expression of extracellular signal-regulated kinase and

Inclusion body myositisExpression of extracellular signal-regulated kinase

and its substrateS Nakano MD PhD A Shinde MD S Kawashima MD PhD S Nakamura MD PhD

I Akiguchi MD PhD and J Kimura MD

Article abstractmdashObjective To assess abnormal intracellular signal transduction in inclusion body myositis (IBM)Background Mitogen-activated protein kinases (MAPKs) play pivotal roles in intracellular signal transduction andregulate cell growth and differentiation Upon their activation MAPKs translocate from the cytoplasm into the nucleusDesignmethods The authors investigated the localization of several forms of the MAPK familymdashextracellular signal-regulated kinase (ERK) c-Jun N-terminal protein kinase (JNK) and p38 MAPK (p38)mdashin 10 patients with sporadic IBMand in 52 control subjects The relationship between the localization of immunopositive deposits and nuclei was testedwith bis-benzimide Results Vacuolated fibers in IBM displayed very strong focal immunoreactivity of ERK but not ofJNK or p38 The ERK-positive deposits in these vacuolated fibers colocalized with the nuclear substrate of ERK Elk-1ERK- and Elk-1ndashpositive deposits were located frequently on the surface of the nuclei in vacuolated fibers in IBM Similarfindings to those of sporadic IBM were observed in three patients with distal myopathy with rimmed vacuoles but not ineight normal or the other 41 disease controls Conclusion There is evidence for impaired molecular transport to thenucleus from the cytoplasm in the vacuolated fibers in IBM This could be due to cytoplasmic aggregation of ERK andElk-1 or to abnormal nuclear pore machinery involved in the transport of ERK and its substrate upon ERK activation

NEUROLOGY 20015687ndash93

Sporadic inclusion body myositis (s-IBM) is the mostcommon muscle disease in patients older than 50years In addition to primary endomysial inflamma-tion muscle biopsies show vacuolated muscle fiberscontaining tubulofilaments in the cytoplasm and nu-clei Conversely hereditary inclusion body myopathy(h-IBM) encompasses hereditary progressive musclediseases with a pathologic process similar to that ofs-IBM except for a lack of lymphocytic inflammation12

It includes myopathy with autosomal dominant inheri-tance of predominantly proximal weakness and autoso-mal recessive quadriceps-sparing myopathy1 Distalmyopathy with rimmed vacuoles which is frequentlyobserved in Japan and the autosomal recessive h-IBMmay be allelic or may be the same disorder34

The vacuolated fibers in IBM express a variety ofproteins that are expressed normally at the neuro-muscular junction and lesions in neurodegenerativediseases5 The ectopic expression of such proteinsshould result from abnormal intracellular events andaltered transcription in IBM Because protein ki-nases play pivotal roles in regulating intracellularsignal transduction and transcription67 studies ofprotein kinases in IBM may help to clarify thechanges of protein expression in this disease Accu-mulation of phosphorylated protein which is immu-noreactive for an antibody directed againstphosphorylated neurofilament proteins (SMI-31) in

vacuolated fibers in IBM8 could indicate abnormalintracellular signal transduction9

Extracellular signal-regulated kinase (ERK) be-longs to the mitogen-activated protein kinase(MAPK) family and plays a central role in transduc-ing extracellular signals to the nucleus After attach-ment to their receptors hormones growth factorsand cytokines evoke several intracellular signaltransduction cascades thereby regulating transcrip-tion in the nucleus610 In one of the major branchesof the cascades these external stimuli induce ty-rosine kinase activity at the cell surface and tran-sient formation of Ras-guanosine 59 triphosphate(GTP) This in turn activates Raf kinase at the mem-brane followed by sequential phosphorylation andactivation of MAPKERK kinase (MEK) and ERKActivated ERK phosphorylates various cytoplasmicmolecules and translocates into the nucleus to phos-phorylate a transcription factor called Elk-1610 Theactivated form of Elk-1 together with serum re-sponse factor (SRF) binds to the serum responseelement (SRE) of the promoter region of immediateearly genes including c-fos (figure 1A)11 Converselyenvironmental stresses and proinflammatory cyto-kines induce other phosphorylation cascades Inthese stress-activated cascades p38 MAPK (p38) andc-Jun N-terminal protein kinase (JNK) two sub-classes of the MAPK family take the equivalent po-sition to ERK in the ERK cascade (see figure 1B)1012

