Possible role of TIEG1 as a feedback regulator of myostatin and TGF-b

in myoblasts
Masato Miyake, Shinichiro Hayashi
1
, Shunsuke Iwasaki, Guozheng Chao, Hideyuki Takahashi,
Kouichi Watanabe, Shyuichi Ohwada, Hisashi Aso, Takahiro Yamaguchi
*
Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi,
Aoba-Ku, Sendai 981-8555, Japan
a r t i c l e i n f o
Article history:
Received 10 February 2010
Available online 18 February 2010
Keywords:
Myoblast
Skeletal muscle
TIEG1
Myostatin
TGF-b
Smad
a b s t r a c t
Myostatin and TGF-b negatively regulate skeletal muscle development and growth. Both factors signal
through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-b signaling
remains unclear. TGF-b inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been
implicated in the modulation of TGF-b signaling. These ndings prompted us to investigate the effect of
TIEG1 on myostatin and TGF-b signaling using C2C12 myoblasts. Myostatin and TGF-b induced the
expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differ-
entiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage
of differentiation. In contrast, TGF-b enhanced TIEG1 expression at an early stage. Overexpression of
TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-b in both proliferating
or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced
expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty
vector in the presence of myostatin or TGF-b. These results demonstrate that TIEG1 is one possible feed-
back regulator of myostatin and TGF-b that prevents excess action in myoblasts.
2010 Elsevier Inc. All rights reserved.
Introduction
Myostatin and TGF-b negatively regulate skeletal muscle devel-
opment, growth and regeneration. Deletion of the myostatin gene
leads to skeletal muscle hypertrophy in mice and cattle [13]. TGF-
b prevents muscle differentiation in vitro and regeneration of skele-
tal muscleinoldmice[4,5]. MyostatinandTGF-bsignal via Smad2/3.
After binding to these receptors (activin receptor type IIB for myost-
atin, TGF-b type II receptor for TGF-b), the co-receptors activin-like
kinase(ALK) 4/5phosphorylate theintracellular signalingmolecules
Smad2/3. Phosphorylated Smad2/3 forms a complex with Smad4
and is translocated into the nucleus, where it binds to specic pro-
moter DNA sequences and regulates gene expression [6]. Smad7
antagonizes the binding of Smad2/3 to their receptors and sup-
presses Smad2/3signaling[6]. MyostatinandTGF-bsignalingis con-
trolled by a feedback loop that depends on the regulation of Smad7
expression[79]. Thus, it seems that the signaling pathways of myo-
statin and TGF-b are similar except for the receptors. The control
mechanism of myostatin and TGF-b signaling remains unclear.
TGF-b inducible early gene (TIEG) 1 has been identied as a
gene inducible by TGF-b in human osteoblasts [10]. TIEG1 is a
ubiquitously expressed zinc nger kruppel-like transcription fac-
tor, and is especially highly expressed in skeletal muscle [11].
TIEG1 has been implicated in the regulation of cell proliferation,
differentiation and apoptosis in osteoblasts, leukocytes, etc. [12
14], but its function in skeletal muscle has yet to be demonstrated.
TIEG1 has an important role in modulating TGF-b signaling in
embryonic broblasts, where TIEG1 overexpression results in the
down-regulation of Smad7 by binding directly to the proximal pro-
moter and increasing Smad-responsive reporter activity [15]. Fur-
thermore, Smad2 expression is regulated by TIEG1 [16]. Overall,
it has been demonstrated that TIEG1 activates Smad signaling by
TGF-b.
Because it shares the same myostatin signaling molecules as
TGF-b (ALK4/5 and Smad2/3), we hypothesized that TIEG1 also
inuences myostatin signaling. In the present study, we detailed
the effects of TIEG1 on myostatin and TGF-b signaling in myoblasts.
Materials and methods
Cell culture. C2C12 mouse myoblasts from the Riken Cell Bank
were kindly gifted by Dr. R. Ngatomi [17] and were maintained
in high-glucose DMEM (Invitrogen, Carlsbad, CA) supplemented
0006-291X/$ - see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.02.077
* Corresponding author. Fax: +81 (0) 22 717 8880.
E-mail address: ty1010@bios.tohoku.ac.jp (T. Yamaguchi).
