Protein Kinase C and Calcium/Calmodulin-activated Protein Kinase II (CaMK II) Suppress Nicotinic Acetylcholine Receptor Gene Expression in Mammalian Muscle
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m109864200
ISSN1083-351X
AutoresPeter Macpherson, Tatiana Y. Kostrominova, Huibin Tang, Daniel Goldman,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoNicotinic acetylcholine receptor (nAChR) gene expression is regulated by both muscle activity and increased intracellular calcium. This regulation is an important developmental event that rids receptors from the extrajunctional region of the developing muscle fiber. In avian muscle, it has been proposed that muscle activity suppresses nAChR gene expression via calcium-activated protein kinase C (PKC)-dependent phosphorylation of the myogenic transcription factor, myogenin. Here, we examined the role that PKC and other kinases play in mediating calcium- and activity-dependent suppression of nAChR genes in rat primary myotubes. We found that although activated PKC could regulate nAChR promoter activity and transiently suppressed both nAChR and myogenin gene expression, it did not appear to be required for calcium- or activity-dependent control of nAChR gene expression in mammalian muscle. Neither depletion of PKC from myotubes nor specific pharmacological inhibition of PKC blocked the suppression of nAChR gene expression produced by calcium or muscle depolarization. In contrast, we provide evidence that calcium/calmodulin-activated protein kinase II participates in mediating the effects of muscle depolarization on nAChR and myogenin gene expression. Nicotinic acetylcholine receptor (nAChR) gene expression is regulated by both muscle activity and increased intracellular calcium. This regulation is an important developmental event that rids receptors from the extrajunctional region of the developing muscle fiber. In avian muscle, it has been proposed that muscle activity suppresses nAChR gene expression via calcium-activated protein kinase C (PKC)-dependent phosphorylation of the myogenic transcription factor, myogenin. Here, we examined the role that PKC and other kinases play in mediating calcium- and activity-dependent suppression of nAChR genes in rat primary myotubes. We found that although activated PKC could regulate nAChR promoter activity and transiently suppressed both nAChR and myogenin gene expression, it did not appear to be required for calcium- or activity-dependent control of nAChR gene expression in mammalian muscle. Neither depletion of PKC from myotubes nor specific pharmacological inhibition of PKC blocked the suppression of nAChR gene expression produced by calcium or muscle depolarization. In contrast, we provide evidence that calcium/calmodulin-activated protein kinase II participates in mediating the effects of muscle depolarization on nAChR and myogenin gene expression. nicotinic acetylcholine receptors tetrodotoxin protein kinase C calcium/calmodulin-dependent protein kinase II phorbol 12-myristate 13-acetate glyceraldehyde-3-phosphate dehydrogenase chloramphenicol acetyltransferase myogenin extracellular signal-regulated kinase Nicotinic acetylcholine receptors (nAChRs)1 mediate communication between motor neurons and skeletal muscle. They are ligand-gated ion channels that are composed of four different subunits with a stoichiometry of α2βγ(ɛ)δ. The nerve plays an important role in regulating the expression and distribution of nAChRs along the surface of the muscle fiber (reviewed in Ref. 1Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1239) Google Scholar). Prior to muscle innervation or after denervation, nAChRs are expressed throughout the surface membrane. In contrast, after innervation, these receptors are localized to the neuromuscular junction. The process of receptor localization involves both neurotrophic influences and nerve-elicited muscle depolarization. Neuronal secretion of agrin and acetylcholine receptor-inducing activity result in receptor clustering and subsynaptic nuclear expression of nAChR genes, whereas muscle depolarization results in suppression of nAChR genes in extrajunctional nuclei. Depolarization-dependent suppression of nAChR gene expression has been attributed to increases in intracellular calcium (2Klarsfeld A. Laufer R. Fontaine B. Devillers-Thiery A. Dubreuil C. Changeux J.P. Neuron. 