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Epilepsy Research (2014) xxx, xxxxxx

journal homepage: www.elsevier.com/locate/epilepsyres

Role of TGF- signaling pathway on

Tenascin C protein upregulation in a
pilocarpine seizure model
Octavio Mercado-Gmez, Jorge Landgrave-Gmez,
Virginia Arriaga-Avila, Adriana Nebreda-Corona,
Rosalinda Guevara-Guzmn
Departamento de Fisiologa, Facultad de Medicina, Universidad Nacional Autnoma de Mxico, 04510
Mxico, DF, Mexico
Received 27 April 2014; received in revised form 29 August 2014; accepted 21 September 2014

KEYWORDS
Tenascin C;
Transforming growth
factor ;
Phospho-Smad2/3;
Hippocampus;
Pilocarpine;
Status epilepticus

Summary Seizures have been shown to upregulate the expression of numerous extracellular matrix molecules. Tenascin C (TNC) is an extracellular matrix protein involved in several
physiological roles and in pathological conditions. Though TNC upregulation has been described
after excitotoxins injection, to date there is no research work on the signal transduction pathway(s) participating in TNC protein overproduction. The aim of this study was to evaluate the
role of TGF- signaling pathway on TNC upregulation. In this study, we used male rats, which
were injected with saline or pilocarpine to induce status epilepticus (SE) and killed 24 h, 3
and 7 days after pilocarpine administration. For evaluating biochemical changes, we measured
protein content of TNC, TGF-1 and phospho-Smad2/3 for localization of TNC in coronal brain
hippocampus at 24 h, 3 and 7 days after pilocarpine-caused SE. We found a signicant increase
of TNC protein content in hippocampal homogenates after 1, 3, and 7 days of pilocarpinecaused SE, together with an enhancement of TNC immunoreactivity in several hippocampal
layers and the dentate gyrus eld where more dramatic changes occurred. We also observed
a signicant enhancement of protein content of both the TGF-1 and the critical downstream
transduction effector phospho-Smad2/3 throughout the chronic exposure. Interestingly, animals
injected with SB-431542, a TGF--type I receptor inhibitor, decreased TNC content in cytosolic
fraction and diminished phospho-Smad2/3 content in both cytoplasmic and nuclear fraction

Corresponding author at: Departamento de Fisiologa, Facultad de Medicina, Universidad Nacional Autnoma de Mxico, Apartado Postal
No. 70250, Delegacin Coyoacn, 04510 Mexico, DF, Mexico. Tel.: +52 55 5623 2396; fax: +52 55 5s623 2155.
E-mail address: rguevara@unam.mx (R. Guevara-Guzmn).

http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019
0920-1211/ 2014 Published by Elsevier B.V.

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019

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O. Mercado-Gmez et al.
compared with pilocarpine vehicle-injected. These ndings suggest the participation of TGF-
signaling pathway on upregulation of TNC which in turn support the idea that misregulation of
this signaling pathway produces changes that may contribute to disease.
2014 Published by Elsevier B.V.

