Wen Guo, John Flanagan, Ravi Jasuja, James

Kirkland, Lan Jiang and Shalender Bhasin

J. Biol. Chem. 2008, 283:9136-9145.
doi: 10.1074/jbc.M708968200 originally published online January 18, 2008

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Supplemental material:
http://www.jbc.org/content/suppl/2008/01/23/M708968200.DC1.html
This article cites 56 references, 26 of which can be accessed free at
http://www.jbc.org/content/283/14/9136.full.html#ref-list-1

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Mechanisms of Signal Transduction:

The Effects of Myostatin on Adipogenic
Differentiation of Human Bone
Marrow-derived Mesenchymal Stem Cells
Are Mediated through
Cross-communication between Smad3 and
Wnt/ -Catenin Signaling Pathways

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 14, pp. 9136 9145, April 4, 2008
2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

The Effects of Myostatin on Adipogenic Differentiation of

Human Bone Marrow-derived Mesenchymal Stem Cells Are
Mediated through Cross-communication between Smad3
and Wnt/-Catenin Signaling Pathways*
S

Received for publication, October 31, 2007, and in revised form, January 18, 2008 Published, JBC Papers in Press, January 18, 2008, DOI 10.1074/jbc.M708968200

Wen Guo1, John Flanagan, Ravi Jasuja, James Kirkland, Lan Jiang, and Shalender Bhasin
From the Sections of Endocrinology, Diabetes, and Nutrition and Geriatrics, Boston University School of Medicine,
Boston Medical Center, Boston, Massachusetts 02118 and the Karolinska Institute, Stockholm SE-171 77, Sweden

Whereas the role of myostatin in the regulation of skeletal

muscle mass in animals has been widely recognized (1 8), its
effects on adipogenesis are poorly understood. Inactivating
mutations of the myostatin gene in a number of mammalian
species are associated with hypermuscularity and decreased fat

* This

work was supported by National Institutes of Health Grants

R01DK59261 (to W. G.) and R01DK70431 and R01DK49296 (to S. B.). The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

S
The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
1
To whom correspondence should be addressed: 670 Albany St., Boston, MA
02118. E-mail: wguo@bu.edu.

9136 JOURNAL OF BIOLOGICAL CHEMISTRY

mass (9 13). Similarly, myostatin knockout mice are characterized by a lower fat mass than wild-type controls (14, 15).
These in vivo observations have led to speculation that myostatin promotes adipogenesis. However, the data on the effects of
myostatin on fat mass and metabolism are conflicting. Transgenic mice that hyperexpress myostatin protein either systemically or in the skeletal muscle have increased fat mass (5),
whereas adipose-specific hyperexpression of myostatin leads to
reduced fat mass and improved insulin sensitivity (16). Mice
bearing tumor cells that hyperexpress myostatin experience
loss of lean as well as fat mass (17); it is unclear whether the loss
of fat mass is a consequence of myostatin hyperexpression or
the tumor-associated cachexia.
In vitro studies using various cell lines also have yielded
inconsistent results. Some studies have reported that myostatin
inhibits adipogenic differentiation of adipocyte precursor cell
lines of murine, bovine, and human origins (16, 18, 19), whereas
others have reported promotion of adipogenic differentiation
by recombinant myostatin in a mouse embryo stem cell line
(20). Differences in cell lines and culture conditions could have
contributed to these discrepancies.
The mechanism by which myostatin affects adipogenic differentiation also remain poorly understood. There is evidence
that myostatin activates the TGF2/activin type II receptor,
which subsequently activates type I receptor Alk5, leading to
phosphorylation of Smad3 (19). It is not known whether this
pathway is activated or how it affects adipogenesis in human
progenitor cells exposed to myostatin. The downstream signaling mechanisms by which the TGF/Smad3 pathway regulates
adipogenesis are also unknown.
The Wnt/-catenin signaling pathway plays an important
role in regulating growth and differentiation of mesenchymal
stem cells (21, 22). Under normal culture conditions, the Wnt
signaling pathway and adipogenic pathway are reciprocally regulated (2326). Factors that activate the Wnt/-catenin pathway, including canonical Wnt ligands, glycogen synthase kinase
3 inhibitors, and tumor necrosis factor , are known to inhibit
2

The abbreviations used are: TGF, transforming growth factor ; hMSC,

human bone marrow-derived mesenchymal stem cells; C/EBP, CCAAT/enhancer-binding protein; IP, immunoprecipitation; Alk5, activin receptorlike kinase 5; BrdU, bromodeoxyuridine; LAP, liver-activated protein; DM,
differentiation medium; PPAR, peroxisome proliferator-activated receptor ; eGFP, enhanced green fluorescent protein; RNAi, RNA interference;
qPCR, quantitative PCR; dn, dominant-negative.

