Title: Association of Active Extracellular Signal-Regulated Protein Kinase with Paired Helical Filaments of Inclusion-Body Myositis Muscle Suggests Its Role in Inclusion-Body Myositis Tau Phosphorylation
Abstract: The possible role of extracellular signal-regulated kinase (ERK) in the pathogenesis of inclusion-body myositis (IBM) was investigated by immunostaining the active phosphorylated form of ERK in muscle biopsies of six IBM and 14 control patients. Between 80% and 90% of IBM vacuolated muscle fibers contained well-defined ERK-immunoreactive inclusions, which were co-localized by light microscopy, with phosphorylated tau in 70 to 80% of those fibers. Immunoelectronmicroscopy colocalized ERK to small amorphous tufts adjacent to the muscle fiber paired-helical filaments. Strong ERK immunoreactivity was also present at the postsynaptic domain of all human neuromuscular junctions. Our study suggests 1) that ERK, a signal transducer, might play a role in IBM pathogenesis, including participation in the pathological phosphorylation of IBM tau; and 2) that signal transduction abnormalities may be a component of the IBM pathogenic cascade. Our novel immunolocalization of ERK at the postsynaptic domain of human neuromuscular junctions supports a role in transcription of junctional-protein genes. The ERK localized in nonjunctional regions of IBM fibers may underlie the known pathological up-regulation of junctional proteins there. The possible role of extracellular signal-regulated kinase (ERK) in the pathogenesis of inclusion-body myositis (IBM) was investigated by immunostaining the active phosphorylated form of ERK in muscle biopsies of six IBM and 14 control patients. Between 80% and 90% of IBM vacuolated muscle fibers contained well-defined ERK-immunoreactive inclusions, which were co-localized by light microscopy, with phosphorylated tau in 70 to 80% of those fibers. Immunoelectronmicroscopy colocalized ERK to small amorphous tufts adjacent to the muscle fiber paired-helical filaments. Strong ERK immunoreactivity was also present at the postsynaptic domain of all human neuromuscular junctions. Our study suggests 1) that ERK, a signal transducer, might play a role in IBM pathogenesis, including participation in the pathological phosphorylation of IBM tau; and 2) that signal transduction abnormalities may be a component of the IBM pathogenic cascade. Our novel immunolocalization of ERK at the postsynaptic domain of human neuromuscular junctions supports a role in transcription of junctional-protein genes. The ERK localized in nonjunctional regions of IBM fibers may underlie the known pathological up-regulation of junctional proteins there. Sporadic inclusion-body myositis (IBM) is the most common, debilitating, and progressive muscle disease of patients 55 years and older.1Askanas V Engel WK Sporadic inclusion-body myositis and its similarities to Alzheimer disease brain. Recent approaches to diagnosis and pathogenesis, and relation to aging.Scand J Rheumatol. 1998; 27: 389-405Crossref PubMed Scopus (55) Google Scholar The characteristic features of IBM muscle pathology are: 1) various degrees of mononuclear-cell inflammation, and 2) vacuolar degeneration of muscle fibers, accompanied by intrafiber amyloid β (Aβ) deposits and focal cytoplasmic accumulations of 15. to 21-nm-diameter, paired-helical filaments (PHFs) containing phosphorylated tau, both of which are congophilic.2Askanas V Engel WK Bilak M Alvarez RB Selkoe DJ Twisted tubulofilaments of inclusion body myositis muscle resemble paired helical filaments of Alzheimer brain and contain hyperphosphorylated tau.Am J Pathol. 1994; 14: 177-187Google Scholar, 3Mirabella M Alvarez RB Bilak M Engel WK Askanas V Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.J Neuropathol Exp Neurol. 1996; 55: 774-786Crossref PubMed Scopus (101) Google Scholar The abnormalities within IBM muscle fibers have many similarities to those in the brain of Alzheimer's disease (AD) patients. In addition to congophilia, tau-containing PHFs, and accumulations of Aβ, they include accumulations of two other epitopes of Aβ-precursor protein (AβPP),4Askanas V Alvarez RB Engel WK Beta-amyloid precursor epitopes in muscle fibers of inclusion body myositis.Ann Neurol. 