1989; 2: 1229-1236Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 3Adams L. Goldman D. J. Neurobiol. 1998; 35: 245-257Crossref PubMed Scopus (21) Google Scholar). When skeletal muscle is made inactive by denervation or pharmacological treatment with drugs such as the sodium channel blocker tetrodotoxin (TTX), calcium concentrations remain low (4Huang C.-F. Flucher B.E. Schmidt M.M. Stroud S.K. Schmidt J. Neuron. 1994; 13: 167-177Abstract Full Text PDF PubMed Scopus (40) Google Scholar), and extrajunctional expression of the nAChR genes is increased dramatically (5Merlie J.P. Isenberg K.E. Russell S.D. Sanes J.R. J. Cell Biol. 1984; 99: 332-335Crossref PubMed Scopus (128) Google Scholar, 6Goldman D. Brenner H.R. Heinemann S. Neuron. 1988; 1: 329-333Abstract Full Text PDF PubMed Scopus (172) Google Scholar). In contrast, when denervated muscle is electrically stimulated, intracellular calcium concentrations are elevated (4Huang C.-F. Flucher B.E. Schmidt M.M. Stroud S.K. Schmidt J. Neuron. 1994; 13: 167-177Abstract Full Text PDF PubMed Scopus (40) Google Scholar), and receptor expression is suppressed (6Goldman D. Brenner H.R. Heinemann S. Neuron. 1988; 1: 329-333Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 7Huang C.-F. Tong J. Schmidt J. Neuron. 1992; 9: 671-678Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 8Witzemann V. Brenner H.R. Sakmann B. J. Cell Biol. 1991; 114: 125-141Crossref PubMed Scopus (209) Google Scholar). Similarly, receptor expression is suppressed in TTX-treated myotubes when they are exposed to calcium-elevating drugs (2Klarsfeld A. Laufer R. Fontaine B. Devillers-Thiery A. Dubreuil C. Changeux J.P. Neuron. 1989; 2: 1229-1236Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 3Adams L. Goldman D. J. Neurobiol. 1998; 35: 245-257Crossref PubMed Scopus (21) Google Scholar, 9Klarsfeld A. Changeux J.P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4558-4562Crossref PubMed Google Scholar, 10Walke W. Staple J. Adams L. Gnegy M. Chahine K. Goldman D. J. Biol. Chem. 1994; 269: 19447-19456Abstract Full Text PDF PubMed Google Scholar). Although muscle depolarization and increases in intracellular calcium can initiate the process of nAChR suppression in extrajunctional nuclei, the signal transduction pathways involved in these processes remain controversial. Experiments performed in avian muscle have implicated protein kinase C (PKC) as the primary mediator of activity-dependent, calcium-induced suppression of nAChR gene expression (2Klarsfeld A. Laufer R. Fontaine B. Devillers-Thiery A. Dubreuil C. Changeux J.P. Neuron. 1989; 2: 1229-1236Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 7Huang C.-F. Tong J. Schmidt J. Neuron. 1992; 9: 671-678Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 11Altiok N. Changeux J.-P. FEBS Lett. 2001; 487: 333-338Crossref PubMed Scopus (11) Google Scholar). The proposed model of suppression involves depolarization-dependent activation of a calcium- and phospholipid-dependent PKC (4Huang C.-F. Flucher B.E. Schmidt M.M. Stroud S.K. Schmidt J. Neuron. 1994; 13: 167-177Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 12Mendelzon D. Changeux J.P. Nghiem H.O. Biochemistry. 1994; 33: 2568-2575Crossref PubMed Scopus (32) Google Scholar). Activated PKC is proposed to phosphorylate myogenin (12Mendelzon D. Changeux J.P. Nghiem H.O. Biochemistry. 1994; 33: 2568-2575Crossref PubMed Scopus (32) Google Scholar, 13Li L. Zhou J. James G. Heller-Harrison R. Czech M.P. Olson E.N. Cell. 1992; 71: 1181-1194Abstract Full Text PDF PubMed Scopus (282) Google Scholar), a basic helix-loop-helix myogenic transcription factor that mediates high level nAChR gene expression in inactive muscle (14Neville C.M. Schmidt M. Schmidt J. Cell. Mol. Neurobiol. 1992; 12: 511-527Crossref PubMed Scopus (63) Google Scholar, 15Kostrominova T.Y. Macpherson P.C.D. Carlson B.M. Goldman D. Am. J. Physiol. 2000; 279: R179-R188Google Scholar, 16Berberich C. Durr I. Koenen M. Witzemann V. Eur. J. Biochem. 1993; 216: 395-404Crossref PubMed Scopus (40) Google Scholar, 17Durr I. Numberger M. Berberich C. Witzemann V. Eur. J. Biochem. 