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J. Biol. Chem., Vol. 277, Issue 18, 15638-15646, May 3, 2002

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Protein Kinase C and Calcium/Calmodulin-activated Protein Kinase II (CaMK II) Suppress Nicotinic Acetylcholine Receptor Gene Expression in Mammalian Muscle

A SPECIFIC ROLE FOR CaMK II IN ACTIVITY-DEPENDENT GENE EXPRESSION^*

Peter Macpherson, Tatiana Kostrominova, Huibin Tang, and Daniel Goldman

From the Mental Health Research Institute and the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, October 12, 2001, and in revised form, January 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 ₂(). The nerve plays an important role in regulating the expression and distribution of nAChRs along the surface of the muscle fiber (reviewed in Ref. 1). 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 (2, 3). 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 (4), and extrajunctional expression of the nAChR genes is increased dramatically (5, 6). In contrast, when denervated muscle is electrically stimulated, intracellular calcium concentrations are elevated (4), and receptor expression is suppressed (6-8). Similarly, receptor expression is suppressed in TTX-treated myotubes when they are exposed to calcium-elevating drugs (2, 3, 9, 10). 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 (2, 7, 11). The proposed model of suppression involves depolarization-dependent activation of a calcium- and phospholipid-dependent PKC (4, 12). Activated PKC is proposed to phosphorylate myogenin (12, 13), a basic helix-loop-helix myogenic transcription factor that mediates high level nAChR gene expression in inactive muscle (14-18). This phosphorylation abrogates myogenin binding to target E-box sequences that regulate nAChR promoter activity (12, 13), 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 (2, 7, 11), there is little evidence supporting this regulatory mechanism in mammalian muscle (10). 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 (11). 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 -subunit gene promoter is robustly regulated by calcium/calmodulin-dependent protein kinase II (CaMK II) activity (19). CaMK II activity increases upon muscle depolarization and reduces binding of a myogenin-containing complex to the 47-bp activity-dependent enhancer of the -subunit gene. Furthermore, overexpression of a dominant-negative CaMK II in contracting primary rat myotubes increased nAChR -subunit promoter activity (19). 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 (2, 7, 11). 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- Rat primary myoblasts were isolated as described previously (20). 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.

Pharmacological Reagents-- 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.

Electrical Stimulation-- 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 (21). Data are presented for myotubes that were electrically stimulated for 6 h.

RNA Isolation and RNase Protection Assay-- 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 -, -, and -subunit RNAs were the same as those described by Chahine et al. (22). RNase protection assays were carried out as previously described (3). The probe for the nAChR -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 -subunit hnRNA, whereas measures of the 240-nucleotide fragment reflected changes in the nAChR -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 (7).² 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.

Subcellular Fractionation and Western Blots-- Cytosolic and membrane fractions from cultured myotubes were prepared by scraping cells from the dishes in homogenization buffer (10 mM Tris-HCl (pH 7.4), 10 mM MgCl_2, 150 mM NaCl, 1 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 µM each 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-PKC/ 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.

Vectors and Transfection Assays-- A constitutively active PKC isoform was created by deleting its inhibitory domain as previously described (23). p-47MEKLuc contains the 47-bp activity-dependent enhancer of the nAChR -subunit gene upstream of the minimal enkephalin promoter (24). 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 pPKC (0.2 µg), p-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 (25). 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.

Statistics-- 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 at p < 0.05. Values are expressed as means ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Calcium-dependent Regulation of nAChR and Myogenin RNAs-- Muscle denervation induces myogenin and nAChR RNA expression, whereas electrical stimulation of denervated muscle suppresses both of these gene activities (14, 15, 26, 27). This effect of muscle activity on gene expression is thought to be mediated by increases in intracellular calcium (2, 3). In avian muscle, activity- and calcium-dependent suppression of myogenin and nAChR RNAs occurs within a few hours after the onset of stimulation (4, 14). Although we had previously documented that increasing intracellular calcium can suppress nAChR gene expression in rat muscle (3, 10), 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 (28, 29); 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 (30). To obtain a more accurate reflection of nAChR gene activity, we assayed nAChR -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 (31). Once mRNA is formed, a variety of factors can have an impact on its stability (32). 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 -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 -subunit hnRNA were also reflected at the level of its mRNA (Fig. 1).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Time course of calcium-dependent suppression of myogenin and nAChR subunit RNAs. Rat primary myotubes were treated with calcium-elevating drugs for 0-48 h and assayed for changes in the levels of expression of Mgn and nAChR -subunit (Alpha) mRNAs and nAChR -subunit hnRNA (hnAlpha). Presented are results from representative RNase protection assays and mean values for Mgn RNA (gray bars), nAChR -subunit hnRNA (white bars), and nAChR -subunit mRNA (black bars) in myotubes after treatment with 1 µM A23187 (n = 3) (A), 1 µM ryanodine (n = 4) (B), and 100 nM thapsigargin (n = 4) (C). The mRNA responses of the nAChR -subunit (gray bars) and -subunit (black bars) were also evaluated after treatment with A23187 (n = 2) (D). All myotubes were treated with 2 µg/ml TTX during myotube formation and for the duration of the experiment. The medium containing calcium drugs was changed after 24 h of treatment. For each protection assay, the levels of the respective RNAs were normalized to the level of expression of GAPDH. Bars represent means ± S.E. *, p < 0.05, significantly different from controls (C).