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The effects of myostatin on adipogenic differentiation are

poorly understood, and the underlying mechanisms are
unknown. We determined the effects of human recombinant
myostatin protein on adipogenesis of bone marrow-derived
human mesenchymal stem cells (hMSCs) and adipose tissuederived preadipocytes. For both progenitor cell types, differentiation in the presence of myostatin caused a dose-dependent
reduction of lipid accumulation and diminished incorporation
of exogenous fatty acid into cellular lipids. Myostatin significantly down-regulated the expression of adipocyte markers
PPAR, C/EBP, leptin, and aP2, but not C/EBP. Overexpression of PPAR, but not C/EBP, blocked the inhibitory effects
of myostatin on adipogenesis. Myostatin induced phosphorylation of Smad3 in hMSCs; knockdown of Smad3 by RNAi or inhibition of its upstream kinase by an Alk5 inhibitor blocked the
inhibitory effect of myostatin on adipogenesis in hMSCs, implying an important role of Smad3 activation in this event. Furthermore, myostatin enhanced nuclear translocation of -catenin and
formation of the Smad3--catenin-TCF4 complex, together with
the altered expression of a number of Wnt/-catenin pathway
genes in hMSCs. The inhibitory effects of myostatin on adipogenesis were blocked by RNAi silencing of -catenin and diminished
by overexpression of dominant-negative TCF4. The conclusion is
that myostatin inhibited adipogenesis in human bone marrow-derived mesenchymal stem cells and preadipocytes. These effects
were mediated, in part, by activation of Smad3 and cross-communication of the TGF/Smad signal to Wnt/-catenin/TCF4 pathway, leading to down-regulation of PPAR.

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

EXPERIMENTAL PROCEDURES
Cell Culture and Supplies hMSC from non-obese young
male donors (BMI 25 kg/m2, age 20 40) were purchased
from Lonza (Allendale, NJ). Human preadipocytes from
abdominal subcutaneous, omental, and mesenteric fat depots
were obtained from the Adipocyte Core of Boston Obesity and
Nutrition Research Center and differentiated as previously
described (38). The effects of myostatin were independently
tested in hMSCs from four different subjects and preadipcotyes
from three different subjects.
Recombinant 110-amino acid human myostatin mature peptide was provided by Amgen (Thousand Oaks, CA), and a mouse
monoclonal anti-myostatin (JA16, Ref. 39) by Wyeth (Cambridge,
MA), respectively. The inhibitor for TGF type I receptor activinlike kinase 5 (Alk5i, cat. 616452) was from Calbiochem.
Adipogenesis was induced by differentiation medium (DM)
that contains IBMX (0.5 mM), dexamethasone (1 M), and insulin (170 nM), as well as proprietary components provided by the
vendor. Unless otherwise noted, cells were incubated in DM
until used for experiments. Differentiation was evaluated by
measuring the lipid content by Oil-red-O staining, lipid synthesis rate, and expression of adipocyte differentiation markers.
Oil-red-O stained lipids were solubilized in isopropyl alcohol
and the relative ratio of lipid contents were quantified by comparing the corresponding absorbance values at 500 nm. Oilred-O staining of undifferentiated hMSC grown in parallel culture served as the blank sample for this assay. The results were
normalized to the DM control.
The lipid synthesis rate was measured by the incorporation
of a fluorescent Bodipy-fatty acid into triglycerides. hMSCs
were differentiated in DM or DM containing different doses of
myostatin for 21 days. Myostatin was then removed by washing
the cells with phosphate-buffered saline and incubated with
fresh DM overnight. Cells were then incubated with 10 M
Bodipy-fatty acid (Invitrogen, D3382) pre-complexed with 3
M bovine serum albumin (Sigma, A2801) in a CO2 incubator
at 37 C. After 4 h of incubation, cells were washed six times
with warm phosphate-buffered saline containing 0.1% albumin,
solubilized in Me2SO, and analyzed using a Safire fluorescence
APRIL 4, 2008 VOLUME 283 NUMBER 14

plate reader (Durham, NC) with excitation at 485 nm and emission at 550 nm. The blank value for this assay was obtained by
treating the cells with the same protocol except that Bodipyfatty acid was washed off immediately after contact (10 s).
Preliminary studies showed that incorporation of Bodipy-fatty
acids into complex lipids is minimal within the first 60 s (data
not shown). The results were normalized to the DM control.
Selected samples were subjected to lipid extraction with
organic solvent (CHCl3/CH3OH, v/v, 1:1) and analyzed by TLC.
Under our experimental conditions, more than 95% of the fluorescent components isolated from the cells migrated with a
similar Rf value as natural triglycerides, implying nearly complete incorporation into cellular triglycerides.
RNAi and Viral VectorsPre-tested duo-pack RNAi oligonucleotides for Smad3, -catenin, and a nonspecific control
oligonucleotide were purchased from Invitrogen (Carlsbad,
CA) and transfected into hMSC using the Lipofectamine protocol (Invitrogen). Type 5 adenovirus encoding wild-type
TCF4, dominant-negative TCF4, PPAR, eGFP, and eGFPtagged -catenin (all human genes) were purchased from Vector
BioLab (Philadelphia, PA) and transfected into hMSCs as previously described (39). Retrovirus vector encoding a constitutively
active C/EBP construct (C/EBP-LAP) and control vector were
provided by Dr. S. R. Farmer (Boston University School of Medicine) and transfected into cells using Lipofectamine.
RNA Isolation, Reverse Transcription, and Real-time PCRTotal RNA was isolated using the RNAeasy isolation kit from Qiagen
(Valencia, CA). First-strand cDNA was synthesized using a Superscript cDNA synthesis kit from Invitrogen. Gene probe/primer
sets for quantitative qPCR of Smad3, -catenin, PPAR, C/EBP,
C/EBP, aP2, and leptin were purchased from ABI. A 96-well
qPCR-based macroarray for the Wnt signaling pathway and relevant Syber Green-based qPCR primers were purchased from
Superarray (Frederick, MD). The PCR array (APHS-043) was precoated with 84 Wnt target genes and 5 housekeeping genes. All
other PCR supplies were purchased from ABI.
All real-time qPCR measurements were performed on an
ABI7500 PCR system (ABI) using the standard temperature
cycling protocol for the relative quantification assay. Each
measurement was run in duplicate with three independent
samples. Selected samples were run after sequential dilution to
confirm that the detected signals were within the linear amplification range. Results were first normalized to the expression level of
an endogenous housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT). Selected samples were tested
against two additional housekeeping genes, 18S and GAPDH,
glyceraldehyde-3-phosphate dehydrogenase, and the results were
no different from the results obtained using HPRT. The final
results were then normalized to DM control except for those
shown in Fig. 3, which were normalized to the basal control.
Western AnalysisPrimary antibodies for phospho-Smad3,
Smad3, aP2, -tubulin, C/EBP, phospho-C/EBP, PPAR,
C/EBP, TCF4, -catenin, and histone-1 were obtained
from Cell Signaling (Danvers, MA), Santa Cruz Biotechnology (Santa Cruz, CA), and Invitrogen. Secondary antibodies
were purchased from Santa Cruz Biotechnology. The cell lysate
was prepared in radioimmune precipitation assay buffer containing a protease inhibitor mixture and a phosphatase mixture
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adipogenesis in vitro (26 28). Similar inverse correlation

