Regulation of skeletal muscle gene expression by p38 MAP kinases

Regulation of skeletal muscle geneexpression by p38 MAP kinasesFrederic Lluıs1, Eusebio Perdiguero1, Angel R. Nebreda2 and Pura Munoz-Canoves1

1Center for Genomic Regulation (CRG), Program on Differentiation and Cancer, Barcelona, Spain2Spanish National Cancer Center (CNIO), Madrid, Spain

The formation of skeletal muscle is a multistep process

orchestrated by the basic helix–loop–helix myogenic

regulatory factors (MRFs). A wide array of proteins can

interact with the MRFs, resulting in either induction or

repression of their myogenic potential and subsequent

MRF-mediated muscle-specific transcription. Findings

published over the past few years have unambiguously

established a key role for the p38 MAP kinase pathway in

the control of muscle gene expression at different stages

of the myogenic process. Here, we discuss the mechan-

isms by which p38 MAP kinase controls skeletal muscle

differentiation by regulating the sequential activation of

MRFs and their transcriptional coactivators, including

chromatin remodeling enzymes.

Introduction

The regulation of skeletal muscle formation (myogenesis)is essential for normal development as well as inpathological conditions such as muscular dystrophiesand inflammatory myopathies. Myogenesis is a dynamicprocess in which mononucleated undifferentiated myo-blasts first proliferate, then withdraw from the cell cycleand finally differentiate and fuse to form the multi-nucleated mature muscle fiber. This process is controlledby members of a family of muscle-specific basic helix–loop–helix (bHLH) proteins that, in concert with membersof the ubiquitous E2A and myocyte enhancer factor-2(MEF2) families, activate the differentiation program byinducing transcription of regulatory and structuralmuscle-specific genes [1] (Figure 1). Additional levels ofregulation impinge on this basic transcriptional model toprovide further versatility to muscle gene expression. Thequestion of how these signals are deciphered by themyogenic effectors has been the center of intensiveinvestigation. A signaling pathway that plays a funda-mental role in the transition of myoblasts to differentiatedmyocytes involves p38 mitogen-activated protein kinase(MAPK). Recent studies have demonstrated that p38MAPK provides a link between the myogenic transcriptionfactors that activate muscle genes directly and thechromatin remodeling activities associated with themuscle differentiation program. Here, we discuss howp38 MAPK controls the transcriptional circuitry that

Corresponding author: Munoz-Canoves, P. ([email protected]).Available online 1 December 2005

www.sciencedirect.com 0962-8924/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved

underlies tissue-specific gene expression, with particularemphasis on skeletal muscle.

Regulation of skeletal muscle gene expression

by myogenic regulatory factors

Activation of muscle differentiation-specific genes iscontrolled by the myogenic regulatory factors (MRFs),which belong to the bHLH family of transcription factors(Figure 2). The MRF family consists of four members:Myf5, MyoD, myogenin and MRF4, all of which bind tosequence-specific DNA elements (E box: .CANNTG.)present in the promoters of muscle genes. Selective andproductive recognition of E boxes on muscle promotersrequires heterodimerization of MyoD with ubiquitouslyexpressed bHLH E proteins, rendering the formation ofthis functional heterodimer the key event in skeletalmyogenesis [2,3]. Preferences for specific sequencesinternal and external to the E box for MRF homodimersand MRF–E-protein heterodimers have been determinedin vitro [4]. Nonetheless, this basic model of muscle-specific transcription by MRF binding to E boxes is overlysimplistic and encounters several pitfalls. For example,canonical E boxes are not exclusively found in theregulatory regions of muscle genes. Other transcriptionfactors, including E protein homodimers and neurogenicbHLH transcription factors such as NeuroD utilize thesame E box as MyoD on B cell- and neuron-specificpromoters, respectively. However, skeletal myoblastsrequire MRFs, lymphocytes require E proteins, andneurons require NeuroD, to activate their correspondingtissue-specific genes. It seems likely that the cell-type-restricted expression of bHLH proteins might avoid theectopic activation of bHLH target genes in the wronglineage. Finally, different muscle genes are expressed atdifferent times during myogenesis, despite all of themhaving E boxes in their promoter regions [1,5]. Thus,promoter-specific and temporal constraints are likely to besuperimposed upon this basic model of myogenic tran-scriptional activation by MRFs.