1994; 224: 353-364Crossref PubMed Scopus (25) Google Scholar, 18Gundersen K. Rabben I. Klocke B.J. Merlie J.P. Mol. Cell. Biol. 1995; 15: 7127-7134Crossref PubMed Scopus (39) Google Scholar). This phosphorylation abrogates myogenin binding to target E-box sequences that regulate nAChR promoter activity (12Mendelzon D. Changeux J.P. Nghiem H.O. Biochemistry. 1994; 33: 2568-2575Crossref PubMed Scopus (32) Google Scholar, 13Li L. Zhou J. James G. Heller-Harrison R. Czech M.P. Olson E.N. Cell. 1992; 71: 1181-1194Abstract Full Text PDF PubMed Scopus (282) Google Scholar), resulting in reduced nAChR gene expression. Although there is ample evidence that PKC participates in mediating the effects of muscle activity on nAChR gene expression in birds (2Klarsfeld A. Laufer R. Fontaine B. Devillers-Thiery A. Dubreuil C. Changeux J.P. Neuron. 1989; 2: 1229-1236Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 7Huang C.-F. Tong J. Schmidt J. Neuron. 1992; 9: 671-678Abstract Full Text PDF PubMed Scopus (70) Google Scholar,11Altiok N. Changeux J.-P. FEBS Lett. 2001; 487: 333-338Crossref PubMed Scopus (11) Google Scholar), there is little evidence supporting this regulatory mechanism in mammalian muscle (10Walke W. Staple J. Adams L. Gnegy M. Chahine K. Goldman D. J. Biol. Chem. 1994; 269: 19447-19456Abstract Full Text PDF PubMed Google Scholar). Moreover, although previous experiments have suggested that a phorbol ester-responsive PKC mediates nAChR gene expression by muscle depolarization in chick muscle, recent experiments have not supported these data and suggest that an atypical PKC may be involved (11Altiok N. Changeux J.-P. FEBS Lett. 2001; 487: 333-338Crossref PubMed Scopus (11) Google Scholar). Therefore, even in chick muscle, the mechanism by which muscle activity suppresses nAChR gene expression remains unclear. We recently showed that the rat muscle nAChR δ-subunit gene promoter is robustly regulated by calcium/calmodulin-dependent protein kinase II (CaMK II) activity (19Tang H. Sun Z. Goldman D. J. Biol. Chem. 2001; 276: 26057-26065Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). CaMK II activity increases upon muscle depolarization and reduces binding of a myogenin-containing complex to the 47-bp activity-dependent enhancer of the δ-subunit gene. Furthermore, overexpression of a dominant-negative CaMK II in contracting primary rat myotubes increased nAChR δ-subunit promoter activity (19Tang H. Sun Z. Goldman D. J. Biol. Chem. 2001; 276: 26057-26065Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). These data suggest that CaMK II may participate in activity-dependent suppression of nAChR gene expression in mammalian muscle. However, the above studies did not determine whether other nAChR subunit genes are also regulated by CaMK II and whether this enzymatic activity is solely responsible for nAChR gene suppression by muscle depolarization. To further evaluate the role that myogenin, PKC, and CaMK II play in regulating mammalian nAChR gene expression by muscle activity and calcium, we have employed a sensitive RNase protection assay for nAChR hnRNA. This assay allows for analysis of rapid changes (3–6 h) in gene expression that may be missed using more conventional mRNA assays and gene transfection studies. These experiments revealed that active PKC can suppress both nAChR and myogenin gene expression in mammalian muscle. However, nAChR gene suppression produced by either calcium-elevating drugs or electrical stimulation did not require PKC activity. This result contrasts with that reported for chick muscle, where PKC enzymatic activity is required for depolarization-dependent gene suppression (2Klarsfeld A. Laufer R. Fontaine B. Devillers-Thiery A. Dubreuil C. Changeux J.P. Neuron. 1989; 2: 1229-1236Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 7Huang C.-F. Tong J. Schmidt J. Neuron. 1992; 9: 671-678Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 11Altiok N. Changeux J.-P. FEBS Lett. 2001; 487: 333-338Crossref PubMed Scopus (11) Google Scholar). Instead, we found that CaMK II activity contributes to the effects of muscle depolarization on nAChR gene expression. In addition, our data suggest that decreased nAChR gene expression caused by muscle depolarization and sustained increases in intracellular calcium is mediated by different signal transduction cascades. Rat primary myoblasts were isolated as described previously (20Goldman D. Carlson B.M. Staple J. Neuron. 