Nerve growth factor mediates activation of the Smad pathway
in PC12 cells
Marion Lutz
1
, Kerstin Krieglstein
2
, Simone Schmitt
1
, Peter ten Dijke
3
, Walter Sebald
1
, Andrea Wizenmann
4
and Petra Knaus
1
1
Department of Physiological Chemistry II, Biocenter, University of Wu
¨
rzburg, Germany;
2
Department of Anatomy, University
of Go
¨
ttingen, Germany;
3
Division of Cellular Biochemistry, the Netherlands Cancer Institute, Amsterdam, the Netherlands;
4
JRG (of) Developmental Neurobiology, Biocenter, University of Wu
¨
rzburg, Germany

factor; transforming growth factor-b.
Proteins of the transforming growth factor b (TGF-b)
family are multifunctional cytokines that display a very
broad range of biological activities including cell prolifer-
ation, differentiation and apoptosis [1]. TGF-bs are ubi-
quitously expressed and act on virtually all tissues, thereby
causing distinct cell-specific effects depending on the present
composition of receptors, Smad proteins and DNA-binding
partners [2,3]. Referring to cell populations of neuronal
origin, TGF-bs are described to possess neurotrophic effects
when acting in concert with other cytokines or neurotro-
phins [4,5]. Signals mediated by TGF-b are propagated by
two receptor serine/threonine kinases designated as TGF-b
type I (TbRI) and type II (TbRII) receptors [6,7]. The type
II receptors comprise TbRII [8] and its alternative splice
variant TbRII-B [9]. The initial binding of TGF-b1to
TbRII is followed by recruitment and activation of TbRI
[10]. Receptor-associated Smads (R-Smads) involved in
TGF-b signaling (Smad2 and Smad3) are phosphorylated
at the C-terminal SSXS-motif [11,12], interact with the
common mediator Smad4 [13] and translocate to the
nucleus to mediate specific transcriptional responses
[14,15]. Although Smad2 and Smad3 share 92% amino
acid identity, they are functionally distinct. A short amino
acid sequence in the MAD homology 1 (MH1) domain of
Smad2 is responsible for its inability to bind DNA [16,17].
However, Smad3 can directly bind to a specific DNA
sequence termed the Smad binding element (SBE). These
distinct properties account for activation of different subsets
of target genes by either Smad2 or Smad3.

tyrosine kinase receptor (TrkA) which initiates multiple
pathways, the most prominent being the Ras/Raf-MAPK
pathway [24,25]. Besides TrkA, NGF can also bind to a
low-affinity neurotrophin receptor, p75
NTR
,whichisa
member of the tumor necrosis factor cytokine receptor
family [26].
It is increasingly evident that signal transduction in
general does not only occur in a linear fashion but rather
comprises a complex network of signaling pathways that
mutually influence their activity [27]. TGF-b family
members are implicated in multiple interdependent signals
between pathways originating from receptor serine/threo-
nine kinases and receptor tyrosine kinases. Cellular
responses induced by bone morphogenetic protein
(BMP) for example, can be impaired by epidermal growth
factor and hepatocyte growth factor, which lead to the
phosphorylation of Smad1 in the linker region and thus
prevent Smad1 nuclear translocation [28]. Direct effects
of signaling intermediates were shown for the c-Jun
N-terminal kinase and protein kinase C. c-Jun N-terminal
kinase phosphorylates Smad3 outside the SSXS-motif,
thus supporting nuclear transport of Smad3 [29]. Protein
kinase C however, abrogates direct DNA binding of
Smad3 by serine phosphorylation in the MH1 domain
[30]. This indicates that Smads are not restricted to TGF-
b/BMP pathways; rather, they represent a point of
convergence of various signals and their activation is a
precise contextually regulated process.

Collagen was isolated from rat tails according to standard
protocols.
1
Appropriate measures were taken to minimize
animal pain and discomfort according to the European
Communities Council Directive of 24 November 1986
(86/609/EEC). Culture dishes were incubated with a
solution of 38 lgÆmL
)1
collagen in 0.1% (v/v) acetic acid
for at least 1 h followed by thorough washing with sterile
dH
2
O and medium without supplements.
Neutralization of TGF-b
All TGF-b isoforms were neutralized by the addition of
either monoclonal antibodies against TGF-b1, -b2, -b3
(20 lgÆmL
)1
) or a 100- or 1000-fold molar excess of the
soluble extracellular domain (ECD) of TbRII-B (TbRII-B-
ECD), kindly provided by J. Nickel (Biocenter, University
of Wu
¨
rzburg, Germany).
DNA constructs
Smad2 and Smad7 constructs were published previously
[20,34]. The Smad3 construct was kindly provided by
R. Derynck (University of California at San Francisco, CA,
USA). Smad1, Smad4 and Smad4 (DSAD) constructs were