between expression of Wnt/-catenin target genes and adipogenic genes has been found in human fat tissue biopsies (29).
Furthermore, adipose tissue-specific hyperexpression of
Wnt10b diminishes fat tissue accumulation in obese mice (30).
Cross-talk between Smad and Wnt signaling has been
reported in some cell types (3137). Little is known about the
interaction between myostatin/Smad and Wnt signaling pathways during adipogenic differentiation. Here, we present an
integrated study of the effect of myostatin on adipogenesis, utilizing both bone marrow-derived mesenchymal stem cells and
fat tissue-derived preadipocytes of human origin. After confirming a similar inhibition in both progenitor cell types, we
used hMSCs to test the hypothesis that the signal generated by
myostatin through the TGF/Smad3 pathway is cross-communicated to the Wnt signaling pathway through -catenin and
TCF4, which mediate the effects of myostatin on adipogenic
differentiation of human adipocyte precursors.

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

(both from Sigma). The protein content was measured by the

Bradford method, and equal amounts of protein (50 g for the
transcription factors, 10 g for aP2) were loaded for electrophoresis on a 4 20% gradient gel. The standard Western analysis protocol was used thereafter. The protein expression was
detected by chemiluminescence, quantified by densitometry,
and normalized to -tubulin, which is not significantly affected
by differentiation or by myostatin treatment.
Nuclear Protein Isolation and ImmunoprecipitationNuclear proteins were isolated using a commercial kit (Active
Motiff, Carlsbad, CA). Protein A/G-Sepharose beads were
washed with the lysis buffer before use. Equal amounts of
nuclear proteins (500 g/reaction) were mixed with 10% (v/v)
protein A/G beads preloaded with rabbit anti-Smad3 or mouse
anti--catenin and rotated at 4 C overnight. After washing three
times with phosphate-buffered saline, the proteins bound to the
beads were eluted by boiling in SDS-based sample-loading buffer,
separated by SDS-PAGE, transferred to the polyvinylidene difluoride membrane, and probed with mouse anti--catenin (IP:
Smad3) or rabbit anti-Smad3 (IP: -catenin). Both membranes
were then re-probed using mouse anti-TCF4. Anti-rabbit and
anti-mouse True Blot from eBioscience (San Diego, CA) were used
as the second antibody for chemiluminescence detection. Equal
protein input for each immunoprecipitation assay was verified by
Western analysis of histone-1 in the input protein mix.

9138 JOURNAL OF BIOLOGICAL CHEMISTRY

Cell Cycle and DNA Synthesis hMSCs were incubated with

DM or DM containing myostatin (0.1 g/ml) and harvested
every 2 h for the first 12 h, every 8 h from 12 to 72 h, and then
once a week for 3 weeks. Cells were fixed in ethanol, stained
with propidium iodide, and analyzed by fluorescence-activated
cell sorting, as previously described (40). For the analysis of
DNA synthesis, hMSCs were treated with basal medium, DM,
or DM containing myostatin (0.1 g/ml) for different days.
BrdU (2 mM) was added 14 h before the termination of incubation. Cells were then fixed, and the incorporation of BrdU was
measured using an ELISA kit from Roche Applied Science
(Indianapolis, IN). The results were normalized to the value in
the DM control harvested at the same time point.
StatisticsAll photomicrographs are representative of at least
three independent experiments. The results are shown as the
mean S.E. (n 3). Multiple group comparisons were performed
using the Duncans test, and comparisons between two independent groups were analyzed using the Students t test. The statistical
significance was inferred from p values 0.05.