1991; 7: 649-658Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Cells were plated on 35-mm collagen-coated culture dishes at a density of 106/ml. Proliferating myoblasts were grown at 37 °C and 8% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 10% horse serum. Between 48 and 72 h post-plating, cultures became confluent, and the medium was adjusted to 5% horse serum to induce myotube formation. At this time, cells were treated with 3 μg/ml cytosine arabinoside for 48 h to inhibit fibroblast proliferation. All primary myotube cultures were treated with 2 μg/ml TTX from the time of myotube formation. With the exception of TTX, pharmacological reagents were added to myotubes between 4 and 6 days after myotube formation. TTX was obtained from Oretek, Inc. (Fremont, CA) and dissolved in phosphate-buffered saline (2 μg/ml). All other drugs were purchased from Sigma or Calbiochem and prepared as stock solutions in Me2SO. The final drug concentrations used in our experiments were as follows: KN-93, 5 μm; A23187, phorbol 21-myristate 13-acetate (PMA), and ryanodine, 1 μm; Go6983, 600 nm; GF109203X, 250 nm; thapsigargin, 100 nm; and staurosporine, 20 nm. Stock solutions were between 500- and 1000-fold concentrated and stored frozen at −20 °C. Treatment of myotubes with 0.2% Me2SO had no effect on either myogenin or nAChR RNA or on cell morphology. For experiments in which myotubes were electrically stimulated, cultures were rinsed twice with TTX-free medium and then returned to the incubator for ∼1 h before commencing with the stimulation protocol. Myotubes were electrically stimulated to contract for up to 24 h using conditions described previously (21Chahine K.G. Walke W. Goldman D. Development. 1992; 115: 213-219Crossref PubMed Google Scholar). Data are presented for myotubes that were electrically stimulated for 6 h. Total RNA was isolated by homogenizing cell cultures in Trizol (Invitrogen), followed by the single-step purification method described in the manufacturer's protocol. Antisense probes used to detect myogenin and nAChR α-, γ-, and ɛ-subunit RNAs were the same as those described by Chahineet al. (22Chahine K.G. Baracchini E. Goldman D. J. Biol. Chem. 1993; 268: 2893-2898Abstract Full Text PDF PubMed Google Scholar). RNase protection assays were carried out as previously described (3Adams L. Goldman D. J. Neurobiol. 1998; 35: 245-257Crossref PubMed Scopus (21) Google Scholar). The probe for the nAChR α-subunit contains 240 nucleotides of exon 8 flanked by ∼310 nucleotides of intron on the 5′-end and 50 nucleotides of intron on the 3′-end. Consequently, measures of the full-length protected probe reflected changes in the nAChR α-subunit hnRNA, whereas measures of the 240-nucleotide fragment reflected changes in the nAChR α-subunit mRNA. The probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Ambion Inc. (Austin, TX). GAPDH probes were included in each experiment and served to normalize for differences in the amount of RNA in each of the samples. GAPDH was chosen for normalization because it was not regulated by any of the conditions employed in this report (7Huang C.-F. Tong J. Schmidt J. Neuron. 1992; 9: 671-678Abstract Full Text PDF PubMed Scopus (70) Google Scholar). 2P. Macpherson and D. Goldman, unpublished data. RNase-resistant hybrids were analyzed on 8 m urea and 6% polyacrylamide gels. After electrophoresis, gels were dried and exposed to x-ray film. Probe signals were quantified by scanning densitometry, and values were normalized to the RNA signal obtained for GAPDH. The specificity of the protected bands was confirmed by hybridizing probes to tRNA, resulting in no protected fragments on the gel. Probe integrity was monitored for each experiment by running an aliquot of non-hybridized probe on each gel. Cytosolic and membrane fractions from cultured myotubes were prepared by scraping cells from the dishes in homogenization buffer (10 mmTris-HCl (pH 7.4), 10 mm MgCl2, 150 mm NaCl, 1 mm β-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, and 1 μmeach leupeptin and pepstatin A). Cells were sheared by passage through a 26.5-gauge needle and centrifuged at 100,000 × g for 1 h. The resulting supernatants were