Interaction of MRFs with transcriptional cofactors

The MRFs have the unique property of converting non-muscle cells to the muscle lineage, strongly suggestingthat they can, directly or indirectly, induce relaxation ofthe otherwise-repressed chromatin on their target genes.The potential of MyoD to stimulate muscle-specific genetranscription derives both from its intrinsic ability to

Review TRENDS in Cell Biology Vol.16 No.1 January 2006

. doi:10.1016/j.tcb.2005.11.002

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MyofiberMyotubeMyocyteMyoblast

Proliferation

MyoD

MRF4

MEF2MEF2

Myogenin

Id

Myf5Id

Id

MyoDId

Id

• α-Actin• MHC• MCK• Others

Skeletal myogenesis

Differentiation(terminal)

Differentiation(late)

Differentiation(early)

EProtein

EProtein

EProtein

EProtein

Induction of muscledifferentiation-specific genes:

TRENDS in Cell Biology

Figure 1. Control of skeletal myogenesis by bHLH and MEF2 transcription factors. Myogenesis is a multi-step process by which new muscle fibers are formed from precursor

muscle cells. Mononucleated undifferentiated myoblasts grow in proliferating conditions, characterized by a high mitogen content (proliferation); upon mitogen withdrawal,

myoblasts differentiate into mononucleated myocytes (early differentiation) that subsequently start to fuse into multinucleated myotubes expressing muscle-specific

proteins (late differentiation), to form the mature muscle fiber (terminal differentiation). Progression through the different myogenic stages is controlled by the sequential

activation of four myogenic regulatory factors (MRFs) belonging to the basic helix–loop–helix (bHLH) family of transcription factors (Myf5, MyoD, myogenin and MRF4),

which cooperate with the ubiquitously expressed E proteins (the E2A gene products E12 and E47, and HEB) and myocyte enhancer factor 2 (MEF2) transcriptional regulators

to activate transcription of muscle-specific genes, coding for structural and enzymatic muscle proteins such as a-actin, myosin heavy chain (MHC) or muscle creatine kinase

(MCK). Studies using both primary cultures of skeletal muscle as well as established muscle cell lines (which partially recapitulate myogenesis, thus being extensively used as

myogenic model systems) have confirmed the expression of MyoD and Myf5 in undifferentiated myoblasts, while myogenin and MRF4 are activated at early and late

differentiation stages, respectively [1]. In proliferating myoblasts, MRFs and E proteins associate with the HLH protein Id (inhibitor of differentiation). Since Id lacks the basic

domain necessary for DNA binding, the resulting E protein–Id and MRF–Id heterodimers cannot bind the E box in the muscle promoters. Id expression is downregulated at the

onset of differentiation, allowing the formation of the functional MRF–E-protein heterodimers (see text for details).

Review TRENDS in Cell Biology Vol.16 No.1 January 2006 37

reorganize chromatin through a region rich in histidineand cysteine residues (H/C domain), which lies N-terminalto the basic region, and a potential amphipathic a-helix(helix III) in the C-terminal region [6–8] (Figure 2) andfrom its capacity to interact with histone acetyltrans-ferases (HATs), especially p300/CBP and PCAF (p300/CBP-associated factor) [9–14] (Box 1). Contrary to HATs,however, histone deacetylases (HDACs) can negativelyregulate MyoD-dependent transcription by interactingdirectly with MyoD [15,16]. For example, in proliferatingmyoblasts, MyoD has been found on the myogeninpromoter in association with HDAC1, acting as atranscriptional repressor [17] (Box 3). The class IIIHDAC Sir2 can also associate with, and deacetylate,both PCAF and MyoD, resulting in the inhibition of

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muscle differentiation upon changes in the redox state ofthe cell [18]. Other studies have shown that the SWI/SNFATP-dependent chromatin remodeling activity is necess-ary for MyoD-mediated activation of endogenous muscledifferentiation-specific loci [19] (Box 1). Together, thesedata suggest that MyoD might direct the chromatin-modifying enzymes to muscle promoters and that,depending on the nature of the E-box-associated enzy-matic activity, MyoD can activate or inhibit muscle-specific gene transcription.

Full activation of muscle gene expression by MRFs isalso dependent on their association with members of theMEF2 family of transcription factors, MEF2A-D. MEF2factors cannot activate muscle genes on their own, butthey do potentiate the activity of the MRFs. Such


HLH

MEF2 MADS MEF2 Activation

Activation

ActivationHIIIH/C HLHBasic

MRF

Activation Activation HLHBasic

E protein

HLH

Id


Figure 2. Functional domains of the bHLH and MEF2 transcription factors. The MRFs share functionally distinct domains: The bHLH region is evolutionary conserved in the

MRFs, being the HLH domain responsible for dimerization of these factors with the E proteins, whereas the basic domain is responsible for the binding to a canonical DNA

sequence, CANNTG, called the E box, within the regulatory regions of muscle genes [1]. Importantly, MRF–E-protein heterodimers, but not the homodimers, are able to bind

to the muscle E box [2]. The N- and C-terminal domains of the MRFs show sequence divergence and are important for transactivation, chromatin remodeling and protein–

protein interactions. MyoD and Myf5 have a higher ability than myogenin to remodel the repressed chromatin at the target loci, owing to two domains conserved in the

former, but not the latter, proteins: a region rich in histidine and cysteine residues (H/C domain), which lies N-terminal to the basic region, and a potential amphipathic a-helix