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 and A23187. 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 -subunit RNA, we also assayed for changes in nAChR - and -subunit RNAs after treatment with A23187 (Fig. 1D). Like the -subunit mRNA, those encoding the - and -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 -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 (4, 7, 12).

Increased Intracellular Calcium Suppresses Myogenin-dependent Gene Activation-- 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 (12, 14). 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 µM A23187 or 0.2 µM ryanodine for 48 h before harvesting cells for luciferase and CAT assays. These concentrations of drugs were previously shown to reduce nAChR -subunit RNA expression by ~50% (A23187) (10) and 80% (ryanodine) (3). Consistent with these results, we found that A23187 and 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 gene expression were observed. However, these higher drug concentrations also reduced expression from the thymidine kinase promoter in a Gal4-Mgn-independent manner.³ These experiments suggest that even though myogenin RNA levels return to normal after a 48-h exposure to calcium-elevating drugs, myogenin protein transactivation function is reduced and may explain the reduced expression of nAChR genes at this time.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Increases in intracellular calcium inhibit myogenin-dependent transactivation. The Gal4TKLuc reporter plasmid, the Gal4 or Gal4-Mgn effector plasmid, and the pCMVCAT plasmid (used for normalization) were cotransfected into rat embryonic muscle cells as described under "Materials and Methods." Transfected cells were treated for 48 h with drugs that increase intracellular calcium (0.2 µM A23187 or 0.2 µM ryanodine). After 48 h of drug treatment, cells were harvested and assayed for luciferase and CAT activities. Note that the calcium drugs had a significant effect on the luciferase activity of cells cotransfected with Gal4-Mgn, but had no effect on cells cotransfected with the Gal4 DNA-binding domain alone. Promoter activity is reported as luciferase activity normalized to CAT activity. Bars represent means ± S.E. (n = 3). *, p < 0.05, significantly different from controls.

Phorbol Ester Stimulation of PKC Suppresses Myogenin and nAChR -Subunit Gene Expression-- Phorbol esters have been used in chick muscle to show that muscle activity suppresses myogenin and nAChR gene expression by a phorbol ester-responsive, PKC-dependent mechanism (2, 4, 7, 12). Whether a similar mechanism operates to regulate mammalian muscle nAChR gene expression in response to increased calcium is not known. Previously, phorbol esters were reported to have little effect on nAChR gene expression in mammalian muscle (10). This lack of response may reflect a difference in gene regulation between birds and mammals or may reflect the experimental design. One possibility is that PKC activation rapidly and transiently regulates nAChR gene expression that is not easily detected in the relatively stable nAChR mRNA transcripts. Therefore, we have readdressed this issue using probes for the relatively unstable myogenin mRNA and nAChR -subunit hnRNA. In addition, a more precise time course of evaluation was performed so that transient changes in gene expression could be revealed.