Introduction
Epilepsy is a chronic neurological disorder that affects people of all ages. It is characterized by recurrent, spontaneous
brain seizures (Aroniadou-Anderjaska et al., 2008). According to the World Health Organization, about 50 million
people may have some kind of epilepsy, being temporal
lobe epilepsy (TLE) the most common form (Engel, 1998).
The temporal lobe structures, such as the hippocampus,
amygdala, and piriform cortex are susceptible to triggering electrical discharges contributing to brain damage and
the epileptogenic mechanism (Aroniadou-Anderjaska et al.,
2008). Furthermore, morphological changes such as cellular
death in the CA1, mossy ber sprouting and the dispersion
of the granule cell layer have been described in both animal
models and TLE patients surgical resections (Houser, 1990;
Blmcke et al., 1999). Thus, these alterations are thought
to be involved in the formation of recurrent excitatory circuits that may contribute to epileptic seizures (Heck et al.,
2004).
Seizures have shown to upregulate the expression of
numerous extracellular matrix molecules (ECM) and extracellular proteases (Dityatev and Fellin, 2008). Heck et al.
(2004) showed a sequential upregulation of the ECM in
the granule cell layer of the dentate gyrus, particularly
Neurocan and Tenascin C proteins after seven days of
kainate-analog domoic acid intrahippocampal injection and
an increase of phosphacan fourteen days post-injection
(Heck et al., 2004). These results are consistent even with
those where Tenascin C had been shown to be upregulated in
the hippocampus of kainate-injected adult rats and patients
with Ammons horn sclerosis (Niquet et al., 1995; Schefer
et al., 1997).
Tenascin C (TNC) is a member of the Tenascin family of multimeric, extracellular matrix glycoproteins (other
members are Tenascin X, R, and W, see Tucker and ChiquetEhrismann, 2009). It is involved in several physiological
roles, such as cell migration, cellcell interaction and
matrix organization during tissue remodeling (ChiquetEhrismann and Tucker, 2011). Likewise, it is implicated in
pathological conditions as cancer invasion in several tissues, i.e. lung, prostate, brain cancer, and wound healing
or inammation (Chiquet-Ehrismann and Chiquet, 2003).
Previous studies in vitro, suggest that the TNC controls
the balance of cell adhesion and de-adhesion, cell motility, proliferation, differentiation, and survival displaying a
developmentally regulated time- and space-dependent tissue distribution (Chiquet-Ehrismann and Chiquet, 2003). In
general, the TNC is transiently expressed during development and either is absent or found in small amounts in adult
tissues; however, in several pathological conditions, it is
markedly increased (Dandachi et al., 2001; Nishioka et al.,
2010). Several studies have recognized numerous molecules
that induce TNC expression in various biological systems,

among them various pro- and anti-inammatory cytokines

and growth factors (Rettig et al., 1994).
The transforming growth factor beta (TGF-) plays a critical role in a wide variety of biological processes including
proliferation, differentiation, apoptosis, and extracellular
matrix production (Taylor and Khachigian, 2000). TGF-
exerts its pleiotropic effects by acting over membranebound serinethreonine kinase TGF- type I and II receptors
(TGF-RI and TGF-RII, respectively) which activate signal
transduction through phosphorylating cytoplasmic proteins
called Smads from the receptors to the nucleus (Heldin
et al., 1997). The receptor-associated Smads, (i.e. Smad1,
Smad2, Smad3, and Smad5), interact directly and are phosphorylated by activated TGF-RI receptor (Nakao et al.,
1997) forming heteromeric complexes with Smad4 and thus
translocating into the nucleus, where they function as transcriptional factors caused by their interaction with other
proteins such as Sp1 (Czuwara-Ladykowska et al., 2002;
Jinnin et al., 2004).
Recently, Verrecchia and colleagues (2001) demonstrated
by microarray analysis that TNC is one of the target genes
of TGF-, and its expression is increased in human dermal broblasts (Verrecchia et al., 2001). In addition, both
TNC gene and protein expressions are upregulated by TGF-
involving the formation of Smad3, Sp1, and Ets1 transcriptionally active complex in human dermal broblasts (Jinnin
et al., 2004). Nevertheless, the signaling pathways that are
involved in TNC expression in the temporal lobe epilepsy
animal model have not been elucidated to date.
The aim of our research work was to elucidate the participation of the TGF- signaling pathway activation over
the protein expression of the extracellular matrix protein
Tenascin C in the pilocarpine-caused seizure model during a
time course.

Methods
Animals
Male Wistar rats weighting 250300 g were used for this
study. The study was approved by the Ethical Committee
of the Faculty of Medicine at the Universidad Nacional
Autnoma de Mxico in accordance with guidelines and
requirements of the World Medical Association Declaration
of Helsinki to minimize animal suffering. The animals had
access to food and water ad libitum and housed in a temperature and humidity controlled room with articial 12 h
light: 12 h dark cycle.