(helix III) in the C-terminal region [1,5]. The H/C and helix 3 domains mediate interaction of MyoD with the Pbx transcription factor, which might be relevant for the initiation of

transcription at the myogenin promoter [6,8,34] (see Box 3).


synergism requires the direct association between thebHLH domain of the MRF and the MADS (MCM1,agamous, deficiens, serum response factor) domain of theMEF2 protein [14,20] (Figure 2). The growing list of MRF-associated factors suggests that these intermolecularinteractions are likely to regulate the specific andtemporal association of MRFs with DNA-regulatoryregions. Moreover, accumulating evidence indicates thatthe activities of the MRFs and associated cofactors mightbe subjected to posttranscriptional regulation by muscle-differentiation-induced signaling pathways.

Box 1. Chromatin modifying and remodeling activities

The fundamental repeating unit of chromatin is the nucleosome,

which consists of 146 base pairs of DNA wrapped around a histone

octamer. Chromatin is generally repressive to extraneous access,

owing to its compact and tight nucleosomal organization. Two main

enzymatic activities induce chromatin modifications and regulate

chromatin access: chromatin modifying complexes and chromatin

remodeling complexes [40,46]. One specific modification of histones

is acetylation, catalyzed by histone acetyltransferase (HAT) enzymes,

which weakens the histone–DNA interaction and has been therefore

associated with transcriptional activation. HAT activity is intrinsic to

numerous transcriptional coactivators, including p300, a functional

homolog of CREB-binding protein (CBP), and p300/CBP-associated

factor (PCAF). HATs can also acetylate certain transcription factors,

thus influencing their activities [46]. Acetylation, however, is a

reversible process, as deacetylation is regulated by histone

deacetylases (HDACs), these being generally associated with

transcriptional repression. Mammalian HDACs are grouped into

three subclasses: class I, class II and class III HDACs [14,46]. On the

other hand, chromatin-remodeling factors use the free energy freed

by ATP hydrolysis to loosen DNA–histone contacts and thus facilitate

the movement of the nucleosomes along a particular DNA sequence

(originally termed ‘sliding’). Common to all chromatin-remodeling

complexes is an ATPase subunit, the motor of the complex. One

important chromatin-remodeling enzyme is SWI/SNF (switching/su-

crose non-fermenting), which is a multisubunit complex that was

first identified in yeast and is highly conserved among eukaryotes.

The mammalian SWI/SNF family consists of complexes that contain

one of two ATPases, either brahma (BRM) or brahma-related gene 1

(BRG1), as the catalytic subunit, which is associated with a variety of

subunits called BRG1-associated factors (BAFs) [40].

Requirement for p38 MAPK activity in skeletal muscle

differentiation

Independent studies have unambiguously demonstratedthat the p38 MAPK signaling pathway (Box 2) is a crucialregulator of skeletal muscle differentiation. Treatmentwith the p38a and p38b inhibitor SB203580 prevented thefusion of myoblasts into myotubes, as well as the inductionof muscle-specific genes [21–24]. Importantly, a recentreport has shown the requirement for p38a/p38b in theactivation of the quiescent satellite cell (the muscle stemcell), although the mechanism underlying this effectremains unknown [25]. Forced activation of p38 MAPKby ectopic expression of a constitutively active mutant ofMKK6 (MKK6-EE) is sufficient to override the inhibitoryfactors present in proliferating cells and to induce both theexpression of differentiation markers and the appearanceof multinucleated myotubes [23,26]. Furthermore, theectopic expression of active MKK6-EE in rhabdomyosar-coma cells, which express MyoD but do not containactivated p38 MAPK, led to the induction of morphologicaland biochemical differentiation of the tumor cells [3],reinforcing the idea that p38 MAPK activity plays anessential role in muscle differentiation. Yet, the mechan-isms that regulate p38 MAPK activity in differentiatingmuscle cells remain unidentified, although these appear tobe different from those involved in the response to stressand cytokines. Thus, the kinetics of p38 MAPK activationin response to the later stimuli are fast and transient,whereas differentiation-induced p38 MAPK activation is


persistent, suggesting the need for constant p38 MAPKactivity during myogenesis [3,21,23].

p38 MAPK-regulated mechanisms controlling muscle-

specific gene transcription

While p38 MAPK plays an essential role in myoblastdifferentiation, the underlying molecular mechanisms ofmuscle-specific transcriptional control by p38 MAPKsremain largely unknown.

