functions in the brain. J. Neurosci.

Res. 51, 139–146.

English, J.M., Cobb, M.H., 2002. Pharmacological inhibitors of MAPK

pathways. Trends Pharmacol. Sci. 23, 40–45.

Farkas, L.M., Jaszai, J., Unsicker, K., Krieglstein, K., 1999. Characteriza-

tion of bone morphogenetic protein family members as neurotrophic

factors for cultured sensory neurons. Neuroscience 92, 227–235.

Favata, M.F., Horiuchi, K.Y., Manos, E.J., Daulerio, A.J., Stradley, D.A.,

Feeser, W.S., van Dyk, D.E., Pitts, W.J., Earl, R.A., Hobbs, F., Cope-

land, R.A., Magolda, R.L., Scherle, P.A., Trzaskos, J.M., 1998. Identi-

fication of a novel inhibitor of mitogen-activated protein kinase kinase.

J. Biol. Chem. 273, 18623–18632.

Forgie, A., Doxakis, E., Buj-Bello, A., Wyatt, S., Davies, A.M., 1999.

Differences and developmental changes in the responsiveness of PNS

neurons to GDNF and neurturin. Mol. Cell. Neurosci. 13, 430–440.

Ghosh Choudhury, G., Jin, D.C., Kim, Y., Celeste, A., Ghosh-Choudhury,

N., Abboud, H.E., 1999. Bone morphogenetic protein-2 inhibits

MAPK-dependent Elk-1 transactivation and DNA synthesis induced

by EGF in mesangial cells. Biochem. Biophys. Res. Commun. 258,

490–496.

Giehl, K., Seidel, B., Gierschik, P., Adler, G., Menke, A., 2000. TGFbeta1

represses proliferation of pancreatic carcinoma cells which correlates

with Smad4-independent inhibition of ERK activation. Oncogene 19,

4531–4541.

Hollnagel, A., Oehlmann, V., Heymer, J., Ruther, U., Nordheim, A., 1999.

Id genes are direct targets of bone morphogenetic protein induction in

embryonic stem cells. J. Biol. Chem. 274, 19838–19845.

Huang, E.J., Reichardt, L.F., 2001. Neurotrophins: roles in neuronal devel-

opment and function. Annu. Rev. Neurosci. 24, 677–736.

Ishida, W., Hamamoto, T., Kusanagi, K., Yagi, K., Kawabata, M., Take-

hara, K., Sampath, T.K., Kato, M., Miyazono, K., 2000. Smad6 is a

Smad1/5-induced Smad inhibitor. Characterization of bone morphoge-

netic protein-responsive element in the mouse Smad6 promoter. J. Biol.

Chem. 275, 6075–6079.

Kaplan, D.R., Miller, F.D., 2000. Neurotrophin signal transduction in the

nervous system. Curr. Opin. Neurobiol. 10, 381–391.

Klinz, F.J., Wolff, P., Heumann, R., 1996. Nerve growth factor-stimulated

mitogen-activated protein kinase activity is not necessary for neurite

outgrowth of chick dorsal root ganglion sensory and sympathetic neu-

rons. J. Neurosci. Res. 46, 720–726.

Knusel, B., Kaplan, D.R., Winslow, J.W., Rosenthal, A., Burton, L.E.,

Beck, K.D., Rabin, S., Nikolics, K., Hefti, F., 1992. K-252b selectively

potentiates cellular actions and trk tyrosine phosphorylation mediated

by neurotrophin-3. J. Neurochem. 59, 715–722.

Kretzschmar, M., Massague, J., 1998. SMADs: mediators and regulators of

TGF-beta signaling. Curr. Opin. Genet. Dev. 8, 103–111.

Kretzschmar, M., Doody, J., Massague, J., 1997. Opposing BMP and EGF

signalling pathways converge on the TGF-beta family mediator Smad1.

Nature 389, 618–622.