Pilocarpine-caused status epilepticus

A total of 60 animals were used in the following experiments. First, we injected them with scopolamine methyl

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019

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nitrate (1 mg/kg, SigmaAldrich) 30 min prior to minimize

the peripheral cholinergic effects of pilocarpine. Pilocarpine
hydrochloride (SigmaAldrich) was dissolved in saline and
animals (n = 24) received a single injection (350 mg/kg, i.p.)
to cause status epilepticus (SE) for 1 h and then received
an injection of benzodiazepine (5 mg/kg) to terminate the
pilocarpine-caused tonic seizures. A modied version of
the seizure scale described by Racine (1972) was used to
identify behavioral stages. Stages: (1) akinesia, staring; (2)
rigid posture; (3) head bobbing, masticatory movements;
(4) rearing, myoclonic movements; and (5) severe tonicclonic seizures. Only animals showing symptoms in stages
4 and 5 were used for experimental procedures. Control
animals (n = 24) were subjected to the same procedure but
with saline injection instead. To investigate the effects of
seizure on the protein expression of Tenascin C, TGF-1,
and phospho-Smad2/3, animals were killed in a time course
(1, 3, 7 days) following the pilocarpine-caused seizures,
and used for biochemical analysis. In additional experiments, rats (n = 4) were injected with TGF--type I receptor
inhibitor SB-431542 (10 mg/kg SigmaAldrich, Cat S4217)
dissolved with dimethyl sulfoxide (DMSO) i.p (Waghabi et al.,
2009) 30 min before the pilocarpine injection and sacriced one day after SE. DMSO was used as vehicle in control
animals or pilocarpine injected animals without inhibitor
(n = 4 each).

anti-mouse IgM or goat anti-rabbit IgG horseradish peroxidase coupled secondary antibody (1:10,000 Cell Signaling
Technology, USA #7074) for 1 h at room temperature and
followed by three additional rinses with TBST for 5 more
minutes each. The band signal was detected by a chemiluminescence Kit (Millipore, Temecula, CA) on Amersham
Hyperlm. For the densitometry measurements of the
detected bands, images were taken with a CCD camera
(DNR Bio-Imaging Systems) and the analysis was made using
an MCID image analysis software (InterFocus Imaging Ltd.,
Cambridge, UK). Cytoplasmic and nuclear samples were
normalized to endogenous control GAPDH or histone 3 proteins, respectively. The relative optical density values were
reported in arbitrary units.

Western blotting analysis

Immunohistochemistry

The rat brains were dissected as quickly as possible and

put in ice-cold phosphate saline buffer for 30 s. Immediately after the hippocampi were dissected and homogenized
in 300 L of lysis buffer (20 mM TRISHCl, 150 mM NaCl,
2 mM EDTA, 1% Triton X-100, 10% glycerol, and a complete
protease inhibitor cocktail (Roche), pH 7.5), centrifuged
at 14,000 rpm for 30 min then after, supernatants were
collected and stored at 70 C for later analysis. Proteins were quantied by Bradford assay (BioRad, USA) and
60 g of protein were loaded in Laemmli buffer and electrophoresed in 420% gradient gels (Mini-PROTEAN TGX
Precast Gels, BioRad, USA) SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes
in a semidry electrophoretic transfer cell system (BioRad,
USA) for 1 h and after that time, Ponceaus reversible
staining was done to conrm a high quality transfer. The
membranes were rinsed twice with TRIS-buffered saline
(TBS), and immediately blocked with solution containing 5% nonfat dry milk in 0.1% TBS-Tween 20 (TBST)
for 1 h at room temperature. Blots were subsequently
probed with rabbit polyclonal anti-Tenascin C recognizing 250350 kDa polypeptides (1:1000, Millipore, Temecula
CA AB19011), monoclonal anti-transforming growth factor
1 (1:1000 Sigma-Aldrich, USA T0438), and anti-phosphoSmad2/3 (S423-425) (Santa Cruz Biotechnology sc-11769)
in the same blocking solution overnight at 4 C; for
loading control of cytoplasmic fraction, a monoclonal antibody against glyceraldehyde 3-phosphate dehydrogenase
(GAPDH, 1:2000, Sigma-Aldrich, USA G8795) and polyclonal
anti-histone 3 (1:1000, Cell Signaling, USA #4499) for nuclear
fraction were used. After three rinses with TBST, 5 min
each, blot membranes were incubated with either goat