From the Department of Neurology Graduate School of Medicine Kyoto University Kyoto JapanReceived March 24 2000 Accepted in final form October 10 2000Address correspondence to Dr Satoshi Nakano Department of Neurology Graduate School of Medicine Kyoto University 54 Shogoin Kawara-cho Sakyo-kuKyoto 606-8507 Japan e-mail snakanoisolakuhpkyoto-uacjp

Copyright copy 2001 by AAN Enterprises Inc 87

These complicated phosphorylation systems providefor a finely tuned rapid regulation of signals at eachlevel of the cascade with cross-talk with other intra-cellular transduction cascades

We previously reported abnormal focal accumula-tion of CDK5 in vacuolated fibers in IBM9 LikeCDK5 MAPKs belong to the family of proline-directed protein kinases which phosphorylate serineor threonine followed by proline in the amino acidsequences of substrate proteins Furthermore likeCDK5 ERK translocates into the nucleus and posi-tively regulates muscle fiber differentiation1314

These observations prompted us to examine the lo-calization of ERK and its association with the nu-cleus in vacuolated fibers in IBM

Materials and methods Patients Limb muscle speci-mens from 10 patients (aged 43 to 76 years nine men onewoman) with sporadic IBM were studied Each musclespecimen contained endomysial inflammatory cell exu-dates rimmed vacuoles and congophilic inclusions There-fore the diagnosis of each patient was definite IBM1 Fourpatients with oculopharyngeal muscular dystrophy(OPMD) three with distal myopathy with rimmed vacu-oles one with colchicine myopathy15 and one with acidmaltase deficiency were studied as non-IBM vacuolar my-opathies Eight muscle specimens deemed free of neuro-muscular disease were used as normal controls Fourteenpatients with polymyositis eight with dermatomyositisfive with Duchenne muscular dystrophy four with mito-chondrial myopathy and four with neurogenic muscularatrophy served as other disease controls The diagnoseswere based on conventional criteria16 Regenerating fiberswere determined by examination of serial hematoxylinndasheosin (H-E) sections17

Primary antibodies The table lists the characteristicsof the primary antibodies against ERK and other sub-classes of MAPKs and against transcription factors used inthis study and the concentrations at which they were ap-plied The anti-ERK antibody reacts with ERK1 andERK218 The anti-JNK antibody binds to JNK1 and otherisoforms of JNK19 The antindashElk-1 antibody has been wellcharacterized20 SMI-31 was obtained from SternbergerMonoclonals (Baltimore MD) and used at 1000-folddilution

Figure 1 Schematic diagram of the (A) extracellularsignal-regulated kinase (ERK) pathway and the (B) stress-activated mitogen-activated protein kinase (MAPK) path-ways MEK 5 MAPK or ERK kinase Elk-1 5 the nuclearsubstrate of ERK SRF 5 serum response factor SRE 5serum response element MEKKs 5 MEK kinases MKK365 MAPK kinase JNK 5 c-Jun N-terminal protein kinasep38 5 p38 MAPK SEK-1 5 SAPKERK kinase-1 For de-tails see references 10 and 12

Table Primary antibodies against mitogen-activated protein kinases (MAPKs) and transcription factors

Antigen Immunogen Antibody CloneID SourceConcentration

or dilution

ERK C-terminus of ERK1 MMAB ERK-7D8 Zymed (San Francisco CA) 2 mgmL

p38 N-terminus of p38 RPAB sc-728 Santa Cruz (Santa Cruz CA) 5 mgmL

JNK C-terminus of JNK1 RPAB sc-474 Santa Cruz (Santa Cruz CA) 5 mgmL

Elk-1 379 to 392 of Elk-1 RPAB 9182 New England Biolabs (Beverly MA) 175

c-Jun 56 to 67 of c-Jundagger RPAB 9261 New England Biolabs (Beverly MA) 175

Phosphorylated at Ser383dagger Phosphorylated at Ser63

ERK 5 extracellular signal-regulated kinase p38 5 p38 mitogen-activated protein kinase JNK 5 c-Jun N-terminal protein kinaseMMAB 5 mouse monoclonal antibody RPAB 5 rabbit polyclonal antibody

