perspective
Signalling pathways that mediate skeletal
muscle hypertrophy and atrophy
David J. Glass
Atrophy of skeletal muscle is a serious consequence of numerous diseases, including cancer and
AIDS. Successful treatments for skeletal muscle atrophy could either block protein degradation
pathways activated during atrophy or stimulate protein synthesis pathways induced during skeletal
muscle hypertrophy. This perspective will focus on the signalling pathways that control skeletal
muscle atrophy and hypertrophy, including the recently identified ubiquitin ligases muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), as a basis to develop targets for pharmacologic
intervention in muscle disease.
T
here are compelling reasons to develop new medicines that can maintain
skeletal muscle mass. Profound atrophy is often a consequence of diseases such
as cancer and AIDS1. Muscle immobilization, as commonly seen when a limb is put
in a cast after an orthopedic injury, causes
rapid muscle loss which may require
months of physical therapy. The effectiveness of glucocorticoid drugs, such as dexamethasone, are limited by muscle wasting,
seen as a side-effect of these agents. Even
during normal ageing there is a gradual loss
of muscle mass and a diminished capacity
to reverse that loss, which results in weakness and morbidity2,3. Currently, there are
no safe and effective medicines available to
treat muscle atrophy.
The rate of protein breakdown increases
during atrophy and has been correlated
with the activation of cellular proteases,
most notably the ATP-dependent ubiquitin
proteasome system1,4. One study which
suggested that activation of ubiquitin-proteasome pathways caused atrophy made use
of inhibitors of the proteasome, which were
shown to reduce the rate of proteolysis in
atrophying skeletal muscles5. However, it
was not known whether there are mediators
of protein breakdown that are unique to
skeletal muscle or whether more general
mechanisms are employed.
Whereas atrophy has been associated
with increases in protein breakdown, an
increase in protein synthesis has been
shown to occur during hypertrophy6. This
protein synthesis results in the addition of
contractile filaments comprising the myofibre, allowing for greater force to be produced by the muscle.
Advances in the understanding of signalling pathways that control skeletal muscle mass will be discussed in an effort to
highlight how progress in basic science
might aid in the identification of novel targets for anti-atrophy drug discovery.
Reciprocally, it will be demonstrated that
the particular requirements making a protein useful as a target for clinical intervention spurs research into fundamental problems in cell biology.
during hypertrophy and is sufficient to
induce hypertrophy12,13. Both the calcineurin pathway and the PI(3)K/Akt pathway will be discussed further below.
IGF-1
An increase in muscle work stimulates the
expression of a protein growth factor
known as insulin-like growth factor 1 (IGF1)7. IGF-1 has been shown to be sufficient
to induce hypertrophy through either
autocrine or paracrine mechanisms7. IGF-1
expression is increased during compensatory hypertrophy7, caused experimentally
by removing several muscles to force those
remaining to take up the resultant increase
in load. Furthermore, transgenic mice engineered to over-express IGF-1 using a muscle-specific promoter have skeletal muscles
that are twofold greater in mass than those
seen in normal mice3,8.
So, could IGF-1 be developed as a medicine to induce muscle hypertrophy? There
are several problems with this approach.
The receptor for IGF-1 is ubiquitously
expressed, so there is the danger that systemic treatment would affect other tissues.
In addition, IGF-1 stimulates proliferation
and survival in mitosis-competent cells.
Therefore, unless genetic therapy using IGF1 could be developed, selective activation of
the IGF-1 pathway in skeletal muscle may
require the determination of downstream
mediators of IGF-1-induced hypertrophy.
The downstream signalling mechanisms
induced by IGF-1 that are required for
hypertrophy have been a matter of some
controversy. It was initially reported that
the serine/threonine protein phosphatase
calcineurin was critical for IGF-1-mediated
hypertrophy9,10. Calcineurin was also
reported to be required for load-induced
hypertrophy11. Other work has focused on
the phosphatidylinositol-3-OH kinase
PI(3)K/Akt (PKB) pathway downstream of
IGF-1, demonstrating that Akt is activated
Calcineurin and the NFATs
Calcineurin is activated by calcium/calmodulin
and mediates the dephosphorylation of the
NFAT (nuclear factor of activated T cells)
transcription factors14. There are four members of the NFAT family, NFATc1–c4
(ref. 14). After dephosphorylation, NFATs
are translocated to the nucleus, resulting in
gene transcription. Therefore, dephosphorylation of NFAT and its subsequent
translocation have been used to gauge calcineurin activity.
