1460 Vol. 36, No. 9Biol. Pharm. Bull. 36(9) 1460–1465 (2013)
© 2013 The Pharmaceutical Society of Japan
Selective Androgen Receptor Modulator, YK11, Regulates Myogenic
Differentiation of C2C12 Myoblasts by Follistatin Expression
Yuichiro Kanno,* Rumi Ota, Kousuke Someya, Taichi Kusakabe, Keisuke Kato, and
Yosh io I nouye
Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan.
Received March 20, 2013; accepted June 19, 2013
The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR)
partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carbox-
ylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen re-
ceptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further in-
vestigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction
of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor
5 (Myf5) and myogenin, was more signiﬁcant in the presence of YK11 than in the presence of DHT. YK11
treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated
myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is
important for the anabolic effect of YK11.
Key words androgen receptor; selective androgen receptor modulator; C2C12 cell; follistatin
Androgens such as testosterone and 5-α-dihydrotestosterone
(DHT) act as agonists of androgen receptor (AR), which is a
member of the nuclear receptor (NR) superfamily of ligand-
dependent transactivation factors. Because androgens show
anabolic effects on skeletal muscles, androgen declining with
age contributes to age-related bone and muscle loss and in-
crease in fat mass.
Thus, the androgen anabolic effect is
attractive for the maintenance of health. However, besides
anabolic effects, various biological effects, such as develop-
ment of male reproductive tissues, sexual development and
spermatogenesis (androgenic effects), are mediated by AR.
Separation of anabolic effects from androgenic effects is criti-
cal for clinical usage of selective androgen receptor modula-
tors (SARMs) in diseases such as sarcopenia, cancer cachexia
However, the detailed mechanism of
selective anabolic action by SARMs in skeletal muscle still
Structurally characterized by an amino-terminal transac-
tivation domain [NTD/activation function 1 (AF1)], a DNA
binding domain (DBD), and a ligand binding domain (LBD)
including a carboxy-terminal transactivation domain [activa-
tion function 2 (AF2)],
AR mediates the expression of
androgen-regulated genes, such as prostate speciﬁc antigen
and FK506-binding protein 51 (FKBP51).
AR is localized in the cytoplasm, where it forms a complex
with chaperones. Upon ligand binding, AR translocates into
the nucleus. Following nuclear translocation, AR binds as a
homodimer to androgen responsive elements (ARE) in the
promoter regions of its target genes. Full activation of AR
requires physical interaction between the NTD/AF1 and LBD/
AF2 (known as the N/C interaction).
Follistatin (Fst) is essential for muscle ﬁber formation and
growth. It is an extracellular protein that acts as an inhibitory
binding partner of activins and selected transforming growth
factor β (TGF-β) family members. As the TGF-β signaling
cascade inhibits myogenic differentiation, inhibition of TGF-β
signaling accelerates myogenic differentiation.
Previously, we have reported the AR partial agonistic
nature of the novel steroidal compound, (17α,20E)-17,20-
diene-21-carboxylic acid methyl ester (YK11), using the
YK11 did not induce the N/C interac-
tion required for the AR full agonist function and was gene-
selective in MDA-MB 453 cells. In the present study, we show
the induction of myogenic differentiation of myoblast C2C12
cells by YK11 in comparison with DHT.
MATERIALS AND METHODS
Chemicals YK11 was prepared as previously reported.
DHT and hydroxyutamide (FLU) were obtained from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan) and Toronto
Research Chemicals (Toronto, Canada), respectively. Anti-
follistatin (Fst) antibody was purchased from GeneTex (San
Antonio, TX, U.S.A.).
Cell Culture Mouse myoblast C2C12 cells were cul-
tured in Dulbecco’s modiﬁed Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) at 37°C
in a humidiﬁed atmosphere with 5% CO
. C2C12 cells were
* To whom correspondence should be addressed. e-mail: firstname.lastname@example.org
The authors declare no conict of interest.