1993; 34: 551-560Crossref PubMed Scopus (140) Google Scholar AβPP mRNA,5Sarkozi E Askanas V Johnson SA Engel WK Alvarez RB Beta-amyloid precursor protein mRNA is increased in inclusion-body myositis muscle.Neuroreport. 1993; 4: 815-818Crossref PubMed Scopus (91) Google Scholar presenilin-1,6Askanas V Engel WK Yang CC Alvarez RB Lee VM Wisniewski T Light and electron microscopic immunolocalization of presenilin 1 in abnormal muscle fibers of patients with sporadic inclusion-body myositis and autosomal-recessive inclusion-body myopathy.Am J Pathol. 1998; 152: 889-895PubMed Google Scholar and several other “Alzheimer characteristic” proteins.1Askanas V Engel WK Sporadic inclusion-body myositis and its similarities to Alzheimer disease brain. Recent approaches to diagnosis and pathogenesis, and relation to aging.Scand J Rheumatol. 1998; 27: 389-405Crossref PubMed Scopus (55) Google Scholar Remaining unknown in both IBM and AD are the factors leading to the abnormal accumulation of various proteins and the molecular pathways responsible for PHF formation in the respective tissues. Oxidative stress, in association with aging, has recently been postulated to be an important component of IBM and AD pathogenesis.1Askanas V Engel WK Sporadic inclusion-body myositis and its similarities to Alzheimer disease brain. Recent approaches to diagnosis and pathogenesis, and relation to aging.Scand J Rheumatol. 1998; 27: 389-405Crossref PubMed Scopus (55) Google Scholar, 7Markesbery WR The role of oxidative stress in Alzheimer disease.Arch Neurol. 1999; 56: 1449-1452Crossref PubMed Scopus (245) Google ScholarIn AD neurons, active extracellular signal-regulated protein kinase (ERK) has been immunocolocalized with both phosphorylated tau8Knowles RB Chin J Ruff CT Hyman BT Demonstration by fluorescence resonance energy transfer of a close association between activated MAP kinase and neurofibrillary tangles: implications for MAP kinase activation in Alzheimer disease.J Neuropathol Exp Neurol. 1999; 58: 1090-1098Crossref PubMed Scopus (58) Google Scholar, 9Perry G Roder H Nunomura A Takeda A Friedlich AL Zhu X Raina AK Holbrook N Siedlak SL Harris PL Smith MA Activation of neuronal extracellular receptor kinase (ERK) in Alzheimer disease links oxidative stress to abnormal phosphorylation.Neuroreport. 1999; 10: 2411-2415Crossref PubMed Scopus (258) Google Scholar and 8-hydroxyguanosine,9Perry G Roder H Nunomura A Takeda A Friedlich AL Zhu X Raina AK Holbrook N Siedlak SL Harris PL Smith MA Activation of neuronal extracellular receptor kinase (ERK) in Alzheimer disease links oxidative stress to abnormal phosphorylation.Neuroreport. 1999; 10: 2411-2415Crossref PubMed Scopus (258) Google Scholar a marker of oxidative damage. Accordingly, ERK was proposed to link oxidative stress to the AD brain pathology.9Perry G Roder H Nunomura A Takeda A Friedlich AL Zhu X Raina AK Holbrook N Siedlak SL Harris PL Smith MA Activation of neuronal extracellular receptor kinase (ERK) in Alzheimer disease links oxidative stress to abnormal phosphorylation.Neuroreport. 1999; 10: 2411-2415Crossref PubMed Scopus (258) Google Scholar In our exploration of the pathogenic cascade leading to IBM, including mechanisms of tau phosphorylation, and the possible influence of oxidative stress, we studied the expression of the active, phosphorylated ERK in IBM muscle biopsies. ERK is a serine/threonine protein kinase that was shown in cell culture to be activated by reactive oxygen species.10Guyton KZ Liu Y Gorospe M Xu Q Holbrook NJ Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury.J Biol Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1138) Google Scholar, 11Bhat NR Zhang P Hydrogen peroxide activation of multiple mitogen-activated protein kinases in an oligodendrocyte cell line: role of extracellular signal-regulated kinase in hydrogen peroxide-induced cell death.J Neurochem. 