First, we determined whether active PKC can regulate nAChR promoter activity. For these experiments, we used a previously characterized -47MEKLuc reporter plasmid that harbors the 47-bp activity-dependent enhancer of the nAChR -subunit gene upstream of the minimal enkephalin promoter (24). The 47-bp enhancer confers calcium- and activity-dependent regulation to the minimal enkephalin promoter (3, 24). This regulation requires a single E-box residing in the 47-bp enhancer that binds myogenin (3, 19, 24). To bypass the transient activation of PKC by phorbol esters, we created a constitutively active PKC by deleting its regulatory domain (23). Cotransfection of primary muscle cells with active PKC and the nAChR -47MEKLuc reporter plasmid showed that PKC activity could suppress gene activation from the 47-bp enhancer (Fig. 3). Furthermore, although mutation of the single E-box (CACCTG) in the 47-bp enhancer to GCCCTG resulted in a significant reduction in promoter activity, the activity was still above background levels and allowed us to evaluate whether active PKC still suppressed -47MEK promoter activity. Indeed, mutation of this single E-box eliminated PKC-dependent regulation (Fig. 3), consistent with the idea that PKC mediates its effect via proteins that bind the E-box in the 47-bp enhancer of the -promoter.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Constitutively active PKC suppresses activation from the 47-bp activity-dependent enhancer of the nAChR -subunit gene. Rat primary myoblasts were cotransfected with the -47MEKLuc (wild-type) or E-box mutant -47MEKLuc reporter plasmid with or without active PKC and CMVCAT. Three days later, cells were harvested and assayed for luciferase and CAT activities. Note that active PKC caused a significant reduction in wild-type luciferase activity, but had no effect on the activity of the E-box mutant reporter. Because the E-box mutant reporter plasmid no longer contains a myogenic transcription factor-binding site, promoter activity is low and was expected to be independent of PKC regulation. Promoter activity is reported as luciferase activity normalized to CAT activity. Bars represent means ± SD (n = 3). *, p < 0.05, significantly different from controls ().

We were next interested in determining whether endogenous PKC can regulate mammalian muscle nAChR and myogenin expression. Phorbol esters such as PMA are potent activators of PKC and have been previously used to show that PKC participates in regulation of nAChR and myogenin gene expression in chick muscle (2, 7). We first examined the effect that PMA had on PKC activity in rat primary myotubes. PKC activity was assayed by measuring its translocation from cytoplasmic to membrane fractions. Within 1 h of PMA treatment, phorbol ester-sensitive PKCs were translocated from the cytoplasmic to the membrane fractions of primary myotubes (Fig. 4A). After 6 h of PMA stimulation, PKC appeared to be depleted from both cytoplasmic and membrane fractions (Fig. 4A). This temporal effect of PMA on PKC translocation is in accord with other experiments demonstrating that PKC activation is associated with its translocation to the membrane and that long-term exposure to PMA results in PKC depletion (33).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Phorbol ester-mediated myogenin and nAChR -subunit gene suppression. Rat primary myotubes were treated with the phorbol ester PMA (1 µM) for 1-24 h. At various times following PMA addition, myotubes were assayed for PKC translocation, Mgn protein and RNA, and nAChR -subunit mRNA (Alpha) and hnRNA (hnAlpha). A, representative Western blot showing PMA-induced PKC translocation from the cytoplasm (c) to the membrane (m). At least three different samples for each time point and drug treatment were evaluated on the Western blots. B, representative RNase protection assays and mean values for Mgn RNA (gray bars), nAChR -subunit hnRNA (white bars), and nAChR -subunit mRNA (black bars) (n = 3). C, representative Western blot and mean values of changes in myogenin protein (n = 5). All myotubes were treated with 2 µg/ml TTX during myotube formation and for the duration of the experiment. Bars represent means ± S.E. *, p < 0.05, significantly different from controls (0 h).

Finally, we determined whether these changes in PKC activity correlate with changes in myogenin or nAChR gene expression. During the first hour of PMA stimulation, no significant changes were observed in the levels of either myogenin mRNA or nAChR -subunit RNA (Fig. 4B). However, by 3 h of PMA treatment, myogenin RNA and -subunit hnRNA were significantly reduced (Fig. 4B). Maximum gene suppression was observed at 6 h of PMA stimulation, with myogenin mRNA and -subunit hnRNA reduced by ~65 and 80%, respectively (Fig. 4B). A small but discernible decrease in -subunit mRNA was observed after 6 h of PMA stimulation; and by 12 h, this RNA was decreased by almost 45%. We assume that the temporal delay and quantitative differences in -subunit mRNA relative to hnRNA reflect differences in nuclear processing and RNA stability.