RESULTS
Myostatin Inhibits the Differentiation of hMSCs into Mature
AdipocytesAdipogenic differentiation in hMSCs was induced
by a hormone mixture (DM) containing graded doses of recombinant human 110-amino acid mature myostatin protein,
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FIGURE 1. Myostatin inhibits adipogenic differentiation of bone marrow-derived hMSC. hMSCs were incubated for 21 days in adipogenic DM with or
without myostatin (myst) and anti-myostatin antibody (JA16). A and B, photomicrograph of Oil-red-O stained hMSCs incubated in DM alone (A, bar 100 m)
or DM containing 1.0 g/ml myostatin (B). C, hMSC distribution among different differentiation stages in DM (open bar) or in DM containing myostatin (1 g/ml,
dark bar, *, p 0.05, n 3). Stage I: elongated fibroblast-like cells without microscopically detectable lipid droplets; stage II: flattened cells without detectable
lipid droplets; stage III: multiple small lipid droplets ( 12 per cell) that are only visible under high magnification (250); stage IV: fewer but larger lipid droplets
(6 12 per cell) that are detected under lower magnification (100); stage V: fewer but larger coalescent lipid droplets (3 6 per cell) that are readily detectable
at low magnification (40); stage VI: 13 very large coalescent lipid droplet(s) that occupy the majority space within a cell. D, lipid synthesis rate assessed by
measuring the incorporation of Bodipy-fatty acid into cellular lipids of hMSCs incubated in DM or DM containing myostatin (abc, p 0.05, n 8).
E, expression of aP2 and leptin mRNAs in hMSCs (abc, p 0.05, n 3); open bar: DM control; dark gray bar: DM containing myostatin (1.0 g/ml), light gray
bar: DM containing myostatin (1.0 g/ml) and anti-myostatin antibody JA16 (10 g/ml). F, lipid content of hMSCs measured by Oil-red-O staining and
quantification at 500 nm, with myostatin (0.1 g/ml) added at each indicated time point (abc, p 0.05, n 3). G, lipid content in hMSCs treated with DM.
Myostatin (0.1 g/ml) was added to hMSCs together with DM at time 0 and withdrawn at the indicated time points, and the incubation was continued in DM
for a total of 21 days. Lipid content was measured by Oil-Red-O staining and quantification at 500 nm (abc, p 0.05, n 3).

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

stained lipids on a grayscale). Cultures treated with myostatin
also had a higher fraction of smaller adipocytes and a lower
fraction of larger adipocytes (Fig. 1C), lower rate of lipid synthesis, assessed by the incorporation of an exogenous fluorescent fatty acid into cellular lipids (Fig. 1D), and reduced mRNA
expression of adipogenic markers, aP2 and leptin (Fig. 1E). Coincubation with a myostatin-neutralizing antibody (41) partially reversed the inhibition of adipocyte gene expression (Fig.
1E) and lipid-filling (not shown), implying that the inhibitory
effects were myostatin-specific.
Suppression of adipogenesis was maximally achieved when
myostatin was added early during differentiation. hMSCs incubated in DM containing myostatin from day 0 to day 21 showed
a 62 8% reduction of cellular lipid content, whereas cells
exposed to myostatin starting day 3 or later were less responsive
(Fig. 1F). In separate experiments, myostatin was added to
hMSC at the initiation of differentiation, removed at different
time points thereafter, and replaced with myostatin-free DM.
The presence of myostatin in the first 48 96 h was sufficient to
cause sustained suppression of lipid accumulation throughout
the 21-day differentiation program, whereas exposure to myostatin for only the first 0 24 h did not affect subsequent lipid
accumulation (Fig. 1G). Thus, early exposure to myostatin is
both required and sufficient to inhibit adipogenesis in hMSCs.
FIGURE 2. Myostatin inhibits adipogenic differentiation of human preaMyostatin Inhibits Differentiation and Lipid Accumulation in
dipocytes. A and B, phase-contrast photomicrographs of human preadipoHuman
PreadipocytesHuman subcutaneous preadipocytes
cytes incubated in differentiating medium (DM) alone (A) or in DM containing
1.0 g/ml myostatin (B, bar 100 m) for 21 days; C, lipid content was meas- were differentiated with and without myostatin. The latter was
ured by Oil-red-O staining on day 21; D, mRNA expression levels of aP2 and
associated with decreased lipid filling (Fig. 2, AC) and reduced
leptin in cells treated for 21 days with DM control (open bar) or DM containing
aP2 and leptin mRNA expression (Fig. 2D). Similar inhibitory
myostatin (1.0 g/ml, dark bar).
effects were also observed in human
preadipocytes derived from omental
*p<0.05
A 600
B
*
and mesenteric fat depots (not shown).
C/EBP
400
Myostatin Did Not Affect Early
Basal
DM
DM/myst
200
Induction of C/EBPThe rapid
C/EBP-LAP
34 kD
and transient induction of tran0
1
3
7
12
24
scription factors C/EBP and - is
Time (h)
aP2
14 kD
one of the earliest events in adipoPPAR
C/EBP
genesis. These transcription factors
PPAR2
*
*
600
55 kD
600
PPAR1
bind to specific sequences in the
400
400
promoter of C/EBP and PPAR to
60 kD
200
200
NS
30 kD
induce
their expression, which then
0
0
1
24
1
24
activates the full differentiation proTime (h)
gram required for adipocyte maturation (42, 43). Incubation of
C
hMSCs in DM up-regulated mRNA
*
aP2
C/EBP
250
expression of C/EBP (Fig. 3A) and
300
* p<0.05
* p<0.05
200
C/EBP (not shown). This effect
150
was attenuated by myostatin in the
200
*
*
100
first hour but not beyond this time
100
50
point (Fig. 3A). Western analysis
0
0
revealed a similar early and tranGFP 100
95
50
0
100 95
50
0 (x 107 Pfu/ml)
sient induction of C/EBP protein
PPAR
0
5
50 100
0
5
50
100 (x 107 Pfu/ml)
and phospho-C/EBP (Thr-235)
FIGURE 3. Myostatin inhibits adipogenic differentiation downstream from C/EBP. A, time course of mRNA within a 314-h incubation with
expression of C/EBP, PPAR, and C/EBP, measured by qPCR in hMSCs incubated in basal medium (not shown),
DM (open bars), or DM containing myostatin (0.1 g/ml, dark bars). B, Western analysis of C/EBP, aP2, and PPAR in DM, and the effect was also insensihMSCs overexpressing a constitutively active C/EBP-LAP construct after being treated for 6 days in basal medium, tive to myostatin (not shown).
DM, or DM containing myostatin (0.1 g/ml). Nonspecific bands (NS) are shown as an indication of equal sample
After 24 h of incubation in DM,
loading. C, hMSCs were transfected with adenovirus encoding eGFP or PPAR, and then treated with DM (open bars)
expression of PPAR and C/EBP
or DM containing myostatin (dark bars). mRNA levels of aP2 and C/EBP were measured by qPCR.
mRNA expression
(% of GFP control)