There is good reason to consider calcineurin as a potential mediator of skeletal
muscle hypertrophy; transgenic animals
that express a constitutively active mutant
of calcineurin or NFATc3 in cardiac
myocytes undergo cardiac hypertrophy15.
However, in skeletal muscle the case for calcineurin as a mediator of hypertrophy is
less certain. Whereas some groups have
reported that cyclosporin A (CsA), a smallmolecule inhibitor of calcineurin, inhibits
compensatory hypertrophy11, others have
seen no effect3,16,17. These discrepancies
could be caused by differences in experimental design; however, in vitro inhibition
of calcineurin-mediated nuclear translocation of NFAT using CsA did not result in an
inhibition of IGF-1-mediated hypertrophy
of myotubes13, and several groups were able
to deliver sufficient levels of CsA in vivo so
as to inhibit other markers of calcineurin
activity in skeletal muscle, and yet inhibition of hypertrophy was not detected3,16,17.
The role of calcineurin in skeletal muscle hypertrophy was also assessed by genetic methods. Transgenic mice that expressed
a constitutively active form of calcineurin
in skeletal muscle did not undergo hypertrophy18,19. Calcineurin activity in the mus-
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© 2003 Nature Publishing Group
87
perspective
PTEN
SHIP2
IGF-1
Ptd Ins(3,4,5)P3
P2
Ptd Ins(4,5)
P
P
IRS1
IRS1
P
P
P
P
PDK
P
P
PI(3)K
Ptd Ins(
P
P
3,4)P
2
P
P
AKT1
P
Tsc1/2
P
GSK3β
mTOR
Rapamycin
eIF2B
p70S6K
PHAS-1
Figure 1 Signalling pathways downstream of IGF-1. This schematic representation emphasizes the primary
role of the PI(3)K/Akt cascade. Proteins that have been shown to have a negative effect on hypertrophy are
shown in red. Proteins that have a positive hypertrophic effect are shown in green. Proteins that have not been
assayed for their role in hypertrophy are shown in blue.
cles obtained from these transgenic animals
increased significantly when compared
with normal levels, indicating that activation of this enzyme is not sufficient to
induce hypertrophy19. It is perhaps hardest
to reconcile the finding that calcineurin
activity does not increase during hypertrophy, as might be expected if it were playing
a causative role12. Although the sum of the
present data does not rule out a role for calcineurin in the hypertrophic response,
more work is required to label calcineurin a
promising target in this field.
The IGF-1/PI(3)K Pathway
Binding of the growth factor IGF-1 induces
a conformational change in the IGF-1
receptor tyrosine kinase, resulting in its
trans-phosphorylation and the subsequent
phosphorylation of insulin receptor substrate 1 (IRS-1). In turn, this results in the
activation of PI(3)K (Fig. 1). In mammalian
muscle, PI(3)K was implicated as a potential
mediator of skeletal muscle hypertrophy in
experiments where a mutant form of the
Ras proto-oncogene that was only competent to activate the PI(3)K pathway was
used20; expression of this mutant caused
skeletal muscle hypertrophy when directly
introduced into muscle and blocked denervation-induced atrophy20, providing early
evidence that activation of the PI(3)K pathway was sufficient to mediate the hypertrophic effect of IGF-1 on skeletal muscle.
The PI(3)K/Akt pathway
Activation of PI(3)K results in the production
of phosphatidylinositol-3,4,5-trisphosphate,
88
which provides a membrane-binding site
for the serine/threonine kinase Akt21
(Fig. 1). Akt is phosphorylated after translocation to the membrane by the kinase Pdk1 (3′-phoshphoinositide-dependent protein
kinase 1)21. Once activated, Akt phosphorylates an array of substrates, including proteins that mediate protein synthesis, gene
transcription, cell proliferation and survival21. In mammals, there are three forms of
Akt, Akt1–3, encoded by distinct genes21.
Expression of a constitutively active form of
Akt1 in skeletal muscle cells, either in vitro22
or in vivo12,23, causes hypertrophy. The
demonstration that Akt1 is activated downstream of PI(3)K stimulation and that Akt1
can recapitulate the hypertrophic effects
observed with PI(3)K suggests that these
two molecules are components of a linear
pathway.