by retroviral transfer
The retroviral construct pMX-GFP-Smad3 was a kind
gift from Y. Henis (Tel Aviv University, Israel) [39].
The construct for N-terminally HA-tagged TbRII-wt
was subcloned into the retroviral vector pczCFG-EGIRT
(D. Lindemann, unpublished results)
3
downstream of a
tetracycline-inducible cytomegalovirus (CMV) minimal
promoter [40]. Cells were transduced by infection with
helper-free VSV-G pseudotyped retroviruses as described
previously [37]. Briefly, 293T cells were cotransfected with
the retroviral construct and plasmids for gag-pol and
Ó FEBS 2004 Crosstalk between NGF and TGF-b pathways (Eur. J. Biochem. 271) 921
VSV-G. Twenty-four hours post-transfection, cells were
treatedwith10m
M
sodium-butyrate for 10 h. Infection of
the target cells was performed 48 h and 72 h after transfec-
tion. Because the retroviral sequences contain the gfp gene,
infected cells could be selected by FACS sorting.
Luciferase reporter gene assays
For reporter gene assays, different TGF-b responsive
elements were used. The p3TP-luc(+) reporter is derived
from the p3TP-luc construct [7] but contains a modified luc
gene (pSP-luc(+), Promega). The pSBE reporter [41] serves
as a readout for TGF-b as well as BMP signaling whereas
p3TP-luc(+) and (CAGA)
12
-luc constructs respond speci-

NaCl, 1% (v/v) Triton
X-100, 1 m
M
EDTA, 1 m
M
phenylmethanesulfonyl fluor-
ide] containing protease inhibitors (Complete
TM
, Roche)
and phosphatase inhibitors (50 m
M
NaF, 10 m
M
Na
4
P
2
O
7
and 1 m
M
Na
3
VO
4
). Equal amounts of cell lysates were
analyzed by immunoblotting using anti-PS2, anti-Smad2,
anti-PS3 or anti-Smad3. Immunoreactive proteins were
visualized by enhanced chemiluminescence
4

NGF or 200 p
M
TGF-b1,
respectively. Control cells were stimulated with 2 n
M
NGF
for 1 h following a 1 h treatment with cycloheximide
(5 lgÆmL
)1
in dimethylsulfoxide) or dimethylsulfoxide only.
Cells were washed with NaCl/P
i
, centrifuged (1000 g,4°C,
10min) and the cell pellet was then resuspended in hypotonic
buffer (10 m
M
Hepes pH 7.9, 1.5 m
M
MgCl
2
,10m
M
KCl,
protease inhibitors). Cells were vortexed thoroughly and cell
lysis was followed by microscopy until 90% of the cells were
lysed. Following centrifugation (1000 g,4°C, 10 min), the
supernatant was referred to as the cytoplasmic fraction. The
pellet containing the nuclei was resuspended in high salt
buffer [20 m
M

i
and 3% (v/v) BSA, fixed in 4% (v/v)
paraformaldehyde and 0.2% (v/v) TX-100 for 10 min at
room temperature. After washing with NaCl/P
i
containing
3% (v/v) BSA, Smad2/3 staining was performed with
antibodies from BD Biosciences. Nuclei were stained by
the addition of 1 lgÆmL
)1
Hoechst 33342 for 2 min. The
subcellular distribution of Smad2/3 was then analyzed by
confocal microscopy.
Results
NGF mediates the activation of Smad-dependent
reporter genes independently of TGF-b
Survival of neuronal cells is described to be synergistically
promoted by TGF-bs and neurotrophic factors (e.g. NGF)
[44,45]. To examine whether NGF has the potential to
modulate the Smad pathway in PC12 cells, we performed
reporter gene assays using luciferase constructs containing
promoter elements that are responsive to proteins of the
TGF-b superfamily, i.e. pSBE-luc, p3TP-luc(+) and
(CAGA)
12
-luc [7,41,42]. As indicated in Fig. 1A, PC12 cells
show a significant increase of transcriptional activation after
stimulation with NGF on all tested Smad-dependent
reporter constructs. In contrast, TGF-b1 is not able to
induce transcription from these reporters in PC12 cells.