Activation of MEF2 by p38 MAPK

A potential explanation for the positive effect of p38MAPK in myogenesis was provided by the finding that p38


Box 2. The p38 MAP kinase signaling pathway

The p38 mitogen-activated protein kinase (MAPK) pathway was

initially described to be preferentially activated by different types of

stress and cytokines, but numerous studies have since implicated this

pathway in the regulation of a wide spectrum of cellular processes,

including cell-cycle arrest, apoptosis, senescence, regulation of RNA

splicing, tumorigenesis or differentiation of various cell types such as

adipocytes, cardiomyocytes, neurons and myoblasts (reviewed in [47–

50]). In vertebrates, there are four p38 MAP kinases, p38a, p38b, p38g

(SAPK3, ERK6) and p38d (SAPK4), which are all phosphorylated and

activated by the MAPK kinase MKK6 (Figure I). Another p38 MAPK

kinase is MKK3, which activates p38a, p38g and p38d, whereas MKK4

can also, in some cases, activate p38a. Once activated, p38 MAPKs

phosphorylate serine/threonine residues of their substrates, which

include transcription factors as well as protein kinases (see [49] and

references therein). The identification of physiological substrates for

p38a and p38b has been facilitated by the availability of specific

pyridinyl imidazole inhibitors such as SB203580/SB202190 and the

recently reported inhibitor of the four p38 isoforms (BIRB0796) [51]

(Figure I). Knockout mice for p38a have been generated, but they die at

midgestation [52–54], whereas tissue-specific knockouts have impli-

cated p38a in cardiomyocyte proliferation and survival [55,56].

Recently, p38b, p38g, p38d and double p38g p38d knockout mice

have been also generated, which appear to be viable and fertile [57,58].

Phosphorylation of MEF2 by p38 MAPK

The substrate specificity of the different p38 isoforms is known to

overlap. Different reports have shown that p38a and p38b phosphor-

ylate and enhance the transcriptional activities of MEF2A and MEF2C,

but not MEF2D [27,28,59,60]. By contrast, p38g only weakly phosphor-

ylates MEF2A, MEF2C and MEF2D in vitro and barely stimulates their

transcriptional activities in vivo, whereas p38d does not phosphorylate

any of them [23,61]. MEF2C can be phosphorylated by p38 MAPKs in

the transactivation domain residues Thr293, Thr300 and Ser387 [27].

Phosphorylation of all these residues is important for MEF2C activation

by p38 MAPK in lymphoid cells [27]. The p38 MAPK phosphorylation

sites in MEF2C are conserved in MEF2A (amino acid relationship

between MEF2C–MEF2A: Thr293–Thr312; Thr300–Thr319; Ser387–

Ser453). However, Ser453 did not play any regulatory role in CHO

and 293 cells, suggesting the existence of cell-type-dependent and

isoform-specific phosphorylation events [59]. In agreement with this,

Thr293 appears to be the only p38 phosphorylation site that is

important for MEF2C regulation in differentiating myocytes

[22,23,28]. Recently, abrogation of p38 MAPK signaling was shown to

block MEF2 activation in a MEF2 transgenic ‘sensor’ mouse, leading to

the inhibition myogenic differentiation in somite cultures and in

embryos in vivo [62].

MKK3/6

SB203580SB202190

ELK-1

MEF2MRF4E47

p53

Protein kinasesMAPKAPK-2/3

PRAKMSK1/2MNK1/2

Other proteins

eEF2KSAKS1Cdc25SAP90SAP97

BIRB0796

Substrates:

MKK kinasesTAK1, ASK1, DLK, MEKK4

Tau

α1-syntrophin

CHOPATF2

NF-κB

Transcription factors


p38α p38β p38γ p38δ

Figure I.


MAPK induces the transcriptional activity of MEF2proteins (Box 2). Han and Ulevitch [27] were the first todemonstrate that, in lipopolysaccharide-stimulatedmacrophages, p38 MAPK directly phosphorylated thetransactivation domain of MEF2C on Thr293, Thr300and Ser387. Importantly, p38 MAPK was also shown tophosphorylate endogenous MEF2A and MEF2C in musclecells in vivo [22,23,28]. However, Thr293 was the onlycrucial MEF2C regulatory phosphorylation site in skeletalmuscle [23], suggesting that MEF2 regulation by p38MAPK might involve selective phosphorylation of distinctresidues in a tissue-restricted manner.

The interactions between MEF2 transcription factorsand MRFs during muscle differentiation [20] raise thepossibility that p38-MAPK-mediated phosphorylation ofMEF2 family members might contribute to the transcrip-tional synergy between MyoD and MEF2. However, severalpieces of evidence argue against this possibility. Indeed, theinteraction between MEF2C and MyoD in a mammaliantwo-hybrid system is not affected by p38 MAPK [23], andmutation of Thr293 to alanine, which prevents phosphoryl-ation and activation of MEF2C in muscle cells, does notaffect MyoD–MEF2C functional synergism [23,29]. More-over, a Gal4–MyoD fusion protein that is impaired in itsassociation with MEF2 proteins can still be activated byMKK6 [3,23]. These findings suggest that distinct


mechanisms might control the stimulation of the intrinsicMEF2C transcriptional activity and the induction offunctional synergism between MEF2C and MyoD.