APRIL 4, 2008 VOLUME 283 NUMBER 14

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mRNA expression
(% of undifferentiated basal)

henceforth referred to simply as myostatin. Myostatin diminished total lipid accumulation compared with cells incubated in
the DM control (Fig. 1, A and B, dark clusters represent the

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

*p<0.05

C
-

DM/Alk5i

62kD

pSmad3

62kD

Smad3

-tub

50kD

DM

DM/Alk5i

34kD

55kD

myst
C/EBP

aP2

50kD

-tub

0
1 50

aP2
2 00
1 00

1 00

D
Myst

14kD

C/EBP

50
0

+ +
- - -

- - - + + - - - + +

- ++
++

RNAi-Smad3

control

62kD

50

30

0
control oligo + + - - - - - RNAi-1
- - + + - - + +
RNAi-2
- - - - + + + +

PPAR

14kD

100

60

Smad3
aP2

FIGURE 4. Smad3 plays a pivotal role in myostatin-induced suppression of adipogenesis. A, Western analysis of Smad3 and phospho-Smad3 in hMSCs grown in
DM or DM containing myostatin (0.1 g/ml) with (DM/Alk5i) or without an Alk5 inhibitor (100 M) for 3 h. -Tubulin served as a loading control. B, Western analysis of
C/EBP, PPAR, and aP2 in hMSCs treated the same as in A but with extended incubation for 48 h. Each blot in both A and B is representative of three independent
experiments. C, expression of Smad3 and adipocyte marker genes in hMSCs after RNAi knockdown of Smad3. hMSCs were treated with two different RNAi oligonucleotides (RNAi-1 and RNAi-2) administered separately or in combination (1:1 mixing). Control cells were transfected with a nonspecific 21-mer oligonucleotide. All
oligonucleotides were added at a final concentration of 100 nM. The cells were then incubated in DM (open bars) or DM plus myostatin (0.1 g/ml, dark bars) for 6 days
and used for qPCR analysis of Smad3, PPAR, aP2, and C/EBP. D, Western analysis of Smad3 and aP2 in cells treated the same as those used for PCR analysis. Two RNAi
oligos were mixed in 1:1 ratio in this experiment. Results are representative of two experiments in duplicates.

in hMSCs was induced 5 6-fold compared with the cells maintained in basal medium. This induction, however, was blunted
by myostatin (Fig. 3A, lower panel). Together, these results suggested C/EBP was not, whereas C/EBP and PPAR were
down-regulated by myostatin, in association with suppression
of adipogenesis.
We then determined whether ectopic expression of C/EBP
reverses the inhibitory effects of myostatin on adipogenic differentiation. We transfected the cells with a retroviral vector
expressing a constitutively active C/EBP construct (C/EBPLAP). After selection with puromycin, the cells were incubated
in DM with or without myostatin. As shown in Fig. 3B, even in
the presence of ectopic C/EBP-LAP, myostatin still significantly down-regulated the expression of PPAR and aP2 proteins. In contrast, when hMSCs were infected with adenovirus
encoding PPAR, the inhibitory effects of myostatin on the
expression of adipogenic markers, C/EBP and aP2, were no
longer detected (Fig. 3C).
Smad3 Mediates the Inhibitory Effect of Myostatin on
AdipogenesisTo investigate the mechanisms that mediate
myostatin effect on adipogenesis, we began with the
upstream steps in myostatin signaling. Myostatin has been
shown to activate TGF/Smad3 signaling pathway in myoblasts and clonal rodent preadipocytes (19, 44). Consistently,
we show that myostatin induced a rapid increase in phos-

9140 JOURNAL OF BIOLOGICAL CHEMISTRY

pho-Smad3 in hMSCs (Fig. 4A), a process which is usually

correlated with Smad3 activation (19). To determine
whether activation of Smad3 is essential for myostatin-mediated suppression of adipogenesis, we first assessed the cellular response to a pharmacological inhibitor of its upstream
kinase, TGF type I receptor Alk5. Incubation with the Alk5
inhibitor (Alk5i) decreased phospho-Smad3 (Fig. 4A),
increased PPAR, C/EBP, and aP2 expression, and blocked
the inhibitory effects of myostatin on each of these adipocyte
markers (Fig. 4B). The cells treated with Alk5i also had
higher expression of PPAR and C/EBP than controls and
greatly increased expression of aP2 (Fig. 4B) as well as cellular lipid
accumulation (not shown). These data suggest that factors other
than myostatin may restrain adipogenesis through the Alk5/
Smad3 pathway under our control conditions.
To evaluate further the role of Smad3 in mediating myostatin
effects on adipogenesis in hMSCs, we used two separate RNAi
oligonucleotides to reduce endogenous Smad3 expression.
Each of the two RNAi oligonucleotides reduced Smad3 expression by 80% (Fig. 4C, upper left panel). Knockdown of Smad3
by each of the two RNAi or their combination blocked myostatin-mediated inhibition of mRNA expression of PPAR,
C/EBP, and aP2 (Fig. 4C). By Western analysis, we confirmed
that Smad3 RNAi also completely reversed myostatin-mediated inhibition of aP2 expression (Fig. 4D).
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PPAR
150