collected (cytosolic fraction), and pellets (membrane fraction) were solubilized in SDS-containing buffer (20 mm Tris-HCl (pH 6.8), 4% (w/v) SDS, 1 mm phenylmethylsulfonyl fluoride, and 1 μm each leupeptin and pepstatin A). Protein concentrations were determined using the Bio-Rad DC protein assay. Protein samples were subjected to SDS-PAGE (10%) and transferred electrophoretically to Immobilon-P membranes (Millipore Corp., Bedford, MA). Gels with identical samples were stained with Coomassie Brilliant Blue and used as an additional control for equilibration of protein loading. After transfer, Immobilon-P membranes were blocked in Blotto buffer containing 5% dry milk in phosphate-buffered saline and 0.2% Tween 20 and then incubated overnight at 4 °C with mouse anti-myogenin monoclonal antibody (clone F5D; obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) or with rabbit anti-phospho-PKCα/β polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA). Immunodetection was done using peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) with subsequent chemiluminescence detection (ECL, Amersham Biosciences). Band intensity was quantified by scanning densitometry. A constitutively active PKCα isoform was created by deleting its inhibitory domain as previously described (23James G. Olson E. J. Cell Biol. 1992; 116: 863-874Crossref PubMed Scopus (82) Google Scholar). pδ-47MEKLuc contains the 47-bp activity-dependent enhancer of the nAChR δ-subunit gene upstream of the minimal enkephalin promoter (24Walke W. Xiao G. Goldman D. J. Neurosci. 1996; 16: 3641-3651Crossref PubMed Google Scholar). pCMVCAT, which harbors the chloramphenicol acetyltransferase (CAT) gene downstream of the cytomegalovirus promoter, was used for normalization. The pCS2Gal4 plasmid, containing the Gal4 DNA-binding domain downstream of the cytomegalovirus promoter, was a kind gift of Dr. Turner (University of Michigan). The pCS2Gal4Mgn plasmid, containing full-length rat myogenin (Mgn) cDNA fused to the Gal4 DNA-binding domain, was made by subcloning myogenin into the EcoRI/XbaI sites of the pCS2Gal4 plasmid. Expression of full-length myogenin using this plasmid was confirmed by Western blotting with anti-myogenin antibody (clone F5D). The pGal4TKLuc reporter plasmid harbors four tandem repeats of the Gal4 DNA-binding sequence upstream of the minimal thymidine kinase promoter driving luciferase expression. Primary embryonic rat muscle cell cultures (80–90% confluence) in 35-mm dishes were transfected with 1.5 μg of DNA mixture containing active pPKCα (0.2 μg), pδ-47MEKLuc (0.3 μg), pCMVCAT (0.5 μg), and Bluescript (BSSK) plasmid (0.5 μg) using FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's directions. Twenty-four hours post-transfection, cells were incubated in differentiation medium. Three days later, cells were harvested and assayed for luciferase and CAT activities as described previously (25Brasier A.R. Tate J.E. Habener J.F. BioTechniques. 1989; 7: 1116-1122PubMed Google Scholar). Alternatively, cells were transfected with pGal4Luc (0.6 μg), pCMVCAT (0.5 μg), and pGal4 (0.4 μg) or pGal4-Mgn (0.4 μg); differentiated; and then treated with buffer or drug (A23187 or ryanodine; 0.2 μm) to raise intracellular calcium levels. Forty-eight hours after drug treatment, cells were harvested and assayed for luciferase and CAT activities. Means ± S.E. were determined for samples from primary cultures. To determine differences in mean values of expression of myogenin and nAChR RNAs and myogenin protein, one-way analyses of variance were performed. If the F statistic of the analysis of variance showed significance, differences among means were detected using the Tukey-Kramer multiple comparisons post-hoc test. The level of significance was set a priori atp < 0.05. Values are expressed as means ± S.E. Muscle denervation induces myogenin and nAChR RNA expression, whereas electrical stimulation of denervated muscle suppresses both of these gene activities (14Neville C.M. Schmidt M. Schmidt J. Cell. Mol. Neurobiol. 1992; 12: 511-527Crossref PubMed Scopus (63) Google Scholar, 15Kostrominova T.Y. Macpherson P.C.D. Carlson B.M. Goldman D. Am. J. Physiol. 2000; 279: R179-R188Google Scholar, 26Duclert A. Piette J. Changeux J.P. Neuroreport. 