S. Althini et al. / Mol. Cell. Neurosci. 25 (2004) 345–354354

Kullander, K., Kaplan, D., Ebendal, T., 1997. Two restricted sites on the

surface of the nerve growth factor molecule independently determine

specific TrkA receptor binding and activation. J. Biol. Chem. 272,

9300–9307.

Levkovitz, Y., Baraban, J.M., 2001. A dominant negative inhibitor of the

Egr family of transcription regulatory factors suppresses cerebellar

granule cell apoptosis by blocking c-Jun activation. J. Neurosci. 21,

5893–5901.

Li, Q.J., Yang, S., Maeda, Y., Sladek, F.M., Sharrocks, A.D., Martins-

Green, M., 2003. MAP kinase phosphorylation-dependent activation

of Elk-1 leads to activation of the co-activator p300. EMBO J. 22,

281–291.

Liberati, N.T., Datto, M.B., Frederick, J.P., Shen, X., Wong, C., Rougier-

Chapman, E.M., Wang, X.F., 1999. Smads bind directly to the Jun

family of AP-1 transcription factors. Proc. Natl. Acad. Sci. U. S. A.

96, 4844–4849.

Maroney, A.C., Sanders, C., Neff, N.T., Dionne, C.A., 1997. K-252b po-

tentiation of neurotrophin-3 is trkA specific in cells lacking p75NTR. J.

Neurochem. 68, 88–94.

Massague, J., Chen, Y.G., 2000. Controlling TGF-beta signaling. Genes

Dev. 14, 627–644.

Mehler, M.F., Mabie, P.C., Zhang, D., Kessler, J.A., 1997. Bone morpho-

genetic proteins in the nervous system. Trends Neurosci. 20, 309–317.

Milbrandt, J., 1987. A nerve growth factor-induced gene encodes a possible

transcriptional regulatory factor. Science 238, 797–799.

Miyazawa, K., Shinozaki, M., Hara, T., Furuya, T., Miyazono, K., 2002.

Two major Smad pathways in TGF-h superfamily signalling. Genes

Cells 7, 1191–1204.

Miyazono, K., Miyazawa, K., 2002. Id: a target of BMP signaling. Sci.

STKE, E40.

Mori, T., Wang, X., Aoki, T., Lo, E.H., 2002a. Downregulation of matrix

metalloproteinase-9 and attenuation of edema via inhibition of ERK

mitogen activated protein kinase in traumatic brain injury. J. Neuro-

trauma 19, 1411–1419.

Mori, T., Wang, X., Jung, J.C., Sumii, T., Singhal, A.B., Fini, M.E., Dixon,

C.E., Alessandrini, A., Lo, E.H., 2002b. Mitogen-activated protein kin-

ase inhibition in traumatic brain injury: in vitro and in vivo effects.

J. Cereb. Blood Flow Metab. 22, 444–452.

Moustakas, A., Souchelnytskyi, S., Heldin, C.H., 2001. Smad regulation in

TGF-h signal transduction. J. Cell Sci. 114, 4359–4369.

Namura, S., Iihara, K., Takami, S., Nagata, I., Kikuchi, H., Matsushita, K.,

Moskowitz, M.A., Bonventre, J.V., Alessandrini, A., 2001. Intravenous

administration of MEK inhibitor U0126 affords brain protection against

forebrain ischemia and focal cerebral ischemia. Proc. Natl. Acad. Sci.

U. S. A. 98, 11569–11574.

O’Donovan, K.J., Wilkens, E.P., Baraban, J.M., 1998. Sequential expres-

sion of Egr-1 and Egr-3 in hippocampal granule cells following electro-

convulsive stimulation. J. Neurochem. 70, 1241–1248.

O’Donovan, K.J., Tourtellotte, W.G., Milbrandt, J., Baraban, J.M., 1999.

The EGR family of transcription-regulatory factors: progress at the

interface of molecular and systems neuroscience. Trends Neurosci.

22, 167–173.