Table 1. Primers for the Target Genes (5′→3′)
Myogenin FW AGCTGTATGAGACATCCCCC
MyoD FW ACTACAGCGGCGACTCCGACGCGTCCAG
Myf5 FW AGCATTGTGGATCGGATCACGTCT
Follistatin FW AGAGGAAATGTCTGCTTCCG
AR FW TACCAGCTCACCAAGCTCCT
β-Actin FW TCCTCCTGAGCGCAAGTACTC
Highlighted Paper selected by Editor-in-Chief
September 2013 1461
seeded on plates and maintained in culture medium for 24 h.
To induce myogenic differentiation, YK11 or DHT in DMEM
supplemented with 2% horse serum (differentiation medium)
was added to the cells on day 0. For the neutralization assay
of Fst (also known as activin-binding protein), C2C12 cells
were maintained in differentiation medium in the presence of
Immunoblotting Cells were harvested and lysed in
sodium dodecyl sulfate (SDS) sample buffer containing
M Tris–HCI, pH 6.8, 4% SDS, 10% sucrose, 10 mM di-
thiothreitol and 0.01% bromophenol blue. Whole-cell lysates
were resolved by SDS-polyachylamide gel electrophoresis
(PAGE) and immunoblotting was performed using anti-myosin
heavy chain (MyHC, eBioscience, San Diego, CA, U.S.A.),
anti-androgen receptor (Epitomics, Burlingame, CA, U.S.A.)
and anti-tubulin antibodies (MBL, Nagoya, Japan) as primary
Fig. 1. Induction of Myogenic Differentiation by YK11 and DHT
(A) C2C12 cells were treated with YK11 (500 nM), DHT (500 nM) or solvent control (EtOH) in differentiation medium for 2 d. Whole-cell lysates were resolved by SDS-
PAGE, and proteins were detected by immunoblotting using antibodies against androgen receptor and tubulin as a loading control. (B) C2C12 cells were treated with
YK11 (500 n
M), DHT (500 nM) or solvent control (EtOH) in differentiation medium for 7 d. Whole-cell lysates were resolved by SDS-PAGE, and proteins were detected by
immunoblotting using antibodies against myosin heavy chain (MyHC) and tubulin as a loading control. (C–H) C2C12 cells were treated with YK11 (C–E: 500 n
M), DHT (C–E: 500 nM, F–H: 1–500 nM) or solvent control (EtOH) in differentiation medium for 2 or 4 d. The mRNA expression of Myf5 (C, F), MyoD (D, G) and
myogenin (E, H) was measured by qRT-PCR. The results were normalized against β-actin and are expressed as mean±S.D. (#, p<0.05 compared with DHT-treated cells;
*, p<0.05 and **, p<0.01 compared with the solvent control; n=3).
1462 Vol. 36, No. 9
antibodies. Horseradish peroxidase-conjugated anti-mouse
or rabbit immunoglobulin G (IgG) antibody (Cell Signaling
Technology, Danvers, MA, U.S.A.) was used as secondary
Knockdown Experiments Cells were transfected with
AR small interfering RNA (SASI_Mm01_00027673; Sigma-
Aldrich, St. Louis, MO, U.S.A.) or control siRNA using Li-
pofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA,
U.S.A.) according to the manufacturer’s instructions.
Real-Time Quantitative Reverse Transcription-Poly-
merase Chain Rection (qRT-PCR) Total RNA was isolated
using ISOGEN II (Nippon Gene Co., Ltd., Toyama, Japan).
cDNA was synthesized using the ReverTra Ace
Kit (TOYOBO, Osaka, Japan). qRT-PCR was conducted using
THUNDERBIRD™ SYBR qPCR Mix (TOYOBO) in a ﬁnal
volume of 25 µL according to the manufacturer’s protocol, and
analyzed with the Applied Biosystems 7500 Fast System SDS
software. The primer pairs used are described in Table 1.
Statistical Analysis Statistically signiﬁcant differences
were determined using the Student’s t-test, and differences
were considered statistically signiﬁcant at p<0.05.
YK11 and DHT Induce Myogenic Differentiation of
C2C12 Cells Firstly, we examined whether AR is expressed
in C2C12 myoblast cells. AR expression was detected in
C2C12 cells during cell differentiation by immunoblot assay
(Fig. 1A). To investigate the effect of YK11 on C2C12 cells,
the expression of the differentiation marker, myosin heavy
chain (MyHC), was examined. C2C12 cells were cultured with
YK11, DHT or solvent in differentiation medium. The MyHC
protein level on Day 7 was enhanced by both YK11 and DHT
treatments (Fig. 1B), suggesting that like DHT, YK11 can in-
duce myogenic differentiation of C2C12 cells.