1
Present address: Mouse Molecular Genetics group, UMR S 787 Groupe
Myologie, INSERM UPMC-Paris VI, 75634 Paris Cedex 13, France.
Biochemical and Biophysical Research Communications 393 (2010) 762766
Contents lists available at ScienceDirect
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j our nal homepage: www. el sevi er . com/ l ocat e/ ybbr c
with 15% fetal bovine serum, 10 U/ml penicillin and 10 mg/ml
streptomycin (proliferation medium; PM) on a collagen type I-
coated ask at 37 C in a humidied incubator under 5% CO
2
. To in-
duce C2C12 differentiation, we used DMEM supplemented with 2%
horse serum, 10 U/ml penicillin and 10 mg/ml streptomycin as the
differentiation medium (DM). Recombinant mouse myostatin and
TGF-b1 were purchased from R&D systems (Minneapolis, MN)
and using at 1 or 2 lg/ml and 5 ng/ml, respectively. Vehicle
(4 mM HCl and 0.1% BSA) was used as a control.
Quantitative RT-PCR. Total RNA was extracted using TRIzol re-
agent (Invitrogen) according to manufacturers protocol. After
extraction, a TURBO DNase kit (Ambion, Austin, TX) was used to
eliminate contaminating DNA. First strand cDNAs were synthe-
sized from 1 lg of the total RNA using SuperScript III reverse
transcriptase (Invitrogen) with oligo (dT) primers (Invitrogen).
Real-time PCR was performed with 1 ll cDNA and SYBR Premix
Ex Taq or SYBR Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) using
a Thermal Cycler Dice Real Time System (TaKaRa Bio). The gene-
specic primer sequences are shown in Supplementary material.
The specicity of the RT-PCR product was examined by dissocia-
tion curve analysis. All values were normalized to that of glyceral-
dehyde-3-phosphate dehydrogenase (GAPDH).
Plasmids. Mouse TIEG1 open reading frame was prepared by RT-
PCR from mouse skeletal muscle using primers containing the
BamH1 and EcoR1 sites shown in Supplementary material (En-
zyme; PrimeStar GXL, TaKaRa Bio). The PCR product was digested
with BamH1 and EcoR1 and cloned into pCMV2-FLAG (Sigma, St.
Louis, MO). The vector was conrmed by sequencing. The pGL3-
(CAGA)
12
-luciferase reporter vector was kindly provided by Drs.
K. Miyazono and K. Tsuchida. pGL4.74 [hRluc/TK] vector was pur-
chased from Promega (Madison, WI).
Luciferase assay. C2C12 cells were seeded on collagen type I-
coated 24-well plates at 0.8 10
4
cells/cm
2
. After 24 h, they were
cotransfected with pGL3-(CAGA)
12
-luciferase reporter vector,
pGL4.74 [hRluc/TK] vector (Promega), and either pCMV2-FLAG or
pCMV2-FLAG-TIEG1 using Lipofectamine 2000 (Invitrogen)
according to manufacturers instructions. To stimulate in PM, the
medium was replaced with fresh medium including myostatin or
TGF-b after 6 h of transfection, then the cells were cultured for
18 h and collected. To stimulate in DM, the medium was replaced
with DM including the stimulants after 24 h of transfection. These
cells were cultured for 24 h and collected. Luciferase activities
were determined using a Dual-Luciferase Reporter Assay System
(Promega). Firey luciferase activity was normalized to renilla
luciferase.
Electroporation. Electroporation was performed using a Neon
Transfection System (Invitrogen) according to manufacturers
instructions. Briey, 1 10
6
cells were suspended in 100 ll elec-
troporation buffer including 5.0 lg of plasmid DNA and electropo-
rated (condition: 1650 V, 10 ms, three times) using a 10 or 100 ll
tip. After electroporation, the cells were seeded in PM excluding
antibiotics in 6-, 12- or 24-well plates. After 24 h, the cells were
harvested as PM-cultured C2C12 cells. To stimulate during differ-
entiation, they were cultured for 24 h after electroporation and re-
placed in DM with or without stimulants. To examine mRNA
expression, the cells were harvested after 24 h. For immunocyto-
chemistry, they were stained after 72 h.
