Induction of Acetylcholine Receptor Gene Expression by ARIA Requires Activation of Mitogen-activated Protein Kinase
1996; Elsevier BV; Volume: 271; Issue: 33 Linguagem: Inglês
10.1074/jbc.271.33.19752
ISSN1083-351X
AutoresJutong Si, Zhijun Luo, Lin Mei,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoTranscription of genes encoding nicotinic acetylcholine receptor (AChR) subunits (α, β, γ or ϵ, and δ) is highest in nuclei localized to the synaptic region of the muscle, which contributes to maintain a high density of AChRs at the postjunctional membrane. ARIA (AChR inducing activity) is believed to be the trophic factor utilized by motor neurons to stimulate AChR synthesis in the subsynaptic area. To elucidate the signaling mechanism initiated by ARIA, we established stable C2C12 cell lines carrying the nuclear lacZ gene under the control of the mouse ϵ subunit promoter or chicken α subunit promoter. ARIA stimulated tyrosine phosphorylation of erbB proteins in these C2C12 cells within 15 s with a peak at 5 min. Immediately following tyrosine phosphorylation of erbB proteins, mitogen-activated protein (MAP) kinase was activated which occurred within 30 s and peaked at 8 min after ARIA stimulation. Concomitantly, expression of AChR genes was induced by ARIA. ARIA-induced AChR subunit transgene expression was observed only in differentiated myotubes and not in myoblasts, suggesting that downstream signaling component(s) are regulated in a manner dependent on the myogenic program. Inhibition of the MAP kinase activity by using a specific MAP kinase kinase inhibitor or by overexpressing dominant negative mutants of Raf or MAP kinase kinase attenuated or abolished the ARIA-induced activation of AChR α and ϵ subunit gene expression. These results indicate that regulation of AChR gene expression by ARIA in C2C12 cells requires activation of the MAP kinase signaling pathway. Transcription of genes encoding nicotinic acetylcholine receptor (AChR) subunits (α, β, γ or ϵ, and δ) is highest in nuclei localized to the synaptic region of the muscle, which contributes to maintain a high density of AChRs at the postjunctional membrane. ARIA (AChR inducing activity) is believed to be the trophic factor utilized by motor neurons to stimulate AChR synthesis in the subsynaptic area. To elucidate the signaling mechanism initiated by ARIA, we established stable C2C12 cell lines carrying the nuclear lacZ gene under the control of the mouse ϵ subunit promoter or chicken α subunit promoter. ARIA stimulated tyrosine phosphorylation of erbB proteins in these C2C12 cells within 15 s with a peak at 5 min. Immediately following tyrosine phosphorylation of erbB proteins, mitogen-activated protein (MAP) kinase was activated which occurred within 30 s and peaked at 8 min after ARIA stimulation. Concomitantly, expression of AChR genes was induced by ARIA. ARIA-induced AChR subunit transgene expression was observed only in differentiated myotubes and not in myoblasts, suggesting that downstream signaling component(s) are regulated in a manner dependent on the myogenic program. Inhibition of the MAP kinase activity by using a specific MAP kinase kinase inhibitor or by overexpressing dominant negative mutants of Raf or MAP kinase kinase attenuated or abolished the ARIA-induced activation of AChR α and ϵ subunit gene expression. These results indicate that regulation of AChR gene expression by ARIA in C2C12 cells requires activation of the MAP kinase signaling pathway. INTRODUCTIONThe AChRs 1The abbreviations used are: AChR(s)nicotinic acetylcholine receptor(s)ARIAAChR inducing activityDMdifferentiation mediumEGFepidermal growth factorGGFglial growth factorMAP kinasemitogen-activated protein kinaseMBPmyelin basic proteinMEKMAP kinase kinasePAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salinePIphosphatidylinositol. mediate postsynaptic depolarization at the neuromuscular junction. This ligand-gated ion channel is a 250-kDa pentameric complex of four different subunits in a stoichiometry of α(2), β, γ or ϵ, and δ (1Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (904) Google Scholar, 2Duclert A. Changeux J.P. Physiol. Rev. 1995; 75: 339-368Crossref PubMed Scopus (191) Google Scholar). The AChRs are highly enriched at the crests of the postjunctional folds at a concentration of 104 receptors/µm2, which is at least 1,000-fold higher than in the extrasynaptic membrane (3Froehner S.C. Annu. Rev. Neurosci. 