Regulation of the MRF transcriptional activity by

p38 MAPK

The requirement for p38 MAPK in the activation ofMyoD-dependent muscle promoter transcription in aMEF2-independent manner suggests that MyoD (andother MRFs) are likely to be direct targets of p38MAPK. Accordingly, p38 MAPK was able to efficientlyphosphorylate Ser5 of MyoD both in vitro and in vivo[23]. However, mutation of Ser5 to alanine did not altersignificantly MyoD transcriptional activity [23], indicat-ing that p38 MAPK stimulates MyoD-dependent tran-scription by, as yet unidentified, indirect mechanisms.One possibility would be that p38 MAPK stimulatesmyogenic transcription by targeting the MRF-associatedcofactors and/or chromatin-modifying enzymes. Consist-ent with this prediction, phosphorylation of the obligateMyoD partner E47 by p38 MAPK has been shown tohave important consequences for muscle gene transcrip-tion [30] (Box 3). In particular, p38 MAPK-mediatedphosphorylation of E47 at Ser140 induced MyoD–E47heterodimer formation, subsequent binding to the E boxon muscle promoters and activation of muscle-specific



gene transcription. By contrast, non-phosphorylatableE47 (Ser140 mutated to Ala) failed to associate withMyoD, displaying reduced myogenic potential. Elegantstudies by Dilworth et al. [31], using forced dimers ofMyoD and/or E47 fusion proteins, have demonstratedthat the role of E proteins is not limited to providing adimerization partner that facilitates MyoD–DNA bind-ing. Thus, forced MyoD homodimers were able to bindto a muscle E box in the context of nucleosomes, but,contrarily to a forced MyoD–E7 heterodimer, they couldnot drive transcription from the E-box-containingpromoter owing to its inability to recruit the necessaryp300 HAT activity to the promoter region [9,10]. Theauthors propose that the heterodimers might provide ascaffolding that is conformationally preferable over thehomodimers for establishing a coactivator bridgebetween the proximal E boxes and the minimalpromoter to activate transcription [31]. In this scenario,p38 MAPK might be favoring both the formation of thefunctional MyoD–E47 transcription factor as well as thesubsequent interaction with DNA and the chromatin-associated proteins.

Nevertheless, some issues remain unclear: should allMyoD-dependent promoters be regulated alike? Doesp38 regulate the expression of all MyoD-dependentgenes? In this regard, a recent chromatin immuno-precipitation (ChIP) coupled with mouse promoter DNAmicroarray hybridization (‘ChIP-on-chip’) analysis hasled to the identification of w100 novel genes bound byMyoD both in myoblasts and in differentiated myocytes[32]; intriguingly, activation of the target genes did notalways correlate with MyoD binding, consequentlyraising additional fundamental questions: what dictatesthe preferential binding of MyoD to different targetpromoters?; what leads to transcriptional activation orinactivation of MyoD-bound genes in myoblasts? In adifferent study, Bergstrom et al. [33] applied expressionarrays and ChIP analysis to a cellular model system of

Box 3. p38 MAP kinases in the control of muscle-specific gene ex

During the proliferation stage, in undifferentiated myoblasts, the

activity of MyoD is repressed by the association of E proteins with Id

(and to a lesser extent by MyoD–Id association), preventing the

formation of functional MyoD–E-protein heterodimers and further

binding to the E box on the muscle promoters [1]. Notably, on the

myogenin promoter, the activity of MyoD can also be repressed by its

association with HDAC1 in myoblasts [16,17]. The transcription factor

Pbx has also been found on the myogenin promoter in undifferentiated

myoblasts, next to a non-canonical E box [6]. These latter results,

concerning exclusively data for the myogenin promoter, are shown

inside a dashed-line box (Figure I).

At early stages of myogenic differentiation, Id levels are transcrip-

tionally downregulated, allowing the potential formation of MyoD–E-

protein heterodimers [1]. Importantly, p38 MAPK is activated at the

onset of muscle differentiation and phosphorylates E47 at Ser140,

promoting the formation of the functional MyoD–E47 heterodimer

rather than the nonfunctional homodimers, and subsequent binding of

the heterodimer to the E box on different muscle promoters [30].