Interestingly, myogenin protein levels were not significantly influenced by PMA (Fig. 4C). However, there was a consistent trend showing an ~25% reduction in myogenin protein at 3 and 6 h after PMA stimulation. This relatively modest effect of PMA on myogenin protein levels, but large effect on myogenin RNA, may be accounted for by differences in their turnover. At 12-24 h of PMA treatment, the levels of myogenin RNA and protein and nAChR RNA either returned to or started to approach control values (Fig. 4, B and C), likely reflecting PKC depletion from the cell (Fig. 4A). Therefore, similar to chick skeletal muscle, PMA-mediated activation of PKC can suppress nAChR and myogenin gene expression in rat skeletal muscle.

Dissociation of PKC Activity from Calcium-dependent nAChR and Myogenin Gene Suppression-- Although the above data showed that activation of PKC could lead to reduced levels of myogenin and nAChR RNAs in mammalian muscle, it was not clear whether PKC activation was also responsible for the reduction of these RNAs in response to increased intracellular calcium. To address this question, we first determined whether increasing intracellular calcium activates PKC in rat primary muscle cells. PKC activity was assayed by translocation from cytoplasmic to membrane fractions. Unlike the translocation observed after 1 h of PMA stimulation (Fig. 4A), a 1-h treatment with calcium drugs had no effect on PKC translocation (Fig. 5). Furthermore, even after 24 h of treatment with calcium drugs, there was no evidence of either PKC translocation or PKC depletion (Fig. 5).

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Elevated intracellular calcium does not cause PKC translocation. Rat primary myotubes were treated with A23187 (1 µM), thapsigargin (100 nM), or ryanodine (1 µM). Control cells were treated with buffer alone. After drug treatment at 1 and 24 h, cells were lysed and separated into cytoplasmic (C) and membrane (M) fractions. Presented are representative Western blots of cell fractions assayed with anti-phospho-PKC polyclonal antibody. Note that calcium drug treatment caused neither PKC translocation (control versus drug treatment) nor PKC depletion (1 h versus 24 h). For each time point and drug treatment, at least three different samples were evaluated.

To further assess whether PKC activity is a necessary component of calcium-induced suppression of myogenin and nAChR RNAs, we performed experiments in which cells were either depleted of PKC or pretreated with PKC inhibitors and then stimulated with the calcium ionophore A23187. Depletion of PKC was accomplished by 24-h treatment with PMA (Fig. 4A). To inhibit PKC, cells were pretreated for 1 h with staurosporine; a potent kinase inhibitor that has some PKC specificity; GF109203X, a highly specific PKC, PKC, PKC, and PKC isoform inhibitor; or Go6983, a specific inhibitor of PKC, PKC, PKC, and PKC isoforms (34). PMA, staurosporine, and GF109203X have been used previously to effectively block suppression of nAChR expression in avian skeletal muscle (2, 7, 12). We were particularly interested in the effects of Go6983 because it will inhibit the atypical PKC isoform that was recently proposed to participate in activity-dependent suppression of nAChR gene expression (11).

Surprisingly, neither depletion nor inhibition of PKC was able to block the reductions in myogenin mRNA or -subunit hnRNA produced by A23187 (Fig. 6). In fact, PMA-mediated depletion of PKC appeared to augment, rather than block, the suppressive effect of A23187 on nAChR -subunit mRNA. Similar results were obtained when cells were treated with ryanodine rather than A23187.² Of the PKC inhibitors used in this study, only staurosporine increased -subunit hnRNA on its own (Fig. 6). Because staurosporine is a relatively nonspecific kinase inhibitor and none of the other PKC inhibitors influenced nAChR gene expression in response to muscle activity, it is most likely that the effect of staurosporine is PKC-independent.

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Suppression of myogenin and nAChR -subunit RNAs during treatment of myotubes with A23187 is not prevented by PKC depletion or inhibition. Rat primary myotubes were pretreated for 24 h with PMA (1 µM) to deplete cells of PKC or for 1 h with staurosporine (Staur; 20 nM), GF109203X (GF; 250 nM), or Go6983 (600 nM) to inhibit PKC activity. Myotubes were then treated with the calcium ionophore A23187 (1 µM) for 6 h. Presented are results from representative RNase protection assays and mean values for Mgn RNA (gray bars), nAChR -subunit hnRNA (hnAlpha; white bars), and nAChR -subunit mRNA (Alpha mRNA; black bars). All myotubes were treated with 2 µg/ml TTX during myotube formation and for the duration of the experiment. Bars represent means ± S.E. (n = 4). *, p < 0.05, significantly different from controls.