1993; 16: 347-368Crossref PubMed Scopus (151) Google Scholar, 4Hall Z.W. Sanes J.R. Cell. 1993; 72 (suppl.): 99-121Abstract Full Text PDF PubMed Scopus (588) Google Scholar). During early development, however, AChRs appear throughout the membrane in myotubes. Innervation of myotubes leads to an increase in AChR synthesis, clustering of AChRs at the synapse, and subsequently to loss of extrasynaptic AChRs (4Hall Z.W. Sanes J.R. Cell. 1993; 72 (suppl.): 99-121Abstract Full Text PDF PubMed Scopus (588) Google Scholar). The adult-type AChR contains an ϵ subunit in place of γ and displays different electrophysiological properties.A description of the molecular mechanisms underlying synapse formation at the neuromuscular junction is far from complete. Synthesis of AChRs is stimulated by ARIA, a factor that was identified initially in chick brain on the basis of its ability to promote AChR synthesis in cultured skeletal muscle cells (5Jessell T.M. Siegel R.E. Fischbach G.D. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5397-5401Crossref PubMed Scopus (172) Google Scholar). This factor is a 42-kDa protein that has sequence homology with the Neu differentiation factor (NDF), heregulin, and glial growth factors (GGF), all of which are recently discovered ligands for erbB receptor tyrosine kinases (6Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (548) Google Scholar, 7Peles E. Bacus S.S. Koski R.A. Lu H.S. Wen D. Ogden S.G. Ben Levy R. Yarden Y. Cell. 1992; 69: 205-216Abstract Full Text PDF PubMed Scopus (476) Google Scholar, 8Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Ben Levy R. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 9Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (677) Google Scholar). ARIA, NDF, heregulin, and GGF are encoded by alternatively spliced transcripts of the same gene (9Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (677) Google Scholar). ARIA is synthesized in motor neurons (6Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (548) Google Scholar) and is thought to be released from motor neurons at the neuromuscular junction. In adult skeletal muscle, ARIA is localized in synaptic basal lamina (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (243) Google Scholar, 11Chu G.C. Moscoso L.M. Sliwkowski M.X. Merlie J.P. Neuron. 1995; 14: 329-339Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 12Goodearl A.D.J. Yee A.G. Sandrock Jr., A.W. Corfas G. Fischbach G.D. J. Cell Biol. 1995; 130: 1423-1434Crossref PubMed Scopus (79) Google Scholar) and remains at original synaptic sites following damage to nerve and muscle (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (243) Google Scholar). The sequence homology between ARIA and heregulins immediately suggested a novel pathway for the regulation of muscle AChR synthesis by motor neurons, i.e. through tyrosine phosphorylation (for review, see Ref. 13Mei L. Si J. Life Sci. 1995; 57: 1459-1466Crossref PubMed Scopus (11) Google Scholar). In fact, ARIA was found to induce tyrosine phosphorylation of 185-kDa proteins in the muscle (14Corfas G. Falls D.L. Fischbach G.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1624-1628Crossref PubMed Scopus (71) Google Scholar). Currently three erbB proteins (erbB2/HER2 (15Bargmann C.I. Hung M.C. Weinberg R.A. Nature. 1986; 319: 226-230Crossref PubMed Scopus (931) Google Scholar, 16King C.R. Kraus M.H. Aaronson S.A. Science. 