1991; 2: 25-28Crossref PubMed Scopus (68) Google Scholar, 27Adams L. Carlson B.M. Henderson L. Goldman D. J. Cell Biol. 1995; 131: 1341-1349Crossref PubMed Scopus (106) Google Scholar). This effect of muscle activity on gene expression is thought to be mediated by increases in intracellular calcium (2Klarsfeld A. Laufer R. Fontaine B. Devillers-Thiery A. Dubreuil C. Changeux J.P. Neuron. 1989; 2: 1229-1236Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 3Adams L. Goldman D. J. Neurobiol. 1998; 35: 245-257Crossref PubMed Scopus (21) Google Scholar). In avian muscle, activity- and calcium-dependent suppression of myogenin and nAChR RNAs occurs within a few hours after the onset of stimulation (4Huang C.-F. Flucher B.E. Schmidt M.M. Stroud S.K. Schmidt J. Neuron. 1994; 13: 167-177Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 14Neville C.M. Schmidt M. Schmidt J. Cell. Mol. Neurobiol. 1992; 12: 511-527Crossref PubMed Scopus (63) Google Scholar). Although we had previously documented that increasing intracellular calcium can suppress nAChR gene expression in rat muscle (3Adams L. Goldman D. J. Neurobiol. 1998; 35: 245-257Crossref PubMed Scopus (21) Google Scholar, 10Walke W. Staple J. Adams L. Gnegy M. Chahine K. Goldman D. J. Biol. Chem. 1994; 269: 19447-19456Abstract Full Text PDF PubMed Google Scholar), we did not know how rapidly this response occurred or whether myogenin was regulated in a similar fashion. To examine the effects of calcium on myogenin and nAChR RNA expression in mammalian muscle, we assayed their RNAs at various times after raising intracellular calcium. Myogenin is a relatively unstable mRNA with a half-life of ∼20 min (28Edmondson D.G. Brennan T.J. Olson E.N. J. Biol. Chem. 1991; 266: 21343-21346Abstract Full Text PDF PubMed Google Scholar, 29Thayer M.J. Tapscott S.J. Davis R.L. Wright W.E. Lassar A.B. Weintraub H. Cell. 1989; 58: 241-248Abstract Full Text PDF PubMed Scopus (348) Google Scholar); and therefore, its level is thought to reflect its gene activity. In contrast, nAChR mRNAs are relatively stable and do not necessarily reflect rapid changes in gene expression (30Neville C. Schmidt M. Schmidt J. Neuroreport. 1991; 2: 655-657Crossref PubMed Scopus (11) Google Scholar). To obtain a more accurate reflection of nAChR gene activity, we assayed nAChR α-subunit hnRNA as well as mRNA levels. In general, hnRNAs are processed rapidly to remove noncoding intronic sequences from the primary RNA transcript prior to mRNA export (31Padgett R.A. Grabowski P.J. Konarska M.M. Seiler S. Sharp P.A. Annu. Rev. Biochem. 1986; 55: 1119-1150Crossref PubMed Google Scholar). Once mRNA is formed, a variety of factors can have an impact on its stability (32Wilusz C.J. Wormington M. Peltz S. Nat. Rev. 2001; 2: 237-246Crossref Scopus (634) Google Scholar). Consequently, the levels of hnRNA more accurately reflect rapid changes in transcription than do measurements of relatively long-lived mRNAs. In our experiments, rat primary myotubes were treated with A23187, a calcium ionophore; ryanodine, an activator of calcium release from the sarcoplasmic reticulum; or thapsigargin, an inhibitor of calcium ATPases. Within 6 h of treatment with either A23187 or ryanodine, the level of myogenin mRNA was reduced by at least 50%, but returned to control values by 48 h of drug treatment (Fig.1, A and B). In contrast, thapsigargin had little effect on myogenin RNA, yet suppressed nAChR hnRNA and mRNA (Fig. 1C). After treatment of cells with A23187, ryanodine, or thapsigargin, reductions in nAChR α-subunit hnRNA occurred within 6–12 h of drug stimulation; but unlike the myogenin response, further reductions were observed through 48 h of stimulation (Fig. 1). Although the time required to produce an initial reduction in mRNA tended to take longer, the changes observed in the nAChR α-subunit hnRNA were also reflected at the level of its mRNA (Fig. 1). The observation that myogenin RNA does not change significantly in response to thapsigargin treatment may indicate that the effects of calcium drugs on myogenin expression are nonspecific. However, we noted a reproducible, but statistically insignificant, 10–15% decrease in myogenin RNA levels at 6 h of thapsigargin treatment (Fig.1C). This small response may reflect the mechanism of thapsigargin action