90

myst
mRNA expression
(% of DM control)

DM

Smad3

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

IB: catenin

121

77

77

48

77

77

48
myst
Alk5i

48

IB: TCF4

+
-

+
+

IB: TCF4
-

+
-

+
+

Input: histone-1

+
-

50

+
+

DM

nuclear

77
121
77

121
77

200

catenin (24h)

100

catenin(48h)

control
RNAi-1
RNAi-2

++ -- - ++
- - --

His-1 (48h)
Myst
121

-tub (48h)

77
19
12

-- -- - ++
+ + ++

control

E
catenin (48h)

C/EBP
400
300
200
100
0

77
48

PPAR

300

39
25

100

DM/myst

*p<0.05

200

121

cytosolic

aP2

300

++
---

-++
--

--++

-++
++

RNAi-cat
-

+
-catenin
aP2

FIGURE 5. -Catenin interacts with Smad3 and acts downstream of Smad3 to mediate the inhibitory effect of myostatin on adipogenesis. A, physical
interaction of Smad3 with -catenin and TCF4. hMSCs were incubated for 3 h in DM, DM plus myostatin (0.1 g/ml), or DM plus myostatin (0.1 g/ml) and Alk5
inhibitor (100 M). Nuclear extracts were immunoprecipitated using anti-Smad3 or anti--catenin antibody, and the resulting protein complex was separated
by electrophoresis and immunoblotted for the expected binding partner of the target protein. After stripping, both membranes were re-probed for the
common binding partner TCF4. Histone-1 was used to demonstrate equal sample input for the immunoprecipitation reactions. B, nuclear distribution of
eGFP-tagged -catenin in hMSCs after 3 h of incubation with DM or DM containing myostatin (0.1 g/ml). C, Western blot analysis of nuclear and cytosolic
-catenin in hMSCs incubated with DM or DM plus myostatin (0.1 g/ml) for 24 and 48 h. Histone-1 and -tubulin were used as loading controls for the nuclear
and cytosolic fractions, respectively. D, effect of -catenin silencing on mRNA expression of adipocyte markers in hMSCs. -Catenin expression was silenced by
two different RNAi oligonucleotides (RNAi-1 and RNAi-2) separately or in combination. Control cells were transfected with a nonspecific 21-mer oligonucleotide. All oligonucleotides were added at a final concentration of 100 nM. hMSCs were then incubated with DM (open bars) or DM plus myostatin (0.1 g/ml, dark
bars) for 6 days and used for qPCR analysis for -catenin, PPAR, C/EBP, and aP2. E, Western analysis of -catenin and aP2 in cells treated the same as those
used for PCR analysis. Two RNAi oligos were mixed in 1:1 ratio in this experiment. Results are representative of two experiments in duplicates.

-Catenin Is Activated by Myostatin through Smad3 and Is a

Key Player in Suppression of AdipogenesisWe tested the
hypothesis that Smad3 mediates the inhibitory effects of myostatin by facilitating -catenin nuclear translocation (45) and
activating the Wnt/-catenin/TCF4 pathway (31, 45, 46). To
evaluate the physical interaction between Smad3, -catenin,
and TCF4, we immunoprecipitated nuclear proteins using antibodies for Smad3 and -catenin, respectively. Equal protein
input in IP experiments was verified by Western analysis of
histone-1 (Fig. 5A). The immunoprecipitated protein complex
was separated by SDS-PAGE, transferred to polyvinylidene
difluoride membrane, and immunoblotted with mouse anti-catenin (IP Smad3) or rabbit anti-Smad3 (IP -catenin). Both
membranes were then stripped and re-probed using mouse
anti-TCF4. As shown in Fig. 5A, the protein complex precipitated by anti-Smad3 contained -catenin and TCF4. Similarly,
the protein complex precipitated by anti--catenin contained
Smad3 and TCF4. Incubation of hMSCs with myostatin
increased the amount of Smad3 co-precipitated with anti-catenin and TCF4 that was co-precipitated using either antiSmad3 or anti--catenin. Co-treatment with Alk5i reduced
APRIL 4, 2008 VOLUME 283 NUMBER 14

the myostatin-mediated association between -catenin and

Smad3, as well as between these two proteins and TCF4
(Fig. 5A).
To confirm the effects of myostatin on the nuclear translocation of -catenin in live cells, we transfected hMSCs with
eGFP-tagged -catenin. Myostatin increased -catenin-associated green fluorescence in the nuclei of hMSCs (Fig. 5B). We
further performed Western analyses of nuclear protein extracts
from hMSCs maintained in DM for 24 or 48 h with or without
myostatin. As shown in Fig. 5C, incubation in DM alone
decreased the amount of -catenin in both nuclear and cytosolic compartments (47). Similarly, we found that hMSCs treated
with myostatin retained strong -catenin immunoreactivity in
both the nuclear and cytosolic compartments after an extended
incubation in DM (24 48 h, Fig. 5C), suggesting that myostatin
stabilizes -catenin in differentiating hMSCs.
To determine whether -catenin is essential for suppression
of adipogenesis by myostatin, we inhibited endogenous -catenin expression by RNAi. As shown in Fig. 5D, each RNAi efficiently suppressed -catenin expression by more than 70%.
Inhibition of -catenin by each RNAi was associated with
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basal