1999; 72: 112-119Crossref PubMed Scopus (222) Google Scholar In vitro, ERK phosphorylates the same residues of tau that are phosphorylated in AD neurofibrillary tangles.12Drewes G Lichtenberg-Kraag B Doring F Mandelkow EM Biernat J Goris J Doree M Mandelkow E Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state.EMBO J. 1992; 11: 2131-2138Crossref PubMed Scopus (492) Google Scholar, 13Illenberger S Zheng-Fischhofer Q Preuss U Stamer K Baumann K Trinczek B Biernat J Godemann R Mandelkow EM Mandelkow E The endogenous and cell cycle-dependent phosphorylation of tau protein in living cells: implications for Alzheimer's disease.Mol Biol Cell. 1998; 9: 1495-1512Crossref PubMed Scopus (272) Google ScholarMaterials and MethodsPatientsImmunocytochemical studies were performed on sections of diagnostic muscle biopsies obtained from 20 patients with these diagnoses: IBM (n = 6), dermatomyositis (n = 2), polymyositis (n = 2), morphologically nonspecific myopathy (n = 2), amyotrophic lateral sclerosis (n = 3), chronic peripheral neuropathy (n = 1), non-IBM vacuolar myopathy (n = 1), and normal muscle (n = 3). Diagnoses were based on clinical and laboratory investigations, including 18-reaction histochemistry of the muscle biopsy.Light Microscopy ImmunocytochemistryImmunocytochemistry was performed on 10-μm-thick sections of the fresh-frozen biopsies. The sections were either unfixed or prefixed with acetone or 2% paraformaldehyde. Peroxidase-antiperoxidase and immunofluorescence procedures were performed as described.2Askanas V Engel WK Bilak M Alvarez RB Selkoe DJ Twisted tubulofilaments of inclusion body myositis muscle resemble paired helical filaments of Alzheimer brain and contain hyperphosphorylated tau.Am J Pathol. 1994; 14: 177-187Google Scholar, 3Mirabella M Alvarez RB Bilak M Engel WK Askanas V Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.J Neuropathol Exp Neurol. 1996; 55: 774-786Crossref PubMed Scopus (101) Google Scholar, 4Askanas V Alvarez RB Engel WK Beta-amyloid precursor epitopes in muscle fibers of inclusion body myositis.Ann Neurol. 1993; 34: 551-560Crossref PubMed Scopus (140) Google Scholar, 6Askanas V Engel WK Yang CC Alvarez RB Lee VM Wisniewski T Light and electron microscopic immunolocalization of presenilin 1 in abnormal muscle fibers of patients with sporadic inclusion-body myositis and autosomal-recessive inclusion-body myopathy.Am J Pathol. 1998; 152: 889-895PubMed Google Scholar To localize active ERK we used two rabbit polyclonal antibodies (New England Biolabs, Beverly, MA; and Promega, Madison, WI), diluted 1:20 to 1:100. Both antibodies specifically recognize dually phosphorylated epitopes, p-Ser202/p-Tyr204 and p-Ser183/p-Tyr185, on ERK1 and ERK2, respectively (8, 9 and immunoblots provided by the companies). Double-immunofluorescence used rabbit polyclonal antibody against ERK combined with: 1) mouse monoclonal antibody AT8 (Innogenetics, Alharetto, GA), a well-characterized specific marker of PHF tau in both AD and IBM that recognizes dually phosphorylated Ser202/Thr205 epitope (according to the sequence of the longest human tau isoform);3Mirabella M Alvarez RB Bilak M Engel WK Askanas V Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.J Neuropathol Exp Neurol. 1996; 55: 774-786Crossref PubMed Scopus (101) Google Scholar, 14Goedert M Jakes R Vanmechelen E Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205.Neurosci Lett. 1995; 189: 167-169Crossref PubMed Scopus (469) Google Scholar 2) a mouse monoclonal antibody against desmin (Chemicon International, Inc., Temecula, CA), which identifies regenerating (positive) and necrotic (negative) muscle fibers; 3) α-bungarotoxin (α-BT) conjugated to fluorescent marker Alexa (Molecular Probes, Eugene, OR), which binds to the postsynaptic nicotinic acetylcholine receptors at neuromuscular junctions (NMJs); 4) a mouse monoclonal antibody against CD163 (clone Ber-MAC3; DAKO, Carpinteria, CA), thereby recognizing human macrophages; or 5) Hoechst 33342 (Molecular Probes), a DNA-binding dye illuminating nuclei.To block nonspecific binding of antibody to Fc receptors, the sections were preincubated with normal goat serum diluted 1:10. Omission of the primary antibody or its replacement with nonimmune serum or irrelevant antibodies were used to control for specificity, as described.2Askanas V Engel WK Bilak M Alvarez RB Selkoe DJ Twisted tubulofilaments of inclusion body myositis muscle resemble paired helical filaments of Alzheimer brain and contain hyperphosphorylated tau.Am J Pathol. 