1985; 229: 974-976Crossref PubMed Scopus (842) Google Scholar), erbB3/HER3 (17Plowman G.D. Whitney G.S. Neubauer M.G. Green J.M. McDonald V.L. Todaro G.J. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4905-4909Crossref PubMed Scopus (323) Google Scholar, 18Kraus M.H. Issing W. Miki T. Popescu N.C. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9193-9197Crossref PubMed Scopus (669) Google Scholar), and erbB4/HER4 (19Plowman G.D. Culouscou J.M. Whitney G.S. Green J.M. Carlton G.W. Foy L. Neubauer M.G. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1746-1750Crossref PubMed Scopus (680) Google Scholar)), of approximately the same molecular mass (∼185 kDa), have been identified in adult skeletal muscle and shown to be tyrosine phosphorylated in response to ARIA (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (243) Google Scholar, 20Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 23: 5842-5848Crossref Scopus (157) Google Scholar). Among them, erbB3 (20Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 23: 5842-5848Crossref Scopus (157) Google Scholar, 21Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (131) Google Scholar) and erbB4 (20Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 23: 5842-5848Crossref Scopus (157) Google Scholar) may be localized at the neuromuscular junction. The mouse C2 muscle cells express erbB2 and erbB3, and little, if any, erbB4 (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (243) Google Scholar). This suggests that one or a combination of these erbB receptor tyrosine kinases may represent the ARIA receptor.The signal transduction pathway initiated by ARIA after activation of the erbB receptor tyrosine kinases has not been elucidated. We established C2C12 cell lines, which stably possessed the nlacZ gene under control of the promoter of the AChR α or ϵ subunit to study the signal transduction mechanism of the ARIA-induced activation of AChR gene expression. In this report, we provide evidence that ARIA-mediated induction of AChR gene expression requires the activation of MAP kinase.DISCUSSIONTo study mechanisms by which ARIA activates AChR gene expression, we have generated stable C2C12 cells which possess the ϵ3500-nlacZ or α-nlacZ transgene. We believe that the C2C12 cells carrying these transgenes are an excellent in vitro model to study the molecular mechanism of AChR gene induction for several reasons. First, the C2C12 muscle cells have been used successfully as an expression system to study transcription of muscle genes and have the unusual capability to activate muscle-specific genes upon fusion (24Blau H.M. Chiu C.P. Webster C. Cell. 1983; 32: 1171-1180Abstract Full Text PDF PubMed Scopus (603) Google Scholar). Second, recent studies demonstrated that the C2C12 muscle cells have the essential elements required for the ARIA-induced activation of AChR gene expression (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (243) Google Scholar). In addition, expression of the ϵ3500-nlacZ and α-nlacZ transgene is spatially restricted in muscle nuclei beneath the motor end plate in transgenic mice (27Sanes J.R. Johnson Y.R. Kotzbauer P.T. Mudd J. Hanley T. Martinou J.C. Merlie J.P. Development. 1991; 113: 1181-1191PubMed Google Scholar), suggesting that these promoters contain the elements necessary to respond to natural cues released from motoneurons. In vitro, ARIA treatment of muscle fibers isolated from transgenic mice carrying ϵ3500-nlacZ transgene induces the AChR ϵ subunit gene expression (11Chu G.C. Moscoso L.M. Sliwkowski M.X. Merlie J.P. Neuron. 