-catenin

100

mRNA expression
(% of control oligo treatment)

25

+ myst

IB: Smad3

39
myst
Alk5i

-myst

IP: catenin

IP: Smad3

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

9142 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 6. Under adipogenic differentiation conditions, myostatin does

not affect cell cycle progression or DNA synthesis. Confluent hMSCs were
treated with basal medium, DM, and DM containing myostatin (0.1 g/ml).
A, cells were harvested after 24 h and subjected to fluorescent-activated cell
sorting (FACS) analysis. The area under each phase (G0/G1 versus G2/M) represents the relative number of cells residing in the corresponding phase. The
majority of the cells fell in the quiescent G0/G1 phase, regardless of the presence of myostatin; B, DNA synthesis assessed by BrdU incorporation in hMSCs
incubated in basal medium, DM, or DM containing myostatin (0.1 g/ml) for
different days, as shown.

CEBP, and aP2 (Fig. 7B). At appropriate concentrations, dnTCF4 largely, but not completely, blocked the inhibitory effect
of myostatin on mRNA expression of each of these adipocyte
gene markers (Fig. 7B).
We next tested whether ectopic dn-TCF4, at its optimal concentration, might diminish the potency of myostatin in inhibiting adipogenesis. Myostatin down-regulated the expression of
aP2 and PPAR at a similarly low concentration range (0.01
0.05 g/ml) in hMSCs transfected with either vector or dnTCF4. However, at any given concentration of myostatin, the
extent of inhibition was consistently attenuated by dn-TCF4.
These results suggest that TCF4 is a major component that
mediates the inhibitory effect of myostatin, whereas other
inhibitory pathways are present that can be activated by myostatin independent of TCF4.

DISCUSSION
Adult hMSC have been well characterized as a renewable
progenitor pool that can differentiate into adipogenic and mulVOLUME 283 NUMBER 14 APRIL 4, 2008

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increased expression of aP2, PPAR, and C/EBP (Fig. 5D).

Silencing of -catenin blocked the inhibitory effect of myostatin on each of these adipocyte marker genes (Fig. 5D) and lipid
accumulation (not shown). Western analysis confirmed the
RNAi effect on the -catenin protein and the reversal of myostatin-mediated suppression of aP2 expression (Fig. 5E). Thus,
-catenin plays an essential role in mediating the inhibitory
effects of myostatin on adipogenesis in hMSCs.
To evaluate whether the increase in nuclear -catenin and
the formation of -catenin /TCF4 complex causes functional
activation of the Wnt/-catenin signaling pathway, we performed a real-time PCR-based macroarray analysis of Wnt/catenin pathway genes (Superarray APHS-043). Myostatin
altered the expression of a number of Wnt/-catenin pathway
genes in hMSCs during differentiation. Among these, PITX2,
DKK1, and Wnt4 were found to be the most responsive to myostatin (2-fold) after confirmation with real-time PCR (data
not shown). Thus, myostatin-induced nuclear translocation of
-catenin and its interaction with TCF4 were associated with
changes in Wnt pathway activity. The increased expression of
PITX2, a progrowth transcription factor, raised the possibility
that myostatin might inhibit adipogenesis by preventing
growth arrest, as previously shown in differentiating myoblasts
(48 50). Accordingly, we evaluate the effect of myostatin on
cell cycle distribution using fluorescence-activated cell sorting
(FACS). As shown in Fig. 6A, the majority of the cells fell in the
Go/G1 phase with a small fraction remaining in the G2/M phase,
in a pattern similar to that found in differentiating mouse clonal
preadipocytes (51). This distribution of cells in different phases
of cell cycle was not significantly different in DM control and
myostatin-treated hMSCs at different time points (from hours
to days, not shown).
We then assessed DNA synthesis in differentiating hMSCs
by measuring the incorporation of bromodeoxyuridine (BrdU).
As shown in Fig. 6B, BrdU incorporation was markedly reduced
within the first 24 h of exposure to DM. Myostatin had no effect
on BrdU incorporation in hMSCs at least within the first 10
days of the differentiation program. Hence, myostatin-mediated activation of Wnt/-catenin/TCF4 pathway was not associated with altered cell cycle regulation under our experimental
conditions. Furthermore, our results show that, unlike murine
preadipocyte cell lines, differentiating hMSCs do not experience early phase clonal expansion.
Inactivation of TCF4 Attenuates Myostatin-mediated Suppression of AdipogenesisAs a central component of the Wnt
signaling pathway, TCF4 has been shown to be an important
regulator of adipogenesis (26). To determine whether TCF4 is a
key player downstream of -catenin, we transfected hMSCs
with adenovirus vector encoding a dominant-negative TCF4
(dn-TCF4) and treated the cells in DM with or without myostatin. Ad5 empty virus was used as control. Adenovirus encoding
wild-type TCF4 was transfected in parallel cultures. The functionality of dn-TCF4 to block TCF4 activity was confirmed by
luciferase activity tested in COS-7 cells transfected with a TCF4
reporter gene (TOPFLASH) and a control Renilla luciferase
gene (Fig. 7D).
Transfection with dn-TCF4 increased lipid accumulation
(Fig. 7A) and increased the mRNA expression of PPAR,