HDAC1 is dissociated from MyoD, whereas HATs (p300/PCAF) are

recruited to regulatory regions through association with MyoD–E47 [9–

13]. Phosphorylation by p38 MAPKs also allows targeting of the

SWI/SNF chromatin-remodeling complex to the muscle promoters, as

well as increased transcriptional activity of MEF2, which, through


MyoD-mediated myogenesis, showing that MyoD wasbound to the regulatory regions of all the muscle genesstudied, whether expressed early or late during muscledifferentiation. Importantly, these authors identifiedspecific loci where MyoD was stably bound but didnot activate transcription without p38 MAPK signaling.Thus, the p38 MAPK signaling pathway, rather thanacting globally on all MyoD-regulated genes, canapparently modulate the activity of MyoD at arestricted subset of promoters, establishing dynamicmodulation of the MyoD-induced programs of geneexpression. Future investigations should clarify whetherthe MyoD–E47 heterodimer is active on all MyoD-responsive promoters and whether the p38-MAPK-mediated phosphorylation of E47 affects its dimerizationwith other MRFs, and hence the expression of thecorresponding target genes.

Regulation of chromatin remodeling activities on

muscle-specific promoters by p38 MAPK

Although all MRFs can bind to the muscle E box withsimilar affinities, their efficiencies in initiating transcrip-tion from normally silent genes differ significantly, withmyogenin being less effective than Myf5 and MyoD. Themain explanation for this might be that MyoD and Myf5,but not myogenin, contain the H/C and helix III domainsendowed with chromatin-remodeling activities (Figure 2).Notably, these two domains of MyoD have been shown tomediate interaction with the transcription factor Pbx onthe myogenin promoter, providing stable binding of MyoDto this promoter [6]. A joint effort between the groups ofTapscott and Imbalzano has subsequently uncovered atwo-step connection between Pbx/MyoD and the chroma-tin remodeling complex SWI/SNF, which might underliethe initiation of myogenin gene transcription. According tothis model, Pbx is constitutively bound to the myogeninpromoter through a Pbx-binding site, located adjacent to anon-canonical E box, and associates with MyoD in a SWI/

pression

interaction with MyoD, contributes to the overall induction of muscle

gene transcription [3,20,22,23,28,29,36]. Finally, p38 also facilitates the

phosphorylation and progression of RNA polymerase II, in a MyoD-

mediated feed-forward circuit [42]. On the myogenin promoter (see

dashed-line box), MyoD–E47 heterodimers have been proposed to

interact with Pbx, through a Pbx-binding site and a non-canonical E

box. Pbx-bound MyoD recruits HATs and the SWI/SNF complex (in a

p38-MAPK-dependent manner), and subsequently MyoD–E47 can

access the canonical E box in the promoter [6,34]. Whether this Pbx-

based model applies to the activation of other muscle genes, in

addition to myogenin, awaits investigation.

In terminal-differentiation stages, the expression of MRF4 is induced

(see also Figure 1). It should be emphasized that MRF4 is the most

abundant MRF in adult muscle tissue. As E47 can heterodimerize with

MRF4, it is proposed that p38 phosphorylation of E47 at Ser140 will

promote the formation of MRF4–E47 heterodimers (as it does with

MyoD–E47 [30]) and the subsequent binding to the E boxes on target

genes. Upon p38 MAPK phosphorylation of Ser31 and Ser42 in the N-

terminal transactivating domain of MRF4, the overall MRF4-mediated

transcription is reduced [26]. While the effect of p38 MAPK on E-

protein–MRF activity can occur throughout myogenesis, the p38-

MAPK–MRF4-mediated repressive mechanism is gene selective and

specifically occurs only at terminal stages of differentiation.


MEF2 E-box

p38

P P

MyoD

IdE47

Id

MEF2 E-box

Pbx

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E-box like

Early myogenesis

Late myogenesis

Proliferation

E-box

E47P

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p38

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E47P

MRF4P P

HATs SWISNF

P

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P

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MEF2P

p38

P P

p38

p38

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SWISNF

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RNA POL II

RNA POL II

RNA POL II


Figure I.


SNF-independent manner. Then, Pbx-associated MyoDrecruits SWI/SNF, which in turn will facilitate directbinding of MyoD to the canonical E box of the myogeninpromoter [6,8,34] (Box 3). Thus, binding sites for cofactorssuch as MEF2 or Pbx might help to expose a crucial E boxor substitute for an E box and facilitate the formation of astable and functional MyoD transcriptional complex[1,8,35]. Given the known ability of p38 MAPK tophosphorylate some of these cofactors (i.e. MEF2) as wellas particular bHLH proteins (i.e. E47, MRF4 – see below),


the regulation of the homo- or heterotypic interactions byp38 MAPK-mediated posttranslational modificationcannot be discarded.