1994; 14: 177-187Google Scholar, 3Mirabella M Alvarez RB Bilak M Engel WK Askanas V Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.J Neuropathol Exp Neurol. 1996; 55: 774-786Crossref PubMed Scopus (101) Google Scholar, 4Askanas V Alvarez RB Engel WK Beta-amyloid precursor epitopes in muscle fibers of inclusion body myositis.Ann Neurol. 1993; 34: 551-560Crossref PubMed Scopus (140) Google Scholar, 6Askanas V Engel WK Yang CC Alvarez RB Lee VM Wisniewski T Light and electron microscopic immunolocalization of presenilin 1 in abnormal muscle fibers of patients with sporadic inclusion-body myositis and autosomal-recessive inclusion-body myopathy.Am J Pathol. 1998; 152: 889-895PubMed Google ScholarImmunoelectronmicroscopyThis was performed on 10-μm, unfixed frozen sections adhered to the bottom of 35-mm Petri dishes.2Askanas V Engel WK Bilak M Alvarez RB Selkoe DJ Twisted tubulofilaments of inclusion body myositis muscle resemble paired helical filaments of Alzheimer brain and contain hyperphosphorylated tau.Am J Pathol. 1994; 14: 177-187Google Scholar, 3Mirabella M Alvarez RB Bilak M Engel WK Askanas V Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.J Neuropathol Exp Neurol. 1996; 55: 774-786Crossref PubMed Scopus (101) Google Scholar, 4Askanas V Alvarez RB Engel WK Beta-amyloid precursor epitopes in muscle fibers of inclusion body myositis.Ann Neurol. 1993; 34: 551-560Crossref PubMed Scopus (140) Google Scholar, 6Askanas V Engel WK Yang CC Alvarez RB Lee VM Wisniewski T Light and electron microscopic immunolocalization of presenilin 1 in abnormal muscle fibers of patients with sporadic inclusion-body myositis and autosomal-recessive inclusion-body myopathy.Am J Pathol. 1998; 152: 889-895PubMed Google Scholar Active ERK was immunolocalized with a HRP-conjugated secondary antibody, or was doubly-immunolocalized with monoclonal SMI-31 antibody (Sternberger Monoclonals, Baltimore, MD), using two appropriate secondary antibodies conjugated either to 10-nm or 5-nm gold particles.2Askanas V Engel WK Bilak M Alvarez RB Selkoe DJ Twisted tubulofilaments of inclusion body myositis muscle resemble paired helical filaments of Alzheimer brain and contain hyperphosphorylated tau.Am J Pathol. 1994; 14: 177-187Google Scholar, 3Mirabella M Alvarez RB Bilak M Engel WK Askanas V Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.J Neuropathol Exp Neurol. 1996; 55: 774-786Crossref PubMed Scopus (101) Google Scholar, 4Askanas V Alvarez RB Engel WK Beta-amyloid precursor epitopes in muscle fibers of inclusion body myositis.Ann Neurol. 1993; 34: 551-560Crossref PubMed Scopus (140) Google Scholar, 6Askanas V Engel WK Yang CC Alvarez RB Lee VM Wisniewski T Light and electron microscopic immunolocalization of presenilin 1 in abnormal muscle fibers of patients with sporadic inclusion-body myositis and autosomal-recessive inclusion-body myopathy.Am J Pathol. 1998; 152: 889-895PubMed Google Scholar SMI-31 antibody cross-reacts with PHF- phosphorylated tau of both IBM muscle3Mirabella M Alvarez RB Bilak M Engel WK Askanas V Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.J Neuropathol Exp Neurol. 