1995; 14: 329-339Abstract Full Text PDF PubMed Scopus (126) Google Scholar). We show here that in the C2C12 myotubes stably carrying these transgenes, ARIA was able to induce the AChR α and ϵ gene expression (Fig. 1, Fig. 2). Interestingly, we found that ARIA treatment of the C2C12 myotubes resulted in activation of MAP kinase (Fig. 5) which followed in time the tyrosine phosphorylation of erbB proteins (Fig. 4). Moreover, the ARIA-induced expression of AChR α and ϵ subunit genes was dependent on activation of MAP kinase. Inhibition of MEK or Raf in C2C12 myotubes blocked activation of AChR gene expression by ARIA. These results suggest a machinery for the ARIA-induced activation of AChR gene expression which begins with stimulation of the erbB receptor tyrosine kinases and requires activation of the Raf/MEK/MAP kinase pathway.ARIA induces tyrosine phosphorylation of 185-kDa erbB proteins in chicken (14Corfas G. Falls D.L. Fischbach G.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1624-1628Crossref PubMed Scopus (71) Google Scholar, 21Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (131) Google Scholar) and C2C12 myotubes (Fig. 4, 10). Within seconds of ARIA stimulation, erbB proteins became tyrosine-phosphorylated (Fig. 4). Treatment of chicken myotubes with erbstatin, a specific inhibitor of protein tyrosine kinase, inhibits ARIA-elicited increase in α subunit mRNA (21Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (131) Google Scholar). Furthermore, vanadate, an inhibitor of protein tyrosine phosphatases, potentiates ARIA-induced AChR gene activation in chicken or C2 myotubes (Ref. 21Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (131) Google Scholar and Fig. 3). The close correlation of ARIA-induced tyrosine phosphorylation of the erbB proteins with activation of AChR gene expression suggests that tyrosine phosphorylation is a key step in the ARIA signaling pathway. NDF/heregulin (or ARIA) was initially identified by its ability to activate erbB2; however, it does not interact directly with the erbB2 protein (41Peles E. Ben-Levy R. Tzahar E. Liu N. Wen D. Yarden Y. EMBO J. 1993; 12: 961-971Crossref PubMed Scopus (210) Google Scholar). Its receptor appears to be erbB3 (42Sliwkowski M.X. Schaefer G. Akita R.W. Lofgren J.A. Fitzpatrick V.D. Nuijens A. Fendly B.M. Cerione R.A. Vandlen R.L. Carraway K.L. J. Biol. Chem. 1994; 269: 14661-14665Abstract Full Text PDF PubMed Google Scholar, 43Carraway K.L. Sliwkowski M.X. Akita R. Platko J.V. Guy P.M. Nuijens A. Diamonti A.J. Vandlen R.L. Cantley L.C. Cerione R.A. J. Biol. Chem. 1994; 269: 14303-14306Abstract Full Text PDF PubMed Google Scholar) and/or erbB4 (44Plowman G.D. Green J.M. Culouscou J.M. Carlton G.W. Rothwell V.M. Buckley S. Nature. 1993; 366: 473-475Crossref PubMed Scopus (436) Google Scholar, 45Tzahar E. Levkowitz G. Karunagaran D. Yi L. Peles E. Lavi S. Chang D. Liu N. Yayon A. Wen D. Yarden Y. J. Biol. Chem. 1994; 269: 25226-25233Abstract Full Text PDF PubMed Google Scholar). Since erbB3 has little, if any, tyrosine kinase activity (46Guy P.M. Platko J.V. Cantley L.C. Cerione R.A. Carraway K.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8132-8136Crossref PubMed Scopus (594) Google Scholar), signaling through erbB3 has been postulated to require ligand-stimulated dimerization of erbB3 with another erbB protein or EGF receptor. In human embryonic kidney 293 cells and mammary carcinoma-derived MCF-7 cells, erbB2 forms heterodimers with erbB3 upon heregulin stimulation (47Wallasch C. Weib F.U. Niederfellner G. Jallal B. Issing W. Ullrich A. EMBO J. 1995; 14: 4267-4275Crossref PubMed Scopus (351) Google Scholar). The erbB2 and EGF receptor can associate into an active heterodimeric tyrosine kinase (48Qian X. LeVea C.M. Freeman J.K. Dougall W.C. Greene M.