Western blotting. After 24 h of electroporation, cells were lysed
in NP-40 buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl
2
, 10 mM
KCl, 0.5 mM DTT, 1% NP-40, 1 mM EDTA-Na
2
, 1 mM EGTA, Com-
plete Mini EDTA Free (Roche Applied Science, Indianapolis, IN),
Phosstop (Roche Applied Science)). After incubation for 30 min at
4 C, the lysate was centrifuged at 18,000 g for 15 min at 4 C and
the supernatant was collected. The protein extracts (2030 lg)
were separated by SDSPAGE and transferred onto an Immuobi-
lon-P membrane (Millipore, Billerica, MA), which was blocked with
0.1% Tween-20, 20 mM TrisHCl, 150 mM NaCl containing 3% or 5%
skim milk. After three washes, the membrane was probed with pri-
mary antibodies at 4 C overnight, then washed and probed with
secondary antibodies. Immunoreactive protein was detected using
ECF reagent (GE Healthcare Biosciences, Piscataway, NJ) and
Molecular Imager FX (Bio-Rad). The primary antibodies used in this
study were rabbit anti-TIEG1 Ab (sc-67062, Santa Cruz Biotechnol-
ogy, CA, 1:200) and mouse anti-FLAG mAb (F1804, Sigma, 1:3000).
The secondary antibodies were goat anti-rabbit IgG conjugated
with alkaline phosphatase (81-6122, Zymed Laboratories Inc.,
South San Francisco, CA, 1:10,000) and goat anti-mouse IgG conju-
gated with alkaline phosphatase (A3562, Sigma, 20,000).
Immunocytochemistry. Cells were washed with PBS and xed in
cold 50% methanol, 50% acetone (20 C) for 20 min. After three
washes with PBS, they were treated with 3% normal goat serum
in PBS for 30 min to block nonspecic staining and incubated with
anti-myosin antibody (MF20, Developmental Studies Hybridoma
Bank, Iowa City, IA, 1:100) overnight in PBS at 4 C. The cells were
then washed with PBS and incubated with Alexa 488-conjugated
goat anti-mouse IgG (A11029, Invitogen, 1:500) for 60 min at room
temperature. Finally, the nuclei were counterstained with propidi-
um iodide (PI). Samples were observed by confocal laser micros-
copy (MRC-1024; Bio-Rad, Hercules, CA).
Statistical analysis. Data are represented as means SD of at
least three independent experiments. Differences were assessed
using an unpaired Students t-test and one-way ANOVA following
Tukeys post-hoc test. P values <0.05 were considered signicant.
Results
Myostatin and TGF-b induce the expression of TIEG1 mRNA
The expression patterns of TIEG1, TIEG2 and Smad7 mRNAs
after stimulation with myostatin or TGF-b were investigated in
proliferating and differentiating C2C12 cells. During proliferation,
TIEG1 expression was signicantly induced by myostatin at 3 h
after stimulation and gradually decreased by 24 h (Fig. 1A). In con-
trast, TIEG2 expression was not inuenced by myostatin (Fig. 1B).
Smad7 expression was up-regulated 36 h after myostatin stimu-
lation (Fig. 1C). TGF-b signicantly induced TIEG1 and Smad7
expression, giving an expression pattern similar to that with myo-
statin (Fig. 1DF). During differentiation, TIEG1 expression in-
creased depending on the time after initiation of C2C12
differentiation. After myostatin stimulation, TIEG1 expression
was signicantly higher than that in control cultures only at 72 h
(Fig. 2). In contrast, TGF-b signicantly enhanced TIEG1 expression
12 and 24 h after stimulation (Fig. 2).
Overexpression of TIEG1 inhibits Smad signaling by myostatin and
TGF-b independently of Smad expression
Smad2/3 signaling was monitored using CAGA-lux assays. Lucif-
erase activity was signicantly induced by myostatin and TGF-b in
PM (Fig. 3A black bars). Overexpression of TIEG1 did not affect the
activity in control cultures, but the increase in luciferase activity
induced by myostatin and TGF-b was blocked (Fig. 3A white bars).