88 NEUROLOGY 56 January (1 of 2) 2001

Immunostaining Consecutive or nonconsecutive 7-mmtransverse cryostat sections were fixed in cold acetone andincubated first in phosphate-buffered saline (PBS) contain-ing 2 bovine serum albumin and 5 nonimmune serumfrom the species in which the secondary antibody wasraised Second the sections were incubated overnight at 4degC in the blocking solution containing the primary antibodyAfter incubation with a biotin-labeled second antibody thesections were developed by the avidinndashbiotin complex (ABC)immunoperoxidase method (Vectastain ABC kit Vector Bur-lingame CA) For double fluorescence staining the sectionswere treated with 1) anti-ERK antibody and antindashElk-1 anti-body or 2) SMI-31 and antindashElk-1 antibody at 4 degC overnightfollowed by incubation with second antibodies consisting ofrhodamine-conjugated goat antimouse immunoglobulin G(IgG) (Protos Immunoresearch San Francisco CA) and fluo-rescein isothiocyanate conjugated (FITC)-labeled swine anti-rabbit IgG (Dako Carpinteria CA) The slides were mountedwith Vectashield (Vector) and viewed with an Olympus pho-tomicroscope (Tokyo Japan) equipped for epifluorescence

Control experiments included omission of the primaryantibody as well as substitution of nonimmune rabbit ormouse IgG in place of the primary antibody

To assess the relationship of ERK- and Elk-1ndashpositivedeposits with nuclei the immunostained sections were fur-ther stained with 1 mgmL Hoechst 33258 (MolecularProbes Eugene OR) a DNA-binding dye The mountedsections were examined under fluorescent microscopy9

Results Localization of MAPKs and Elk-1 in s-IBM Ins-IBM 60 to 96 of vacuolated fibers displayed very

strong vacuolar and cytoplasmic immunoreactivity of ERK(figure 2 A C and E) The ERK-positive deposits wereround or irregular In vacuolated fibers no or few depositspositive for p38 or JNK were observed

Diffuse moderate ERK p38 and JNK immunoreactivitywas observed in regenerating fibers and in some degener-ating fibers

In vacuolated fibers in IBM there were strong Elk-1ndashimmunopositive deposits (figure 3 A C and E) Theirlocalization and contours were identical with those ofERK-positive deposits We confirmed this in a double im-munofluorescence study (figure 4 upper row) We nextexamined the relationship between the Elk-1ndashpositive de-posits and those of SMI-31 a marker of IBM filaments8

The distribution and configuration of the Elk-1ndashpositivedeposits were the same as those of SMI-31 (see figure 4lower row) in vacuolated fibers Although SMI-31 alsostained axons of IM nerves as described previously21

antindashElk-1 did notThere were no immunoreactive deposits for c-Jun the

nuclear substrate of JNK in vacuolated fibers in IBMComparison of ERK- or Elk-1ndashpositive deposits with the

localization of nuclei in vacuolated fibers in s-IBM ERK-or Elk-1ndashpositive deposits were often observed on the ex-ternal surface of the nuclei but were sometimes alsopresent in the cytoplasm unrelated to the nuclear localiza-tion (see figure 2 B D and F and figure 3 B D and F)There were sometimes overlaps of the positive depositsand nuclei In rare fibers protrusions of the positive depos-its into nuclei were observed

A quantitative study of the relationship between ERK-

Figure 2 Extracellular signal-regulated kinase immunostaining (A C E) in vacuolated fibers in inclusion body myositisand nuclear DNA staining with Hoechst 33258 in the same sections (B D F) The bright spots represent nuclei (3680before reduction)