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1500-1504Crossref PubMed Scopus (139) Google Scholar). The C2 cells express erbB2 and erbB3 but not erbB4 (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (243) Google Scholar, 20Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 23: 5842-5848Crossref Scopus (157) Google Scholar), suggesting that the ARIA-triggered signaling in C2 cells is initiated by erbB3. Wright et al. (49Wright J.D. Reuter C.W. Weber M.J. J. Biol. Chem. 1995; 270: 12085-12093Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) have shown that EGF can stimulate a kinase-dead EGF receptor to activate MAP kinase via association with erbB2. In C2C12 cells, presumably, ARIA binds to erbB3 which has little kinase activity and stimulates MAP kinase by interacting with erbB2 or erbB2-related tyrosine kinase.The activation of receptor tyrosine kinases by ligands initiates signal transduction cascades essential for cell proliferation and differentiation. The 21-kDa Ras proteins function as signaling mediators for activated receptor tyrosine kinases (50Marshall M.S. FASEB J. 1995; 9: 1311-1318Crossref PubMed Scopus (271) Google Scholar). Binding of adaptor proteins such as Grb2 and/or Shc to tyrosine-phosphorylated intracellular domains recruits guanine nucleotide exchanger proteins to the receptor complex, which promotes exchange of GTP for GDP and thus activates Ras proteins. There are several consensus binding sites for Grb2 and Shc in erbB2, erbB3, and erbB4 (43Carraway K.L. Sliwkowski M.X. Akita R. Platko J.V. Guy P.M. Nuijens A. Diamonti A.J. Vandlen R.L. Cantley L.C. Cerione R.A. J. Biol. Chem. 1994; 269: 14303-14306Abstract Full Text PDF PubMed Google Scholar). In fact, erbB2 and erbB3 have been shown to bind to Grb2 (51D'souza B. Taylor-Papadimitriou J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7202-7206Crossref PubMed Scopus (134) Google Scholar, 52Segatto O. Pelicci G. Giuli S. Digiesi G. Di Fiore P.P. McGlade J. Pawson T. Pelicci P.G. Oncogene. 1993; 8: 2105-2112PubMed Google Scholar) and Shc (17Plowman G.D. Whitney G.S. Neubauer M.G. Green J.M. McDonald V.L. Todaro G.J. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4905-4909Crossref PubMed Scopus (323) Google Scholar, 51D'souza B. Taylor-Papadimitriou J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7202-7206Crossref PubMed Scopus (134) Google Scholar, 52Segatto O. Pelicci G. Giuli S. Digiesi G. Di Fiore P.P. McGlade J. Pawson T. Pelicci P.G. Oncogene. 1993; 8: 2105-2112PubMed Google Scholar). Overexpression of constitutively active erbB2 in NIH3T3 cells activates Ras by forming complex with Shc/Grb2/Sos (53Xie Y. Pendergast A.M. Hung M.-C. J. Biol. Chem. 1995; 270: 30717-30724Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). NDF has been recently found to stimulate growth and proliferation of mammary epithelial cells, which was accompanied by activation of MAP kinase (36Marte B.M. Graus-Porta D. Jeschke M. Fabbro D. Hynes N.E. Taverna D. Oncogene. 1995; 10: 167-175PubMed Google Scholar). We found that ARIA activated MAP kinase via the Raf/MEK pathway in terminally differentiated C2C12 myotubes, and such an activation is required for ARIA-induced AChR gene expression. This suggests an important role of MAP kinase even in highly differentiated cells. In addition to Raf, Ras also stimulates many signaling proteins including PI 3-kinase, protein kinase C ζ, and MEK kinase (50Marshall M.S. FASEB J. 1995; 9: 1311-1318Crossref PubMed Scopus (271) Google Scholar). Stimulation of cells with EGF results in the tyrosine phosphorylation of erbB3 and the recruitment of PI 3-kinase to this receptor (38Soltoff S.P. Carraway K.L. Prigent S.A. Gullick W.G. Cantley L.C. Mol. Cell. Biol. 1994; 14: 3550-3558Crossref PubMed Scopus (460) Google Scholar). Moreover, NDF activates the p70/p85 S6 kinase pathway in mammary epithelial cells (36Marte B.M. Graus-Porta D. Jeschke M. Fabbro D. Hynes N.E. Taverna D. Oncogene. 