YK11 and DHT Upregulate the mRNA Expression of
MRFs Next, we analyzed the mRNA expression of the
MRFs, Myf5, MyoD and myogenin. Cells were treated with
YK11, DHT or solvent, and the expression of Myf5, MyoD
and myogenin was analyzed by qRT-PCR on Day 2 and 4.
Myf5 and myogenin mRNA expression on Day 4 was more
signiﬁcantly enhanced by YK11 treatment than by DHT treat-
ment (Figs. 1C, E). Interestingly, upregulation of Myf5 and
myogenin mRNA on Day 4 by YK11 treatment required high
YK11 concentrations (100 n
M or 500 nM; Figs. 1F–H), whereas
DHT increased the expression of these genes at lower concen-
Co-treatment with the AR antagonist, FLU, suppressed this
upregulation, suggesting that YK11-induced MRFs expression
may be mediated by AR (Figs. 2A–C).
YK11 Induces Myf5 mRNA Expression via Fst mRNA
Upregulation by AR It has been reported that androgen-
induced myogenic differentiation is regulated by Fst, which
stimulates Myf5 expression.
Fst modulates the function of
a number of TGF-β family members, such as myostatin, which
is a negative regulator of myogenic differentiation, by directly
binding to them. We speculated that the AR-dependent Fst
induction may be responsible for the functional difference
between YK11 and DHT. To verify this hypothesis, Fst ex-
pression was measured on Day 2 and Day 4 after the addition
of YK11 or DHT. Fst mRNA expression was signiﬁcantly
induced by YK11 treatment, whereas it was not affected by
DHT treatment (Fig. 3A). The YK11-induced upregulation of
Fst mRNA was signiﬁcantly reduced by co-treatment with
FLU, supporting the AR-dependence of the YK11-mediated
upregulation of Fst expression (Fig. 3B). Furthermore, we car-
ried out an AR knockdown experiment. Similarly to the FLU
treatment, the YK11-induced upregulation of Fst mRNA was
signiﬁcantly reduced by knockdown of AR (Fig. 3C).
To see whether the YK11-induced Fst upregulation is re-
sponsible for the induction of Myf5 mRNA expression, C2C12
cells were treated with YK11 in the presence of neutralizing
anti-Fst antibody for 2 d in differentiation medium. The in-
crease in Myf5 mRNA level induced by YK11 treatment was
abolished when cells were treated with the antibody against
Fst (Fig. 4).
In this study, we showed that the AR partial agonist, YK11,
Fig. 2. Effect of the AR Antagonist, FLU, on the YK11-Induced Upregulation of MRFs (A–C)
C2C12 cells were treated with YK11 (500 nM) or solvent control (EtOH) in the presence or absence of FLU (10 µM) in differentiation medium for 4 d. The mRNA ex-
pression of Myf5 (A), MyoD (B) and myogenin (C) was measured by qRT-PCR. The results were normalized against β-actin and are expressed as mean±S.D. (*, p<0.05
compared with solvent control or FLU alone; n=3).
September 2013 1463
induced myogenic differentiation of C2C12 myoblast cells.
Key MRFs such as MyoD, Myf5 and myogenin are known
to be required for myogenic differentiation. MyoD and Myf5
are important for myogenic determination, whereas myogenin
is important for terminal differentiation and lineage mainte-
Here, we demonstrated that YK11 signiﬁcantly in-
creased the mRNA levels of these MRFs compared with DHT,
an AR full agonist. Based on these ﬁndings, YK11 may be
more potent in inducing myogenic differentiation than DHT.
Recent reports have shown that testosterone treatment up-
regulates Fst expression and promotes myogenic differentia-
tion in mesenchymal multipotent C3H 10T1/2 cells
isolated satellite cells.