1996; 55: 774-786Crossref PubMed Scopus (101) Google Scholar and AD brain,15Ksiezak-Reding H Dickson DW Davies P Yen SH Recognition of tau epitopes by anti-neurofilament antibodies that bind to Alzheimer neurofibrillary tangles.Proc Natl Acad Sci USA. 1987; 84: 3410-3414Crossref PubMed Scopus (153) Google Scholar, 16Nukina N Kosik KS Selkoe DJ Recognition of Alzheimer paired helical filaments by monoclonal neurofilament antibodies is due to crossreaction with tau protein.Proc Natl Acad Sci USA. 1987; 84: 3415-3419Crossref PubMed Scopus (165) Google Scholar, 17Lichtenberg-Kraag B Mandelkow EM Biernat J Steiner B Schroter C Gustke N Meyer HE Mandelkow E Phosphorylation-dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau.Proc Natl Acad Sci USA. 1992; 89: 5384-5388Crossref PubMed Scopus (178) Google Scholar and it recognizes the dually phosphorylated epitope Ser 396/Ser 404.17Lichtenberg-Kraag B Mandelkow EM Biernat J Steiner B Schroter C Gustke N Meyer HE Mandelkow E Phosphorylation-dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau.Proc Natl Acad Sci USA. 1992; 89: 5384-5388Crossref PubMed Scopus (178) Google ScholarResultsLight Microscopy ImmunocytochemistryIBMIn all IBM biopsies, 80 to 90% of the vacuolated muscle fibers and 10 to 15% of nonvacuolated ones had strong ERK immunoreactivity (IR). ERK-IR was in the form of numerous, well-defined squiggly, linear, or dotty inclusions (Figure 1, A–C, and Figure 2, A and C). Occasional muscle fibers, in addition to the immunoreactive inclusions, had a slight diffuse cytoplasmic ERK-IR. These patterns of immunostaining were present in both unfixed and fixed sections. In unfixed or acetone-fixed sections, ERK-IR in the myonuclei was very weak. Paraformaldehyde fixation significantly increased ERK-IR of the myonuclei in all IBM and control biopsies. In IBM, paraformaldehyde combined with Hoechst counterstaining revealed that only a minority of the ERK-immunoreactive inclusions in abnormal muscle fibers were associated with nuclei (Figure 2, F–I, arrows), most being in the cytoplasm.Figure 2Double-labeled fluorescence. A–I: IBM; ERK-immunoreactive inclusions (A and C) closely co-localize with AT8-immunoreactive inclusions (B, D, and E, double exposure), the latter indicating phosphorylated tau on IBM-PHFs. In some muscle fibers (lower fiber in C), ERK immunoreactivity is much stronger than that of AT8 (lower fiber in D). F–I: Co-localization of ERK with the Hoechst nuclear marker. Only a minority of the ERK-immunoreactive inclusions are associated with nuclei. Original magnification, all ×1,200. J–N: Two normal human NMJs. There is a co-localization of ERK (J–M) with α-bungarotoxin (α-BT. bound to the postsynaptic acetylcholine receptors (K and N). J and K. ERK and α-BT are closely overlapping as indicated by the yellow fluorescence in (L). Original magnification, ×600.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In 70 to 80% of muscle fibers containing ERK-immunoreactive inclusions, there was close co-localization of them with AT8-immunoreactive inclusions (indicating phosphorylated tau on PHFs. Figure 2, A–E). However, in some muscle fibers, ERK-IR was either much more abundant than AT8-IR (Figure 2, C–E, lower fiber), or there was no detectable AT8 immunoreactivity.When primary antibody was omitted or replaced with an irrelevant antibody the above-described immunoreactions were not detectable.Control Muscle BiopsiesNone of the control biopsies contained ERK-immunoreactive inclusions characteristic of the IBM abnormal muscle fibers.At the postsynaptic domain of the neuromuscular junctions in all control and IBM biopsies that contained them, there was strong ERK-IR. Depending on the plane of section, ERK-IR either strictly overlapped with the bound α-BT or occupied a slightly larger territory (Figure 2, J–N). In all myopathies, macrophages invading necrotic muscle fibers had increased ERK-IR (Figure 1, D and E). Occasionally, weak diffuse immunoreactivity was present in the cytoplasm of necrotic, desmin-negative muscle fibers, independently from ERK-immunoreactive macrophages. The cytoplasm of regenerating fibers was negative, but after paraformaldehyde fixation slightly increased ERK-IR was occasionally present in their nuclei.ImmunoelectronmicroscopyIn IBM abnormal muscle fibers, ERK immunolocalized to the clusters of PHFs. When HRP-labeled secondary antibody was used, the HRP reaction product labeled PHFs entirely (Figure 3A). However, gold immunolabeling revealed that ERK was very closely associated with the PHFs but its