Experimental animals were killed with an injection of an

overdose of sodium pentobarbital and perfused transcardially with a volume of 250 mL of ice-cold saline solution
followed by a volume of 200 mL of 4% paraformaldehyde in
phosphate buffer (pH 7.4) xative solution. The brains were
removed and immersed in the xative solution overnight.
The brains were then dehydrated in alcohol solutions and
xylene and embedded in parafn wax. Coronal sections
of 10 m thickness were cut in a microtome and placed
in poly-L-lysine coated slides. The parafn was removed
and sections were pretreated with a heat retrieval solution (Citrate buffer 10 mM, pH 6) and placed in an electric
pressure cooker (decloaking chamber, Biocare Medical, USA)
for 5 min. The coronal sections were rinsed with distilled
water and phosphate buffered saline (PBS) and permeabilized with PBS containing 0.3% Triton X-100 and 0.3%
H2 O2 solution for 30 min and left with the blocking solution (Background Sniper, Biocare Medical, USA) for 1 h
to reduce the background staining. Then the brain sections were incubated with rabbit polyclonal anti-Tenascin
C (1:1000, Millipore, Temecula, CA) overnight at 4 C and
rinsed three times for 5 min with PBS. Immediately after,
the sections were incubated with Trekkie Universal Link for
1 h at room temperature rinsed with PBS again and then
with TrekAvidin-HRP (Starr Trek Universal HRP Detection,
Biocare Medical, USA) for 1 h, and rinsed one more time with
PBS. Afterwards, coronal sections were revealed by using
3,3-diaminobenzidine (Betazoid DAB Chromagen Kit, Biocare
Medical, USA) dehydrated, mounted, and observed under a
Brighteld microscope (Leica Microsystem, Germany). Negative controls consisted of eliminating the primary antibodies
for the procedure.

Cytoplasmic and nuclear fractions

Rat hippocampi, from control, pilocarpine and pilocarpine
plus TGF--type I receptor inhibitor SB-431542, were dissected as described before. Hippocampal tissue was put
in cold PBS and subcellular protein fractions kit for
tissue (Thermo Scientic, USA) were used to obtain cytoplasmic and nuclear fractions following manufacturers
instruction.

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019

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Statistical analysis
The values of relative optical units were examined for testing normality of a data set. Then a two-tailed unpaired
Students t-test, MannWhitney test or KruskalWallis with
Dunns post hoc test were run using the Prism GraphPad
statistics software version 5 (GraphPad Software, San Diego,
CA). P < 0.05 was considered statistically signicant.

Results
Tenascin C protein is upregulated after pilocarpine
administration throughout time-course
To examine whether a pilocarpine model may have similar results upon TNC upregulation as observed with kainic
or domoic acid injection, we administrated the muscarinic
receptor agonist to a group of animals during a few day
exposure.
After 24 h of pilocarpine-caused status epilepticus, we
measured a statistically signicant increase (MannWhitney
U = 8.00, P = 0.013) of TNC protein content by Western
blot analysis compared with the control hippocampus
homogenate samples (Fig. 1 A and B). Interestingly, the
increase in the amount of TNC protein also occurred through
3 and 7 days post-pilocarpine caused SE (Fig. 1A and
B, t = 7.52, df = 14, P = 0.0001; MannWhitney U = 12.00,
P = 0.04). These results are consistent with previous reports
where the generation of a state of persistent seizures
can trigger an increase of the extracellular matrix protein
Tenascin C content (Nakic et al., 1996).
To further examine whether the increase of the TNC protein was taking place in a specic region of the hippocampus,
we made an immunohistochemical analysis. In a coronal section of the hippocampus of control rats, there was slight
immunoreactivity in regions such as stratum radiatum and
stratum lacunosum-moleculare, though in the pilocarpineinduced SE rats, we observed a strong immunstaining in all
regions (data not shown), being the dentate gyrus the area
where the most evident changes occurred. As we can see
in Fig. 2, the granular cell layer from the dentate gyrus
has a markedly strong TNC immunoreactivity surrounding
cell bodies resembling brous-like structures in all pilocarpine injected animals along the experimental time course
(Fig. 2B, D, and F, insets) compared with their respective
controls (Fig. 2A, C, E). We can also observe an increase
of TNC immunostaining in the subgranular zone throughout
the time course, being more evident after 7 days of pilocarpine administration where the granular cell dispersion
begins (Fig. 2F, arrowhead). Thus, our results showed that
pilocarpine administration produces an excessive increase
(overproduction) of TNC protein content similarly to kainic
acid injection indicating seizures per se increase this extracellular matrix protein.