A breakthrough in these studies came from Simoneet al. [36], who showed that, in the absence of p38 MAPKsignaling, the chromatin of the myogenin and MCKpromoters was not remodeled under differentiation-promoting conditions. Most interestingly, pharmacologicalinhibition of p38a and p38b activity prevented theassociation, on these muscle promoters, between MyoD



and the ATPase subunits of the SWI/SNF complex, BRG1and BRM, although neither the acetylation status ofhistones nor the recruitment of p300 and PCAF wereaffected. These results demonstrate a crucial and specificrole for p38 MAPK in the recruitment of SWI/SNF tomuscle gene promoters, providing an additional mechan-ism to account for the positive effect of p38 MAPK inmyogenesis [36]. The SWI/SNF subunit BAF60 could bephosphorylated by p38 MAPK in vitro, although thefunctional relevance has yet to be established. It isnoteworthy, however, that several BAF60 isoforms havebeen implicated as the surface for interactions betweenthe SWI/SNF complex and transcription factors [37,38].Further studies by de la Serna et al. [34] have shown thatBRG1 can interact with both MyoD and Pbx on themyogenin promoter. Taken together, it is reasonable tosuggest that p38 MAPK might control not only the BRG1-based association of SWI/SNF to the myogenin promoterbut also its interactions with acetylated chromatin, MyoDand Pbx (Box 3).

The work by Simone et al. [36] further showed that thep38 MAPK-mediated activation of a chromatin-integratedGal4-responsive promoter by a Gal4–MyoD(N-terminal)fusion protein required BRG1 and BRM. Intriguingly, theGal4–MyoD(N-terminal) neither binds MEF2 nor con-tains any of the domains previously reported to mediateactivation of genes in repressive chromatin [5,20]. Itseems therefore unlikely that regulation of MyoD-depen-dent transcription by the p38-MAPK–SWI/SNF pathwayrelies exclusively on p38-MAPK-mediated phosphoryl-ation of MEF2 or on MyoD–Pbx interactions. Theseresults are in conflict, at least in part, with the model inwhich the interaction of MyoD with Pbx (through theMyoD C/H and helix3 domains) would be necessary forSWI/SNF recruitment [6,34]. As a reconciling alternative,SWI/SNF could interact with the acidic transactivationdomains or other regions of sequence-specific transcrip-tion factors on the target loci [39,40]. Whether the effectsof the p38 MAPK pathway on E47–MyoD heterodimerformation and on the SWI/SNF complex recruitment areexerted exclusively on MyoD-dependent promoters or alsoaffect promoters regulated by other MRFs, and whetherthey are mediated by interactions with Pbx or othercofactors, awaits further investigation.

Regulation of RNA polymerase II recruitment to

muscle-specific promoters by p38 MAPK

Studies from several groups have indicated that p38MAPK might contribute to the recruitment of the activeRNA polymerase II to muscle-specific promoters throughat least three different mechanisms.

First, recruitment of the SWI/SNF complex has beenlinked to the engagement of the active fraction of RNApolymerase II [41]. Accordingly, the inhibition of BRG1/BRM recruitment to muscle promoters by treatment withthe p38a/p38b inhibitor SB203580 correlated withreduced levels of active RNA polymerase II at themyogenin and MCK promoters [36].

Second, a muscle gene transcription circuit initiated byMyoD in association with MEF2 and RNA polymerase II isfacilitated by p38 MAPK [42]. MyoD has been shown to


initiate the expression of specific MEF2 isoforms at theonset of differentiation and to activate (through anunknown mechanism) the p38 MAPK pathway, which inturn will facilitate MyoD and MEF2 binding at genesexpressed late in the myogenic program. Importantly, thebinding of MEF2D has been shown to recruit RNApolymerase II, correlating with the transcription of thesegenes. Conversely, expression of late-activated genescould be advanced by precocious activation of p38 MAPKand expression of MEF2D, demonstrating a mechanismfor temporally patterning muscle gene expression througha MyoD-mediated feed-forward circuit involving p38MAPK [42]. Two distinct roles have been proposed forp38 MAPK in this circuit. On one side, p38 MAPK mightfunction as a rate-limiting factor in promoting the bindingof MEF2D and MyoD to muscle promoters, most likelythrough an effect on chromatin – i.e. through targeting theSWI/SNF complex to muscle loci. Furthermore, p38MAPK might facilitate the progression of RNA polymer-ase II, probably through the phosphorylation of MEF2D.Despite the original nature of this model, the latterassumption is in discrepancy with results from othergroups showing that MEF2D, in particular, is not a goodphosphorylation substrate for p38a and p38b (see Box 2).These differences might be explained by the use ofdifferent experimental approaches, strongly suggestingthe need to reconfirm the phosphorylation of the MEF2isoforms by p38 MAPK in the muscle context. None-theless, the proposal of a MyoD-initiated feed-forwardcircuit involving p38 MAPK and MEF2 is appealing.

Third, the yeast p38 MAPK, Hog1, has been shown tointeract with, and recruit, the RNA polymerase II complexto yeast stress-responsive promoters such as STL1, byassociation with the Hot1 transcription factor [43].