measured. Data were normalized as described
in Materials and methods, and error bars
represent the SD evaluated from three inde-
pendent experiments. (B) Transfection and
starvation of PC12 cells was carried out as
described in (A) using pSBE-luc as the repor-
ter construct. Subsequently, cells were treated
with 2 n
M
NGF (black bars) or 200 p
M
TGF-
b1 (gray bars) either in presence or in absence
of 20 lgÆmL
)1
TGF-b1, -b2, -b3antibody.
After 24 h, luciferase activity was recorded
and the data were evaluated as described
above. (C) L6 cells stably expressing
GFP-Smad3 were starved for 4 h followed by
treatment with either 20 lgÆmL
)1
anti-
(TGF-b1, -b2, -b3) (lane 2), 200 p
M
TGF-b1
(lane 3) or 200 p
M
TGF-b1 together with
20 lgÆmL

neutralizing capacity of the TGF-b1, -b2and-b3 antibodies
was verified in L6 cells stably transduced with a GFP–
Smad3 construct (Fig. 1C). In the absence of TGF-b1, -b2
and -b3 antibodies, stimulation with TGF-b1leadsto
C-terminal phosphorylation of Smad3, whereas treatment
with TGF-b1 together with neutralizing antibodies impedes
the phosphorylation of Smad3. Second, we performed a
complementary experiment using the soluble ECD of
TbRII-B, a TGF-b type II receptor splice variant that
binds all three TGF-b isoforms [9]. TbRII-B-ECD was
added in two different concentrations (100- and 1000-fold
molar excess) to PC12 cells transfected with the pSBE-luc
reporter in the presence of NGF or TGF-b1(Fig.1D,
upper panel). The TbRII-B-ECD did not abolish NGF-
mediated Smad activation in PC12 cells. Next, we harvested
the supernatant of PC12 cells treated in this way and placed
it on the TGF-b sensitive L6 myoblast cell line that was
equally transfected with pSBE-luc (Fig. 1D, lower panel).
As there was no detectable increase in luciferase activity in
TGF-b-sensitive L6 cells that were treated with the super-
natant from NGF stimulated PC12 cells (Fig. 1D, lower
panel, bar 2), we can conclude that NGF treatment of PC12
cells does not lead to the production of active TGF-b.In
contrast, the TGF-b-treated PC12 cell supernatant results in
reporter activation in L6 cells (Fig. 1D, lower panel, bar 3).
This activation is almost completely blocked by a 1000-fold
molar excess of TbRII-B-ECD (Fig. 1D, lower panel, bar 9).
Furthermore, we measured the amount of TGF-b that
is produced in response to NGF and checked whether this
TGF-b is present in an active or latent form. The

signaling in PC12 cells.
Mechanism of Smad activation by NGF
The mechanism of Smad reporter activation was investigated
by using luciferase constructs that allowed us to distinguish
between signals originating from different R-Smads, i.e.
pSBE-luc, p3TP(+)-luc and (CAGA)
12
-luc [7,41,42] (see
Materials and methods). Using p3TP-luc(+), NGF-induced
reporter activation was investigated after ectopic expression
of various R-Smad constructs (Fig. 3A). From all R-Smads
tested, Smad3 shows the most prominent induction of
transcription after NGF stimulation. Similar results were
obtained with the (CAGA)
12
-luc reporter (data not shown).
Given that in TGF-b signaling, phosphorylation of the
C-terminal serine residues (SSXS) is essential for dissoci-
ation from the type I receptor and for heteromeric complex
formation with Smad4 [34,49], we investigated C-terminal
phosphorylation in response to TGF-b1aswellasNGF.
The phosphorylation pattern of Smad3 resulting from
stimulation with either TGF-b or NGF was analysed in
Fig. 2. PC12 cells express low levels of endogenous TbRII. (A)
C-terminal phosphorylation of Smad2 was investigated in either par-
ental PC12 cells (lanes 1–3) or PC12 cells stably expressing TbRII-wt
(lanes 4–6). Cells were kept in media containing 1 lgÆmL
)1
doxycycline
for 3 days to induce expression of TbRII-wt. Following starvation,