January (1 of 2) 2001 NEUROLOGY 56 89

positive deposits and 275 nuclei in 61 randomly selectedand photographed ERK-positive fibers indicated that782 of the nuclei were closely associated with the depos-its 32 of the 275 nuclei had ERK-positive deposits occu-pying more than half of their area and 750 of the nuclei

were touched penetrated or partially covered by thedeposits

Findings in other vacuolar myopathies Distal myop-athy with rimmed vacuoles As in s-IBM strong focalimmunoreactivity of ERK and Elk-1 but not of p38 JNKor c-Jun were observed in vacuolated fibers (figure 5 Aand B) As in s-IBM 28 of the nuclei in ERK-positivefibers were closely associated with the immunopositivedeposits

Acid maltase deficiency Immunoreactivity for MAPKswas observed on the boundaries of vacuoles (see figure 5C)Elk-1 and c-Jun were negative

Oculopharyngeal muscular dystrophy No ERK-positive dots or few small ERK-positive dots were found invacuolated fibers (see figure 5D)

Colchicine myopathy Focal cytoplasmic deposits con-taining ERK p38 JNK Elk-1 and c-Jun were observed ina fraction of vacuolated and nonvacuolated fibers (see fig-ure 5E)

Findings in other control subjects Specimens withoutpathologic findings were negative or weakly positive forMAPKs and the transcription factors No focal depositswere observed Target formations showed strong activityfor MAPKs Elk-1 and c-Jun (see figure 5F) Regeneratingdegenerating fibers showed positive immunoreactivity forMAPKs and Elk-1 (see figure 5 G and H) These fiberswere variably positive for c-Jun

Discussion Several growth factors and hormoneshave been shown to be associated with the growthand differentiation of muscle cells22 However untilrecently little was known about the consequent in-tracellular mechanisms that finally activate the

Figure 3 Elk-1 immunoreactivity (A C E) and nuclear staining (B D F) in the same sections in vacuolated fibers ininclusion body myositis (3680 before reduction)

Figure 4 Two-color immunofluorescence Localization in thesame sections and fibers Upper row extracellular signal-regulated kinase (orange) and Elk-1 (green) Lower rowSMI-31 (orange) and Elk-1 (green) (3400)


MyoD family of transcription factors which regulatedifferentiation of muscle cells Recent studies haveindicated involvement of MAPKs in the control of themyogenic transcription factors In a study of C2 mus-cle cell cultures ERK was activated during musclefiber terminal differentiation and it positively regu-lated the activity of MyoD while the high JNK activ-ity of myoblasts was downregulated14 Also p38 mayplay a positive role in muscle fiber differentiationearlier than ERK via another myogenic transactiva-tion factor MEF223 Regenerating fibers consist ofreplicating myoblasts and differentiating myo-tubes17 Thus the presence of MAPKs in regenerat-ing fibers in diseased muscles may correlate with thefindings of the cell culture studies Because SRF thepartner of activated Elk-1 in the nucleus (see figure1A) is also essential for muscle fiber differentia-tion24 ERK probably takes part in myogenesis viaphosphorylation of Elk-114

Whereas ERK-positive immunoreactivity was notfound in normal fibers and was diffuse in the cyto-plasm of regenerating fibers very strong immunore-active deposits of ERK were found in vacuolatedfibers in IBM These deposits were round or irregu-lar in shape and were present in the cytoplasm or in

vacuoles A fraction of the ERK-positive deposits wasclosely associated with the nuclei of the vacuolatedfibers and was frequently located on the surface ofthe nuclei The abnormal cytoplasmic condensationof ERK protein may result from an abnormality ofthe ERK molecule itself or of another molecule thatprevents abnormal aggregation of ERK Because thenuclear transcription factor Elk-1 showed similar cy-toplasmic aggregation and perinuclear localizationwe suspect a defect in a chaperone-like molecule in-volved in the folding and nuclear transport of Elk-1and ERK

Both cytoplasmic and nuclear proteins includingElk-1 are synthesized in the endoplasmic reticulumNewly synthesized proteins destined for the nucleusor some activated enzymes such as MAPKs aretransported through the nuclear membrane This im-port is a selective and mediated process in which setsof chaperone molecules vehicle proteins and energysuppliers take part Inhibition of any component ofthe nuclear transport system results in cytoplasmicand perinuclear accumulation of karyophilic pro-teins25 For example heat shock cognate protein 70(HSC70) which belongs to the family of heat shockproteins is a carrier protein in nuclear transport In