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

PPAR

1.0

1.5

1.5
1.0

* *

0.5
0.0

-- 5 -- -- - 5 -- -- -- --- -- 2.5 5 10

C
aP2
1.0
0.8

0.6

0.4
0.0

0.1

0.2

0.0
-- 5 -- -- -- x108 Pfu/ml
5 -- -- -- -- x108 Pfu/ml
-- -- 2.5 5
10 x108 Pfu/ml

-- 5 -- -- -5 -- -- -- -- - -- 2.5 5 10

*p<0.05
PPAR

Ad5
dn-TCF4

0.5

0.9

0.6

0.3
0.0

myst (g/ml)

0.1

0.2

TOPFLASH/Renilla

0.0

aP2

2.0

1.0

0.5

Ad-5
wt-TCF4
dn-TCF4

2.5

2.0 C/EBP

1.5

mRNA expression
(% of DDM control)

DM/myst

DM

DM/myst

Ad5-Vector

2.0

*p<0.05

1 .0

0 .8
0 .6
0 .4
0 .2
0 .0

10

15

dn-TCF4 (x 108 pfu/ml)

FIGURE 7. Dominant-negative TCF4 (dn-TCF) attenuates the inhibitory effect of myostatin on adipogenesis. hMSCs were transfected with an empty
control vector (Ad5-Vector), an adenovirus vector encoding a dn-TCF4, or wild-type TCF4. Cells were then induced to differentiate with or without myostatin.
A, lipid accumulation in hMSCs treated with Ad5 or dn-TCF4 (final concentration of 5 108 pfu/ml for each) after 6 days of incubation with DM or DM containing
myostatin (0.1 g/ml). The cells were stained with Texas Red and photographed under a fluorescent microscope. B, hMSCs were transfected with different
doses of dn-TCF4 and then treated with DM (open bars) or DM containing myostatin (0.1 g/ml, dark bars) for 6 days and the mRNA levels of PPAR, C/EBP,
and aP2 were measured by qPCR. C, hMSCs transfected with Ad-5 or dn-TCF4 (5 108 pfu/ml) were incubated for 6 days with DM or DM containing myostatin
at different doses. The mRNA levels of PPAR, C/EBP, and aP2 were measured by qPCR. D, ratio between TCF4 reporter gene activity (TOPFLASH luciferase) and
the transfection control Renilla luciferase in COS-7 cells in proportion to dn-TCF4 virus concentration (n 3, mean S.E.).

tiple other lineages. Adipocytes derived from hMSCs have gene

expression profiles similar to that of primary adipocytes (52),
rendering them a good model for studying the effects of myostatin on adipogenic differentiation. In this study, we provide
the first evidence that recombinant human myostatin protein
inhibits adipogenic differentiation of hMSCs and human adipose tissue-derived preadipocytes. The inhibitory effects of
myostatin require the participation of the Alk5 receptor and
Smad3. Our data show that Smad3 interacts with -catenin to
form a complex that includes TCF4. Thus, the signal from
Smad3 is cross-communicated to the Wnt/-catenin pathway,
resulting in activation of the Wnt/-catenin pathway and inhibition of C/EBP and PPAR, the two principal regulators of
terminal adipogenesis. -Catenin plays an obligatory role in the
cross-communication between Smad3 signaling and Wnt/
TCF4 signaling pathways and in mediating the inhibitory
effects of myostatin on adipogenesis.
Myostatin did not affect the early transient induction of
C/EBP, which has been shown to facilitate mitotic clonal
expansion and to transactivate C/EBP and PPAR in murine
preadipocytes. In hMSC, we did not observe clonal expansion.
However, myostatin markedly down-regulated the expression
of PPAR and C/EBP, which was not reversed by ectopic
APRIL 4, 2008 VOLUME 283 NUMBER 14

C/EBP. In contrast, ectopic PPAR completely blocked the

inhibitory effects of myostatin.
Several lines of evidence demonstrate that activation and
cross-communication of Smad signal to Wnt/-catenin pathway are essential for mediating the effects of myostatin on adipogenic differentiation. First, the inhibitory effect of myostatin
was blocked by an Alk5/Smad3 inhibitor and by RNAi of
Smad3. Second, myostatin promotes -catenin association
with Smad3, its nuclear translocation, and its association with
TCF4, a hallmark of Wnt pathway activation. Finally, myostatin-mediated suppression of adipogenesis was completely
blocked by RNAi silencing of -catenin and also greatly attenuated by dn-TCF4, a binding partner of -catenin.
The mechanisms by which TCF4 and -catenin interfere
with adipogenesis remain unclear. Both have been shown to
associate with PPAR in different cell systems (23). Because
PPAR is auto-regulated and also cross-regulates C/EBP,
inactivation of this transcription factor down-regulates its own
expression and suppresses essentially all other adipogenic
genes. Other Wnt/TCF4 pathway proteins, such as c-Myc and
cyclin D1, have also been shown to bind and inactivate PPAR
(23, 53). Although myostatin did not alter the steady-state
mRNA levels of these genes (not shown), it is not known
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Ad5-dn-TCF4

mRNA expression
(% of Ad-5 in DM)

B
DM

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

AcknowledgmentsWe thank Dr. Steven Farmer for critical reading

of this manuscript and for the generous gift of the retroviral vector of
C/EBP mutant, and Amgen Inc. and Wyeth Inc. for providing recombinant human myostatin peptide and antibody, respectively. We
thank the Adipocyte Core of the Boston Obesity Nutrition Research
Center (DK42600) for providing human preadipocytes.
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fat mass.

Mechanism of Myostatin Action on Adipogenic Differentiation of hMSCs

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