Activation of Smad signaling by myostatin and TGF-b was also
inhibited in differentiating cultures (Fig. 3B). Previously, it has
been shown that overexpression of TIEG1 suppresses Smad7 and
enhances Smad2 expression [15,16]. We therefore examined
Smad2 and Smad7 expression in TIEG1-overexpressing C2C12 cells
using electroporation. The transfection efciency was approxi-
mately 6080% as monitored by the GFP expression vector (data
not shown). The forced expression of TIEG1 was conrmed by
Western blotting using anti-TIEG1 and FLAG antibody (Fig. 3C).
Unexpectedly, Smad2 and Smad7 expression was unchanged dur-
M. Miyake et al. / Biochemical and Biophysical Research Communications 393 (2010) 762766 763
ing both the proliferation and differentiation stages despite TIEG1
overexpression (Fig. 3D).
Overexpression of TIEG1 prevents myogenic differentiation but does
not alter the effects of myostatin and TGF-b
To examine the effect of TIEG1onthe inhibitionof myogenesis by
myostatin and TGF-b, TIEG1-overexpressing cells were differenti-
ated to formmyotubes with or without myostatin and TGF-b stimu-
lation. Myostatin and TGF-b substantially prevented myogenic
differentiation (Fig. 4AC, G). TIEG1 overexpression also resulted
in the inhibition of myogenic differentiation (Fig. 4D and G). How-
ever, no further inhibition of myotube formation by TIEG1-over-
expressing cells was induced by myostatin and TGF-b (Fig. 4EG).
Discussion
The expression of TIEG1 mRNA was enhanced by myostatin and
TGF-b in C2C12 cells. Several researchers have shown that TGF-b
upregulates TIEG1 expression in osteoblasts, T cells and bone mar-
row stroma cells [10,13,18]. Bone morphogenic protein 2 also en-
hances TIEG1 expression in osteoblasts and cerebellar granule
cells [10,19]. From these ndings, it may be inferred that TGF-b
family proteins are important in regulating TIEG1 expression in
many type cells. On the other hand, epidermal growth factor,
which does not activate Smad signaling, also induces TIEG1 expres-
sion [10]. This shows that other signaling pathway besides Smad2/
3 signaling may control TIEG1 expression. Indeed, myostatin and
TGF-b modulate Akt/mTOR or MAPK signaling in myoblasts [20
22]. These non-Smad pathways may regulate TIEG1 expression.
Our results showed that TGF-b and myostatin induced the
expression of TIEG1 mRNA by different patterns in differentiating
C2C12 cells but the same pattern during proliferation. Both TGF-
b and myostatin negatively regulate myogenesis [1,4], but the de-
tailed functions and actions are individually distinguished in
C2C12 in vitro [23] and in muscle progenitor cells during regener-
ation in vivo [5]. In addition, Rios et al. have shown that exogenous
myostatin is less effective than endogenous myostatin [24], indi-
cating that the effect of the former is suppressed by unknown fac-
tors. Thus, there are distinct differences between the effects of
myostatin and TGF-b. Actually, TGF-b increases CAGA-lux activity
more than myostatin does. The different changes in TIEG1 expres-
sion caused by myostatin and TGF-b stimulation described in the
present study may be evidence of their different effects, but further
studies are required to clarify this.
Unexpectedly, overexpression of TIEG1 did not lead to inhibi-
tion of Smad signaling in C2C12 cells. In embryonic broblasts,
TIEG1 enhances Smad-responsive reporter activity [15]. This dis-
crepancy is not surprising because the function of a transcription
factor depends on cell type. To understand the mechanistic differ-
ences, the proteins interacting with TIEG1 in broblasts and myo-
blasts need to be identied.
The fact that Smad2 and Smad7 expression was not inuenced
by TIEG1 overexpression suggests that Smad signaling is differ-
ently regulated by TIEG1 in C2C12 myoblasts and broblasts. It
has been shown that TIEG1 deletion in T cells reduces Smad2 phos-
phorylation by TGF-b through the down-regulation of TGF-b
expression, with no changes in the expression of Smads or TGF-b
Fig. 1. Effects of myostatin and TGF-b on TIEG1, TIEG2 and Smad7 expression in proliferating C2C12 cells. C2C12 cells were seeded and stimulated by myostatin (AC) or TGF-
b (DF) for the indicated times. After stimulation, the cells were harvested and the expression of TIEG1 (A, D), TIEG2 (B, E) and Smad7 (C, F) mRNA was analyzed by
quantitative RT-PCR. (ac:) P < 0.05.