Dissociation of PKC Activity from Activity-dependent nAChR and Myogenin Gene Suppression-- In chick muscle, PMA-mediated PKC depletion blocks the suppressive effects of muscle activity on nAChR and myogenin gene expression (2, 7). It is generally assumed that increased intracellular calcium mediates the effects of muscle activity, but it is also possible that the sustained increase in calcium used in our studies does not reflect the effects of muscle depolarization. Therefore, we examined the effect that PMA has on activity-dependent regulation of nAChR gene expression in primary rat myotubes. Electrical stimulation of myotubes for 6 h resulted in a 60% reduction in myogenin mRNA, an 80% reduction in -subunit hnRNA, and a 40% reduction in -subunit mRNA (Fig. 7). PMA pretreatment for 24 h did not prevent activity-dependent suppression of myogenin and nAChR RNAs (Fig. 7). Consequently, in mammalian muscle, it appears that although activated PKC can reduce levels of myogenin and nAChR RNAs (Figs. 3 and 4), a PMA-regulated PKC is not required as an intermediary during calcium- or activity-dependent suppression of these molecules (Figs. 6 and 7). We confirmed this result by also evaluating the effects of staurosporine, GF109203X, and Go6983 on activity-dependent suppression of nAChR and myogenin RNAs. None of these PKC inhibitors were able to block the effects of muscle activity on nAChR and myogenin RNA expression (Fig. 7).

View larger version (45K): [in this window] [in a new window]   Fig. 7.   Suppression of myogenin and nAChR -subunit RNAs during electrical stimulation of myotubes is not prevented by PKC depletion or inhibition. Rat primary myotubes were pretreated for 24 h with PMA (1 µM) to deplete cells of PKC or for 1 h with staurosporine (Staur; 20 nM), GF109203X (GF; 250 nM), or Go6983 (600 nM) to inhibit PKC activity and then electrically stimulated for 6 h. All myotubes were treated with 2 µg/ml TTX during myotube formation and for the duration of the experiment with the exception of electrically stimulated myotubes, which were washed three times with TTX-free medium prior to the onset of electrical stimulation. Presented are results from representative RNase protection assays and mean values for Mgn RNA (gray bars), nAChR -subunit hnRNA (hnAlpha; white bars), and nAChR -subunit mRNA (Alpha mRNA; black bars) RNA. Bars represent means ± S.E. (n = 2). *, p < 0.05, significantly different from controls.

CaMK II-dependent Regulation of nAChR Gene Expression-- Because neither depletion nor inhibition of PKC was able to block calcium- or activity-dependent suppression of myogenin or nAChR gene expression, we investigated the role of other signaling molecules in this process. One candidate regulatory molecule is CaMK II. We recently showed that overexpression of an activated version of CaMK II can suppress nAChR -subunit promoter activity, whereas overexpression of a dominant-negative version of CaMK II can partially block activity-dependent suppression of this promoter (19). Although this effect may be specific to the -promoter, we thought it was likely that increased CaMK II activity would suppress other subunit genes that are also regulated by muscle depolarization.

KN-93 is a potent and specific CaMK II inhibitor (35). Surprisingly, KN-93 had only a minor effect on A23187- or thapsigargin-mediated suppression of nAChR hnRNA or myogenin RNA (A23187 data shown in Fig. 8A). In contrast, pretreatment of electrically stimulated myotubes with KN-93 resulted in a significant block (~50%) of nAChR -subunit hnRNA suppression (Fig. 8B) that is typically produced during 6 h of electrical stimulation. This result further implicates CaMK II as a mediator of activity-dependent regulation of nAChR. However, the observation that KN-93 only partially blocks the suppression produced by electrical stimulation suggests that activity-dependent regulation involves a considerable degree of complexity. Indeed, if activity-dependent regulation does involve multiple signals, one possible explanation for the difference between the KN-93 responses after treatment with A23187 and electrical stimulation may be that continuous exposure to A23187 results in more effective activation of additional signaling pathways that mask an effect of KN-93.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   The CaMK II inhibitor KN-93 differentially affects calcium- and activity-dependent gene expression. Rat primary myotubes were pretreated with KN-93 (5 µM) for 1 h prior to a 6-h exposure to A23187 (1 µM) or electrical stimulation. A, results from representative RNase protection assays and mean values for Mgn RNA (gray bars), nAChR -subunit hnRNA (hnAlpha; white bars), and nAChR -subunit mRNA (Alpha mRNA; black bars) from myotubes treated with buffer (Control), A23187 (A23), KN-93, or a combination of KN-93 and A23187 (KN93/A23) (n = 4). B, results from representative RNase protection assays and mean values for Mgn RNA (gray bars), nAChR -subunit hnRNA (white bars), and nAChR -subunit mRNA (black bars) obtained from myotubes that were electrically stimulated (Estim) or pretreated with KN-93 and then electrically stimulated (KN93/Estim) (n = 5). All myotubes were treated with 2 µg/ml TTX during myotube formation and for the duration of the experiment with the exception of electrically stimulated myotubes, which were washed three times with TTX-free medium prior to the onset of electrical stimulation. Bars represent means ± S.E. *, p < 0.05, significantly different from controls.