Activation of the TGF- signaling pathway is

involved in Tenascin C upregulation
Previous reports have shown that both, the TNC gene expression and protein levels, are upregulated by TGF- either in

human dermal broblasts, carcinoma cell lines, or pathological conditions such as human nasal polyp tissues and
myocardial infarction (Sakai et al., 1994; Jinnin et al.,
2004; Liu et al., 2006; Dobaczewski et al., 2010). To determine whether the TGF- signaling pathway is implicated
in the increase of the TNC protein content, we made an
immunoblotting analysis using antibodies against TGF-1.
Our results showed a statistically signicant increase of a
specic band of 25 kDa corresponding to TGF-1 mature
protein 24 h (MannWhitney U = 0.00, P = 0.0002) after the
pilocarpine injection compared with that of saline-injected
animals (Fig. 3A and B) (Liao et al., 2008). Surprisingly, this
increase endures through 3 days and with a slight increase
at 7 days in the pilocarpine-injected animals (Fig. 3A
and B, MannWhitney U = 8.00, P = 0.0104; t = 353, df = 14,
P = 0.003). To learn whether the increase of TGF-1 could
activate the signaling pathway, we made a Western blot
analysis of phospho-Smad2/3 phosphorylated (pS423-425),
a crucial downstream signal transducer of the signaling
pathway, which together with Smad4 forms a heteromeric
complex translocating into the nucleus where they regulate
a number of genes (Penn et al., 2012). Fig. 4 also shows
a statistically signicant increase of phospho-Smad2/3 protein content in rats that were injected with pilocarpine
after 24 h (t = 14.05, df = 12, P = 0.0001) post-injection compared with that of their respective saline-injected animals
(Fig. 4A and B). Similarly, an increase in phospho-Smad2/3
was observed after 3 and 7 days of pilocarpine-caused
seizures compared with that of their controls (Fig. 4A
and B; MannWhitney U = 8.00, P = 0.0104; t = 3.40, df = 14,
P = 0.00042). Even though the increase was observed in
the detergent-soluble fraction (supernatant) from the hippocampal homogenates, this suggests that phosphorylation
of phospho-Smad2/3 was occurring at the cytosolic compartment where it normally occurs to transduce the canonical
TGF- signaling pathway, conrming the involvement of the
TGF- signaling pathway.

SB-431542, a specic TGF--type I receptor

inhibitor, partially reverts the increase of TNC
protein levels but fully decreases the translocation
of phospho-Smad2/3 from cytoplasm to nucleus
To further investigate whether the inhibition of TGF-
signaling can prevent the increase of TNC protein content
and alter the translocation of the downstream signal transducer as phospho-Smad2/3 from cytoplasm to the nucleus,
we performed an additional experiment using the compound
SB-431542, a specic inhibitor TGF--type I receptor (Halder
et al., 2005). Our data showed a reduction of TNC protein
concentration in cytoplasmic fraction of rat hippocampus
homogenates treated with TGF--type I receptor inhibitor
30 min before pilocarpine-caused SE compared with pilocarpine vehicle-treated rats thereafter 24 h; however, it
was not statistically signicant (Fig. 5A, F = 13.66, P > 0.05).
Nevertheless, TNC protein levels did not return to baseline as control. As seen by nuclear/cytoplasmic ratio of
phospho-Smad2/3 content, we observed a statistically signicant reduction of phospho-Smad2/3 translocated to the
nucleus in pilocarpine-treated rats injected with SB-431542
compound from hippocampus homogenates (Fig. 5B and C,

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019

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Role of TGF- signaling pathway on Tenascin C protein upregulation in a pilocarpine seizure model

Figure 1 Effect of pilocarpine-caused seizures on Tenascin C protein expression. Immunoblots from adult hippocampal rat brain
homogenates 24 h after the pilocarpine injection reveal a signicant increase of 250 kDa TNC protein content compared with their
respective control (A, B). The protein content was also enhanced at 3 and 7 days postpilocarpine-caused status epilepticus (A, B).
Samples were normalized to endogenous control 37 kDa glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. Data are the
mean SEM from each determination. *P < 0.05; ***P < 0.001. Representative blots from 4 independent animals.