rather than a nonspecific effect of other calcium-elevating drugs. Unlike A23187 and ryanodine, which cause rapid and large changes in intracellular calcium, thapsigargin inhibits the ATPase responsible for calcium re-uptake by the sarcoplasmic reticulum. It is plausible that in an inactive myotube (TTX-treated), where depolarization-dependent release of calcium is blocked, thapsigargin would only inhibit the re-uptake of calcium leaking out of the sarcoplasmic reticulum. This would result in a much smaller increase in cytoplasmic calcium levels compared with ryanodine andA23187. This reduced elevation in calcium may be approaching the threshold of myogenin responsiveness. To ensure that the rapid effect of calcium stimulation was not limited to the nAChR α-subunit RNA, we also assayed for changes in nAChR γ- and ɛ-subunit RNAs after treatment with A23187 (Fig. 1D). Like the α-subunit mRNA, those encoding the γ- and ɛ-subunits were reduced relatively rapidly and were further reduced with continued exposure to the drug. Similar results were obtained when cells were treated with ryanodine and thapsigargin, except that the ɛ-subunit was less responsive to thapsigargin treatment.2 These data indicate that as in avian muscle, calcium-dependent suppression of myogenin and nAChR-encoding RNAs occurs relatively rapidly in mammalian muscle. However, the apparent dissociation between the return of myogenin RNA to pre-stimulus levels and the continued suppression of nAChR hnRNA and mRNA during extended periods of elevated calcium (Fig. 1,A and B) suggests a more complex regulatory pathway at work in mammalian muscle compared with that previously proposed for chick muscle (4Huang C.-F. Flucher B.E. Schmidt M.M. Stroud S.K. Schmidt J. Neuron. 1994; 13: 167-177Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 7Huang C.-F. Tong J. Schmidt J. Neuron. 1992; 9: 671-678Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 12Mendelzon D. Changeux J.P. Nghiem H.O. Biochemistry. 1994; 33: 2568-2575Crossref PubMed Scopus (32) Google Scholar). Based on studies in chick muscle, calcium is proposed to mediate its effects on nAChR gene expression via inactivation of myogenin function by PKC-dependent phosphorylation (12Mendelzon D. Changeux J.P. Nghiem H.O. Biochemistry. 1994; 33: 2568-2575Crossref PubMed Scopus (32) Google Scholar, 14Neville C.M. Schmidt M. Schmidt J. Cell. Mol. Neurobiol. 1992; 12: 511-527Crossref PubMed Scopus (63) Google Scholar). In addition, because myogenin is proposed to autoregulate its own gene, this suppression of myogenin function should be reflected in reduced myogenin gene expression and therefore RNA levels. Surprisingly, we found that although calcium could initially suppress myogenin RNA levels, this effect was transient. Therefore, myogenin may not participate in nAChR gene suppression in response to sustained elevated levels of intracellular calcium. Alternatively, myogenin protein function may be affected by this increased calcium that is not reflected in its RNA. To directly assay myogenin protein function, we employed a Gal4TKLuc reporter and a Gal4-Mgn fusion protein. The Gal4-Mgn fusion harbors the Gal4 DNA-binding domain fused to the N terminus of myogenin. The reporter Gal4TKLuc contains four Gal4-binding sites upstream of the minimal thymidine kinase promoter driving luciferase expression. Primary muscle cells were cotransfected with these vectors along with CMVCAT for normalization purposes. Transfected myotubes were then treated with either 0.2 μmA23187 or 0.2 μmryanodine for 48 h before harvesting cells for luciferase and CAT assays. These concentrations of drugs were previously shown to reduce nAChR α-subunit RNA expression by ∼50% (A23187) (10Walke W. Staple J. Adams L. Gnegy M. Chahine K. Goldman D. J. Biol. Chem. 1994; 269: 19447-19456Abstract Full Text PDF PubMed Google Scholar) and 80% (ryanodine) (3Adams L. Goldman D. J. Neurobiol. 1998; 35: 245-257Crossref PubMed Scopus (21) Google Scholar). Consistent with these results, we found that A23187and ryanodine suppressed myogenin-dependent reporter gene activation by 38 and 70%, respectively (Fig.2). When higher concentrations of drug were employed, larger decreases in Gal4-Mgn-dependent reporter
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