Figure 5 Extracellular signal-regulated kinase (ERK) and Elk-1immunolocalization in control sub-jects Positive deposits of (A) ERKand (B) Elk-1 in distal myopathywith rimmed vacuoles (C) Small cir-cular ERK-positive immunoreactivityin acid maltase deficiency (D) ERKimmunostaining in oculopharyngealmuscular dystrophy only a smallpositive dot is observed in the vacu-

oles (E) Cytoplasmic ERK-positive deposits in colchicine myopathy (F) ERK-positive target formations in neurogenicmuscular atrophy (G) ERK and (H) Elk-1 in regenerating fibers in dermatomyositis Elk-1 shows strong nuclear reactiv-ity (3340 before reduction)


human cell cultures cytoplasmic injection of anti-bodies against HSC70 strongly inhibits the nuclearimport of several karyophilic proteins and causestheir cytoplasmic aggregation26 In yeast cells HOG1MAPK translocates into the nucleus upon activationMutations in transport proteins result in impairedcytonuclear transport and cytoplasmic accumulationof HOG1 MAPK27 We found cytoplasmic and perinu-clear accumulation of ERK in vacuolated fibers ofIBM but not of p38 or JNK the two kinases whichshow strong activity in regenerating fibers ERK isthe last MAPK that becomes activated during mus-cle fiber differentiation1423 Therefore the defect thatcauses abnormal ERK accumulation may occur inthe late phase of differentiation after JNK and p38activities have declined Chaperone proteins or heatshock proteins show regulated expression during dif-ferentiation and development28 a chaperone proteinthat is specifically involved in this phase of differen-tiation might be responsible for the abnormality inthe vacuolated fibers in IBM The same abnormalmechanism may also cause the perinuclear accumu-lation of Elk-1

We showed focal deposits of Elk-1 colocalized withSMI-31 immunoreactivity in vacuolated fibers inIBM SMI-31 has been developed for phosphorylatedepitopes of neurofilament proteins It can also reactwith other proteins such as microtubule-associatedprotein (MAP) tau and MAP-229 Because SMI-31stains IBM filaments8 ERK and its target Elk-1 maybe associated with the filaments or might be compo-nents of the filaments Electron microscopic studiesindicated that IBM filaments are largely cytoplas-mic and only occasional nuclei contain those fila-ments Rarely electron micrographs revealed adisruption of nuclear membranes by filamentous in-clusions30 These observations are in accord with theresults of the current study of double staining ofERK- or Elk-1ndashpositive deposits and nuclei in thisstudy 32 of the nuclei in the ERK-positive fibersharbored or were overlaid by immunopositive depos-its and few nuclei were penetrated by the depositsConversely ERK- and Elk-1ndashpositive deposits wereoften larger than nuclei and they sometimes occu-pied almost entire vacuoles Therefore these mole-cules do colocalize with IBM filaments but they mayalso be present in other neighboring regions as hasbeen indicated in SMI-31 immunoreactivity30

In distal myopathy with rimmed vacuoles the lo-calization of ERK and Elk-1 was similar to that seenin s-IBM in vacuolated fibers though the associationof ERK-positive deposits with nuclei was weakerthan that in s-IBM (28 versus 78) Nevertheless arelevant mechanism related to that in s-IBM mayoperate in this disease and possibly in its allelic dis-order autosomal recessive h-IBM34

Acid maltase deficiency showed immunopositivityfor MAPKs on the vacuolar boundaries but not fortheir nuclear substrates The substrates for MAPKson the boundaries could be cytoskeletal proteins

such as dystrophin which is present on the vacuolarboundaries31 and can be phosphorylated by MAPKs32

In vacuolated fibers in OPMD only a few focaldeposits of ERK were observed Therefore the mech-anism of formation of inclusions in OPMD may bedifferent from that in IBM In OPMD a short expan-sion of triplet repeats in the poly(A) bindingprotein-2 gene may cause intranuclear accumulationof tubulofilamentous inclusions33

Colchicine disrupts microtubular networksthereby preventing intracellular vesicular traffick-ing15 Microtubular blocking agents induce stress-activated protein kinases34 Furthermore MAPKsand transcription factors are physically associatedwith microtubules1835 Cytoplasmic accumulation ofMAPKs and transcription factors in colchicine myop-athy may result from a combination of these effects

Target formations showed the same immunoposi-tivity as colchicine myopathy This result suggeststhat MAPKs in the center of the fibers induced bydenervation stress are stagnated in the original posi-tion The impairment of molecular trafficking might bemore proximal in colchicine myopathy and in targetformations than the cytonuclear transport in IBM

In a previous report we showed CDK5-positivedeposits in vacuolated fibers in IBM A high propor-tion of the CDK5-positive deposits abutted on thenucleus like ERK in the current study9 CDK5 tran-siently appears in the nucleus during the terminaldifferentiation and promotes the process13 Thus twoprotein kinases both normally activated and translo-cated into the nucleus during some phase of differen-tiation accumulate in the cytoplasm and around thenuclei in vacuolated fibers in IBM We suspect thatthe induction of ERK and CDK5 is part of the intrin-sic program of muscle fiber differentiation and thatthe abnormally high concentration of these enzymesresults from their aggregation in the cytoplasm andinability to enter the nucleus The study of moleculesinvolved in molecular folding36 and nuclear translo-cation of ERK and CDK5 during differentiationshould clarify the molecular mechanism that under-lies IBM

A recent study showed abnormal expression of aB-crystallin which belongs to the small heat shock pro-tein family in IBM37 Because aB-crystallin mayplay a role during myogenesis38 this molecule mightbe the defective chaperone protein we postulatedabove or it might be induced to compensate for thefunction of another chaperone protein

References1 Griggs RC Askanas V DiMauro S et al Inclusion body myo-

sitis and myopathies Ann Neurol 199538705ndash7132 Mikol J Engel AG Inclusion body myositis In Engel AG

FranzinindashArmstrong C ed Myology 2nd ed New York NYMcGraw-Hill 19941384ndash1398

3 MitranindashRosenbaum S Argov Z Blumenfeld A Seidman CESeidman JG Hereditary inclusion body myopathy maps tochromosome 9p1-q1 Hum Mol Genet 19965159ndash163

4 Nonaka I Murakami N Suzuki Y Kawai M Distal myopathywith rimmed vacuoles Neuromuscul Disord 19988333ndash337


5 Askanas V Engel WK New advances in the understanding ofsporadic inclusion-body myositis and hereditary inclusion-body myopathies Curr Opin Rheumatol 19957486ndash496

6 Hill C Treisman R Transcriptional regulation by extracellu-lar signals mechanisms and specificity Cell 199580199ndash211

7 Hunter T Protein kinases and phosphatases the Yin andYang of protein phosphorylation and signaling Cell 199580225ndash236

8 Askanas V Alvarez RB Mirabella M Engel WK Use of anti-neurofilament antibody to identify paired-helical filaments ininclusion-body myositis Ann Neurol 199639389ndash391

9 Nakano S Akiguchi I Nakamura S Satoi H Kawashima SKimura J Aberrant expression of cyclin-dependent kinase 5 ininclusion body myositis Neurology 1999531671ndash1676

10 Force T Bonventre JV Growth factors and mitogen-activatedprotein kinases Hypertension 199831152ndash161

11 Treisman R Ternary complex factors growth factor regulatedtranscriptional activators Curr Opin Genet Dev 1994496ndash101

12 Kyriakis J Avruch J Sounding the alarm protein kinasecascades activated by stress and inflammation J Biol Chem199627124313ndash24316

13 Lazaro J Kitzmann M Poul MA Vandromme M Lamb NJFernandez A Cyclin dependent kinase 5 cdk5 is a positiveregulator of myogenesis in mouse C2 cells J Cell Sci 19971101251ndash1260

14 Gredinger E Gerber AN Tamir Y Tapscott SJ Bengal EMitogen-activated protein kinase pathway is involved in thedifferentiation of muscle cells J Biol Chem 199827310436ndash10444

15 Shinde A Nakano S Abe M Kohara N Akiguchi I ShibasakiH Accumulation of microtubule-based motor protein in a pa-tient with colchicine myopathy Neurology 2000551414ndash1415

16 Arahata K Engel AG Monoclonal antibody analysis of mono-nuclear cells in myopathies I Quantitation of subsets accord-ing to diagnosis and sites of accumulation and demonstrationand counts of muscle fibers invaded by T cells Ann Neurol198416193ndash208

17 Banker B Engel AG Basic reactions of muscle In Engel AGFranzinindashArmstrong C ed Myology 2nd ed New York NYMcGraw-Hill 1994832ndash888

18 Reszka A Seger R Diltz CD Krebs EG Fischer EH Associa-tion of mitogen-activated protein kinase with the microtubulecytoskeleton Proc Natl Acad Sci USA 1995928881ndash8885

19 Clerk A Sugden PH Cell stress-induced phosphorylation ofATF2 and c-Jun transcription factors in rat ventricular myo-cytes Biochem J 1997325801ndash810

20 Sgambato V Vanhoutte P Pages C et al In vivo expressionand regulation of Elk-1 a target of the extracellular-regulatedkinase signaling pathway in the adult rat brain J Neurosci199818214ndash226

21 Mirabella M Alvarez RB Bilak B Engel WK Askanas VDifference in expression of phosphorylated tau epitopes be-tween sporadic inclusion-body myositis and hereditary

inclusion-body myopathies J Neuropathol Exp Neurol 199655774ndash786

22 Olsen EN Signal transduction pathways that regulate skeletalmuscle gene expression Mol Endocrinol 199371369ndash1378

23 Zetser A Gredinger E Bengal E p38 mitogen-activated pro-tein kinase pathway promotes skeletal muscle differentiationJ Biol Chem 19992745193ndash5200

24 Soulez M Rouviere CG Chafey P et al Growth and differen-tiation of C2 myogenic cells are dependent on serum responsefactor Mol Cell Biol 1996116065ndash6074

25 Goumlrlich D Mattaj IW Nucleocytoplasmic transport Science19962711513ndash1518

26 Imamoto N Matsuoka Y Kurihara T et al Antibodiesagainst 70-kD heat shock cognate protein inhibit mediatednuclear import of karyophilic proteins J Cell Biol 19921191047ndash1061

27 Ferringno P Psoas F Koepp D Saito H Silver PA Regulatednucleocytoplasmic exchange of HOG1 MAPK requires the im-portin homologs NMD5 and XPO1 EMBO J 1998175606ndash5614

28 Heikkila J Heat shock gene expression and development IIAn overview of mammalian and avian developmental systemsDev Genet 19931487ndash91

29 Nukina N Kosik KS Selkoe DJ Recognition of Alzheimerpaired helical filaments by monoclonal neurofilament antibod-ies is due to crossreaction with tau protein Proc Natl Acad SciUSA 1987843415ndash3419

30 Carpenter S Inclusion body myositis a review J NeuropatholExp Neurol 1996551105ndash1114

31 De Bleecker JL Engel AG Winkelmann JC Localization ofdystrophin and spectrin in vacuolar myopathies Am J Pathol19931431200ndash1208

32 Shemanko CS Sanghera JS Milner RE Pelech S MichalakM Phosphorylation of the carboxyl terminal region of dystro-phin by mitogen-activated protein (MAP) kinase Mol CellBiochem 199515263ndash70

33 Brais B Rouleau GA Bouchard JP Fardeau M Tomeacute FMOculopharyngeal muscular dystrophy Semin Neurol 19991959ndash66

34 Wang T-H Wang H-S Ichijo H Giannakou P Foster JS FojoT Wimalasena J Microtubule-interfering agents activatec-Jun N-terminal kinasestress-activated protein kinasethrough both ras and apoptosis signal-regulating kinase path-ways J Biol Chem 19982734928ndash4936

35 Gundersen GG Cook TA Microtubules and signal transduc-tion Curr Opin Cell Biol 19991181ndash94

36 Hendrick JP Hartl FU The role of molecular chaperones inprotein folding FASEB J 199591559ndash1569

37 Banwell BL Engel AG aB-crystallin immunolocalizationyields new insight into inclusion body myositis Neurology2000541033ndash1041

38 Benjamin IJ Shelton J Garry DJ Richardson JA Temporalexpression of the small HSPaB-crystallin in cardiac and skel-etal muscle during mouse development Dev Dyn 199720875ndash84


DOI 101212WNL5618720015687-93 Neurology

S Nakano A Shinde S Kawashima et al substrate

Inclusion body myositis Expression of extracellular signal-regulated kinase and its

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These complicated phosphorylation systems providefor a finely tuned rapid regulation of signals at eachlevel of the cascade with cross-talk with other intra-cellular transduction cascades

We previously reported abnormal focal accumula-tion of CDK5 in vacuolated fibers in IBM9 LikeCDK5 MAPKs belong to the family of proline-directed protein kinases which phosphorylate serineor threonine followed by proline in the amino acidsequences of substrate proteins Furthermore likeCDK5 ERK translocates into the nucleus and posi-tively regulates muscle fiber differentiation1314

These observations prompted us to examine the lo-calization of ERK and its association with the nu-cleus in vacuolated fibers in IBM

Materials and methods Patients Limb muscle speci-mens from 10 patients (aged 43 to 76 years nine men onewoman) with sporadic IBM were studied Each musclespecimen contained endomysial inflammatory cell exu-dates rimmed vacuoles and congophilic inclusions There-fore the diagnosis of each patient was definite IBM1 Fourpatients with oculopharyngeal muscular dystrophy(OPMD) three with distal myopathy with rimmed vacu-oles one with colchicine myopathy15 and one with acidmaltase deficiency were studied as non-IBM vacuolar my-opathies Eight muscle specimens deemed free of neuro-muscular disease were used as normal controls Fourteenpatients with polymyositis eight with dermatomyositisfive with Duchenne muscular dystrophy four with mito-chondrial myopathy and four with neurogenic muscularatrophy served as other disease controls The diagnoseswere based on conventional criteria16 Regenerating fiberswere determined by examination of serial hematoxylinndasheosin (H-E) sections17

Primary antibodies The table lists the characteristicsof the primary antibodies against ERK and other sub-classes of MAPKs and against transcription factors used inthis study and the concentrations at which they were ap-plied The anti-ERK antibody reacts with ERK1 andERK218 The anti-JNK antibody binds to JNK1 and otherisoforms of JNK19 The antindashElk-1 antibody has been wellcharacterized20 SMI-31 was obtained from SternbergerMonoclonals (Baltimore MD) and used at 1000-folddilution

Figure 1 Schematic diagram of the (A) extracellularsignal-regulated kinase (ERK) pathway and the (B) stress-activated mitogen-activated protein kinase (MAPK) path-ways MEK 5 MAPK or ERK kinase Elk-1 5 the nuclearsubstrate of ERK SRF 5 serum response factor SRE 5serum response element MEKKs 5 MEK kinases MKK365 MAPK kinase JNK 5 c-Jun N-terminal protein kinasep38 5 p38 MAPK SEK-1 5 SAPKERK kinase-1 For de-tails see references 10 and 12

Table Primary antibodies against mitogen-activated protein kinases (MAPKs) and transcription factors

Antigen Immunogen Antibody CloneID SourceConcentration

or dilution

ERK C-terminus of ERK1 MMAB ERK-7D8 Zymed (San Francisco CA) 2 mgmL

p38 N-terminus of p38 RPAB sc-728 Santa Cruz (Santa Cruz CA) 5 mgmL

JNK C-terminus of JNK1 RPAB sc-474 Santa Cruz (Santa Cruz CA) 5 mgmL

Elk-1 379 to 392 of Elk-1 RPAB 9182 New England Biolabs (Beverly MA) 175

c-Jun 56 to 67 of c-Jundagger RPAB 9261 New England Biolabs (Beverly MA) 175

Phosphorylated at Ser383dagger Phosphorylated at Ser63

ERK 5 extracellular signal-regulated kinase p38 5 p38 mitogen-activated protein kinase JNK 5 c-Jun N-terminal protein kinaseMMAB 5 mouse monoclonal antibody RPAB 5 rabbit polyclonal antibody





































































































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