KruskalWallis table = 8.70, P < 0.012) compared with that

of pilocarpine vehicle-treated rats where a non-signicant
increase is observed. These results indicate that blocking
the signal transduction of TGF- by using the SB-431542
compound prevents the translocation of downstream transduction effector phospho-Smad2/3 into nucleus and, thus,
partially avoiding the overproduction of TNC protein.

Discussion
Tenascin C (TNC) is an extracellular matrix glycoprotein implicated in several physiological roles, such as
cellcell interaction, cell migration, matrix organization
that also plays an important role in pathological processes
such as tumorigenesis, wound healing, or inammation

(Chiquet-Ehrismann and Chiquet, 2003; Midwood and Orend,

2009). Several reports have shown that TNC mRNA is upregulated after kainic acid injections into rats as a model of TLE
(Ferhat et al., 1996; Nakic et al., 1996); however, they do
not explain which biochemical mechanisms are involved in
Tenascin C upregulation seen after seizures and most importantly, there are no reports about the increase of the TNC
protein caused by pilocarpine injection. Our data show that
the TNC protein is overexpressed in the widely described
pharmacological pilocarpine model of TLE. The increase of
the TNC protein content was continuous throughout the time
course. As a matter of interest, microarray analysis made
in our laboratory showed an upregulated expression of the
TNC gene in hippocampal samples from the TLE patients
(unpublished results) suggesting that both gene expression
and protein content are elevated either in pharmacological

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019

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Figure 2 Tenascin C upregulation after pilocarpine-caused status epilepticus. Photomicrographs showing an increase of the extracellular matrix protein TNC immunoreactivity on the dentate gyrus, especially in the subgranular cell layer (arrowhead) 24 h after
pilocarpine-caused seizures (B) compared with that of their control (A). This enhancement of the TNC immunostaining endures
throughout 3 (D) and 7 (F) days after the pilocarpine injection compared with their respective controls (C and E). Insets depict high
magnications of the zone marked by arrowheads from each photomicrograph. Scale bar = 50 M and for high magnications = 10 m.
Representative immunohistochemistry from 4 independent experiments.

animal models or in brain tissue from the TLE patients (Nakic

et al., 1996; Schefer et al., 1997).
Our data are also consistent with those reports where
authors have shown an increase in the TNC protein content
by systemic injection of kainic acid into rats suggesting a
transcriptional and translational disequilibrium in the extracellular matrix components after seizures (Nakic et al.,
1996). Moreover, in coronal hippocampal sections, we also
detected an increase of immunoreactivity of TNC in several strata in pilocarpine-caused SE rats over the time
course. Most importantly, dramatic changes were observed
in the dentate gyrus, such as strong TNC immunoreactivity surrounding granular cell bodies resembling brous-like

structures at all times with pilocarpine-injected animals at

day 7 after the SE induction. Furthermore, we observed the
appearance of granular cell dispersion (data not shown),
an important histopathological feature observed in TLE
patients with hippocampal sclerosis (HS) (Houser, 1990;
Blmcke et al., 1999). Even though several mechanisms
could be involved in the granular cell dispersion, it has been
described that a decrease of extracellular glycoproteins,
such as Reelin, can be involved in the development of
this pathological feature (Haas et al., 2002). Therefore,
we suggest that TNC could also play an important role
through inhibition of cell adhesion in cell culture broblasts
(Chiquet-Ehrismann et al., 1988; Lotz et al., 1989); however,

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
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Role of TGF- signaling pathway on Tenascin C protein upregulation in a pilocarpine seizure model

Figure 3 Increase of mature transforming growth factor 1 protein expression after pilocarpine injection through the temporal
course. Treatment of pilocarpine causes a signicant increase of 25 kDa TGF-1 protein 24 h postpilocarpine-caused status epilepticus
on rat hippocampal samples, compared with that of their respective controls (A). As shown, TGF-1 immunolabeling is also enhanced
in protein cytoplasmic fraction during the time-course. Samples were normalized to endogenous control 37 kDa GAPDH protein. Data
are the mean SEM from each determination. *P < 0.05; **P < 0.01; ***P < 0.001. Representative blots from 4 independent animals.