1995; 10: 167-175PubMed Google Scholar). However, the inability of wortmannin and rapamycin, well characterized inhibitors of PI 3-kinase and S6 kinase, respectively, to affect the ARIA-induced activation of AChR gene expression in C2C12 myotubes (Fig. 8) suggests that activation of these enzymes is probably not involved in this event.Activated MAP kinase is believed to translocate into nuclei and activate gene expression by phosphorylating respective transcriptional factors (30Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1195) Google Scholar). It is worth noting that ARIA-induced activation of AChR α and ϵ gene expression in the C2C12 cells appeared to be dependent on the state of cell differentiation. Treatment with ARIA had little, if any, effect on the reporter gene expression in ϵ1 myoblasts (Fig. 1) and in α1 myoblasts (data not shown). Only after differentiation into myotubes did the C2C12 cells respond to ARIA with increased expression of the reporter gene. However, ARIA was able to elicit tyrosine phosphorylation of the erbB proteins and activation of MAP kinase in C2C12 myoblasts. Moreover, ARIA-activated MAP kinase appeared to be able to translocate into nuclei in C2C12 myotubes. 3J. Si and L. Mei, unpublished data. These results suggest that downstream signaling component(s) are regulated in a manner dependent on the myogenic program.Following formation of neuromuscular junctions, in addition to the ARIA pathway, motor neurons control the AChR gene transcription also by eliciting AChR-evoked muscle depolarization, which represses AChR gene expression in extrasynaptic nuclei (54Martinou J.C. Merlie J.P. J. Neurosci. 1991; 11: 1291-1299Crossref PubMed Google Scholar; for review, see Refs. 2Duclert A. Changeux J.P. Physiol. Rev. 1995; 75: 339-368Crossref PubMed Scopus (191) Google Scholar and 4Hall Z.W. Sanes J.R. Cell. 1993; 72 (suppl.): 99-121Abstract Full Text PDF PubMed Scopus (588) Google Scholar). The electric activity-dependent repression of AChR gene transcription in extrasynaptic nuclei probably involves activation of protein kinase C, a serine/threonine kinase (55Huang C.-F. Tong J. Schmidt J. Neuron. 1992; 9: 671-678Abstract Full Text PDF PubMed Scopus (70) Google Scholar). Interestingly, it was reported recently that treatment of chick myotubes with phorbol 12-myristate-13-acetate, a stimulator of protein kinase C, blocks heregulin/NDF (or ARIA)-activated AChR α subunit expression, presumably via inhibiting the heregulin-dependent tyrosine phosphorylation of the erbB2 and erbB3 proteins (21Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (131) Google Scholar). These data suggest a coordination among different regulatory mechanisms of AChR gene expression by the ARIA pathway, the myogenic factors, and the electric activity-dependent regulation.In summary, we have shown that ARIA activates MAP kinase in C2C12 myotubes which is required for ARIA-induced activation of AChR gene expression. Activation of MAP kinase occurs after tyrosine phosphorylation of ARIA receptors. Inhibition of MAP kinase activation blocks ARIA-dependent stimulation of AChR gene expression. We propose a pathway for ARIA-induced activation of AChR gene expression which begins with stimulation of the erbB protein tyrosine kinases and requires activation of the Raf/MEK/MAP kinase pathway. The C2C12 cells stably possessing AChR transgenes should prove to be a good model to further elucidate regulatory mechanisms of AChR gene expression. INTRODUCTIONThe AChRs 1The abbreviations used are: AChR(s)nicotinic acetylcholine receptor(s)ARIAAChR inducing activityDMdifferentiation mediumEGFepidermal growth factorGGFglial growth factorMAP kinasemitogen-activated protein kinaseMBPmyelin basic proteinMEKMAP kinase kinasePAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salinePIphosphatidylinositol. mediate postsynaptic depolarization at the neuromuscular junction. This ligand-gated ion channel is a 250-kDa pentameric complex of four different subunits in a stoichiometry of α(2), β, γ or ϵ, and δ (1Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (904) Google Scholar, 2Duclert A. Changeux J.P. Physiol. Rev. 1995; 75: 339-368Crossref PubMed Scopus (191) Google Scholar). The AChRs are highly enriched at the crests of the postjunctional folds at a concentration of 104 receptors/µm2, which is at least 1,000-fold higher than in the extrasynaptic membrane (3Froehner S.C. Annu. Rev. Neurosci. 