phosphorylation at the C-terminal
SSXS-motif.
Involvement of Smad4 in NGF-triggered activation
of the Smad signaling cascade
In TGF-b1-induced signaling, R-Smads form heteromeric
complexes with Smad4 following the activation by TbRI.
Reporter gene assays using the p3TP(+)-luc construct were
performed to determine whether NGF-mediated activation
of Smad response elements is also Smad4-dependent. PC12
cells were transfected with Smad3, Smad4 or a functionally
inactive Smad4 variant – Smad4(DSAD) – either alone or
in the indicated combinations. Smad4(DSAD) lacks amino
acids 274–321 which encode the Smad activation domain
(SAD) [35,50]. Figure 4A demonstrates that ectopic expres-
sion of Smad3 results in efficient transcriptional activation
of Smad-dependent reporter genes, whereas neither Smad4
nor the mutant Smad4 show an effect on luciferase
induction when expressed alone. Coexpression of Smad3
and Smad4, however, enhances the Smad3 effect. In
Fig. 3. Mode of Smad activation by NGF. (A) Induction of specific
R-Smads was investigated using the p3TP-luc(+) reporter. PC12 cells
were transiently transfected with the indicated Smad constructs. Fol-
lowing starvation, cells were left untreated (white bars) or were stimu-
lated with 2 n
M
NGF (black bars). Data were normalized as described
inMaterialsandmethods.Errorbarswerecalculatedfromthree
independent measurements. (B) Phosphorylation of Smad3 was tested
in TGF-b responsive L6 rat myoblasts (lanes 1–4) and PC12 cells (lanes
5–8) that were transduced with pMX-GFP-Smad3 (lanes 2–4 and 6–8)

variant – Smad4(DSAD) – largely prevents Smad3-
mediated reporter gene activation. These results were also
confirmed by using the (CAGA)
12
-luc reporter (data not
shown).
Furthermore, co-immunoprecipitation studies confirmed
that NGF stimulation of PC12 cells leads to interaction
between Smad3 and Smad4 (Fig. 4B). Whereas in the
absence of ligand there is no heteromeric complex forma-
tion, NGF treatment triggers association of the Smad
proteins within 30 min, indicating that NGF directly
activates Smad signaling.
NGF stimulation rapidly initiates nuclear accumulation
of Smad3
To assess whether Smad3 translocates to the nucleus in
response to NGF treatment, nuclear extracts were investi-
gated for the content of Smad3 protein and the cellular
distribution of Smad3
6
was determined in whole cells.
Nuclear extracts were prepared from L6 rat myoblasts that
were stimulated with TGF-b1 (Fig. 5, upper panel) and
from PC12 cells at several time points after NGF treatment
(Fig. 5
7
, middle panel). In both cell lines, Smad3 can be
detected in the nuclear fraction after 5 min of ligand
stimulation and the amount of nuclear Smad3 increases
with prolonged growth factor treatment, reaching a maxi-

elevated already but the signal can be potently enhanced by
stimulation with NGF. Transfection of the TrkA variant
(TrkA–K538A) that carries a mutation resulting in the
inactivation of the tyrosine kinase activity causes a signifi-
cant reduction of responsiveness. An even stronger inhi-
bitory effect can be observed after cotransfection of the
wild-type TrkA receptor together with Smad7. The antago-
nizing impact of Smad7 becomes additionally apparent by
the strong inhibitory effect on endogenous signaling that is
elicited following expression of ectopic Smad7 (Fig. 7, lanes
2 and 4). These results suggest functional TrkA receptors
to be necessary for NGF-mediated activation of Smad-
dependent reporter genes and demonstrate the inhibitory
role of Smad7 on this NGF-mediated effect.
Discussion
Originally, Smad proteins were exclusively attributed to
pathways activated by TGF-b family members but it
becomes increasingly evident that multiple signaling cas-
cades originating from other receptor systems are involved
in modulating Smad signaling [14,27,30,51,52]. In the
present work, we demonstrate that in PC12 cells NGF-
stimulated signaling via the TrkA receptor leads to activa-
tion of the Smad pathway. NGF-mediated Smad activation
is independent of TGF-b ligand and occurs by a mechanism
which is different from that induced by TGF-b.
PC12 rat pheochromocytoma cells represent a widely
used model system to investigate neuronal differentiation
that is initiated following stimulation with NGF [36].
Fig. 5.
10