Taken together, these studies demonstrate that p38MAPK plays a role in chromatin remodeling through itsaction on different transcriptionally competent molecules,and these mechanisms might be shared by differentorganisms ranging from mammals to yeast. Moreover,these findings establish a link between differentiation-activated p38 MAPK and recruitment of chromatin-remodeling complexes to transcriptionally active lociduring skeletal myogenesis. This also extends the functionof p38 MAPK beyond its ability to activate gene expressionby direct phosphorylation of transcription factors.

Selective repression of myogenesis by p38 MAPK

A novel and unexpected inhibitory function of p38 MAPKat late stages of myogenesis has been recently reported intwo independent studies. Weston et al. [44] showed thattreatment of primary limb mesenchymal cultures, whichshould differentiate to cartilage, with p38a/p38b inhibi-tors enhances muscle formation rather than promotingchondrogenesis. In addition, G8 and C2C12 muscle cellsco-cultured with the primary limb mesenchymal cells alsoshowed enhanced expression of myogenic markers andmyotube formation upon treatment with the p38 MAPKinhibitors. Likewise, the transcriptional activity ofMEF2–GAL4 fusion proteins expressed in primarycultures was also enhanced by treatment with the p38a/p38b inhibitors [44]. Using a different experimental


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approach, Suelves et al. [26] showed that inhibition of p38MAPK activity at late stages of C2C12 cell differentiationresulted in increased expression of certain skeletal musclegenes. In particular, p38 MAPK phosphorylated in vitroand in vivo Ser31 and Ser42 located in the N-terminaltransactivating domain of MRF4, leading to downregula-tion of its transcriptional activity, which induced arepressive, but selective, effect on the expression of musclegenes during terminal differentiation [26] (Box 3).Recently, elegant studies by Kassar-Duchossoy et al.[45], using Myf5:MyoD double-null mutants whoseMRF4 expression is unaffected, have demonstrated anunexpected role for MRF4 in the early stages of mousemyogenesis, challenging its established role in terminaldifferentiation. Thus, the specific function played byMRF4 during myogenesis is more complex than previouslyanticipated – MyoD and Myf5 seem to be required forcommitment to the myogenic lineage, whereas myogeninplays a role in the expression of the differentiatedphenotype, and MRF4 can partly exert both functions.

Concluding remarks

The formation of skeletal muscle is a well-orchestratedmultistep process controlled by the MRF family oftranscription factors. Several cofactors enhance or repressthe myogenic potential of the MRFs, either directly orindirectly, thus influencing the expression of muscle-specific genes. Phosphorylation and activation of MEF2,a co-activator of the MRF family member MyoD, was formany years the sole explanation for the promyogenic effectof p38 MAPK. However, it is now clear that thestimulation of MyoD-dependent transcription by p38MAPK also involves MEF-2-independent mechanisms.These include the p38-MAPK-mediated phosphorylationof the MyoD dimerization partner E47, which promotesformation of functional MyoD–E47 heterodimers andinitiation of muscle-specific transcription, as well as therecruitment of chromatin-remodeling SWI/SNF activityand RNA polymerase II to muscle gene promoters.

Overall, a new model for myogenic differentiation isemerging in which the MRFs display unique functions andactivate different subsets of muscle genes in a distinctspatial and temporal fashion. In addition to the promyo-genic role of p38 MAPK at early myogenic stages describedabove, an unexpected repressive p38 MAPK function hasalso been identified, which operates selectively at latestages of muscle differentiation.

Although p38 MAPK emerges as a pivotal moleculeorchestrating sequential events in the myogenic pathway,many details of p38-MAPK-induced myogenesis remain tobe elucidated – for example, the relative contribution ofthe four p38 MAPK family members to muscle differen-tiation. Most of the work that demonstrates the require-ment for p38 MAPK in myogenesis is based on the use ofsynthetic compounds such as SB203580, which onlyinhibit the activity of p38a and p38b. Whether differentp38 MAPK family members specifically regulate theexpression of particular subsets of genes, at differentstages of differentiation, and whether they possessinducing or repressing activities, remain to be deter-mined. The identification of new myogenic substrates for


the different p38 MAPK family members as well as theupstream signaling mechanisms will undoubtedlyincrease our understanding of how p38 MAPK regulatesmyogenesis. Finally, it is important to note that the cell-culture studies outlined here require verification usingin vivo models, including the generation of mice withmuscle-specific inactivation of the individual p38 MAPKfamily members. These mice should provide powerfulbiological models to address the regulation of muscleformation by p38 MAPK.

AcknowledgementsWe apologize to the authors whose original work is not included in thereferences owing to space limitations. Work in the authors’ laboratories issupported by the Spanish Ministry of Education and Science (SAF2004–06983, HF2004–0185), the Muscular Dystrophy Association, Marato-TV3and AFM. E.P. was a recipient of a Novartis postdoctoral fellowship.

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Regulation of skeletal muscle gene expression by p38 MAP kinases