Mice that lack Akt1 are smaller than control littermates, supporting a role for Akt1 in
cell growth24. Interestingly, deletion of Akt2
results in a distinct phenotype. Akt2−/− animals are resistant to insulin and undergo a
diabetes-like syndrome, supporting other
experiments that implicate the PI(3)K/Akt
pathway in glucose transport25. The distinction between Akt1 and Akt2 function highlights the possibility of selectively activating
Akt1 and therefore stimulating protein synthesis without interfering with glucose
homeostasis.
Lipid phosphatases that inhibit Akt
The demonstration that activation of Akt1 is
sufficient to induce hypertrophy mandates
consideration of Akt1 as a target for drug
discovery. As a practical matter, however, it is
difficult to produce small-molecule activators of cytoplasmic enzymes; therefore, in
cases where activation of an enzyme is
desired, a more fruitful approach may be
to find those proteins that down-regulate
the enzyme of interest and target those
inhibitory proteins. In the case of Akt,
activity can be controlled by regulating the
levels of its lipid-binding site, phosphatidylinositol-3,4,5-trisphosphate,
which is dephosphorylated by two different types of phosphatases, PTEN and
SHIP (for SH2-domain-containing inositol 5′-phosphatase; there are two mammalian SHIPs, SHIP1 and SHIP2).
Overexpression of PTEN results in the
inactivation of Akt1 (ref. 26) and a subsequent decrease in cell size27,28.
Overexpression of SHIP2 has been shown
to inhibit hypertrophy in muscle12,13. A
dominant-negative mutant of SHIP2 that
activates Akt1 by inhibiting the function of
endogenous SHIP2 (ref. 29) causes hypertrophy in skeletal myotubes13.
Could PTEN or SHIP2 be useful targets
for drug discovery in the search for activators of skeletal muscle hypertrophy?
Unfortunately, genetic loss of PTEN results
in an increased propensity for cancer
through activation of the Akt pathway in
cells capable of proliferation30. A knockout
mouse of SHIP2 has been generated, but
SHIP2−/− animals die soon after birth as a
result of hypoglycemia31. It is not clear,
however, whether the SHIP2−/− mice are
genetically null for SHIP2. Of concern is the
fact that the first 18 exons were left intact in
these animals. In addition, a phenotype was
observed in the SHIP2+/− animals consistent
with the possibility that some portion of
the protein is being made and is functioning
as a dominant-negative molecule; however,
data suggested that the SHIP2 mRNA and
protein are absent in the knockouts31. Taken
at face value, this experiment indicates that
SHIP2 might be an attractive target for
insulin sensitization in diabetics; however
its use as a target for hypertrophy awaits
experiments that can discriminate between
effects on glucose transport through Akt2
and potential stimulation of protein synthesis through activation of Akt1.
The PI(3)K/Akt/Tsc/mTOR pathway
Indications that there was a particular pathway downstream of Akt relevant to the control of cell size came from experiments in
Drosophila melanogaster, where loss or inhibition of either PI(3)K32, mTOR33 or p70 S6
kinase34 (p70S6K) resulted in decreases in
cell size. These signalling molecules comprise members of a pathway from IRS-1 to
p70S6K (Fig. 1).
In mammalian cells, the precise pathway
connecting PI(3)K to the activation of
p70S6K is a matter of some dispute. The
Akt1 activator PDK1 can phosphorylate
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© 2003 Nature Publishing Group
perspective
p70S6K directly35, implying that Akt may be
dispensable for signalling to p70S6K.
However, there are also data that point to
Akt and the mammalian target of rapamycin
(mTOR; also known as FRAP or RAFT-1) as
necessary intermediates between PI(3)K and
p70S6K (Fig. 1). mTOR was so-named
because it is inhibited by a chemical called
rapamycin; rapamycin initially binds a protein called FK506-binding protein
(FKBP12), and the complex of rapamycin
and FKBP inhibits mTOR function23. Akt
phosphorylates36 and activates mTOR37, and
phosphorylation of both Akt12,23 and mTOR
are increased during muscle hypertrophy38.
In mouse myotubes, rapamycin blocks
both Akt- and IGF-1-mediated activation
of p70S6K (refs 13, 23), and this blockade
decreases both hypertrophy13 and muscle
growth23. Inhibition of mTOR activity with
rapamycin blocks compensatory hypertrophy in vivo12. In that same system,
rapamycin blocks activation of p70S6K, but
not Akt. These data are consistent with the
concept that Akt and mTOR are requisite
members of a linear signalling pathway
upstream of p70S6K (Fig. 1). However, data
obtained with rapamycin may not be dispositive, as there may be concerns about the
specificity of any chemical agent. Recently,
genetic support for a linear pathway came
from reports demonstrating that the tuberous sclerosis complex 1 (Tsc1) and Tsc2
proteins can inhibit mTOR-mediated signalling. Akt directly phosphorylates Tsc2,
activating mTOR in part by inactivating
Tsc2 (ref. 39). Presumably, if PDK could
circumvent Akt and mTOR, Tsc2-mediated
inactivation of mTOR would not be sufficient to block activation of p70S6K downstream of insulin- or serum-mediated stimulation. However, p70S6K and PHAS-1
phosphorylation are inhibited by Tsc2 in
insulin- or serum-treated cells39,40. A definitive answer as to whether PDK1 might be
sufficient to activate p70S6K in mammalian
systems awaits the generation of appropriate genetic mutants. It is also possible that
there may be differences in signalling that
are dependent on cell context.
Activation of mTOR results in an
increase in protein translation by two
mechanisms: first, mTOR activates p70S6K, a
positive regulator of protein translation41;
second, mTOR inhibits the activity of
PHAS-1 (also known as 4E-BP1), a negative
regulator of the protein initiation factor
eIF-4E42. Signalling from Drosophila TOR
to PHAS-1 was also confirmed genetically
in Drosophila42. Thus, a search for targets in
this pathway should focus on inhibitors of
p70S6K, or whether it is possible to directly
block the activity of PHAS-1.
β pathway
The PI(3)K/Akt/GSK3β
Glycogen synthase kinase 3β (GSK3β) is
another substrate of Akt that has been
shown
to
mediate
hypertrophy.
Phosphorylation of Akt results in the inactivation of GSK3β44. Therefore, expression
of a dominant-negative form of GSK3β is a
way to mimic Akt activity. This dominantnegative form of GSK3β has been shown to
induce hypertrophy in skeletal myotubes13.
GSK3β might be an attractive target for
drug discovery because inhibition of this
kinase produces the desired effect.
However, there is the concern that inhibition of GSK3β would have pleiotropic
effects, as it has multiple substrates and is
expressed in multiple tissues. However, it
will still be interesting to perform the
experiment.
β2 adrenergic receptor
It is difficult to point to modulators of
skeletal muscle hypertrophy downstream of
the PI(3)K/Akt pathway whose expression
is specific to muscle; those rare candidates
may be difficult to target pharmacologically. Therefore, one may need to look for
receptors other than the ubiquitously
expressed IGF-1 receptor that signal
through the PI(3)K/Akt pathway and have a
more restricted expression pattern. For
example, agonists of the β2-adrenergic
receptor, such as clenbuterol, have been
shown to induce skeletal muscle hypertrophy and block atrophy45. Most of the
β adrenergic receptor signalling studies
have focused on activation of cAMP pathways mediated by the Gαs protein.
However, it was reported that the Gβ/γ proteins activate the PI(3)K/Akt pathway46. It is
therefore of interest to know whether an
agent such as rapamycin would inhibit the
hypertrophic effects of clenbuterol.
Clenbuterol and related compounds
could be used to block skeletal muscle atrophy if not for their effects on the circulatory system; stimulation of the β2 adrenergic
receptor induces changes in blood pressure
and heart rate, and so far these effects have
been inseparable from their hypertrophic
effects on skeletal muscle.
Ubiquitin ligases
Perhaps the most direct strategy for identifying useful anti-atrophy targets is to determine the signalling pathways that are
required for atrophy. The involvement of
the ubiquitin-proteasome pathway in skeletal muscle atrophy has been previously
established. For example, inhibitors of the
proteasome block increases in protein
breakdown normally seen in atrophy5, the
level of ubiquitinated conjugates increase
during atrophy47 and genes that encode
various components of the ubiquitin pathway increase during atrophy48,49. Although
there was little basis to expect that musclespecific mediators of atrophy would be
found, especially given the ubiquitous
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expression of the cytoplasmic proteins that
induce skeletal muscle hypertrophy, a
search for markers of the atrophy process
identified two genes that are up-regulated
in multiple models of skeletal muscle atrophy and are expressed specifically in skeletal
and cardiac muscle. The two genes are
called MuRF1 (ref. 50; for muscle ring finger protein 1) and MAFbx50 (for muscle
atrophy F-box protein, also called Atrogin1 (ref. 49). Both genes encode ubiquitin ligases, proteins that bind and mediate ubiquitination of specific substrates.
MuRF1−/− and MAFbx−/— mice have been
generated50. Under normal conditions, these
animals appear to be phenotypically identical to wild-type littermates. After muscle
denervation, however, significantly less
muscle mass is lost in both MuRF1−/− and
MAFbx−/− animals, by comparison to denervated control animals50. MuRF1 has been
shown to bind the giant sarcomeric protein
titin51 and overexpression of MuRF1 disrupts the portion of titin that was shown to
bind MuRF1, suggesting that MuRF1 regulates the stability of this large structural protein52. If MuRF1 ubiquitinates structural
components of the sarcomere, this would
provide a clear mechanism for how MuRF1
might mediate skeletal muscle atrophy.
The genetic demonstration that MuRF1
and MAFbx are required for skeletal muscle atrophy identifies them as potential targets for drug discovery. Unlike some of the
other signalling components, MuRF1 and
MAFbx fit the criteria for small-molecule
drug targets for several reasons. First, their
enzymatic activity seems to be required for
muscle atrophy; second: they are expressed
specifically by muscle cells; third, they do
not seem to be required for normal muscle
growth or function. Ongoing work is
aimed at determining whether the muscle
that is preserved in the knockout animals
maintains a normal functional capacity.
Conclusion
Significant progress has been made in the
understanding of signalling pathways that
induce both skeletal muscle atrophy and
hypertrophy, pointing to potential targets
for pharmaceutical intervention. Activation
of the PI(3)K/Akt pathway has been shown
to be necessary and sufficient to induce
hypertrophy and to block skeletal muscle
atrophy. Akt mediates its hypertrophic
effects at least in part by stimulating protein
synthesis pathways downstream of mTOR
and GSK3β. In addition, the muscle-specific ubiquitin ligases MuRF1 and MAFbx
have been found to be essential for muscle
atrophy. Thus, cytoplasmic proteins in both
the Akt and the ubiquitin pathways may
potentially serve as clinical targets. Also, the
potent hypertrophic effects of both IGF-1
and clenbuterol make the search for useful
89
perspective
muscle-restricted receptors attractive.
The demonstration that the muscle-specific genes MAFbx and MuRF1 are upregulated in multiple models of skeletal muscle
atrophy helps to suggest that atrophy is not
simply the converse of hypertrophy.
Although there are apparently muscle-specific atrophy pathways, during hypertrophy
more general protein synthesis pathways
are strongly upregulated. It is of interest to
determine whether atrophy and hypertrophy pathways communicate with each
other. If so, and if one pathway can dominantly suppress the other, then this would
help in the search for the primary control
mechanism of the atrophy process. It is critical to continue studying the cellular pathways mediating skeletal muscle hypertrophy and atrophy to discover promising targets for the development of effective new
treatments for skeletal muscle disease.
David Glass is in Regeneron Pharmaceuticals, 777
Old Saw Mill River Road, Tarrytown NY, 105916707, USA
e-mail: david.glass@regeneron.com
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ACKNOWLEDGEMENTS
Thanks to L. S. Schleifer, P. R. Vagelos and G. D. Yancopoulos for
enthusiastic support and critical guidance, and to the Regeneron
community for their support. Thanks to colleagues at Procter &
Gamble Pharmaceuticals for their support and for enthusiastic scientific collaboration, particularly R. Isfort. Particular thanks to
G. Thurston for editing this manuscript. T. Stitt, M. Gonzalez,
E. Latres, S. Stevens, M. Sleeman and K-L Lai also provided useful
edits. Sincere apologies to scientific colleagues whose work was
omitted from this perspective as a result of space constraints.
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