containing 0.2% (v/v) horse serum for 4 h and stimulated for with 2 n
M
NGF for 30 min (second row), 3 h (third row) or were left untreated (first
row). Control cells were stimulated with 2 n
M
NGFfor30minfollowinga1htreatmentwithcycloheximide(CHX;fourthrow)ordimethyl-
sulfoxide (DMSO; fifth row). Cells were fixed, nuclei were stained with Hoechst 33342 for 2 min and the cells were analysed by confocal
microscopy. The projection of multiple sections is seen on the left for each panel to visualize the morphology of the cells. The middle row shows
staining of Smad3 and on the right an overlay of Smad3 and Hoechst staining is seen.
Ó FEBS 2004 Crosstalk between NGF and TGF-b pathways (Eur. J. Biochem. 271) 927
can be activated by NGF is mainly important for other
cellular responses.
PC12 cells show transcriptional activation of TGF-
b-responsive reporter genes upon NGF but not TGF-b1
stimulation (Fig. 1). Low amounts of TbRII expressed in
these cells can account for TbRII being the limiting factor
for proper TGF-b1 signaling, which is in accordance with
results showing that ectopic expression of TbRII restores
TGF-b responsiveness (Fig. 2). As an earlier report shows
upregulation of TGF-b by NGF [46], we investigated
whether NGF-triggered Smad activation is caused by
autocrine action of TGF-b. Considering the limiting
amount of TbRII discussed above, PC12 cells are equally
resistant to signals evoked by either exogenous or autocrine
TGF-b. Furthermore, even if all TGF-b isoforms are
neutralized by the addition of antibodies or the soluble
extracellular domain of TbRII-B (TbRII-B-ECD), NGF is
still capable of activating Smad-dependent reporter genes
(Fig. 1B,D). Concerning the amounts of secreted TGF-b,we
found that besides latent (i.e. biologically inactive) TGF-b,

lation of Smad3 at sites other than the SSXS motif. This
modification in turn promotes TbRI-dependent C-terminal
phosphorylation of Smad3 [29]. The mitogen-activated
protein kinase kinase kinase was shown to trigger
phosphorylation outside the C-terminal motif, which
results in enhanced transcriptional activity of Smad2 in
endothelial cells [54]. These examples support our findings
that activation of Smad proteins can occur independently
of C-terminal phosphorylation. Besides direct phosphory-
lation events, NGF potentially triggers other modifications
of R-Smads resulting in Smad nuclear translocation and
transcriptional activation.
As the NGF-initiated processes were shown to be
dependent on functional Smad4 proteins (Fig. 4A) and to
lead to heteromeric complex formation between Smad3
and Smad4 (Fig. 4B), we assume that the presence of the
SSXS-motif is crucial to allow interaction between
R-Smads and Smad4, even if the C-terminal serines are
not phosphorylated. Recent reports show that phosphory-
lation of the SSXS-motif enhances heteromeric complex
formation and stabilizes the assembly of the Smad homo-
and hetero-oligomers. Nevertheless, Smad3 and Smad4
were shown to heterotrimerize in the absence of phos-
phorylation [55,56].
Thus it remains to be elucidated whether phosphorylation
of other residues or different modifications causes the same
or even a distinct oligomerization pattern of Smads.
Nuclear translocation of Smad3 could be confirmed by
the appearance of the Smad3 protein in nuclear extracts
following NGF stimulation (Fig. 5) and by investigation of

calculated from three independent measurements.
928 M. Lutz et al.(Eur. J. Biochem. 271) Ó FEBS 2004
function of the TrkA receptor is required for the activation
of Smad-dependent reporter constructs by NGF (Fig. 7).
Interestingly, expression of Smad7 results in an almost
complete loss of transcriptional activity, even when it is
coexpressed with functional TrkA receptors. This demon-
strates that Smad7 functions downstream of TrkA to block
Smad signaling. Different scenarios of Smad7-mediated
signal abrogation have been previously described. Smad7 is
capable of blocking Smad signaling at the receptor level by
interaction with activated TbRI [19] or by recruiting the
E3 ubiquitin ligases Smurf1 and Smurf2 to the receptors,
resulting in enhanced turnover of TGF-b receptors [21,22].
Furthermore, Smad7 was shown to interfere with signal
transduction by interaction with cytoplasmic proteins such
as TAB1 [61] or mitogen-activated protein kinase kinase
kinase [54]. These distinct antagonizing mechanisms of
Smad7 open up the question whether Smad7 blocks NGF-
induced Smad signaling at the receptor level or by
interaction with other proteins. As dominant-negative
TGF-b receptor mutants did not block NGF-induced
Smad activation (data not shown), they seem to be
dispensible for NGF-mediated signals, and therefore a
mechanism that involves Smad7 interaction with cytoplas-
micproteinsisfavored.
The Alk7 type I receptor is highly similar in its
intracellular domain to TbRI and the constitutively active
form of Alk7 was shown also to induce Smad2/3 phos-
phorylation. Studies in PC12 cells have indicated that Alk7

fer and M. Sammar for helpful discussions. This
work was supported by the Deutsche Forschungsgemeinschaft (DFG)
grant Kn332/3–2 to P. Knaus and EEC, Project 171R ERB-
FMRXCT980216 to P. ten Dijke. M. Lutz was supported by GK 181.
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