In addition to KN-93, other kinase inhibitors we tested included U0126 for ERK inhibition, SB202190 for p38 inhibition, wortmannin and rapamycin for phosphatidylinositol 3-kinase inhibition, and genistein for tyrosine kinase inhibition. In each case, the inhibitor appeared to have no effect on calcium- or activity-dependent suppression of nAChR or myogenin RNA.²

	DISCUSSION

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The studies reported here address the mechanism by which muscle calcium and depolarization regulate nAChR gene expression in mammals. Muscle activity regulates expression of many genes and is crucial to proper formation of the neuromuscular junction (1). In addition, muscle activity has a profound effect on expression of many structural and contractile proteins. Much of this regulation resides at the level of gene expression. Therefore, it is important to identify the mechanism by which muscle activity mediates these changes.

Our current understanding of the mechanism by which nAChR gene expression is regulated by muscle activity comes largely from studies of avian muscle. These investigations have identified PKC as an important regulator of nAChR gene expression. In chick muscle, PKC is activated by muscle depolarization (2, 7) and phosphorylates myogenin (12). This phosphorylation is proposed to inactivate myogenin by blocking its ability to bind target E-box sequences (13), ultimately resulting in reduced nAChR gene expression. Surprisingly, there are no reports suggesting that PKC mediates calcium- and activity-dependent nAChR gene expression in mammalian muscle. Therefore, we set out to test whether the model that has been proposed for activity-dependent gene expression in chick muscle also explains changes in nAChR gene expression in mammalian muscle. We report here that during sustained elevations in calcium and muscle activity, mammalian muscles utilize a mechanism of regulation independent of PKC to control nAChR gene expression. In addition, our data are consistent with and extend recent results showing that CaMK II participates in mediating activity-dependent regulation of the nAChR -subunit gene promoter (19).

Myogenin autoregulation is an important component of the current model of nAChR gene regulation by calcium and muscle activity (12, 14). In this model, PKC-dependent phosphorylation of myogenin blocks myogenin function, resulting in reduced levels of myogenin RNA and protein expression, ultimately reducing nAChR gene expression. Interestingly, we found that elevated calcium only transiently suppressed myogenin RNA, whereas nAChR RNAs were suppressed throughout the time course of the experiment (Fig. 1). These results are at odds with an autoregulatory mechanism of myogenin expression accounting for reduced nAChR gene expression, but do not preclude myogenin mediating nAChR gene expression.

It is worth noting that we are not the first to obtain data that are inconsistent with myogenin autoregulation as the mechanism for control of myogenin gene expression. Two different transgenic mouse models have demonstrated that myogenin autoregulation is not an essential component of myogenin gene regulation (18, 36). In one case, a myogenin lacZ transgene appeared to be robustly activated during development in myogenin null mice (36); and in the other case, transgenic overexpression of myogenin had no effect on the level of endogenous myogenin transcription (18).

If myogenin mediates nAChR gene expression, and increased calcium does not decrease myogenin protein levels, yet suppresses nAChR gene activity, then a post-transcriptional mechanism of myogenin regulation must exist. In fact, there is substantial literature demonstrating that post-transcriptional modifications of myogenic transcription factors regulate their activity (12, 13, 19, 37, 38). Notably, Li et al. (13) and Tang et al. (19) showed that phosphorylation of myogenin by PKC and CaMK II, respectively, decreases myogenin activity through decreased DNA binding. Here, we used a Gal4-Mgn fusion protein to show that calcium can also regulate myogenin-dependent transactivation of reporter gene expression (Fig. 2). Therefore, as reported for chick muscle, post-transcriptional modification of myogenin appears to participate in calcium-dependent control of nAChR gene expression in mammals.