Fig. 2. Effects of myostatin and TGF-b on TIEG1 expression in differentiating C2C12
cells. C2C12 cells were cultured in proliferating medium and the medium was
replaced with differentiating medium containing either myostatin or TGF-b. Cells
were harvested at each indicated time and the expression of TIEG1 mRNA was
analyzed by quantitative RT-PCR.
*
P < 0.05 compared to control medium.
764 M. Miyake et al. / Biochemical and Biophysical Research Communications 393 (2010) 762766
Fig. 4. Myogenic suppressionbymyostatinandTGF-binTIEG1-overexpressingC2C12cells. C2C12cells wereelectroporatedwithvector anddifferentiatedfor 72 hinthepresence
of myostatin or TGF-b, followed by immunostaining for myosin heavy chain (MyHC) (AF). Bars show 100 lm. The ratio of nuclei in MyHC positive cells was determined by
countingat least 600nuclei ineachexperiment (G).
*
P < 0.05versus emptyvector usingthesamestimulants,
#
P < 0.05versus control amongcells transfectedwiththesamevector.
Fig. 3. Inhibition of Smad-responsive reporter activity by TIEG1 overexpression in C2C12 cells. Cells were transfected with vectors and stimulated by myostatin or TGF-b in
PM (A) or DM (B). The cells were then harvested and luciferase activity was measured as described in Materials and methods.
*
P < 0.05 versus control in empty vector,
#
P < 0.05 versus myostatin or TGF-b in empty cells. Overexpression of TIEG1 in electroporated C2C12 cells was conrmed by Western blotting using the antibody indicated
(C). Electroporated cells were harvested and the expression of Smad2 and Smad7 mRNA was analyzed by quantitative RT-PCR (D).
M. Miyake et al. / Biochemical and Biophysical Research Communications 393 (2010) 762766 765
receptors [14]. Therefore, auto-regulated expression of TGF-b and
myostatin may be associated with the reduction of Smad2/3 sig-
naling activity. However, because both myostatin and TGF-b sig-
naling was blocked, it seems likely that a common intracellular
molecule between myostatin and TGF-b (e.g. smurf or arkadia) is
more important for TIEG1 in C2C12.
Our results showed that stimulation with myostatin and TGF-b
prevented myogenic differentiation, as previously shown [4,25].
Overexpression of TIEG1 also inhibited differentiation (detailed
data are being submitted). If TIEG1 and these cytokines function
independently of each other, stimulation of TIEG1 overexpression
by myostatin or TGF-b would lead to strong suppression of myo-
genic differentiation. However, myostatin and TGF-b acted simi-
larly on myotube formation in empty- and TIEG1-vector
electroporated cells. This could be explained by alteration of Smad
signaling. In TIEG1-overexpressing cells, the effects of myostatin
may be canceled and the inhibition of myogenic differentiation
may depend upon the effect of TIEG1. The action of TGF-b may
have been partly canceled because Smad transcription was not
completely repressed. Therefore, myogenic inhibition in TIEG1-
overexpressing cells stimulated by TGF-b may derive from both
TIEG1 and the attenuated action of TGF-b. Our results give valuable
information about the relationship of TIEG1 to myostatin and TGF-
b action in myoblasts.
In conclusion, we demonstrated that myostatin and TGF-b reg-
ulate the expression of TIEG1 mRNA in C2C12 myoblasts and our
results indicate that TIEG1 modulates Smad signaling and the ac-
tions of myostatin or TGF-b. The results suggest a function of TIEG1
as a possible negative feedback regulator of myostatin and TGF-b.
Acknowledgments
We thank Dr. Ryoichi Nagatomi, Dr. Kunihiro Tsuchida and
Dr. Kohei Miyazono for kindly providing cells or materials. We
are grateful to Dr. Takafumi Uchida and Dr. Hirotada Akiyama for
helpful discussions, technical advice and provision of reagents.
We are also grateful to all members of the laboratory of functional
morphology for helpful discussions. This work was supported by
the Research Fellowship for Young Scientists Program from the
Japan Society for the Promotion of Science (JSPS) and the Pro-
gramme for Promotion of Basic and Applied Research for
Innovations in Bio-oriented Industry.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2010.02.077.
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