Partlow, L.M., Larrabee, M.G., 1971. Effects of a nerve-growth factor,

embryo age and metabolic inhibitors on growth of fibers and on syn-

thesis of ribonucleic acid and protein in embryonic sympathetic ganglia.

J. Neurochem. 18, 2101–2118.

Patapoutian, A., Reichardt, L.F., 2001. Trk receptors: mediators of neuro-

trophin action. Curr. Opin. Neurobiol. 11, 272–280.

Pollack, S., Young, L., Bilsland, J., Wilkie, N., Ellis, S., Hefti, F., Brought-

on, H., Harper, S., 1999. The staurosporine-like compound L-753,000

(NB-506) potentiates the neurotrophic effects of neurotrophin-3 by act-

ing selectively at the TrkA receptor. Mol. Pharmacol. 56, 185–195.

Rosenthal, A., 1999. The GDNF protein family: gene ablation studies

reveal what they really do and how. Neuron 22, 201–203.

Sgambato, V., Pages, C., Rogard, M., Besson, M.J., Caboche, J., 1998.

Extracellular signal-regulated kinase (ERK) controls immediate early

gene induction on corticostriatal stimulation. J. Neurosci. 18,

8814–8825.

Sukhatme, V.P., Cao, X.M., Chang, L.C., Tsai-Morris, C.H., Stamenkovich,

D., Ferreira, P.C., Cohen, D.R., Edwards, S.A., Shows, T.B., Curran, T.,

1988. A zinc finger-encoding gene coregulated with c-fos during

growth and differentiation, and after cellular depolarization. Cell 53,

37–43.

Swain, C., Harper, S., Pollack, S., Smith, R., Hefti, F., 1999. Neurotrophic

factor mimetics. In: Hefti, F. (Ed.), Neurotrophic Factors. Handbook of

Experimental Pharmacology. Springer, Berlin, pp. 281–309.

ten Dijke, P., Miyazono, K., Heldin, C.H., 2000. Signaling inputs con-

verge on nuclear effectors in TGF-h signaling. Trends Biochem. Sci.

25, 64–70.

Thiel, G., Cibelli, G., 2002. Regulation of life and death by the zinc finger

transcription factor Egr-1. J. Cell. Physiol. 193, 287–292.

Tice, D.A., Soloviev, I., Polakis, P., 2002. Activation of the Wnt pathway

interferes with serum response element-driven transcription of immedi-

ate early genes. J. Biol. Chem. 277, 6118–6123.

Unsicker, K., Krieglstein, K., 2000. Co-activation of TGF-hs and cytokine

signaling pathways are required for neurotrophic functions. Cytokine

Growth Factor Rev. 11, 97–102.

Verrecchia, F., Tacheau, C., Schorpp-Kistner, M., Angel, P., Mauviel, A.,

2001. Induction of the AP-1 members c-Jun and JunB by TGF-beta/

Smad suppresses early Smad-driven gene activation. Oncogene 20,

2205–2211.

Virdee, K., Tolkovsky, A.M., 1996. Inhibition of p42 and p44 mitogen-

activated protein kinase activity by PD98059 does not suppress nerve

growth factor-induced survival of sympathetic neurones. J. Neurochem.

67, 1801–1805.

von Bubnoff, A., Cho, K.W., 2001. Intracellular BMP signaling regulation

in vertebrates: pathway or network? Dev. Biol. 239, 1–14.

Wiklund, P., Ekstrom, P.A., Edstrom, A., 2002. Mitogen-activated protein

kinase inhibition reveals differences in signalling pathways activated by

neurotrophin-3 and other growth-stimulating conditions of adult mouse

dorsal root ganglia neurons. J. Neurosci. Res. 67, 62–68.

Zhang, Y., Feng, X.H., Derynck, R., 1998. Smad3 and Smad4 cooperate

with c-Jun/c-Fos to mediate TGF-h-induced transcription. Nature 394,

909–913.