their possible role on granular cell dispersion, if any, needs

to be further investigated.
On the other hand, it is well-known that TNC expression is down-regulated in adult CNS with the exception of
neurogenic zones and regions of plasticity in the hypothalamus (Theodosis et al., 1997). However, in pathological
conditions as wounds, a subset of GFAP-positive reactive
astrocytes responds to injury by up-regulating TNC protein
(Dobbertin et al., 2010). In this sense, it has been described
that pilocarpine-caused status epilepticus produces reactive
gliosis in dentate gyrus of hippocampus after 12 h and continuing to 3 weeks in mice (do Nascimento et al., 2012);
therefore, we suggest that the enhanced production of TNC

protein in the present work could be mediated by this type

of glial cells.
The canonical TGF- signaling pathway plays an important role because of its involvement in a wide variety of
biological processes being mediated through its binding to
a transmembrane receptor with serinethreonine kinase
activity (Taylor and Khachigian, 2000). In addition, activation of the receptor phosphorylates receptor-activated
Smads (R-Smads), Smad2 and Smad3, translocating them
into the nucleus, where they regulate transcription of a variety of target genes (Kawabata et al., 1998). In the present
study, we showed an increase of protein content of TGF-1
at 24 h, which is maintained for 3 days; however at day 7,

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019

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Figure 4 Pilocarpine-caused seizures increase the protein expression of the downstream component TGF- signaling pathway
phospho-Smad2/3. (A) Immunoblot staining of 52 kDa phospho-Smad2/3 in hippocampal protein samples from rats killed 24-h after
injection of pilocarpine show a signicant increase of protein expression compared with their control (A). Similarly, the increasing
protein was also noted at 3 and 7 days after status epilepticus was produced. (B) Histograms are the semiquantitative optical densities
of bands immunostained. Samples were normalized to endogenous control 37 kDa GAPDH protein. Data are the mean SEM from
each determination. *P < 0.05; **P < 0.01; ***P < 0.001. Representative blots from 4 independent animals.

the increase of mature TGF-1 is less pronounced compared

to that of other time points. These results might suggest
two possibilities; (1) the TGF- signaling pathway became
less efcient over time: or, (2) the maintenance of TNC protein described in a chronic exposure (30 days, Nakic et al.,
1996) is probably caused by an imbalance-inhibition in the
regulating proteases that degrade and regulate Tenascin C.
It is well-known that metalloproteinases (MMPs) regulate the
TNC-expression (Jones et al., 1997) and that the inhibition
of MMPs attenuates TNC production (Vyavahare et al., 2000);
however, more experiments are needed to elucidate these
assumptions in our model.

In accordance, several reports have shown the participation of TGF- on the TNC protein upregulation in
cell culture; like injury models, or transformed cells
(Mukaratirwa et al., 2005; Liu et al., 2006; Carey et al.,
2010). However, there are no studies demonstrating the
involvement of TGF-1 in the production of extracellular
matrix proteins in a model of temporal lobe epilepsy.
Recently, Okamoto et al. (2010) showed, by means of
gene expression proling, that the Tgfb1 gene was found signicantly over-expressed in the hippocampi of rats treated
with pilocarpine at 3 and 7 days post-status epilepticus
(latency period) and after the rst spontaneous seizure

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019

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Role of TGF- signaling pathway on Tenascin C protein upregulation in a pilocarpine seizure model

Figure 5 Inhibition of TGF- signaling partially prevents TNC upregulation. Representative immunoblots show that SB-431542
compound, a specic inhibitor of TGF--type I receptor, reduces 250 kDa TNC protein content in cytosolic fraction compared with
pilocarpine vehicle-treated rats after 24 h post-status epilepticus. However, it was not statistically signicant (A). Interestingly,
52 kDa phospho-Smad2/3 shows a statistically signicant decrease in translocation to the nucleus in pilocarpine-treated rats injected
with SB-431542 compound (B and C) compared with pilocarpine DMSO-treated rats where a non-signicant increase is observed
after 24 h post-status epilepticus. The ratio values represent the protein content of phospho-Smad2/3 in nuclear extracts divided
by phospho-Smad2/3 protein content in cytoplasmic extracts. Cytoplasmic and nuclear samples were normalized to endogenous
control 37 kDa GAPDH or 17 kDa histone 3 proteins respectively. Data are the mean SEM from each determination. *P < 0.05 Control
vs Pilocarpine; # P < 0.05 Pilocarpine vs Pilocarpine plus SB-431542. Representative blots from 4 independent animals.