Because PKC-dependent phosphorylation of myogenin plays a key role in current models of nAChR gene regulation (2, 7, 12, 14), we explored whether PKC participates in regulating myogenin and nAChR gene expression in mammalian muscle. Consistent with PKC-dependent regulation of these genes, we found that a constitutively active version of PKC can suppress nAChR -promoter activity and that this effect is mediated by a critical E-box sequence within the activity-dependent enhancer of the -promoter (Fig. 3). This result was confirmed in a more in vivo setting using PMA to activate endogenous PKC in rat primary myotubes, which also reduced nAChR and myogenin RNA expression (Fig. 4). The observation that myogenin protein is only slightly affected by PKC activation (Fig. 4), whereas its mRNA is dramatically reduced, may simply reflect differences in their stability.

Although PKC can suppress mammalian muscle nAChR and myogenin gene expression, a direct link between PKC and calcium- and/or activity-dependent gene regulation remained to be established. Our data tend to argue against this possibility. First, we were not able to observe a translocation of PKC from the cytoplasm to the membrane in response to increased intracellular calcium (Fig. 5). Second, PMA-mediated depletion of PKC did not influence calcium-dependent gene expression (Fig. 6). Third, pharmacological inhibition of PKC had no significant effect on calcium-dependent gene expression (Fig. 6). Fourth, neither PKC depletion nor inhibition blocked the reductions in nAChR and myogenin gene expression produced by electrical stimulation (Fig. 7). These results suggest that although activated PKC can suppress myogenin and nAChR gene activities, it does not appear to play a physiological role in the calcium- or activity-dependent regulation of these genes in mammalian muscle.

We recently reported that overexpression of a constitutively active CaMK II can suppress cotransfected nAChR -subunit promoter activity via inhibition of myogenin binding to the 47-bp enhancer of the -promoter (19). Those results prompted us to examine a role for CaMK II in mediating the effects of calcium and muscle activity on endogenous nAChR gene expression. Based on our experience with PKC, we also felt that it was important to determine whether endogenous CaMK II activity participates in mediating the effects of calcium and muscle activity on nAChR and myogenin gene expression. Surprisingly, inhibition of CaMK II with the potent and specific inhibitor KN-93 (35) had no significant effect on calcium-induced suppression of nAChR and myogenin gene expression, yet significantly inhibited the suppression produced by muscle depolarization (Fig. 7).

The reason for this difference is not known, but may suggest that the sustained increases in intracellular calcium, brought about by pharmacological agents, activate different or additional signaling cascades compared with muscle depolarization. In addition, the incomplete block of muscle activity by CaMK II inhibition suggests that multiple signaling cascades may participate in controlling gene expression in response to muscle depolarization. Indeed, we have previously shown that a cAMP-dependent signal transduction cascade can block the effects of muscle activity on nAChR and myogenin gene expression (22). These results are not surprising in light of recent experiments demonstrating that specific features of calcium influx into the cytoplasm may dictate the nuclear response. The cellular location of calcium influx and the amplitude, kinetics, or frequency of the cytoplasmic rise in calcium appear to direct specific nuclear responses by activating specific signal transduction cascades (39, 40-42). For example, differences in calcium influx amplitude and kinetics in B-lymphocytes lead to different patterns of transcription factor activation and gene expression (42). Our results showing a role for CaMK II in activity-dependent gene expression, but not in calcium ionophore-mediated gene expression, may represent another example.

	FOOTNOTES

^* This work was supported by NINDS Grant RO1 NS25153 and NIA Grant PO1 AG10821 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Mental Health Research Inst., University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109. Tel.: 734-936-2057; Fax: 734-647-4130; E-mail: neuroman@umich.edu.

Published, JBC Papers in Press, February 27, 2002, DOI 10.1074/jbc.M109864200

² P. Macpherson and D. Goldman, unpublished data.

³ T. Kostrominova and D. Goldman, unpublished data.

	ABBREVIATIONS

The abbreviations used are: nAChRs, nicotinic acetylcholine receptors; TTX, tetrodotoxin; PKC, protein kinase C; CaMK II, calcium/calmodulin-dependent protein kinase II; PMA, phorbol 12-myristate 13-acetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyltransferase; Mgn, myogenin; ERK, extracellular signal-regulated kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES