IKK/NF- B regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis

Abstract

Nuclear factor kappaB (NF-kappaB) is involved in multiple skeletal muscle disorders, but how it functions in differentiation remains elusive given that both anti- and promyogenic activities have been described. In this study, we resolve this by showing that myogenesis is controlled by opposing NF-kappaB signaling pathways. We find that myogenesis is enhanced in MyoD-expressing fibroblasts deficient in classical pathway components RelA/p65, inhibitor of kappaB kinase beta (IKKbeta), or IKKgamma. Similar increases occur in myoblasts lacking RelA/p65 or IKKbeta, and muscles from RelA/p65 or IKKbeta mutant mice also contain higher fiber numbers. Moreover, we show that during differentiation, classical NF-kappaB signaling decreases, whereas the induction of alternative members IKKalpha, RelB, and p52 occurs late in myogenesis. Myotube formation does not require alternative signaling, but it is important for myotube maintenance in response to metabolic stress. Furthermore, overexpression or knockdown of IKKalpha regulates mitochondrial content and function, suggesting that alternative signaling stimulates mitochondrial biogenesis. Together, these data reveal a unique IKK/NF-kappaB signaling switch that functions to both inhibit differentiation and promote myotube homeostasis.

Full text

THE JOURNAL OF CELL BIOLOGY
JCB: ARTICLE
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 180, No. 4, February 25, 2008 787–802
http://www.jcb.org/cgi/doi/ JCB 787
10.1083/jcb.200707179
Correspondence to Denis C. Guttridge: denis.guttridge@osumc.edu
Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; DM, dif-
ferentiation medium; GM, growth medium; IKK, inhibitor of  B kinase; MEF,
mouse embryonic fi broblast; MSCV, murine stem cell virus; MyHC, myosin
heavy chain; NF-  B, nuclear factor  B; NIK, NF-  B – inducing kinase; Rb, retino-
blastoma; SR, superrepressor; TA, transactivation domain; Tn, troponin.
The online version of this article contains supplemental material.
Introduction
Nuclear factor  B (NF-  B) is a ubiquitously expressed tran-
scription factor and, in mammals, consists of  ve family mem-
bers: RelA/p65, c-Rel, RelB, p50 (the processed form of p105),
and p52 (the processed form of p100; Hayden and Ghosh,
2004 ). These subunits contain a DNA-binding protein dimer-
ization domain and nuclear localization signal, but only RelA/
p65 (hereafter referred to as p65), c-Rel, and RelB possess
transactivation domains (TAs). NF-  B forms homo- and het-
erodimers, with the p50 – p65 complex being the most common.
In most cells, NF-  B is bound to I  B inhibitor proteins that mask
its nuclear signal and sequester it in the cytoplasm ( Huxford
et al., 1998 ).
NF-  B is regulated by a variety of factors, such as in am-
matory cytokines that direct NF-  B by what is now referred to
as the classical pathway ( Ghosh and Karin, 2002 ). This occurs
through stimulation of the inhibitor of  B kinase (IKK) com-
plex consisting of two catalytic subunits, IKK  and IKK  , and
a regulatory subunit, IKK  /NF-  B essential modulator/IKKAP1
( Karin, 1999 ). Once activated, IKK  phosphorylates I  B pro-
teins, targeting them for ubiquitination and proteasomal degra-
dation. This releases p50 – p65 or p50 – c-Rel dimers to translocate
to the nucleus and bind DNA, where they induce gene expres-
sion. Mice null for IKK  , IKK  , or p65 are embryonic lethal as
a result of massive liver apoptosis, and cells derived from these
embryos are unresponsive to classical NF-  B inducers ( Beg et al.,
1995b ; Tanaka et al., 1999; Rudolph et al., 2000 ), demonstrat-
ing a signaling link between p65, IKK  , and IKK  subunits.
In response to a second set of factors that include CD40L,
B cell – activating factor, and lymphotoxin  , NF-  B is activated
through an alternative pathway independent of IKK  ( Pomerantz
and Baltimore, 2002 ). Instead, activation proceeds through the
Nuclear factor  B (NF-  B) is involved in multiple
skeletal muscle disorders, but how it functions in
differentiation remains elusive given that both
anti- and promyogenic activities have been described.
In this study, we resolve this by showing that myogenesis
is controlled by opposing NF-  B signaling pathways.
We fi nd that myogenesis is enhanced in MyoD-expressing
fi broblasts defi cient in classical pathway components
RelA/p65, inhibitor of  B kinase  (IKK  ), or IKK  .
Similar increases occur in myoblasts lacking RelA/p65
or IKK  , and muscles from RelA/p65 or IKK  mutant
mice also contain higher fi ber numbers. Moreover, we
show that during differentiation, classical NF-  B signal-
ing decreases, whereas the induction of alternative
members IKK  , RelB, and p52 occurs late in myogenesis.
Myotube formation does not require alternative signal-
ing, but it is important for myotube maintenance in re-
sponse to metabolic stress. Furthermore, overexpression
or knockdown of IKK  regulates mitochondrial content
and function, suggesting that alternative signaling stim-
ulates mitochondrial biogenesis. Together, these data
reveal a unique IKK/NF-  B signaling switch that func-
tions to both inhibit differentiation and promote myo-
tube homeostasis.
IKK/NF-  B regulates skeletal myogenesis via
a signaling switch to inhibit differentiation and
promote mitochondrial biogenesis
Nadine Bakkar ,
1,2 Jingxin Wang ,
1 Katherine J. Ladner ,
1 Huating Wang ,
1 Jason M. Dahlman ,
1 Micheal Carathers ,
1
Swarnali Acharyya ,
1 Michael A. Rudnicki ,
4 Andrew D. Hollenbach ,
5 and Denis C. Guttridge
1,2,3
1 Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics,
2 Molecular, Cellular, and Developmental Biology Graduate
Program, and
3 Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210
4 Molecular Medicine Program, Ottawa Health Research Institute, Ottawa K1Y 4E9, Ontario, Canada
5 Department of Genetics, Louisiana State University, New Orleans, LA 70112

JCB • VOLUME 180 • NUMBER 4 • 2008 788
was also determined that p38 MAPK-induced myogenesis func-
tions through IL-6 synthesis in an NF-  B – dependent manner
( Baeza-Raja and Munoz-Canoves, 2004 ).
Collectively, these studies show that NF-  B function in
skeletal muscle differentiation remains at best enigmatic. Resolving
this will not only provide insight into the involvement of NF-  B
during muscle development and repair but may also increase our
understanding of its participation in a growing list of muscle-
wasting disorders, including cachexia ( Guttridge et al., 2000 ;
Cai et al., 2004 ; Mourkioti et al., 2006 ), disuse atrophy ( Hunter
and Kandarian, 2004 ), muscular dystrophies ( Baghdiguian et al.,
1999 ; Kumar and Boriek, 2003; Acharyya et al., 2007 ), and in-
 ammatory myopathies ( Monici et al., 2003 ). To this end, we
used a genetic approach to decipher the role of NF-  B/IKK sub-
units during myogenic differentiation. Our results provide an
explanation for the previously reported anti- and promyogenic
activities of NF-  B by revealing that myogenesis involves both
classical and alternative NF-  B pathways. Although constitutive
activation of the classical pathway functions in myoblasts to in-
hibit differentiation, NF-  B signaling switches to the alternative
pathway late in the myogenic program to promote mitochondrial
biogenesis and myotube homeostasis.
Results
Myogenic activity is enhanced in p65
ⴚ / ⴚ
MEFs expressing MyoD
To extend our understanding of NF-  B in skeletal myogenesis,
we used established mouse embryonic  broblasts (MEFs) that
were wild type or null for individual NF-  B subunits converted
to skeletal muscle by exogenous expression of MyoD ( Davis
et al., 1987 ). We initiated this analysis with p65 because this sub-
unit is constitutively active in myoblast nuclei ( Guttridge et al.,
1999 ). Results showed that myogenic activity derived from a
troponin (Tn) I enhancer reporter plasmid (TnI-luc) was signi -
cantly enhanced in p65
⫺ / ⫺
MEFs compared with wild-type cells
( Fig. 1 A ). Similar  ndings were obtained with plasmids con-
taining the acetylcholine receptor promoter (AchR-luc) or mul-
timerized muscle regulatory factor binding sites (4RTK-luc),
arguing that this effect was not reporter speci c. To verify the
speci city of p65 regulation, reporter assays were repeated in
early passaged primary  broblasts prepared from embryonic
day (E) 13.5 p65
+/+ , p65
+/ ⫺
, and p65
⫺ / ⫺
embryos. Myogenic ac-
tivity was again elevated in MEFs lacking p65, which occurred
in a gene dosage – dependent manner ( Fig. 1 B ). This con rmed
that p65 regulation of myogenesis was not a consequence of cell
immortalization. As a control, myogenesis was also assessed in
primary MEFs that were wild type or null for the retinoblas-
toma (Rb) protein, a cell cycle checkpoint required for skeletal
muscle differentiation ( Novitch et al., 1999 ). As predicted, reporter
activity was signi cantly impaired in Rb
⫺ / ⫺
MEFs ( Fig. 1 B ),
thus supporting the relevance of our  ndings with p65. To fur-
ther address p65 speci city, myogenic assays were extended
to MEFs lacking other NF-  B subunits. Results showed that
activity from cRel
⫺ / ⫺
or p50
⫺ / ⫺
MEFs was considerably lower
than that for p65
⫺ / ⫺
cells ( Fig. 1 C ). In addition, we used MEFs
de cient in I  B  that contain constitutive levels of nuclear p65
NF-  B – inducing kinase (NIK) that phosphorylates and activates
IKK  homodimers and, in turn, phosphorylates p100 in com-
plex with RelB. This leads to ubiquitin-dependent processing
of p100 to p52 and translocation of p52 – RelB to the nucleus
( Senftleben et al., 2001 ; Xiao et al., 2001 ). B cell – activating
factor, NIK, and p100 – p52 knockout mice have similar pheno-
types ( Gerondakis et al., 1999 ), con rming that these molecules
are also part of the same linear nonclassical signaling cascade.
In addition, the classical and alternative pathways are thought to
regulate distinct genes in response to their various activators
( Dejardin et al., 2002; Bonizzi et al., 2004 ).
Aside from its more commonly accepted role as a regu-
lator of innate immunity and cell survival, NF-  B is also
prominent in regulating cellular differentiation. In hematopoi-
etic cells, c-Rel and RelB are essential for B cell lymphopoie-
sis and T cell maturation ( Weih et al., 1996; Gerondakis et al.,
1999 ). NF-  B is also required for osteoclastogenesis, and
mice lacking p50 and p52 display severe osteopetrosis ( Iotsova
et al., 1997 ). Furthermore, IKK  is important for skin differ-
entiation as well as skeletal and craniofacial morphogenesis
( Takeda et al., 1999; Hu et al., 2001 ; Sil et al., 2004 ), a func-
tion thought to be independent of its kinase activity.
Over the past years, an increasing number of studies have
also implicated NF-  B in skeletal muscle differentiation, a pro-
cess regulated by transcription factors MyoD, Myf5, myogenin,
MRF4/Myf6/herculin, and MEF2A-D ( Naya and Olson, 1999 ;
Sabourin and Rudnicki, 2000; Pownall et al., 2002 ). These fac-
tors regulate myoblasts to undergo growth arrest and fuse into
multinucleated myotubes. However, in contrast to hematopoiesis,
the function of NF-  B in myogenesis is less de ned, and results
have con icted as to whether NF-  B promotes or inhibits this
differentiation process. On the one hand, studies demonstrate that
NF-  B DNA binding and transcriptional activities decrease dur-
ing differentiation ( Lehtinen et al., 1996; Guttridge et al., 1999 )
and that inhibition of NF-  B via expression of the I  B  super-
repressor (SR) mutant accelerates myogenesis ( Guttridge et al.,
1999 ). In addition, activators of NF-  B such as TNF  , IL-1  , or
the receptor-interacting protein (RIP) homologue RIP2 act as po-
tent inhibitors of differentiation ( Guttridge et al., 2000 ; Langen
et al., 2001 ; Munz et al., 2002 ), which together support the notion
that NF-  B functions as an inhibitor of myogenesis. NF-  B me-
diates this regulation through the induction of cyclin D1 ( Guttridge
et al., 1999 ) or by suppressing MyoD synthesis through a de-
stabilization element in the MyoD transcript ( Guttridge et al., 2000 ;
Sitcheran et al., 2003 ). More recent data suggest that NF-  B can
also inhibit myogenesis by stimulating expression of the Poly-
comb group protein YY1 ( Wang et al., 2007 ).
In contrast, similarly performed studies have reported that
NF-  B activity increases during myogenesis in response to
insulin-like growth factor ( Kaliman et al., 1999; Conejo et al.,
2002 ). Insulin-like growth factor activation is mediated, in part,
through the classical pathway, causing I  B  degradation and
p65 nuclear translocation, although the alternative pathway also
appears to be involved because the overexpression of IKK  or
NIK was seen to enhance myogenesis ( Canicio et al., 2001 ).
In addition, the expression of I  B  -SR in L6 rat myoblasts was
found to inhibit terminal differentiation markers, and recently it

789 IKK/NF-  B SIGNALING SWITCH IN SKELETAL MYOGENESIS • BAKKAR ET AL.
p65 (1 – 551) or TA mutant (1 – 521) strongly repressed myogen-
esis, whereas the expression of mutant (1 – 313) was effective in
partially rescuing this regulation ( Fig. 2 D ). To verify these
results, MyoD was stably expressed in p65
⫺ / ⫺
MEFs along
with wild-type or mutant forms of p65 ( Fig. 2 E ). Consistent
with reporter assays, myotubes were completely absent in
p65
⫺ / ⫺
MEFs expressing wild-type p65, whereas some myotubes
formed in cells reconstituted with the (1 – 313) mutant ( Fig. 2 F ).
This suggested that residues within TA (521 – 551) are largely
dispensable for repressing myogenesis, whereas residues
(313 – 521) play a more signi cant role in this regulation.
However, because the (1 – 313) mutant only partially rescued
myogenesis, it further suggested that residues within the Rel
domain contributed to p65-suppressive activity. Because the
phosphorylation of serine 276 is required for p65 transactiva-
tion ( Zhong et al., 1998 ), we examined the involvement of this
residue in regulating myogenesis. Reconstitution of p65
⫺ / ⫺
cells with p65 containing a 276 serine to alanine mutation was
less effective in inhibiting myogenic activity, whereas muta-
tions outside the Rel domain in residues 529 and 536 had no
effect ( Fig. 2 G ). This is consistent with the aforementioned
data showing that the deletion of TA (521 – 551) is not required
for this regulation. In addition, the generation of serine to ala-
nine 276 in the p65 (1 – 313) mutant caused a full rescue of
myogenic activity ( Fig. 2 H ), demonstrating that NF-  B regu-
lation of myogenesis is dependent on p65 transcriptional activ-
ity mediated from both serine 276 and other residues lying
within the 313 – 521 domain.
( Beg et al., 1995a ). Electrophoretic mobility shift assay con-
 rmed that NF-  B binding was higher in I ␬ B ␣ ⫺ / ⫺
MEFs, which
correlated with lower myogenic activity ( Fig. 1 D ). Together,
these genetic data indicated that p65 functions as a negative
regulator of MyoD-induced myogenesis.
MEFs null for p65 are accelerated in their
myogenic program
To examine how the absence of p65 exerts its effects on the
myogenic program, MyoD was stably expressed in p65
+/+ and
p65
⫺ / ⫺
MEFs using a murine stem cell virus (MSCV) – MyoD-
IRES-GFP retrovirus. After selection, cells were sorted by  ow
cytometry for GFP to ensure equal levels of MyoD ( Fig. 2 A ).
Cells were then differentiated, and myogenic markers were ana-
lyzed over a 4-d period. This analysis revealed that both the
induction and overall expression of markers muscle creatine
kinase, Tn, myosin heavy chain (MyHC), and tropomyosin
were greater in cells lacking p65 ( Fig. 2 B ). Myotube formation
was also strikingly higher in p65
⫺ / ⫺
cells ( Fig. 2 C ), which
together supported the aforementioned reporter data that p65
functions as an inhibitor of myogenesis.
The transcriptional activity of p65 derives from three TAs
located in its C terminus ( Schmitz et al., 1994 ). To determine
whether the regulation of myogenesis was dependent on p65
transcriptional activity, reporter assays were repeated in p65
⫺ / ⫺
MEFs reconstituted with either p65 wild type (1 – 551 amino
acids) or mutants truncated in TA1 (1 – 521) or all three (1 – 313)
TA domains. Compared with vector, the addition of wild-type
Figure 1. Loss of p65 enhances myogenic activity in MEFs. (A) p65
+/+ and p65
⫺ / ⫺
MEFs were cotransfected with cytomegalovirus-MyoD and either of the
following reporter constructs: TnI-luc, AchR-luc, or 4RTK-luc. The next day, cells were switched to differentiation medium (DM), and, after 48 h, lysates were
prepared and assayed for luciferase activity. (B) p65
+/+ , p65
+/ ⫺
, p65
⫺ / ⫺
and pRb
+/+ , and pRb
⫺ / ⫺
primary MEFs were transfected with MyoD and TnI-luc.
Cells were differentiated as in A, and luciferase assays were performed. (C) p65
⫺ / ⫺
, cRel
⫺ / ⫺
, and p50
⫺ / ⫺
MEFs were transfected with MyoD and TnI-luc,
differentiated, and monitored for luciferase activity. (D) Myogenic assays similar to those described in A – C were performed in I ␬ B ␣
+/ ⫺
and I ␬ B ␣
⫺ / ⫺
cells.
(inset) Electrophoretic mobility shift assay analysis of I ␬ B ␣
+/ ⫺
and I ␬ B ␣
⫺ / ⫺
MEFs. Error bars represent SEM.

JCB • VOLUME 180 • NUMBER 4 • 2008 790
TNF  ( Doi et al., 1999 ). Thus, TNF ␣ ⫺ / ⫺
;p65 +/ ⫺
mice were
crossed, and primary myoblasts were prepared from 2 – 4-d-old
neonates ( Fig. 3 A ). Transfections with TnI or MyHCIIB report-
ers showed that myogenic activity was substantially elevated in
p65
⫺ / ⫺
myoblasts, and, as in primary MEFs, this regulation ap-
peared to be gene dosage dependent ( Fig. 3 B ). In comparison,
Myogenesis is accelerated in p65-defi cient
myoblasts
To determine the physiological relevance of our  ndings, myo-
genesis was further explored in p65
⫺ / ⫺
myoblasts. Although
mice lacking p65 are embryonically lethal ( Beg et al., 1995b ),
this phenotype can be rescued with the additional deletion of
Figure 2. Loss of p65 accelerates the myogenic program in MEFs. (A) p65
+/+ and p65
⫺ / ⫺
MEFs were infected with MSCV-MyoD and, after puromycin
selection, were sorted for GFP to ensure equal MyoD levels. Cells were then probed for p65 and MyoD (45 kD) protein.  -Tubulin (55 kD) was used as a
loading control. (B) p65
+/+ and p65
⫺ / ⫺
MEFs stably expressing MyoD were differentiated, and lysates were then probed for the indicated myogenic differ-
entiation markers. (C) Cells were differentiated as in B, and MyHC immunofl uorescence was performed. (D) p65
⫺ / ⫺
MEFs were transfected with TnI-luc and
either vector plasmid, wild-type p65 (1 – 551), or p65 TA mutants (1 – 521 and 1 – 313) along with MyoD. Cells were then differentiated, and lysates were
prepared for luciferase assays. RLU, relative light units. (E) p65
⫺ / ⫺
MEFs were reconstituted with either vector and full-length or truncated p65 along with
MSCV-MyoD. After selection, whole cell lysates were prepared and probed for p65, MyoD, and  -tubulin. (F) Cells were infected as in E, differentiated for
72 h, fi xed, and stained for MyHC. (G) p65
⫺ / ⫺
MEFs were transfected with MyoD, TnI-luc, and either vector control, wild-type p65 (WT), or p65 constructs
containing S/A mutation at positions 276, 529, and 536. MEFs were differentiated and harvested after 48 h for luciferase assays. (H) Relative luciferase
activities from p65
⫺ / ⫺
MEFs transfected with MyoD, TnI-luc, and either vector control, wild-type p65, or p65 (1 – 313) containing the S276A mutation. Error
bars represent SEM. Bars: (C) 200  m; (F) 80  m.

791 IKK/NF-  B SIGNALING SWITCH IN SKELETAL MYOGENESIS • BAKKAR ET AL.
 brillar genes are silent in myoblasts, this suggested that p65
functions as a transcriptional repressor of Tn, which is consis-
tent with our recent report that NF-  B is capable of silencing Tn
enhancers through the production of YY1 and recruitment of
Ezh2 and HDAC-1 ( Wang et al., 2007 ). Because TNF  has re-
cently been shown to be required for muscle regeneration ( Chen
et al., 2007 ), admittedly it was possible that the increase in
muscle differentiation in p65
⫺ / ⫺
myoblasts occurred secondary to
myogenic activity in p50
⫺ / ⫺
myoblasts was not signi cantly
different from wild-type cells. Furthermore, under differentia-
tion conditions, p65
⫺ / ⫺
myoblasts formed 58% more myotubes
( Fig. 3, C and D ) and expressed higher levels of myo brillar
proteins ( Fig. 3 E ) compared with p65
+/+ cells. It is also note-
worthy that the modest but reproducible expression of Tn was
detectable in p65
⫺ / ⫺
myoblasts even under growth conditions
(growth medium [GM]; Fig. 3 E , asterisk). Given that myo-
Figure 3. Loss of p65 enhances the differentiation of primary myoblasts. (A) Primary myoblasts were prepared from 2 – 4-d-old TNF ␣
⫺ / ⫺
;p65
+/+ ,
TNF ␣
⫺ / ⫺
;p65
+/ ⫺
, and TNF ␣
⫺ / ⫺
;p65
⫺ / ⫺
neonates, and genotypes were verifi ed by Western blots for p65. (B) p65 and p50 primary myoblasts were
transfected with TnI-luc or MyHC-luc plasmids, differentiated for 48 h, and subsequently harvested for luciferase assays. RLU, relative light units.
(C) TNF ␣
⫺ / ⫺
;p65
+/+ and TNF ␣
⫺ / ⫺
;p65
⫺ / ⫺
myoblasts were differentiated for 0 h (GM) or 48 h (DM) and subsequently stained for MyHC. (D) Quantifi cation
of myogenesis was performed by scoring MyHC-positive cells from a minimum of 25 fi elds normalized to total cell number as determined by Hoechst staining.
(E) Myoblasts were differentiated for 0 (GM) and 48 h (DM), and lysates were probed for MyHC and Tn. The asterisk indicates Tn expression under
GM conditions. (F) Primary or C2C12 myoblasts were transfected with siControl (siCont) or siRNA against p65 (sip65) along with Tn-luc reporter. Cells
were switched to DM, and luciferase assays were performed. (G) C2C12 myoblasts were transfected with siControl or siRNA against p65 and switched
to DM for 48 h, after which lysates were prepared and Western blots were performed. Error bars represent SEM. Bar, 80  m.

JCB • VOLUME 180 • NUMBER 4 • 2008 792
 bers that were noticeably smaller in size than their wild-type
counterpart ( Fig. 4 A ). In p65-de cient tibialis anterior muscles
from 4-wk-old male or female mice, mean  ber diameter was
reduced by 39% as compared with wild-type littermates (25.6  m
for wild type vs. 15.5  m for null; n = 5; Fig. 4 B ). This pheno-
type was common to multiple hind limb muscles and was se-
lective to p65 because no such differences were observed in
p50
⫺ / ⫺
mice ( Fig. 4 A ). Slow MyHC staining from gastroc-
nemius muscles con rmed that the absence of p65 did not af-
fect  ber type (Fig. S1 A, available at http://www.jcb.org/cgi/
content/full/jcb.200707179/DC1). Although muscle atrophy is
the loss of this cytokine. However, siRNA knockdown of p65 in
primary myoblasts and C2C12 cells again led to increases in
myogenic activity and muscle markers ( Fig. 3, F and G ), sup-
porting results that negative regulation of myogenesis is speci c
to p65 and is unlikely to be related to the absence of TNF  .
The absence of p65 enhances myogenesis
in vivo
Next, we explored muscles from TNF ␣ ⫺ / ⫺
;p65 ⫺ / ⫺
mice in an at-
tempt to correlate our in vitro  ndings with an in vivo phenotype.
To our surprise, p65-null muscles displayed a large number of
Figure 4. Myogenesis is enhanced in p65-defi cient mice. (A) Hematoxylin- and eosin-stained cryosections of tibialis anterior (TA), gastrocnemius (Gastroc),
and quadriceps (Quad) muscles from TNF ␣
⫺ / ⫺
;p65
+/+ and TNF ␣
⫺ / ⫺
;p65
⫺ / ⫺
mice or p50
+/+ and p50
⫺ / ⫺
gastrocnemius. (B) Fiber diameters were mea-
sured from gastrocnemius muscle sections from a total of 1,500 fi bers ( n = 5 mice per group). (C) Fiber numbers were determined in whole cross sections
from tibialis anterior muscles from TNF ␣
⫺ / ⫺
;p65
+/+ and TNF ␣
⫺ / ⫺
;p65
⫺ / ⫺
mice ( n = 3). (D) Fiber numbers were recorded from premeasured randomly
selected areas (minimum of 25 per animal) throughout the tibialis anterior, gastrocnemius, and quadriceps muscles ( n = 5 mice per genotype). Error bars
represent SEM. Bar, 50  m.

793 IKK/NF-  B SIGNALING SWITCH IN SKELETAL MYOGENESIS • BAKKAR ET AL.
7 and 9 neonates (Fig. S1, C and D). These  ndings are consis-
tent with cell culture data suggesting that p65 absence in vivo
leads to enhanced myogenesis, a phenotype highly reminiscent
of dystrophin-de cient or acutely injured muscles depleted of
p65 ( Acharyya et al., 2007 ).
p65 regulates myogenesis through multiple
mechanisms
Next, we sought to address the mechanism by which p65 nega-
tively regulates myogenesis. Previous use of the I  B  -SR in-
hibitor revealed that p65 can inhibit C2C12 differentiation
through the suppression of MyoD synthesis ( Guttridge et al.,
2000 ). Such analysis also revealed that NF-  B is capable of in-
hibiting myogenesis through cyclin D1 ( Guttridge et al., 1999 ),
an underlying cause of  ber reduction associated with induction
of the E3 ubiquitin ligases MuRF1 and atrogene-1/MAFbx,
muscles depleted for p65 displayed no evidence of this regula-
tion (Fig. S1 B). However, given our  ndings with primary myo-
blasts lacking p65, we considered the possibility that changes in
p65
⫺ / ⫺
muscles might result from an increase in overall myotube
number. Indeed,  ber counts from entire cross sections of tibi-
alis muscles revealed a 76% increase in p65
⫺ / ⫺
mice compared
with control littermates ( Fig. 4 C ). Similar results were found
when counts were extended to gastrocnemius and quadriceps,
demonstrating that this regulation is not muscle type speci c
( n = 5; Fig. 4 D ). In addition, the p65
⫺ / ⫺
phenotype was not re-
lated to a compromised immune function in adult mice because
increases in  ber number were also observed in postnatal day (P)
Figure 5. p65 regulation of myogenesis occurs through multiple mechanisms. (A) C2C12 myoblasts were transfected with control and p65 siRNA, and lysates
were harvested for Western analysis. (B) MyoD
⫺ / ⫺
myoblasts were transfected with TnI-luc along with either an empty vector (CMV-Vect) or a p65 expression
plasmid (CMV-p65) or were transfected with vector and subsequently treated with 5 ng/ml TNF  . Cells were differentiated for 2 d, and luciferase assays
were performed. RLU, relative light units. (inset) Western blot for MyoD wild-type and null myoblasts. (C) MyoD
⫺ / ⫺
myoblasts were infected with pBabe-Puro or
pBabe-p65 retroviruses. After selection and differentiation for 3 and 5 d, protein lysates were prepared for Western analysis. (D) MyoD
⫺ / ⫺
myoblasts stably
expressing pBabe-Puro and pBabe-p65 were differentiated, fi xed, and stained for MyHC (red) and nuclei (Hoechst; blue). (E) p65
+/+ and p65
⫺ / ⫺
MEFs were
transfected with TnI-luc and either MyoD or myogenin plasmids. As a control, transfections were also performed with a p53 expression plasmid and responsive
reporter (pGL13-luc). Cells were subsequently differentiated, and luciferase assays were performed. Error bars represent SEM. Bar, 80  m.

JCB • VOLUME 180 • NUMBER 4 • 2008 794
( Fig. 6 E ), a time when cell fusion and contractile myo brillar
expression is well underway. This activity was speci c to IKK
because phosphorylation was undetectable when assays were re-
peated with a mutated substrate, and the increase in activity was
not a consequence of altered protein expression because IKK
subunits remained unchanged during myogenesis ( Fig. 6 E ).
Next, we analyzed endogenous IKK substrates to ascer-
tain which IKK complex became activated during late stage
myogenesis. As part of the classical pathway, IKK  activation
results in the phosphorylation of I  B  and p65 ( Hayden and
Ghosh, 2004 ). On the other hand, IKK  predominantly phos-
phorylates p100, leading to its proteolysis and conversion to
p52. Results revealed that levels of phosphorylated I  B  and
p65 decreased during C2C12 differentiation, whereas total pro-
tein levels remained unchanged ( Fig. 6 F ). Consistent with this
decrease, nuclear p65 levels declined, and, by chromatin immuno-
precipitation (ChIP), p65 binding activity on the I  B  pro-
moter was also lost ( Fig. 6 F ). In comparison, the processing of
p100 to p52 was induced during myogenesis, with similar kinetics
to C2C12 as well as primary myoblast differentiation ( Fig. 6 G
and not depicted). Because IKK  activation results in the for-
mation of RelB – p52, we also investigated the contribution of
these NF-  B subunits by repeating myogenic assays in RelB
⫺ / ⫺
and p52
⫺ / ⫺
MEFs. Consistent with  ndings in IKK ␣ ⫺ / ⫺
MEFs,
myogenic activities were reduced in both p52
⫺ / ⫺
and RelB
⫺ / ⫺
 broblasts ( Fig. 6 H ). Together, these data suggest that skeletal
myogenesis is characterized by a temporal switch in NF-  B sig-
naling pathways whereby the reduction of classical NF-  B is
followed by activation of the alternative pathway relatively late
in the myogenic program.
IKK ␣ functions as a regulator of myotube
maintenance under metabolic stress
The aforementioned data suggested that components of the alter-
native pathway might function to promote myogenesis. How-
ever, stable expression of an HA-tagged version of IKK  in
C2C12 myoblasts did not affect induction of the early and late
myogenic markers myogenin and Tn, respectively ( Fig. 7 A ), nor
was myogenic activity affected when IKK  or a kinase-dead
version (K/M) of this kinase was overexpressed in MyoD-
expressing 10T1/2  broblasts (Fig. S3 A, available at http://www
.jcb.org/cgi/content/full/jcb.200707179/DC1). In comparison,
the expression of classical signaling components IKK  , IKK  ,
or p65 led to clear reductions in myogenic activity in these same
cells. Furthermore, no differences in skeletal muscle gene expres-
sion were detected by Affymetrix microarray between control
and HA-IKK  – expressing C2C12 myotubes (not depicted), and
siRNA-mediated depletion of IKK  from differentiating myo-
blasts also revealed little change in myogenic markers ( Fig. 7 B ).
Therefore, although results from IKK ␣ ⫺ / ⫺
, RelB
⫺ / ⫺
, and p52
⫺ / ⫺
MEFs suggested that the alternative pathway is promyogenic, over-
all, the data do not support that activation of this NF-  B signaling
pathway is necessary for myotube formation (see Discussion).
However, under long-term differentiation conditions (6 d)
without medium replenishment, we observed that myotubes ex-
pressing HA-IKK  were better maintained compared with con-
trol cells ( Fig. 7 C ). Speci cally, IKK  -expressing myotubes
limiting myoblasts from exiting the cell cycle or through YY1
to silence myo brillar promoters in myoblasts ( Wang et al.,
2007 ). Consistent with these  ndings, MyoD was elevated in
p65
⫺ / ⫺
myoblasts, whereas both YY1 and cyclin D1 levels de-
clined ( Fig. 5 A ). Thus, it is likely that p65 negatively regulates
myogenesis through multiple mechanisms.
To determine whether these mechanisms could function
independently of each other, we examined the regulation of
myogenesis by p65 in MyoD
⫺ / ⫺
myoblasts. Although myotube
formation is impaired in these cells ( Sabourin et al., 1999 ),
myogenic activity was nevertheless retained under differen-
tiation conditions ( Fig. 5 B ). However, the addition of p65 or
TNF  strongly repressed this activity. Likewise, retroviral ex-
pression of p65 in MyoD
⫺ / ⫺
myoblasts caused a noticeable
reduction of myogenic markers ( Fig. 5 , C and D), demonstrat-
ing that p65 can inhibit myogenesis independently of MyoD.
To substantiate this  nding, reporter assays were repeated in
p65
+/+ and p65
⫺ / ⫺
MEFs where MyoD was substituted with
myogenin. Like MyoD, myogenin is capable of converting  bro-
blasts to a muscle lineage, albeit with lower ef ciency ( Gerber
et al., 1997 ). Indeed, myogenin-induced myogenic activity was less
than that for MyoD, but these levels were nonetheless greater in
p65
⫺ / ⫺
MEFs compared with wild-type cells ( Fig. 5 E ). This
regulation also appeared speci c to these muscle regulatory fac-
tor proteins because a p53-responsive reporter was not affected
by the absence of p65.
Myogenesis is regulated by a temporal
switch in IKK signaling pathways
Having gained insight into the role of p65 in muscle differentia-
tion, we now turned our attention to its upstream regulator, the
IKK complex. Recently, our group elucidated that chronic acti-
vation of IKK  in mdx muscles inhibits muscle differentiation
( Acharyya et al., 2007 ). Interestingly, Mourkioti et al. (2006)
have also reported that skeletal muscle deletion of IKK  in-
creased intermediate  ber numbers in 4-mo-old mice, a pheno-
type that closely matched that of younger p65
⫺ / ⫺
mice ( Fig. 4 ).
Such results suggested that p65 and IKK  share overlapping
functions in skeletal muscle differentiation. Indeed, analogously
to p65, myogenic activity was increased in primary  broblasts
and myoblasts deleted for IKK   oxed (f/f) alleles using Cre
recombinase ( Fig. 6 A and Fig. S2, A and B; available at http://www
.jcb.org/cgi/content/full/jcb.200707179/DC1). Likewise, 4-wk-
old mice lacking skeletal muscle IKK  exhibited an increase in
total  ber number ( Fig. 6, B and C ), reaf rming that IKK  , like
p65, functions as a negative regulator of myogenesis. Because
IKK  and p65 are components of classical NF-  B signaling,
myogenesis was also tested in IKK ␥ ⫺ / ⫺
MEFs to address
whether this pathway acts as a general inhibitor of differentia-
tion. Consistent with this thinking, myogenic activity was in-
creased in IKK ␥ ⫺ / ⫺
MEFs but decreased in MEFs lacking
IKK  , the latter of which is not considered part of the classical
pathway ( Fig. 6 D ; Senftleben et al., 2001 ).
To further explore the myogenic functions of IKK, we
measured its activity in differentiating myoblasts. Results
showed that IKK activation was relatively low in undifferenti-
ated cells but became induced at 48 h into the myogenic program

795 IKK/NF-  B SIGNALING SWITCH IN SKELETAL MYOGENESIS • BAKKAR ET AL.
expressing myotubes were also more resistant to low glucose
but not to heat shock, oxidative stress, or DNA damage ( Fig. 7 C
and not depicted), suggesting a selective resistance to metabolic
were 48% less atrophic (26.0 ± 5.7  m in  ber diameter com-
pared with 13.5 ± 3.5  m in control cells) and overall exhibited
a healthier morphological appearance. In addition, IKK  -
Figure 6. IKK signaling is temporally regulated and functionally distinct in myogenesis. (A) IKK  (f/f) MEFs or myoblasts prepared from E13.5 embryos
or 3-d-old pups, respectively, were infected with pBabe-Puro or pBabe-Cre retrovirus. After selection, cells were transfected with MyoD and TnI-luc, and
luciferase assays were performed after 2 d in DM. (B) Hematoxylin- and eosin-stained cryosections from tibialis anterior muscles of 4 – 6-wk-old IKK  f/f and
IKK  f/f;muscle creatine kinase – Cre mice. (C) Fiber numbers were determined from premeasured randomly selected areas (minimum of 25 per animal)
throughout the tibialis anterior muscle ( n = 3 mice per genotype). *, P = 0.005. (D) IKK wild-type and null MEFs were transiently transfected with MyoD and
TnI-luc, and, after 2 d in DM, lysates were prepared for luciferase assays. (E) C2C12 myoblasts were differentiated, and, at the indicated times, cells were
harvested, and lysates were prepared for IKK kinase assays using wild-type or serine to alanine mutant I  B  proteins as substrates (KA, kinase assay; WB,
Western blot). (F) C2C12 cells were differentiated, and, at the indicated time points, extracts were prepared to probe for phosphorylated I  B  , total I  B  ,
phosphorylated p65, and total p65. Parallel samples were prepared for nuclear extraction, and Western blots were performed for nuclear p65. Parallel
differentiated C2C12 cells were immunoprecipitated with a p65 antibody and processed for chromatin immunoprecipitation (ChIP). Fragments from the
I  B  promoter were amplifi ed by PCR before (input) or after immunoprecipitation. (G) Lysates from differentiating C2C12 cells were prepared and used
to probe for p100 – p52 and  -tubulin. (H) MEFs wild type or null for p52 and RelB were transfected with MyoD and TnI-luc. Cells were differentiated for
48 h and prepared for luciferase assays. Error bars represent SEM. Bar, 0.5  m.

JCB • VOLUME 180 • NUMBER 4 • 2008 796
alternative pathway during myogenesis functions to maintain
myotubes in response to metabolic stress.
IKK ␣ regulates mitochondrial biogenesis
Finally, we attempted to address the process by which IKK 
controls myotube maintenance. Because IKK  regulation ap-
peared selectively linked to starvation stress, we speculated that
this kinase was involved in regulating the energy capacity of dif-
ferentiating muscle. Energy production during myogenesis occurs
through a switch from glycosidic to oxidative phosphorylation
stress. This effect was dependent on the kinase activity of IKK 
because myotube maintenance was lost upon expression of a
kinase-dead mutant ( Fig. 7 D ). Moreover, siRNA deletion of the
 but not the  subunit of IKK negated this protective effect
upon glucose deprivation, con rming the speci city of IKK  in
this regulation ( Fig. 7 E ). Furthermore, 6-d starved IKK  -
expressing myotubes displayed higher levels of the myogenic
markers myogenin, Tn, MyHC, and activated p38, whereas
these markers were reduced upon IKK  knockdown ( Fig. 7 F ).
Together, these data suggest that activation of IKK  and the
Figure 7. IKK ␣ regulates myotube maintenance. (A) C2C12 cells were transfected with vector or HA-IKK  expression plasmids. After selection, cells
were differentiated and harvested for Western analysis probing for HA and myogenic markers. (B) Myoblasts were transfected with siControl or siIKK 
oligonucleotides and differentiated, and Western blotting was performed as in A. (C) 3-d differentiated myotubes stably expressing vector or IKK  were
subjected to varying stress conditions, including no media replenishment for 6 d (6 d in DM) or low glucose (1 g/L glucose in DM for 48 h). Cells were
then fi xed and photographed by phase contrast at 20 × magnifi cation. (D) Differentiated myotubes stably expressing wild-type (WT) or a kinase-dead (KD)
version of IKK  were switched to low glucose for 24 h and photographed by phase contrast. (E) Myotubes expressing siControl, siIKK  , or siIKK  were
differentiated for 3 d and switched to low glucose for 20 h before fi xation. Parallel samples were harvested for Western blots to confi rm knockdown ef-
fi ciency. (F) C2C12 cells expressing vector or IKK  were differentiated for 6 d. Lysates were subsequently prepared for Western blots probing for IKK  and
myogenic markers. Bars, 200  m.

797 IKK/NF-  B SIGNALING SWITCH IN SKELETAL MYOGENESIS • BAKKAR ET AL.
ferentiating myoblasts also revealed signi cantly higher levels
of MTCO1 DNA compared with vector cells, whereas the depletion
of IKK  led to a reduction of MTCO1 ( Fig. 8 B ). This sug-
gested the possible novel  nding that IKK  is a regulator of
mitochondrial biogenesis. Consistent with this notion, myotubes
resulting from an increase in mitochondrial content ( Moyes et al.,
1997; Lyons et al., 2004 ). Using semiquantitative PCR and the
mitochondrial marker gene cytochrome oxidase 1 (MTCO1),
we readily detected an increase in mitochondrial DNA during
C2C12 myogenesis ( Fig. 8 A ). Examination of HA-IKK  dif-
Figure 8. IKK ␣ regulates mitochondrial biogenesis. (A) DNA was prepared from GM or 3-d DM C2C12 cells, diluted, and used to amplify a 648-bp
fragment from MTCO1. Separate PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize for loading. (B) C2C12 cells stably
expressing vector (Vect) or IKK  were differentiated, and DNA samples were prepared for the determination of mitochondrial number as in A. (C) Vec-
tor and IKK  -overexpressing cells were differentiated for 3 d and stained for mitochondria with MitoTracker green. Staining was viewed by fl uorescence
at 20 × magnifi cation. (D) Mitochondrial and cytoplasmic extracts were prepared from HA-IKK  or IKK  -depleted myotubes, and lysates were probed
for cytochrome c . (E) IKK  -expressing or -depleted cells were differentiated for 3 d and lysed, and ATP production was measured by luminescence.
An ATP standard curve was generated in parallel to convert luminescence readings into [ATP] (*, P = 0.02; **, P = 0.001). (F) Vector and IKK  cells were
differentiated, lysed, and prepared for a citrate synthase assay. All experiments were initiated with equal protein and were performed during the linear
phase of the reaction to ensure adequate substrate amounts (*, P = 0.01). A parallel set of myotubes expressing vector or IKK  was cultured and switched
to DM, and dehydrogenase activity was measured by the conversion of MTS tetrazolium into aqueous formazan. Readings were taken at 15 min before
saturation (**, P = 0.001). (G) C2C12 cells were transfected with vector, p52 – RelB, or p50 – p65 and differentiated for 3 d. DNA was prepared for the
determination of mitochondrial numbers as in A or was processed for the determination of ATP production as in E (*, P = 0.001; **, P = 0.03). Error bars
represent SEM. Bar, 40  m.

JCB • VOLUME 180 • NUMBER 4 • 2008 798
chondria is linked to myotube maintenance, we treated C2C12
myotubes with mitochondrial inhibitors chloramphenicol and
oligomycin under glucose deprivation. This led to visibly less
numbers of preserved myotubes (Fig. S3 B), a phenotype strik-
ingly similar to IKK  -depleted cells. Collectively, our results
strongly support that activation of NF-  B alternative signaling
during myogenesis does not function to promote myotube for-
mation but rather is important for regulating mitochondrial bio-
genesis and myotube homeostasis.
Discussion
Recent studies have shown that chronic activation of NF-  B is
detrimental to muscle function. In skeletal muscles, NF-  B has
been linked with disease states such as cachexia and various
forms of muscular dystrophies and in ammatory myopathies
( Baghdiguian et al., 1999 ; Kumar and Boriek, 2003 ; Monici et al.,
2003; Hunter and Kandarian, 2004 ; Acharyya et al., 2007 ).
Although such studies implicate NF-  B as a therapeutic target,
mechanistically, relatively little is known about how this tran-
scription factor mediates its pathological effects. Elucidation
of these mechanisms might be better achieved by studying
NF-  B function in basic models of skeletal myogenesis. How-
ever, even in tissue culture systems, reports have con icted as to
whether NF-  B acts as a repressor or promoter of myogenesis
( Lehtinen et al., 1996 ; Guttridge et al., 1999 ; Kaliman et al., 1999 ;
Canicio et al., 2001 ; Langen et al., 2001 ; Conejo et al., 2002 ;
Munz et al., 2002 ; Baeza-Raja and Munoz-Canoves, 2004 ).
In the present study, we describe what we believe to be a new
understanding for the role of NF-  B in skeletal muscle differ-
entiation. Our  ndings reveal that NF-  B is capable of func-
tioning as both a repressor of differentiation and a promoter of
overexpressing IKK  contained higher levels of the mitochon-
drial dye MitoTracker ( Fig. 8 C ). In addition, mitochondrial
fractions from these cells were also enriched in cytochrome c ,
which were, in turn, reduced upon the expression of IKK 
siRNA ( Fig. 8 D ). Furthermore, total ATP was elevated by 72%
in IKK  -expressing myotubes (P = 0.001), whereas IKK  knock-
down caused an 18% reduction in ATP levels (P = 0.02; Fig. 8 E ).
To address whether the increase in mitochondrial content by
IKK  re ected mitochondrial function, biochemical assays were
performed for citrate synthase and dehydrogenase enzymes.
Results showed that enzyme activities were signi cantly increased
in IKK  -expressing myotubes ( Fig. 8 F ). This function appeared
selective to the NF-  B alternative pathway because p52 – RelB
but not p50 – p65 increased MTCO1 and ATP ( Fig. 8 G ). Further-
more, ATP levels were also increased in MEF and HeLa cells
overexpressing IKK  (unpublished data), suggesting that IKK 
regulation of mitochondria is not speci c to skeletal muscle.
To further investigate this regulation, ultrastructural anal-
ysis was performed in IKK  overexpression and knockdown
conditions. Remarkably, HA-IKK  – expressing myotubes dis-
played elongated networks of mitochondria, a hallmark of ex-
tensive proliferation ( Fig. 9 A ). Conversely, IKK  knockdown
resulted in degenerating organelles, which was indicated by
swelling and the absence of cisternae in the mitochondrial ma-
trix ( Fig. 9 B ). In addition, genome-wide L2L analysis ( Newman
and Weiner, 2005 ) of microarray data from IKK  mytotubes
identi ed 126 selectively enriched biological processes in genes
that were up-regulated ( > 1.5-fold). From these, 48% were in-
volved in mitochondrial and metabolic regulation ( Fig. 9 C ),
whereas signi cantly fewer IKK  regulated genes associated
with transcription/translation (12%) or even skeletal myogenic
processes (6%). To examine whether the regulation of mito-
Figure 9. IKK ␣ controls mitochondrial structure. (A) Ultrathin sections from vector or IKK  -expressing myotubes were analyzed by EM at 18,500 × direct
magnifi cation. (B) Myotubes expressing control or IKK  siRNA were sectioned and visualized by EM as in A. (C) Microarray analysis was performed on
vector and IKK  -expressing myotubes using the murine MG430.20 Affymetrix chip. Genes up-regulated in IKK  myotubes as compared with vector were
analyzed by L2L analysis (http://depts.washington.edu/l2l/) for statistically signifi cant enriched biological processes. Bars, 500 nm.

799 IKK/NF-  B SIGNALING SWITCH IN SKELETAL MYOGENESIS • BAKKAR ET AL.
in skin differentiation ( Hu et al., 2001 ; Sil et al., 2004 ) or
NF-  B – dependent gene expression ( Anest et al., 2003 ; Yamamoto
et al., 2003; Hoberg et al., 2004 ), we were unable to detect
nuclear IKK  in myoblasts or myotubes (unpublished data).
Although our current results do not rule out the possibility that
IKK  might still phosphorylate an unknown target to modulate
myogenic gene expression, we favor instead that IKK  function
in skeletal muscle differentiation is represented by the alterna-
tive pathway requiring the cytoplasmic form of IKK  to acti-
vate p52 – RelB complexes.
Evidence from IKK ␣ ⫺ / ⫺
, p52
⫺ / ⫺
, and RelB
⫺ / ⫺
MEFs indi-
cated that alternative activation of NF-  B is required for myo-
genic activity. These results appear consistent with previous
 ndings implicating IKK  as a positive regulator of myogene-
sis ( Canicio et al., 2001 ). However, in contrast to these  ndings,
we were unable to demonstrate by either forced expression or
RNAi depletion that IKK  is essential for the induction of myo-
genic genes or myotube formation. Although genetic evidence
from p65 and IKK  knockout MEFs was consistent with how
these classical signaling components were found to function in
muscle cells, we do not yet understand why this same consis-
tency was not present between IKK ␣ ⫺ / ⫺
MEFs and C2C12 cells
depleted of IKK  with siRNA. Possibly, the fraction of IKK 
that remains in cells after siRNA depletion is suf cient to mask
a phenotype that otherwise requires its complete absence, or
perhaps the increase in myogenic activity derived from estab-
lished IKK ␣ ⫺ / ⫺
MEFs might be an indirect consequence of
immortalization and continued subculturing. We suspect that
additional myogenic reporter assays in primary IKK ␣ ⫺ / ⫺
MEFs
and myoblasts will be needed to clarify this issue.
Nevertheless, our observations led to the novel discovery
that IKK  acts as a regulator of mitochondrial biogenesis.
Although the mechanism remains unknown, we predict that IKK 
activation functions through p52 – RelB to promote mitochondrial
biogenesis and meet the metabolic needs of newly formed con-
tractile myotubes. The inhibitor compounds of mitochondria were
also seen to decrease myotube maintenance, suggesting that IKK 
regulation of mitochondria is necessary for myotube homeostasis
in response to changing metabolic conditions.
A model for IKK/NF- ␬ B signaling in skeletal
muscle differentiation
Collectively, our data support a model whereby IKK/NF-  B
signaling both inhibits and promotes the differentiation state of
muscle cells ( Fig. 10 ). This model helps unify the literature on
the contradictory functions of NF-  B in myogenesis and pre-
dicts that during differentiation, a temporal switch occurs be-
tween NF-  B classical and alternative signaling pathways.
In myoblasts, classical signaling is constitutively active and func-
tions to maintain cells in an undifferentiated state. This function
is regulated through the control of MyoD as well as other
MyoD-independent mechanisms involving cyclin D1 and YY1.
Once differentiation cues are initiated, classical signaling is
turned down, whereas the alternative pathway is induced late in
the myogenic program. In turn, the activation of IKK  leading
to p52 – RelB association regulates myogenesis by mediating the
production of mitochondria necessary to satisfy the metabolic
myotube maintenance depending on speci c activities of IKK
and NF-  B subunits.
p65 and the classical NF- ␬ B signaling
pathway function as negative regulators
of myogenesis
Utilization of knockout MEFs demonstrated that myogenic ac-
tivity was enhanced in cells lacking p65, and comparisons with
all  ve NF-  B subunits showed that this activity was highest in
p65
⫺ / ⫺
cells. Therefore, although myoblast nuclei have been
shown to contain constitutive activity for p50 and p65 ( Guttridge
et al., 1999 ), our current data argue that suppression of myogen-
esis by NF-  B is mediated speci cally through p65. This notion
is consistent with results in primary myoblasts in which myo-
genic activity was also elevated in p65- but not p50-null cells.
Together, these genetic data reaf rm that p65 activity in prolif-
erating myoblasts functions as a negative regulator of myo-
genesis. This function of p65 is evident in muscle injury, in which
the lack of p65 enhances myogenesis in mdx and toxin-treated
mice ( Acharyya et al., 2007 ). Given that p65 de ciency corre-
lated with increases in overall  ber numbers in young and adult
mice, it suggests that p65 is also relevant during postnatal mus-
cle growth, as indicated by the high levels of NF-  B activity in
muscles from neonates ( Acharyya et al., 2007 ). Why p65 would
function in this capacity at this stage of development is not yet
known, and whether it functions in a similar manner during em-
bryonic or fetal myogenesis remains to be investigated.
Our current results demonstrate that regulation of myo-
genesis is dependent on p65 transcriptional activity. This notion
is in line with our previous  ndings that NF-  B inhibits myogen-
esis through the transcriptional activation of cyclin D1 ( Guttridge
et al., 1999 ). Repression of myogenesis by p65 has also been
seen in response to TNF  , leading to the loss of MyoD ( Guttridge
et al., 2000 ) and, more recently, to the gain of YY1, resulting in
silencing of myo brillar genes ( Wang et al., 2007 ). Thus, p65
requires its transactivation function to suppress muscle differ-
entiation, and results from MyoD
⫺ / ⫺
myoblasts support that this
can occur via multiple mechanisms.
Similar to p65, we discovered that myogenic activity was
enhanced in MEFs lacking classical components IKK  and
IKK  . Like p65, IKK  deletion in muscle led to increases in  -
ber number and to enhanced myogenesis, as recently reported
in mdx mice ( Acharyya et al., 2007 ). Collectively, these data
argue strongly that classical NF-  B signaling functions as a
negative regulator of muscle differentiation in both physiological
and disease processes.
IKK ␣ signaling promotes myotube
maintenance through mitochondrial
biogenesis
With respect to alternative NF-  B signaling, our results showed
that activation of IKK during myogenesis is selective to IKK  ,
as this activity tightly correlated with p100 processing. Such acti-
vation was preceded by a decline in classical pathway activity,
which is depicted by decreases in I  B  and p65 phosphoryla-
tion, as well as p65 nuclear and DNA-bound levels. In contrast
to recent  ndings that nuclear localization of IKK  is required

JCB • VOLUME 180 • NUMBER 4 • 2008 800
tein extraction reagent solution (Thermo Fisher Scientifi c), and assays were
performed as previously reported ( Guttridge et al., 1999 ). IKK  , IKK  ,
and p65 siRNAs were obtained from Dharmacon, Inc., and transfections
were performed using Lipofectamine 2000. Retrovirus production and in-
fection were performed as previously described ( Guttridge et al., 1999 ).
Mice and genotyping
Animals were housed in the animal facility at The Ohio State University
Heart and Lung Research Institute under sterile conditions maintaining
constant temperature and humidity and were fed a standard diet. Treat-
ment of mice was in accordance with the institutional guidelines of the
Animal Care and Use Committee. Mice null for p65 were generated as
previously described ( Doi et al., 1999 ). p50 mice were obtained from
Jackson ImmunoResearch Laboratories, and IKK  fl ox mice ( Li et al.,
2003 ) were crossed to muscle creatine kinase – Cre mice to delete IKK  in
skeletal muscle. Mice genotypes were confi rmed by PCR analysis from
prepared tail DNA.
Cell culture
C2C12 murine myoblasts and fi broblasts were cultured as previously de-
scribed ( Guttridge et al., 2000 ). Primary myoblasts were prepared from
2-d-old neonates adopted from the described procedures ( Rando and
Blau, 1994 ). In brief, limbs from pups were skinned and incubated with
collagenase/dispase mixture at 37 ° C for 1 h. Then, the cell suspension
was further homogenized by pipetting and preplated on uncoated cell
culture plates in F10 media (Invitrogen) to selectively enrich for myoblasts.
After two rounds of preplating, the cell suspension was plated on gelatin-
precoated plates in the presence of 20% FBS and 6 ng/ml basic FGF.
Primary myoblasts were used at passage 3 – 5 after isolation.
Immunoblotting, Northern blots, ChIP, and kinase assays
Western and Northern blots and kinase analyses were performed as de-
scribed previously ( Hertlein et al., 2005 ). For ChIP, assays were performed
as recommended by the manufacturer (Millipore).
Histology, electron microscopy, and immunofl uorescence
For muscle analysis, tissues were sectioned at 10  m on a cryostat (Leica)
and stained with hematoxylin and eosin or processed for immunohisto-
chemistry. The internal diameters (shortest diameter) from 1,200 fi bers in
random fi elds throughout the muscle were recorded using a microscope
(BX50; Olympus) and MetaVue 6.2r6 software (MDS Analytical Tech-
nologies). Fiber number was recorded in 25 randomly selected fi elds
throughout the muscle and averaged for comparisons. Muscles from three
to fi ve different animals per group were used. Immunostaining proce-
dures on cell lines and muscle sections were performed as described pre-
viously ( Acharyya et al., 2004, 2005 ), and all images were captured
with a fl uorescent microscope (Axioskop 40; Carl Zeiss, Inc.) using a
needs of contractile muscle cells. Although cooperative func-
tions of NF-  B signaling pathways are important for mam-
mary and osteoclast tissue development ( Demicco et al., 2005 ;
Ruocco et al., 2005 ), skeletal muscle is, to the best of our knowl-
edge, the  rst example of a differentiation system regulated
through a functional switch of classical and alternative NF-  B
signaling pathways.
Materials and methods
Materials
Antibodies to p100 – p52, I  B  (C21), IKK  , IKK  (FL419), myogenin (M-225),
p38, MyoD (M-318), and p65 (N terminal) were obtained from Santa
Cruz Biotechnology, Inc. MyHCIIB (MY-32), MyHC slow (NOQ7.5.4D),
Tn T (JLT-12), sarcomeric tropomyosin (CH1), and  -sarcomeric actin (5C5)
were purchased from Sigma-Aldrich. p65 antibody was obtained from
Rockland Immunochemicals, Inc., HA was purchased from Covance, and
IKK  was purchased from Imgenex. Phospho-I  B  , p38, and p65 were
obtained from Cell Signaling Technology, and cytochrome c was pur-
chased from BD Biosciences. Bovine insulin, collagen type I, and gelatin
were obtained from Sigma-Aldrich, whereas TNF  was purchased from
Roche. Both collagenase P and dispase (grade II) were obtained from
Boehringer Mannheim. Basic human FGF was purchased from Promega,
and oligomycin was obtained from Alexis Biochemicals. MitoTracker green
and secondary antibodies for immunofl uorescence were obtained from
Invitrogen, whereas other materials for immunohistochemical analysis were
obtained from Vector Laboratories.
Plasmids
Reporter and p65 expression plasmids were previously described ( Guttridge
et al., 1999 ; Acharyya et al., 2004 ; Hertlein et al., 2005 ) with the excep-
tion of the p65(1 – 313; S276A) mutant, which was generated by mutating
serine 276 to alanine in the p65(1 – 313) plasmid. MSCV-MyoD was gener-
ated by subcloning the MyoD cDNA from a pBabepuroMyoD retroviral
construct ( Guttridge et al., 2000 ). IKK plasmids were designed by subclon-
ing IKK  , IKK  , and IKK  into the pBSx-HSAvpA plasmid, whereby trans-
gene expression is driven from the human skeletal actin promoter.
Transfections, luciferase assays, and retrovirus infections
Subconfl uent C2C12 cells were transfected in low serum Opti-MEM using
Lipofectamine (Invitrogen) according to the manufacturer. For luciferase as-
says, cells were transiently transfected using Superfect (QIAGEN) for MEFs
or Lipofectamine for primary myoblasts. All transfections were normalized
to cytomegalovirus-  Gal expression. Cells were lysed in mammalian pro-
Figure 10. A model for IKK/NF- ␬ B signaling and function in skeletal myogenesis. The model depicts different phases of myogenesis from proliferating myo-
blasts to differentiated myotubes. In proliferating myoblasts, classical NF-  B signaling mediated by IKK  and IKK  leads to the activation of p65 that binds
DNA and regulates gene expression to inhibit myogenesis. During differentiation, classical NF-  B is down-regulated, whereas the alternative signaling be-
comes activated. Activation of alternative signaling occurs late in the myogenic program to regulate mitochondrial biogenesis and myotube maintenance.

801 IKK/NF-  B SIGNALING SWITCH IN SKELETAL MYOGENESIS • BAKKAR ET AL.
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camera (AxioCam HRc; Carl Zeiss, Inc.) and AxioVision 3.1 software
(Carl Zeiss, Inc.). Ultrastructural analysis was performed on fi xed cells
and sectioned using a microtome (EM UC6; Leica) at 70 nm. Sections
were then stained and visualized using a transmission electron micro-
scope (Spirit Tecnai; FEI) at 80 kV, and images were captured with a
camera (XR60; Advanced Microscopy Techniques).
Mitochondrial assays
Both CellTiter-Glo Luminescent Assay for ATP determination and MTS cell
viability assays were obtained from Promega and performed according to
the manufacturer ’ s recommendations. Citrate synthase activity was deter-
mined by using Ellman ’ s reagent with acetyl-CoA and oxaloacetate ( Leek
et al., 2001 ). Procedures for primer design and PCR of MTCO1 as well as
mitochondrial extraction for identifi cation of cytochrome c were followed
as described previously ( Huo and Scarpulla, 2001 ; Liu et al., 2004 ).
Statistical analysis
All quantitative data are represented as means ± SEM. Analysis was per-
formed between different groups using a two-tailed t test. Statistical signifi -
cance was set at P < 0.05.
Online supplemental material
Fig. S1 shows that the absence of p65 in young mice leads to increases
in fi ber numbers that is independent of fi
ber type and muscle atrophy.
Fig. S2 shows evidence for the conditional deletion of IKK  in primary
fi broblasts and adult muscles. Fig. S3 shows that the NF-  B classical
pathway inhibits myogenesis in 10T1/2 fi broblasts, whereas C2C12
myotubes are not maintained with compounds that inhibit mitochondria.
Online supplemental material is available at http://www.jcb.org/cgi/
content/full/jcb.200707179/DC1.
We thank U. Siebenlist and F. Weih for MEF knockout cell lines, A. Beg, M. Karin,
and R. Kahn for mice, J. Didonato for expression plasmids, J. Rafael-Fortney for
advice, and K. Wolken at The Ohio State University Campus Microscopy
Core for assistance with EM analysis.
This work was supported by National Institutes of Health grants
CA098466 and AR052787.
Submitted: 26 July 2007
Accepted: 29 January 2008
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