immunoreactivity was present on very small tufts of amorphous material adjacent to, and sometimes apparently attached to, the PHFs (Figure 3B, arrows). Therefore, it appeared that ERK was not a component of the PHF backbone itself.Figure 3Immunoelectronmicroscopy of ERK in IBM vacuolated muscle fibers. A: Lower-power electronmicrograph of peroxidase reaction demonstrates the dark reaction product (top, left. covering PHFs exclusively and entirely, whereas the adjacent portion of the myofiber (bottom, right) is not immunostained (original magnification, ×26,400). B: Higher-power double-labeled gold immunoelectronmicroscopy illustrating co-localization of ERK (5-nm gold particles) and AT8 (10-nm gold particles). AT8 is present on the PHFs, whereas most of the ERK immunoreactivity is present on very small tufts of amorphous material adjacent to, and often touching, the PHFs but not directly aligned on them (original magnification, ×67,200).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionERK is a member of a family of mitogen-activated protein kinases (MAPKs), which are serine/threonine kinases involved in the intracellular transduction of signals regulating cell proliferation, growth, differentiation, survival, and death.18Widmann C Gibson S Jarpe MB Johnson GL Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human.Physiol Rev. 1999; 79: 143-180Crossref PubMed Scopus (2249) Google Scholar, 19Lewis TS Shapiro PS Ahn NG Signal transduction through MAP kinase cascades.Adv Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar Two isoforms of ERK, ERK1 and ERK2, have ∼90% structural homology and seem to have the same functions.18Widmann C Gibson S Jarpe MB Johnson GL Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human.Physiol Rev. 1999; 79: 143-180Crossref PubMed Scopus (2249) Google Scholar, 19Lewis TS Shapiro PS Ahn NG Signal transduction through MAP kinase cascades.Adv Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar ERK can be activated by many different types of receptors, including receptors for growth factors (eg, receptor tyrosine kinases such as EGFR and PDGFR). Ligand-binding to a growth-factor receptor initiates a sequence of signaling events involving the Ras pathway and kinases Raf and MEK, which lead to phosphorylation activation of ERK.18Widmann C Gibson S Jarpe MB Johnson GL Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human.Physiol Rev. 1999; 79: 143-180Crossref PubMed Scopus (2249) Google Scholar, 19Lewis TS Shapiro PS Ahn NG Signal transduction through MAP kinase cascades.Adv Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar Other receptors, such as cytokine receptors, G-protein-coupled receptors and integrins, initially use somewhat different signaling steps, but subsequently their signals converge on the cascade initiated by Ras. ERK is activated by phosphorylation of its specific threonine and tyrosine residues. Active ERK, which is the form immunolocalized in the present study, phosphorylates cytosolic enzymes and cytoskeletal proteins, including tau, and also translocates to the nucleus, where it phosphorylates transcription factors that influence expression of various genes.18Widmann C Gibson S Jarpe MB Johnson GL Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human.Physiol Rev. 1999; 79: 143-180Crossref PubMed Scopus (2249) Google Scholar, 19Lewis TS Shapiro PS Ahn NG Signal transduction through MAP kinase cascades.Adv Cancer Res. 1998; 74: 49-139Crossref PubMed Google ScholarIn a various cell types, ERK was shown to be activated by oxidative stress. Reactive oxygen species are thought to activate ERK through modification of the physiological ERK-activation pathways.10Guyton KZ Liu Y Gorospe M Xu Q Holbrook NJ Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury.J Biol Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1138) Google Scholar For example, Ras, a physiological ERK inducer, can be activated by nitric oxide through nitrotyrosylation of one of its cysteine residues.20Lander HM Hajjar DP Hempstead BL Mirza UA Chait BT Campbell S Quilliam LA