We found that the Fst mRNA level
was enhanced by YK11 treatment in C2C12 cells in an AR-
dependent manner, as this effect was signiﬁcantly reduced by
co-treatment with an AR antagonist. The differentiation state
may be involved in the agonist-speciﬁc Fst mRNA regula-
tion, as unlike C3H 10T1/2 and isolated satellite cells, which
are poorly differentiated compared with C2C12 cells, we did
not observe a DHT induction of Fst mRNA in C2C12 cells.
Recent reports have suggested that testosterone induces Fst
expression via the non-canonical Wnt signaling.
terone promotes nuclear translocation of the AR/β-catenin
complex, which interacts with the Wnt pathway downstream
factor, T-cell factor 4 (TCF-4). The Fst promoter has a TCF
binding site and Fst mRNA is induced by Wnt.
ports have shown crosstalk between AR and Wnt signaling,
however the molecular mechanism is not clear. In contrast,
the Wnt3a-induced canonical Wnt signal decreases myogenic
and promotes osteoblastic differentiation
through upregulation of inhibitor of differentiation-3 (Id3)
in C2C12 cells.
Using various N/C interaction deﬁcient
mutants, it has been shown that the N/C interaction of AR
is important for AR-dependent gene regulation. The N/C in-
teraction contributes to selective gene activation by cofactor
recruitment and chromatin binding.
Previously, we have
shown by ARE-luciferase reporter assay that YK11 acts as a
partial AR agonist without inducing N/C interaction, whereas
DHT strongly induced N/C interaction. In fact, a different
regulation of AR target genes has been observed between
YK11 and DHT treatment in MDA-MB453 cells.
more, mutations of AR, which impair the N/C interaction,
were found in incompletely virilized patients with partial
In Fig. 2, a slight upregulation
of MRFs mRNA was observed by treatment of FLU alone.
Since FLU inhibits N/C interaction of AR, FLU may increase
MRFs expression. A recent report has demonstrated that the
SARM, S-101479, which shows low N/C interaction, induced
recruitment of fewer cofactors than DHT.
suggests that the difference in Fst mRNA regulation between
YK11 and DHT stems from cofactor recruitment differences.
In conclusion, YK11 induces myogenic differentiation via
AR-dependent induction of Fst expression. Although DHT
did not increase Fst mRNA, both YK11 and DHT elevated
the expression of MyHC protein and MRFs’ mRNA, imply-
ing that DHT enhances myogenic differentiation through an
Fig. 3. YK11-Induced Myogenic Differentiation Is Mediated by Induction of Fst Expression via AR in C2C12 Cells
(A) C2C12 cells were cultured with YK11, DHT (500 nM each) or solvent control (EtOH) in differentiation medium for 2 or 4 d. (**, p<0.01 compared with each solvent
control on Day 2. (B) C2C12 cells were cultured in differentiation medium for 24 h. After 30 min of pretreatment with FLU (10 µ
M), cells were treated with YK11, DHT
M each) or solvent control (EtOH) for 6 h. (*, p<0.05). (C) C2C12 cells were treated with siRNA against AR or negative control siRNA. After 24 h the medium was
changed to differentiation medium. After another 24 h, cells were treated with YK11 or solvent for an additional 6 h. (**, p<0.01). The mRNA expression of Fst was mea-
sured by qRT-PCR. The results were normalized against β-actin and are expressed as mean±S.D. (n=3).
Fig. 4. Neutralization of Fst Inhibits YK11-Induced Myogenic Differen-
tiation of C2C12 Cells
C2C12 cells were treated with YK11 (500 nM) or solvent control (EtOH) in dif-
ferentiation medium for 4 d in the presence or absence of anti-Fst antibody. The
mRNA expression of Myf5 was measured by qRT-PCR. The results were normal-
ized against β-actin and are expressed as mean±S.D. (*, p<0.05; n=3).
1464 Vol. 36, No. 9
Fst-independent pathway. In addition to the Fst pathway, YK11
may share this Fst-independent pathway with DHT.
In this report, YK11 was shown to be an appropriate
anabolic SARM. However, further investigation is required to
elucidate the mechanisms of the differential activation of the
Fst pathway by YK11 and DHT.
Acknowledgments This study was partially supported by
the Ministry of Education, Culture, Sports, Science and Tech-
nology of Japan, a Grant-in-Aid for Young Scientists (B), and
the “Open Research Center” Project.
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