(chronic period) while the TgfbRI gene was only upregulated

at the third day after post-pilocarpine injection (Okamoto
et al., 2010). Furthermore, there are data supporting the
participation of the TGF- signaling pathway in epileptogenesis. Cacheaux et al. (2009) demonstrated that direct
activation of the TGF- signaling pathway by TGF-1 results
in epileptiform activity and that albumin causes similar
activity by binding of albumin through the TGF--type II
receptor on brain slice preparations. Moreover, TGF- blockers (neutralizing the TGF-RII antibody and SB-431542 a
TGF-RI kinase activity inhibitor) could prevent epileptiform
activity suggesting that TGF- per se or an entrance of albumin by breakdown of the blood brain barrier can activate this
signaling cascade impacting on epileptogenesis (Cacheaux

et al., 2009). These data corroborate those found where

there is an increase in protein synthesis of both the TGFRI and TGF-1 protein in the brain tissue of TLE patients
(Lu et al., 2009; Das et al., 2012). In addition, we also
detected an increase of important downstream effectors of
the signaling pathway such as Smad2/3 proteins over the
time course, supporting that the canonical TGF- signaling
pathway is being activated (Penn et al., 2012). Interestingly
in our results, SB-431542-injected rats before pilocarpinecaused status epilepticus did not revert the increase of
TNC protein to basal levels even though the translocation
of phospo-Smad2/3 was reduced to the nucleus suggesting
that there may be other signaling pathways that are being
activated and are involved in the production of protein TNC.

Please cite this article in press as: Mercado-Gmez, O., et al., Role of TGF- signaling pathway on Tenascin C protein
upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019

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10
In this regard, it has been shown in previous investigations that the TNC protein increment can be caused
by inammatory processes (Midwood and Orend, 2009).
Fibroblast cell cultures treated with tumor necrosis factor alpha, interleukin-4, and interleukin-1 showed up to
>100-fold increase in Tenascin production (Rettig et al.,
1994). Furthermore, current evidence indicates that neuroinammation plays an important role in the mechanism
of ictogenesis in epileptic tissue apart from contributing to
neuronal death (for reviews see Vezzani and Baram, 2007).
Vezzanis research group has recently demonstrated a Tolllike receptor 4 and its agonist high-mobility group box 1
are involved in the generation of seizures (Maroso et al.,
2010). Furthermore, Tenascin C has been found to act as
an endogenous activator of TLR4-causing proinammatory
cytokine synthesis in macrophage and synovial broblast
cells (Midwood et al., 2009). These ndings suggest that
the protein itself could be involved in its own upregulation
and participate in neuroinammatory processes in epilepsy
at the same time; however, its contribution in pathology is
still unknown. Most importantly, these assertions may partly
explain our results where the inhibition of TGF- signaling
pathway did not decrease completely the overproduction of
the Tenascin C protein seizures.

Conclusion
In conclusion, our results demonstrate that TNC upregulation is mediated in part by activation of TGF- signaling
pathway, which support the idea of this signaling pathway
is involved in the overproduction of extracellular matrix
proteins responsible for the production of morphological
changes which might be involved in the formation of recurrent excitatory circuits or to the generation of epileptiform
activity that may contribute to epileptic seizures.

Conict of interest
None of the authors has any conict of interest to disclose.
We conrm that we have read the Journals position on issues
involved in ethical publication and afrm that this report is
consistent with those guidelines.

Acknowledgments
This work was supported by grants from Programa de Apoyo
a Proyectos de Investigacin e Innovacin Tecnolgica,
DGAPA-PAPIIT (IN200110) and Consejo Nacional de Ciencia
y Tecnologa, CONACyT (152613). The authors want to thank
Ellis Glazier (RIP) and Mrs. Josena Bolado for editing this
English-language text manuscript and Felix Recillas for his
time reviewing this manuscript.

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upregulation in a pilocarpine seizure model. Epilepsy Res. (2014), http://dx.doi.org/10.1016/j.eplepsyres.2014.09.019