A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction.J Biol Chem. 1997; 272: 4323-4326Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar Although activation of ERK protects various cells in culture from oxidant-mediated death,10Guyton KZ Liu Y Gorospe M Xu Q Holbrook NJ Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury.J Biol Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1138) Google Scholar in other cells ERK promotes death induced by reactive oxygen species.11Bhat NR Zhang P Hydrogen peroxide activation of multiple mitogen-activated protein kinases in an oligodendrocyte cell line: role of extracellular signal-regulated kinase in hydrogen peroxide-induced cell death.J Neurochem. 1999; 72: 112-119Crossref PubMed Scopus (222) Google ScholarIn the present study, we demonstrated accumulation of active ERK in the IBM abnormal muscle fibers, in activated muscle macrophages, and at the postsynaptic domain of normal human neuromuscular junctions.The anti-ERK antibodies used in our studies specifically recognize the double-phosphorylated, active form of ERK. In the IBM muscle fibers, at the light-microscopic level ERK-immunoreactive cytoplasmic inclusions were co-labeled with antibodies against phosphorylated tau-containing PHFs. Our immunoelectronmicroscopy with the peroxidase technique demonstrated a high concentration of ERK in the large clusters of PHFs, but the gold technique showed ERK to be in small amorphous tufts among, and often touching, PHFs, but not directly aligned on the PHFs themselves. That appearance suggested that ERK may be associated with formation of nascent PHFs in the tufts, which would be harmonious with the known ability of ERK to phosphorylate tau, which is considered critical to the transformation of tau into PHFs.12Drewes G Lichtenberg-Kraag B Doring F Mandelkow EM Biernat J Goris J Doree M Mandelkow E Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state.EMBO J. 1992; 11: 2131-2138Crossref PubMed Scopus (492) Google Scholar, 13Illenberger S Zheng-Fischhofer Q Preuss U Stamer K Baumann K Trinczek B Biernat J Godemann R Mandelkow EM Mandelkow E The endogenous and cell cycle-dependent phosphorylation of tau protein in living cells: implications for Alzheimer's disease.Mol Biol Cell. 1998; 9: 1495-1512Crossref PubMed Scopus (272) Google Scholar The phosphoepitopes of tau phosphorylated by ERK in vitro are the same ones that are recognized by the AT8 and SMI-31 antibodies12Drewes G Lichtenberg-Kraag B Doring F Mandelkow EM Biernat J Goris J Doree M Mandelkow E Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state.EMBO J. 1992; 11: 2131-2138Crossref PubMed Scopus (492) Google Scholar, 13Illenberger S Zheng-Fischhofer Q Preuss U Stamer K Baumann K Trinczek B Biernat J Godemann R Mandelkow EM Mandelkow E The endogenous and cell cycle-dependent phosphorylation of tau protein in living cells: implications for Alzheimer's disease.Mol Biol Cell. 1998; 9: 1495-1512Crossref PubMed Scopus (272) Google Scholar used by us to co-localize phosphorylated tau (on PHFs) with ERK, by light microscopy and gold immunoelectronmicroscopy. In some IBM fibers, ERK-IR was either more abundant than that of phosphorylated tau, or there was no detectable tau-IR, suggesting that over-expression of ERK may precede tau phosphorylation. Therefore, our results suggest that in IBM muscle ERK may participate in the pathological phosphorylation of PHF tau. Accordingly, activation of ERK in IBM may be at least one of the underlying causes of PHF formation.The ERK-immunoreactive inclusions in IBM vacuolated muscle fibers do not reflect macrophages, because our previous study21Askanas V Bilak M Engel WK Alvarez RB Tome F Leclerc A Prion protein is abnormally accumulated in inclusion-body myositis.Neuroreport. 1993; 5: 25-28Crossref PubMed Scopus (56) Google Scholar using double-labeling with a macrophage-specific marker, and the present study using the macrophage marker anti-CD163, demonstrated that virtually none of the IBM vacuolated muscle fibers contained invading macrophages.Visualization of the nu