1993; 16: 347-368Crossref PubMed Scopus (151) Google Scholar, 4Hall Z.W. Sanes J.R. Cell. 1993; 72 (suppl.): 99-121Abstract Full Text PDF PubMed Scopus (588) Google Scholar). During early development, however, AChRs appear throughout the membrane in myotubes. Innervation of myotubes leads to an increase in AChR synthesis, clustering of AChRs at the synapse, and subsequently to loss of extrasynaptic AChRs (4Hall Z.W. Sanes J.R. Cell. 1993; 72 (suppl.): 99-121Abstract Full Text PDF PubMed Scopus (588) Google Scholar). The adult-type AChR contains an ϵ subunit in place of γ and displays different electrophysiological properties.A description of the molecular mechanisms underlying synapse formation at the neuromuscular junction is far from complete. Synthesis of AChRs is stimulated by ARIA, a factor that was identified initially in chick brain on the basis of its ability to promote AChR synthesis in cultured skeletal muscle cells (5Jessell T.M. Siegel R.E. Fischbach G.D. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5397-5401Crossref PubMed Scopus (172) Google Scholar). This factor is a 42-kDa protein that has sequence homology with the Neu differentiation factor (NDF), heregulin, and glial growth factors (GGF), all of which are recently discovered ligands for erbB receptor tyrosine kinases (6Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (548) Google Scholar, 7Peles E. Bacus S.S. Koski R.A. Lu H.S. Wen D. Ogden S.G. Ben Levy R. Yarden Y. Cell. 1992; 69: 205-216Abstract Full Text PDF PubMed Scopus (476) Google Scholar, 8Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Ben Levy R. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 9Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (677) Google Scholar). ARIA, NDF, heregulin, and GGF are encoded by alternatively spliced transcripts of the same gene (9Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (677) Google Scholar). ARIA is synthesized in motor neurons (6Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (548) Google Scholar) and is thought to be released from motor neurons at the neuromuscular junction. In adult skeletal muscle, ARIA is localized in synaptic basal lamina (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. 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A. 1993; 90: 1746-1750Crossref PubMed Scopus (680) Google Scholar)), of approximately the same molecular mass (∼185 kDa), have been identified in adult skeletal muscle and shown to be tyrosine phosphorylated in response to ARIA (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (243) Google Scholar, 20Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 23: 5842-5848Crossref Scopus (157) Google Scholar). Among them, erbB3 (20Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 23: 5842-5848Crossref Scopus (157) Google Scholar, 21Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (131) Google Scholar) and erbB4 (20Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 23: 5842-5848Crossref Scopus (157) Google Scholar) may be localized at the neuromuscular junction. The mouse C2 muscle cells express erbB2 and erbB3, and little, if any, erbB4 (10Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (243) Google Scholar). This suggests that one or a combination of these erbB receptor tyrosine kinases may represent the ARIA receptor.The signal transduction pathway initiated by ARIA after activation of the erbB receptor tyrosine kinases has not been elucidated. We established C2C12 cell lines, which stably possessed the nlacZ gene under control of the promoter of the AChR α or ϵ subunit to study the signal transduction mechanism of the ARIA-induced activation of AChR gene expression. In this report, we provide evidence that ARIA-mediated induction of AChR gene expression requires the activation of MAP kinase.
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